Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise.

PubMed

Soliman, Taha; Yang, Sung-Yin; Yamazaki, Tomoko; Jenke-Kodama, Holger

2017-01-01

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil ® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin ® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P  < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

RNA sample preparation applied to gene expression profiling for the horse biological passport.

PubMed

Bailly-Chouriberry, Ludovic; Baudoin, Florent; Cormant, Florence; Glavieux, Yohan; Loup, Benoit; Garcia, Patrice; Popot, Marie-Agnès; Bonnaire, Yves

2017-09-01

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgene® Blood RNA Kit for horse gene expression analysis in blood collected on PAXgene® tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Forensic interlaboratory evaluation of the ForFLUID kit for vaginal fluids identification.

PubMed

Giampaoli, Saverio; Alessandrini, Federica; Berti, Andrea; Ripani, Luigi; Choi, Ajin; Crab, Roselien; De Vittori, Elisabetta; Egyed, Balazs; Haas, Cordula; Lee, Hwan Young; Korabecná, Marie; Noel, Fabrice; Podini, Daniele; Tagliabracci, Adriano; Valentini, Alessio; Romano Spica, Vincenzo

2014-01-01

Identification of vaginal fluids is an important step in the process of sexual assaults confirmation. Advances in both microbiology and molecular biology defined technical approaches allowing the discrimination of body fluids. These protocols are based on the identification of specific bacterial communities by microfloraDNA (mfDNA) amplification. A multiplex real time-PCR assay (ForFLUID kit) has been developed for identifying biological fluids and for discrimination among vaginal, oral and fecal samples. In order to test its efficacy and reliability of the assay in the identification of vaginal fluids, an interlaboratory evaluation has been performed on homogeneous vaginal swabs. All the involved laboratories were able to correctly recognize all the vaginal swabs, and no false positives were identified when the assay was applied on non-vaginal samples. The assay represents an useful molecular tool that can be easily adopted by forensic geneticists involved in vaginal fluid identification. Copyright © 2013 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods.

PubMed

Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N; Hoflund, Anders; Mogensen, Helle S; Hansen, Anders J; Morling, Niels

2013-05-01

The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were processed. The highest DNA recovery was obtained with the PrepFiler Express kit on an AutoMate Express while the lowest DNA recovery was obtained using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen. Extraction using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen resulted in the lowest percentage of PCR inhibition (0%) while extraction using manual Chelex resulted in the highest percentage of PCR inhibition (51%). The largest number of reportable STR-profiles was obtained with DNA from samples extracted with the PrepFiler Express kit (75%) while the lowest number was obtained with DNA from samples extracted using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen (41%). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Paternity testing in case of brother-sister incest.

PubMed

Macan, Marijana; Uvodić, Petra; Botica, Vladimir

2003-06-01

We performed a paternity test in a case of incest between brother and sister. DNA from blood samples of the alleged parents and their two children was obtained with Chelex DNA extraction method and quantified with Applied Biosystems QuantiBlot quantitation kit. Polymerase chain reaction (PCR) amplification of DNA samples was performed with AmpFlSTR SGM Plus PCR amplification kit and GenePrint PowerPlex PCR amplification kit. The amplified products were separated and detected by using the Perkin Elmer's ABI PRISM trade mark 310 Genetic Analyser. DNA and data analysis of 17 loci and Amelogenin confirmed the suspicion of brother-sister incest. Since both children had inherited all of the obligate alleles from the alleged father, we could confirm with certainty of 99.999999% that the oldest brother in the family was the biological father of both children. Calculated data showed that even in a case of brother-sister incest, paternity could be proved by the analysis of Amelogenin and 17 DNA loci.

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

PubMed Central

Buttner, Mark P.; Cruz, Patricia; Stetzenbach, Linda D.; Klima-Comba, Amy K.; Stevens, Vanessa L.; Emanuel, Peter A.

2004-01-01

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m2 for wet sampling and 100.5 ± 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site. PMID:15574898

Knowledge, actual and potential use of HIV self-sampling testing kits among MSM recruited in eight European countries.

PubMed

Hoyos, J; Maté, T; Indave, B I; Agustí, C; Chanos, S; Pichon, F; Kuske, M; Cigan, B; Fuertes, R; Ooms, L; Stefanescu, R; Cabeza de Vaca, C; Arranz, B; de la Fuente, L; Belza, M J

2018-02-01

To describe the knowledge as well as current and potential use of self-sampling kits among men who have sex with men (MSM) and to analyse their preferred biological sample and result communication method. We analyse data of MSM of HIV negative or unknown serostatus from an online survey conducted in eight countries (Belgium, Denmark, Germany, Greece, Portugal, Romania, Slovenia and Spain) between April and December 2016. It was advertised mainly in gay dating websites. We conduct a descriptive analysis of the main characteristics of the participants, and present data on indicators of knowledge, use and potential use of HIV self-sampling as well as their preferences regarding blood or saliva sample and face or non-face-to-face result communication by country of residence. A total of 8.226 participants of HIV negative or unknown serostatus were included in the analysis. Overall, 25.5% of participants knew about self-sampling (range: 18.8-47.2%) and 1.1% had used it in the past (range: 0.3-8.9%). Potential use was high, with 66.6% of all participants reporting that they would have already used it if available in the past (range: 62.1-82.1%). Most (78.6%) reported that they would prefer using a blood-based kit, and receiving the result of the test through a non-face-to-face-method (70.8%), even in the case of receiving a reactive result. The high potential use reported by MSM recruited in eight different European countries suggests that self-sampling kits are a highly acceptable testing methodology that could contribute to the promotion of HIV testing in this population. © 2018 British HIV Association.

Development of an add-on kit for scanning confocal microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Guo, Kaikai; Zheng, Guoan

2017-03-01

Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.

High-quality and -quantity DNA extraction from frozen archival blood clots for genotyping of single-nucleotide polymorphisms.

PubMed

Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke; Vogel, Ulla; Andersen, Paal Skytt

2013-06-01

The recovery of biological samples for genetic epidemiological studies can be cumbersome. Blood clots are routinely collected for serological examinations. However, the extraction of DNA from blood clots can be difficult and often results in low yields. The aim was to compare the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. Serum tubes with clotted blood were stored at -20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp DNA Micro kit (Qiagen), and the Nucleospin 96 Blood kit (Macherey-Nagel). Furthermore, blood clots from 1055 inflammatory bowel disease patients were purified using the Maxwell 16 Blood purification kit (Promega). The DNA was extracted according to the manufacturers` instructions and real-time PCR and the A(260)/A(280) ratio were used to evaluate the quality of the extracted DNA. The highest DNA yield was obtained by the Maxwell 16 Blood purification kit (Promega) with a median of 4.90 μg (range 0.8-25 μg) pr 300 μL total blood. PureGene with glycogen (Qiagen) had the second highest yield with a median of 0.65 μg (range 0.5-2.6 μg) pr 300 μL total blood. The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots, even after prolonged storage. The recovered DNA served as a reliable PCR template for single-nucleotide polymorphism assays.

Kits in Motion

ERIC Educational Resources Information Center

Gee, Maureen

1975-01-01

Discusses three kits developed by museums in British Columbia for use in rural classrooms. The science kit on marine biology consists of modules which included specimens, books, audiovisual materials and student activities. (BR)

Deployment of the DosiKit System Under Operational Conditions: Experience From a French Defense National Nuclear Exercise.

PubMed

Entine, F; Bensimon Etzol, J; Bettencourt, C; Dondey, M; Michel, X; Gagna, G; Gellie, G; Corre, Y; Ugolin, N; Chevillard, S; Amabile, J-C

2018-07-01

Estimation of the dose received by accidentally irradiated victims is based on a tripod: clinical, biological, and physical dosimetry. The DosiKit system is an operational and mobile biodosimetry device allowing the measurement of external irradiation directly on the site of a radiological accident. This tool is based on capillary blood sample and hair follicle collection. The aim is to obtain a whole-body and local-surface dose assessment. This paper is about the technical evaluation of the DosiKit; the analytical process and scientific validation are briefly described. The Toulon exercise scenario was based on a major accident involving the reactor of a nuclear attack submarine. The design of the scenario made it impossible for several players (firefighters, medical team) to leave the area for a long time, and they were potentially exposed to high dose rates. The DosiKit system was fully integrated into a deployable radiological emergency laboratory, and the response to operational needs was very satisfactory.

Comparison of Methods for miRNA Extraction from Plasma and Quantitative Recovery of RNA from Cerebrospinal Fluid

PubMed Central

McAlexander, Melissa A.; Phillips, Maggie J.; Witwer, Kenneth W.

2013-01-01

Interest in extracellular RNA (exRNA) has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific exRNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Toward these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and quantitative recovery of RNA is observed from increasing volumes of cerebrospinal fluid. PMID:23720669

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: might c-KIT represent a therapeutic target?

PubMed

Giantin, M; Aresu, L; Aricò, A; Gelain, M E; Riondato, F; Martini, V; Comazzi, S; Dacasto, M

2013-04-15

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials. Copyright © 2013 Elsevier B.V. All rights reserved.

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Analysis of Platelet-Rich Plasma Extraction

PubMed Central

Fitzpatrick, Jane; Bulsara, Max K.; McCrory, Paul Robert; Richardson, Martin D.; Zheng, Ming Hao

2017-01-01

Background: Platelet-rich plasma (PRP) has been extensively used as a treatment in tissue healing in tendinopathy, muscle injury, and osteoarthritis. However, there is variation in methods of extraction, and this produces different types of PRP. Purpose: To determine the composition of PRP obtained from 4 commercial separation kits, which would allow assessment of current classification systems used in cross-study comparisons. Study Design: Controlled laboratory study. Methods: Three normal adults each donated 181 mL of whole blood, some of which served as a control and the remainder of which was processed through 4 PRP separation kits: GPS III (Biomet Biologics), Smart-Prep2 (Harvest Terumo), Magellan (Arteriocyte Medical Systems), and ACP (Device Technologies). The resultant PRP was tested for platelet count, red blood cell count, and white blood cell count, including differential in a commercial pathology laboratory. Glucose and pH measurements were obtained from a blood gas autoanalyzer machine. Results: Three kits taking samples from the “buffy coat layer” were found to have greater concentrations of platelets (3-6 times baseline), while 1 kit taking samples from plasma was found to have platelet concentrations of only 1.5 times baseline. The same 3 kits produced an increased concentration of white blood cells (3-6 times baseline); these consisted of neutrophils, leukocytes, and monocytes. This represents high concentrations of platelets and white blood cells. A small drop in pH was thought to relate to the citrate used in the sample preparation. Interestingly, an unexpected increase in glucose concentrations, with 3 to 6 times greater than baseline levels, was found in all samples. Conclusion: This study reveals the variation of blood components, including platelets, red blood cells, leukocytes, pH, and glucose in PRP extractions. The high concentrations of cells are important, as the white blood cell count in PRP samples has frequently been ignored, being considered insignificant. The lack of standardization of PRP preparation for clinical use has contributed at least in part to the varying clinical efficacy in PRP use. Clinical Relevance: The variation of platelet and other blood component concentrations between commercial PRP kits may affect clinical treatment outcomes. There is a need for standardization of PRP for clinical use. PMID:28210651

Population genetic analyses of the Powerplex(®) Fusion kit in a cosmopolitan sample of Chubut Province (Patagonia Argentina).

PubMed

Parolin, María Laura; Real, Luciano E; Martinazzo, Liza B; Basso, Néstor G

2015-11-01

Allele frequencies and forensic parameters for 22 autosomal STR loci and DYS391 locus included in the PowerPlex(®) Fusion System kit were estimated in a sample of 770 unrelated individuals from Chubut Province, southern Patagonia. No significant deviations from Hardy-Weinberg equilibrium were observed after Bonferroni's correction. The combined power of discrimination and the combined probability of exclusion were >0.999999 and 0.999984, respectively. Comparisons with other worldwide populations were performed. The MDS obtained show a close biological relation between Chubut and Chile. The estimated interethnic admixture supports a high Native American contribution (46%) in the population sample of Chubut. These results enlarge the Argentine databases of autosomal STR and would provide a valuable contribution for identification tests and population genetic studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Comparison of three DNA extraction kits to establish maximum yield and quality of coral-associated microbial DNA

USGS Publications Warehouse

Baker, Erin J.; Kellogg, Christina A.

2014-01-01

Coral microbiology is an expanding field, yet there is no standard DNA extraction protocol. Although many researchers depend on commercial extraction kits, no specific kit has been optimized for use with coral samples. Both soil and plant DNA extraction kits from MO BIO Laboratories, Inc., have been used by many research groups for this purpose. MO BIO recently replaced their PowerPlant® kit with an improved PowerPlantPro kit, but it was unclear how these changes would affect the kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit.

Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.

PubMed

Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

2013-07-01

Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpFℓSTR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples.

Current molecular profile of juvenile nasopharyngeal angiofibroma: First comprehensive study from India.

PubMed

Pandey, Praveen; Mishra, Anupam; Tripathi, Ashoak Mani; Verma, Veerendra; Trivedi, Ritu; Singh, Hitendra Prakash; Kumar, Sunil; Patel, Brijesh; Singh, Vinay; Pandey, Shivani; Pandey, Amita; Mishra, Subhash Chandra

2017-03-01

An attempt is made to analyze the molecular behavior of juvenile nasopharyngeal angiofibroma (JNA). Case Series METHODS: Quantification of mRNAs expression was undertaken through real-time polymerase chain reaction in JNA (9-24) samples for VEGF-A, basic fibroblast growth factor (b-FGF), platelet-derived growth factor PDGF-A, KIT proto-oncogene receptor tyrosine kinase (c-Kit), Avian myelomatosis viral oncogene homolog (c-Myc), Harvey rat sarcoma viral oncogene homolog (H-Ras), tumor suppressor gene TP53, and androgen receptor and interleukin 6 (IL-6). The β-catenin expression was evaluated by western blot in 16 samples. Nasal polyp was taken as control. A significantly increased (P < 0.01) expression of c-myc, VEGFA, bFGF, PDGFA, c-kit, and TP53 was seen, along with enhanced expression of β-catenin. A massive enhancement of H-Ras expression was seen for the first time. Androgen receptor expression was no different, whereas IL-6 despite showing upregulation trend was not significant. The enhanced expressions of various markers suggest their potential role in JNA. Although the biological significance of c-kit, c-myc, and one of the novel markers H-Ras has yet to be defined, their significant expression may have a therapeutic importance. NA. Laryngoscope, 127:E100-E106, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples.

PubMed

Holmes, Amy S; Houston, Rachel; Elwick, Kyleen; Gangitano, David; Hughes-Stamm, Sheree

2018-05-01

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate.

PubMed

Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscilla Emiko; Palmieri, Chiara; Laufer-Amorim, Renée

2017-12-06

c-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines. Eleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive. The present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

Clinical forensic sample collection techniques following consensual intercourse in volunteers - cervical canal brush compared to conventional swabs.

PubMed

Joki-Erkkilä, Minna; Tuomisto, Sari; Seppänen, Mervi; Huhtala, Heini; Ahola, Arja; Rainio, Juha; Karhunen, Pekka J

2014-10-01

The purpose of the research was to evaluate gynecological evidence collection techniques; the benefit of cervical canal brush sample compared to vaginal fornix and cervical swab samples and the time frame for detecting Y-chromosomal material QiAmp DNA Mini Kit(®) and Quantifiler Y Human Male DNA Quantification Kit(®) in adult volunteers following consensual intercourse. Eighty-four adult female volunteers following consensual intercourse were recruited for the study. By combining all sample collecting techniques, 81.0% of the volunteers were Y-DNA positive. Up to 60 h the conventional swab sampling techniques detected more Y-DNA positive samples when compared to the brush technique. However, after 60 h, the cervical canal brush sample technique showed its benefit by detecting 27.3% (6/22) of Y-DNA positive samples, which were Y-DNA negative in both conventional swab sampling techniques. By combining swab and brush techniques, 75% of the volunteers were still Y-DNA positive in 72-144 post-coital hours. The rate of measurable Y-DNA decreased approximately 3% per hour. Despite reported consensual intercourse, 6.8% (3/44) of volunteers were Y-DNA negative within 48 h. Y-DNA was not detected after 144 post-coital hours (6 days). In conclusion, the brush as a forensic evidence collection method may provide additional biological trace evidence from the cervical canal, although the best biological trace evidence collection can be obtained by combining all three sampling techniques. The time frame for gynecological forensic evidence sample collection should be considered to be at least a week if sexual violence is suspected. Copyright © 2014 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Evaluation of digital PCR for absolute RNA quantification.

PubMed

Sanders, Rebecca; Mason, Deborah J; Foy, Carole A; Huggett, Jim F

2013-01-01

Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.

[Membrane-filtration immunoassay: reagents, methods and the diagnostic and technical means for detection].

PubMed

Khramov, E N; Osin, N S; Pomelova, V G; Vikha, I V; Bychenkova, T A; Smirnova, V G; Grakina, G I; Kas'ianova, T A

1999-01-01

The comprehensive development of dot-EIA made at the State Research Institute of Biological Instrument-Making Industry has provided devices KIMF-02 and KIMF-03), a base of chemical reagents, immunoassays, test systems for detection of a wide range of causative agents of viral and bacterial infections, that of serodiagnosis of their related diseases. The KIMF-02 kit has undergone engineering and medical tests and recommended for the Ministry of Health of the Russian Federation to produce them in stock. The kit includes all required for analysis even in an ill-equipped laboratory, a set of attached agents ensures a valid visual recording of results. The developed procedures and test systems allow the immunoassay to be as sensitive as TIFA; however, they are laborious and much simpler in design. The simple and rapid procedures of dot-EIA are recommended for incorporation into the a package of laboratory methods for verification of the accumulation of virus-specific antigens in various biological substrata, environmental samples, for control of the activity of antigens and antibodies used in serological tests, for detection of specific antigens in the clinical samples, and for serodiagnosis of infections.

[Comparison and evaluation of the Binax EIA and Biotest EIA urinary antigen kits for detection of Legionella pneumophila antigen in urine samples].

PubMed

Rastawicki, Waldemar; Rokosz, Natalia; Jagielski, Marek

2011-01-01

The Binax and the Biotest urinary antigen kits for detection of L. pneumophila antigen were compared by testing of selected 67 urine samples obtained from EWGLI as reference samples in External Quality Assessment Scheme. Thirty nine were positive with the Binax kit (100% of sensitivity), and 33 were positive with the Biotest (84.6% of sensitivity). The test specificities were 100% for the both kits. It was concluded that the Binax kit was more suitable for the routine diagnosis of Legionella infections than the Biotest kit.

The immunophenotype of mast cells and its utility in the diagnostic work-up of systemic mastocytosis.

PubMed

Teodosio, Cristina; Mayado, Andrea; Sánchez-Muñoz, Laura; Morgado, José M; Jara-Acevedo, María; Álvarez-Twose, Ivan; García-Montero, Andrés C; Matito, Almudena; Caldas, Caldas; Escribano, Luis; Orfao, Alberto

2015-01-01

SM comprises a heterogeneous group of disorders, characterized by an abnormal accumulation of clonal MCs in 1 or more tissues, frequently involving the skin and BM. Despite the fact that most adult patients (>90%) carry the same genetic lesion (D816V KIT mutation), the disease presents with multiple variants with very distinct clinical and biologic features, a diverse prognosis, and different therapeutic requirements. Recent advances in the standardization of the study of BM MC by MFC allowed reproducible identification and characterization of normal/reactive MCs and their precursors, as well as the establishment of the normal MC maturational profiles. Analysis of large groups of patients versus normal/reactive samples has highlighted the existence of aberrant MC phenotypes in SM, which are essential for the diagnosis of the disease. In turn, 3 clearly distinct and altered maturation-associated immunophenotypic profiles have been reported recently in SM, which provide criteria for the distinction between ISM patients with MC-restricted and multilineage KIT mutation; thus, immunphenotyping also contributes to prognostic stratification of ISM, particularly when analysis of the KIT mutation on highly purified BM cells is not routinely available in the diagnostic work-up of the disease. © Society for Leukocyte Biology.

Comparative sensitivity and inhibitor tolerance of GlobalFiler® PCR Amplification and Investigator® 24plex QS kits for challenging samples.

PubMed

Elwick, Kyleen; Mayes, Carrie; Hughes-Stamm, Sheree

2018-05-01

In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, been buried, decomposed, and/or contain inhibitory substances. This study compares the performance of a relatively new STR kit in the US market (Investigator® 24plex QS kit; Qiagen) with the GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) when genotyping highly inhibited and low level DNA samples. In this study, DNA samples ranging from 1 ng to 7.8 pg were amplified to define the sensitivity of two systems. In addition, DNA (1 ng and 0.1 ng input amounts) was spiked with various concentrations of five inhibitors common to human remains (humic acid, melanin, hematin, collagen, calcium). Furthermore, bone (N = 5) and tissue samples from decomposed human remains (N = 6) were used as mock casework samples for comparative analysis with both STR kits. The data suggest that the GlobalFiler® kit may be slightly more sensitive than the Investigator® kit. On average STR profiles appeared to be more balanced and average peak heights were higher when using the GlobalFiler® kit. However, the data also show that the Investigator® kit may be more tolerant to common PCR inhibitors. While both STR kits showed a decrease in alleles as the inhibitor concentration increased, more complete profiles were obtained when the Investigator® kit was used. Of the 11 bone and decomposed tissue samples tested, 8 resulted in more complete and balanced STR profiles when amplified with the GlobalFiler® kit. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

PubMed

Liu, Jason Yingjie

2014-11-01

The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

HNU-HANBY PCP IMMUNOASSAY TEST KIT - INNOVATIVE TECHNOLOGY EVALUATION REPORT

EPA Science Inventory

The HNU-Hanby pentachlorophenol (PCP) test kit rapidly analyzes for PCP in soil samples. The test kit can only detect those PCP carriers that contain aromatic compounds. The test kit estimates PCP concentrations in soil samples indirectly by measuring petroleum hydrocarbon carrie...

DEMONSTRATION BULLETIN: HNU-HANBY PCP IMMUNOASSAY TEST KIT - HNU - SYSTEMS, INC.

EPA Science Inventory

The HNU-Hanby test kit rapidly analyzes for petroleum hydrocarbons in soil and water samples. The test kit can be used to estimate pentachlorophenol (PCP) concentrations in samples when the carrier solvent is a petroleum hydrocarbon. The test kit estimates PCP concentrations in ...

Development of an anti-EPO antibody detection kit based on lab-on-a-chip and bridging antibody technologies.

PubMed

Oh, Jin-Gyo; Seong, Jihyun; Han, Sunmi; Heo, Tae-Hwe

2018-05-17

Immunogenicity is a major concern in the use of biological drugs. In particular, antibody-mediated pure red cell aplasia (PRCA) is a rare condition that is caused by administration of recombinant erythropoietin. There are numerous assay platforms for detect EPO anti-drug antibody (ADA), and most have appropriate assay sensitivity, but in need of improvement in terms of assay turnaround time and user accessibility. Here, the new method was developed based on lab-on-a-chip technology and bridging ELISA. The FREND™ Cartridge is equipped with a microfluidic lateral flow channel, enabling easy, fast and accurate immunoassays with small sample volumes. Biotinylated EPO was immobilized on the avidin-coated solid phase of the test zone in the FREND™ cartridge. Initially, ADA in the serum sample binds to the detector conjugate (EPO-HRP-anti HRP antibody-FL bead) in the conjugation zone, and it flows into the test zone prepared with capture complex (avidin-biotinylated EPO). Unbound detector complexes are captured in the reference zone. The FREND™ system detects and quantifies the fluorescence signals in each zone and then calculates the concentration of EPO ADA in the sample. The FREND™ EPO ADA kit may be useful in local clinics as a rapid method for monitoring patients administered recombinant erythropoietin. Copyright © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

An evaluation of suspicious powder screening tools for first responders.

PubMed

Poore, Carrie; Clark, Paul; Emanuel, Peter A

2009-12-30

Field screening tools are required which would allow first responders to quickly ascertain if a suspicious powder poses a potential threat necessitating additional testing for biological pathogens such as Bacillus anthracis. In this study, three commercially available generic screening technologies were evaluated for the effectiveness to accurately differentiate between a hoax powder and a true biological threat. The BioCheck Kit was able to detect the following biological agents 1 x 10(8)CFU of B. anthracis Sterne (washed 4 times), 1x10(7)CFU of B. anthracis DeltaSterne (washed 2 times), 1 x 10(7)CFU of Yersinia pestis A1122, and 100 microg of ricin. The Prime Alert kit was able to detect 2 x 10(10)CFU of B. anthracis DeltaSterne 4x, 1 x 10(9)CFU of B. anthracis DeltaSterne 2x, and 1 x 10(8)CFU of Y. pestis A1122. The Prime Alert kit was not able to detect ricin. The Profile-1 kit was able to detect 1 x 10(4)CFU of B. anthracis DeltaSterne 4x and B. anthracis DeltaSterne 2x, and 1 x 10(6)CFU of Y. pestis A1122. The Profile-1 kit was not able to detect ricin. All of the kits showed positive results for powders containing components specifically targeted by the particular technology being used. Each technology assessed in this evaluation employs a different mechanism for the detection of biological materials and it is important that first responders are aware of the strengths and the limitations of each system so that they can effectively employ the technology to protect the homeland.

[Validation of Differential Extraction Kit in forensic sexual assault cases].

PubMed

Wu, Dan; Cao, Yu; Xu, Yan; He, Bai-Fang; Bi, Gang; Zhou, Huai-Gu

2009-12-01

To evaluate the validity of Differential Extraction Kit in isolating spermatozoa and epithelial cell DNA from mixture samples. Selective lysis of spermatid and epithelial cells combined with paramagnetic particle method were applied to extract the DNA from the mock samples under controlled conditions and forensic case samples, and template DNA were analyzed by STR genotype method. This Differential Extraction Kit is efficient to obtain high quality spermatid and epithelial cell DNA from the mixture samples with different proportion of sperm to epithelial cell. The Differential Extraction Kit can be applied in DNA extraction for mixed stain from forensic sexual assault samples.

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

PubMed

Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

2014-01-01

Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.

A Novel Tool to Facilitate the Learning of Buffering Mechanism by Undergraduate Students of the Biological Area

ERIC Educational Resources Information Center

Carvalho, Eduardo O.; Nantes, Iseli L.

2008-01-01

In this study, the application and evaluation of a novel didactic tool (buffer kit) is described to make it easy for students in the biological area to overcome their conceptual deficiencies that render the learning of the buffering mechanism difficult. The buffer kit was constructed with double-face EVA cards with a conjugated acid formula…

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity.

PubMed

Veerasami, Maroudam; Venkataraman, K; Karuppannan, Chitra; Shanmugam, Arun Attur; Prudhvi, Mallepaddi Chand; Holder, Thomas; Rathnagiri, Polavarapu; Arunmozhivarman, K; Raj, Gopal Dhinakar; Vordermeier, Martin; Mohana Subramanian, B

2018-03-01

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria . To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit's performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.

Comparison of methods for the extraction of DNA from formalin-fixed, paraffin-embedded archival tissues.

PubMed

Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.

DNA-PK assay

DOEpatents

Anderson, Carl W.; Connelly, Margery A.

2004-10-12

The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

76 FR 3119 - Availability for Exclusive, Non-Exclusive, or Partially-Exclusive Licensing of an Invention...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-19

... Partially-Exclusive Licensing of an Invention Concerning a Urinary Field Sampling Kit for the Determination.... Provisional Patent Application Serial No. 61/458,797, entitled ``Urinary Field Sampling Kit for the... INFORMATION: The invention relates to a urinary field sampling kit and method of its use for collecting and...

Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR

PubMed Central

Desneux, Jérémy; Pourcher, Anne-Marie

2014-01-01

Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631

15 CFR 742.2 - Proliferation of chemical and biological weapons.

Code of Federal Regulations, 2011 CFR

2011-01-01

... medical, analytical, diagnostic, and food testing kits that consist of pre-packaged materials of defined... health purposes: (1) Test kits containing no more than 300 grams of any chemical controlled by ECCN 1C350... part 745 of the EAR). Such test kits are controlled by ECCN 1C395 for CB and CW reasons, to States not...

15 CFR 742.2 - Proliferation of chemical and biological weapons.

Code of Federal Regulations, 2013 CFR

2013-01-01

... medical, analytical, diagnostic, and food testing kits that consist of pre-packaged materials of defined... health purposes: (1) Test kits containing no more than 300 grams of any chemical controlled by ECCN 1C350... part 745 of the EAR). Such test kits are controlled by ECCN 1C395 for CB and CW reasons, to States not...

15 CFR 742.2 - Proliferation of chemical and biological weapons.

Code of Federal Regulations, 2014 CFR

2014-01-01

..., analytical, diagnostic, and food testing kits that consist of pre-packaged materials of defined composition... purposes: (1) Test kits containing no more than 300 grams of any chemical controlled by ECCN 1C350.b or .c... the EAR). Such test kits are controlled by ECCN 1C395 for CB and CW reasons, to States not Party to...

Comparison of DNA extraction methods for human gut microbial community profiling.

PubMed

Lim, Mi Young; Song, Eun-Ji; Kim, Sang Ho; Lee, Jangwon; Nam, Young-Do

2018-03-01

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

PubMed Central

2010-01-01

Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research. PMID:20180960

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

PubMed

Moore, Simon J; Lai, Hung-En; Kelwick, Richard J R; Chee, Soo Mei; Bell, David J; Polizzi, Karen Marie; Freemont, Paul S

2016-10-21

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.

Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy.

PubMed

Vigneron, Nicolas; Meryet-Figuière, Matthieu; Guttin, Audrey; Issartel, Jean-Paul; Lambert, Bernard; Briand, Mélanie; Louis, Marie-Hélène; Vernon, Mégane; Lebailly, Pierre; Lecluse, Yannick; Joly, Florence; Krieger, Sophie; Lheureux, Stéphanie; Clarisse, Bénédicte; Leconte, Alexandra; Gauduchon, Pascal; Poulain, Laurent; Denoyelle, Christophe

2016-08-01

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross-studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin(®) kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter-sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT-qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two-fold lower inter-sample variability than the widely used endogenous miR-16-5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up-to 0.97 between the microarrays and individual RT-qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues

PubMed Central

Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE. PMID:24688314

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.

PubMed

Helwa, Inas; Cai, Jingwen; Drewry, Michelle D; Zimmerman, Arthur; Dinkins, Michael B; Khaled, Mariam Lotfy; Seremwe, Mutsa; Dismuke, W Michael; Bieberich, Erhard; Stamer, W Daniel; Hamrick, Mark W; Liu, Yutao

2017-01-01

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40-150 nm). The three kits, though, produced a significantly higher yield (80-300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.

Genoviz Software Development Kit: Java tool kit for building genomics visualization applications.

PubMed

Helt, Gregg A; Nicol, John W; Erwin, Ed; Blossom, Eric; Blanchard, Steven G; Chervitz, Stephen A; Harmon, Cyrus; Loraine, Ann E

2009-08-25

Visualization software can expose previously undiscovered patterns in genomic data and advance biological science. The Genoviz Software Development Kit (SDK) is an open source, Java-based framework designed for rapid assembly of visualization software applications for genomics. The Genoviz SDK framework provides a mechanism for incorporating adaptive, dynamic zooming into applications, a desirable feature of genome viewers. Visualization capabilities of the Genoviz SDK include automated layout of features along genetic or genomic axes; support for user interactions with graphical elements (Glyphs) in a map; a variety of Glyph sub-classes that promote experimentation with new ways of representing data in graphical formats; and support for adaptive, semantic zooming, whereby objects change their appearance depending on zoom level and zooming rate adapts to the current scale. Freely available demonstration and production quality applications, including the Integrated Genome Browser, illustrate Genoviz SDK capabilities. Separation between graphics components and genomic data models makes it easy for developers to add visualization capability to pre-existing applications or build new applications using third-party data models. Source code, documentation, sample applications, and tutorials are available at http://genoviz.sourceforge.net/.

[Determination of Hair Shafts by InnoTyper® 21 Kit].

PubMed

Li, F; Zhang, M; Wang, Y X; Shui, J J; Yan, M; Jin, X P; Zhu, X J

2017-12-01

To explore the application value of InnoTyper® 21 kit in forensic practice. Samples of hair shafts and saliva were collected from 8 unrelated individuals. Template DNA was extracted by AutoMate Express™ forensic DNA automatic extraction system. DNA was amplified by InnoTyper® 21 kit and AmpFℓSTR™ Identifiler™ Plus kit, respectively, and then the results were compared. After the amplification by InnoTyper® 21 kit, complete specific genotyping could be detected from the saliva samples, and the peak value of genotyping profiles of hair shafts without sheath cells was 57-1 219 RFU. Allelic gene deletion could be found sometimes. When amplified by AmpFℓSTR™ Identifiler™ Plus kit, complete specific genotyping could be detected from the saliva samples, and the specific fragment was not detected in hair shafts without sheath cells. The InnoTyper® 21 kit has certain application value in the cases of hair shafts without sheath cells. Copyright© by the Editorial Department of Journal of Forensic Medicine

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food.

PubMed

Park, Douglas L; Coates, Scott; Brewer, Vickery A; Garber, Eric A E; Abouzied, Mohamed; Johnson, Kurt; Ritter, Bruce; McKenzie, Deborah

2005-01-01

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.

Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay.

PubMed

McKenzie, Jennifer Helen; Alwis, K Udeni; Sordillo, Joanne E; Kalluri, Kesava Srinivas; Milton, Donald Kirby

2011-06-01

Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.

Limits of diagnostic accuracy of anti-hepatitis C virus antibodies detection by ELISA and immunoblot assay.

PubMed

Suslov, Anatoly P; Kuzin, Stanislav N; Golosova, Tatiana V; Shalunova, Nina V; Malyshev, Nikolai A; Sadikova, Natalia V; Vavilova, Lubov M; Somova, Anna V; Musina, Elena E; Ivanova, Maria V; Kipor, Tatiana T; Timonin, Igor M; Kuzina, Lubov E; Godkov, Mihail A; Bajenov, Alexei I; Nesterenko, Vladimir G

2002-07-01

When human sera samples are tested for anti-hepatitis C virus (HCV) antibodies using different ELISA kits as well as immunoblot assay kits discrepant results often occur. As a result the diagnostics of HCV infection in such sera remains unclear. The purpose of this investigation is to define the limits of HCV serodiagnostics. Overall 7 different test kits of domestic and foreign manufacturers were used for the sampled sera testing. Preliminary comparative study, using seroconversion panels PHV905, PHV907, PHV908 was performed and reference kit was chosen (Murex anti-HCV version 4) as the most sensitive kit on the base of this study results. Overall 1640 sera samples have been screened using different anti-HCV ELISA kits and 667 of them gave discrepant results in at least two kits. These sera were then tested using three anti-HCV ELISA kits (first set of 377 samples) or four anti-HCV ELISA kits (second set of 290 samples) at the conditions of reference laboratory. In the first set 17.2% samples remained discrepant and in the second set - 13.4%. "Discrepant" sera were further tested in RIBA 3.0 and INNO-LIA immunoblot confirmatory assays, but approximately 5-7% of them remained undetermined after all the tests. For the samples with signal-to-cutoff ratio higher than 3.0 high rate of result consistency by reference, ELISA routing and INNO-LIA immunoblot assay was observed. On the other hand the results of tests 27 "problematic" sera in RIBA 3.0 and INNO-LIA were consistent only in 55.5% cases. Analysis of the antigen spectrum reactive with antibodies in "problematic" sera, demonstrated predominance of Core, NS3 and NS4 antigens for sera, positive in RIBA 3.0 and Core and NS3 antigens for sera, positive in INNO-LIA. To overcome the problem of undetermined sera, methods based on other principles, as well as alternative criteria of HCV infection diagnostics are discussed.

30 CFR 7.405 - Critical characteristics.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Cable Splice Kits § 7.405 Critical characteristics. (a) A sample from each production run, batch, or lot of manufactured electric cable, signaling cable, or splice made from a splice kit shall be flame... cable or splice and a sample of the cable or splice kit assembly shall be visually inspected or tested...

30 CFR 7.405 - Critical characteristics.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Cable Splice Kits § 7.405 Critical characteristics. (a) A sample from each production run, batch, or lot of manufactured electric cable, signaling cable, or splice made from a splice kit shall be flame... cable or splice and a sample of the cable or splice kit assembly shall be visually inspected or tested...

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

PubMed Central

Leite, D.C.A.; Balieiro, F.C.; Pires, C.A.; Madari, B.E.; Rosado, A.S.; Coutinho, H.L.C.; Peixoto, R.S.

2014-01-01

Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar.

PubMed

Leite, D C A; Balieiro, F C; Pires, C A; Madari, B E; Rosado, A S; Coutinho, H L C; Peixoto, R S

2014-01-01

Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.

Correlation Between Iron and alpha and pi Glutathione-S-Transferase Levels in Humans

DTIC Science & Technology

2012-09-01

assays were performed as described in the Biotrin High Sensitivity Alpha GST EIA kit protocol. First, serum samples were diluted 1:10 with wash solution...immunosorbent assays were performed as described in the Biotrin Pi GST EIA kit protocol. First, plasma samples were diluted 1:5 with sample diluent...immunosorbent assays were performed as described in the AssayMax Human Transferrin ELISA kit protocol. First, serum samples were diluted 1:2000 with MIX

Facilitators and Barriers to Naloxone Kit Use Among Opioid-Dependent Patients Enrolled in Medication Assisted Therapy Clinics in North Carolina.

PubMed

Khatiwoda, Prasana; Proeschold-Bell, Rae Jean; Meade, Christina S; Park, Lawrence P; Proescholdbell, Scott

2018-01-01

BACKGROUND Naloxone-an opioid antagonist that reverses the effects of opioids-is increasingly being distributed in non-medical settings. We sought to identify the facilitators of, and barriers to, opioid users using naloxone kits in North Carolina. METHODS In 2015, we administered a 15-item survey to a convenience sample of 100 treatment seekers at 4 methadone/buprenorphine Medication Assisted Therapy (MAT) clinics in North Carolina. RESULTS Seventy-four percent of participants reported having ever gotten a naloxone kit; this percentage was higher for females (81%) than males (63%) ( P = .06). The primary reason given for not having a kit was not knowing where to get one. Only 6% had heard of kits from the media and only 5% received one from a medical provider. Among kit recipients, 56% of both females and males reported mostly or sometimes carrying the kit, with additional participants reporting always. Reasons for not carrying a kit were no longer being around drugs, forgetting it, and the kit being too large. Men discussed the difficulties of carrying the naloxone kits, which are currently too large to fit in a pocket. Ninety-four percent of naloxone users reported intending to call emergency services in case of an overdose emergency. LIMITATIONS Study limitations included a small sample, participants limited to MAT clinics, and a predominantly white sample. CONCLUSIONS MAT treatment seekers reported a willingness to carry and use naloxone kits. Education, outreach, media, and medical providers need to promote naloxone kits. A smaller kit may increase the likelihood of men carrying one. ©2018 by the North Carolina Institute of Medicine and The Duke Endowment. All rights reserved.

Evaluation of the LIVE/DEAD BacLight Kit for Detection of Extremophilic Archaea and Visualization of Microorganisms in Environmental Hypersaline Samples

PubMed Central

Leuko, Stefan; Legat, Andrea; Fendrihan, Sergiu; Stan-Lotter, Helga

2004-01-01

Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites. PMID:15528557

Disposable collection kit for rapid and reliable collection of saliva.

PubMed

Yamaguchi, Masaki; Tezuka, Yuki; Takeda, Kazunori; Shetty, Vivek

2015-01-01

To describe and evaluate disposable saliva collection kit for rapid, reliable, and reproducible collection of saliva samples. The saliva collection kit comprised of a saliva absorbent swab and an extractor unit was used to retrieve whole saliva samples from 10 subjects. The accuracy and precision of the extracted volumes (3, 10, and 30 μl) were compared to similar volumes drawn from control samples obtained by passive drool. Additionally, the impact of kit collection method on subsequent immunoassay results was verified by assessing salivary cortisol levels in the samples and comparing them to controls. The recovered volumes for the whole saliva samples were 3.85 ± 0.28, 10.79 ± 0.95, and 31.18 ± 1.72 μl, respectively (CV = 8.76%) and 2.91 ± 0.19, 9.75 ± 0.43, and 29.64 ± 0.91 μl, respectively, (CV = 6.36%) for the controls. There was a close correspondence between the salivary cortisol levels from the saliva samples obtained by the collection kit and the controls (R(2)  > 0.96). The disposable saliva collection kit allows accurate and repeatable collection of fixed amounts of whole saliva and does not interfere with subsequent measurements of salivary cortisol. The simple collection process, lack of elaborate specimen recovery steps, and the short turnaround time (<3 min) should render the kit attractive to test subjects and researchers alike. © 2015 Wiley Periodicals, Inc.

What's Living in Your World?

ERIC Educational Resources Information Center

Powell, Karen; Stiller, John

2005-01-01

The average biotech catalog contains a dizzying array of kits offering tried-and-true protocols in molecular biology and biochemistry. Prepackaged experiences, ranging from DNA fingerprinting to protein purification, are now available to high school students. Although commercial kits designed for education provide excellent hands-on experiences in…

Hypocretin measurement: shelf age of radioimmunoassay kit, but not freezer time, influences assay variability.

PubMed

Keating, Glenda; Bliwise, Donald L; Saini, Prabhjyot; Rye, David B; Trotti, Lynn Marie

2017-09-01

The hypothalamic peptide hypocretin 1 (orexin A) may be assayed in cerebrospinal fluid to diagnose narcolepsy type 1. This testing is not commercially available, and factors contributing to assay variability have not previously been comprehensively explored. In the present study, cerebrospinal fluid hypocretin concentrations were determined in duplicate in 155 patient samples, across a range of sleep disorders. Intra-assay variability of these measures was analyzed. Inter-assay correlation between samples tested at Emory and at Stanford was high (r = 0.79, p < 0.0001). Intra-assay correlation between samples tested in duplicate in our center was also high (r = 0.88, p < 0.0001); intra-assay variability, expressed as the difference between values as a percentage of the higher value, was low at 9.4% (SD = 7.9%). Although both time the sample spent in the freezer (r = 0.16, p = 0.04) and age of the kit used for assay (t = 3.64, p = 0.0004) were significant predictors of intra-kit variability in univariate analyses, only age of kit was significant in multivariate linear regression (F = 4.93, p = 0.03). Age of radioimmunoassay kit affects intra-kit variability of measured hypocretin values, such that kits closer to expiration exhibit significantly more variability.

Protein kinase C-δ-mediated recycling of active KIT in colon cancer.

PubMed

Park, Misun; Kim, Won Kyu; Song, Meiying; Park, Minhee; Kim, Hyunki; Nam, Hye Jin; Baek, Sung Hee; Kim, Hoguen

2013-09-15

Abnormal signaling through receptor tyrosine kinase (RTK) moieties is important in tumorigenesis and drug targeting of colorectal cancers. Wild-type KIT (WT-KIT), a RTK that is activated upon binding with stem cell factor (SCF), is highly expressed in some colon cancers; however, little is known about the functional role of SCF-dependent KIT activation in colon cancer pathogenesis. We aimed to elucidate the conditions and roles of WT-KIT activation in colon cancer tumorigenesis. Colorectal cancers with KIT expression were characterized by immunoblotting and immunohistochemistry. The biologic alterations after KIT-SCF binding were analyzed with or without protein kinase C (PKC) activation. We found that WT-KIT was expressed in a subset of colon cancer cell lines and was activated by SCF, leading to activation of downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways. We also showed that KIT expression gradually decreased, after prolonged SCF stimulation, due to lysosomal degradation. Degradation of WT-KIT after SCF binding was significantly rescued when PKC was activated. We also showed the involvement of activated PKC-δ in the recycling of WT-KIT. We further showed that a subset of colorectal cancers exhibit expressions of both WT-KIT and activated PKC-δ and that expression of KIT is correlated with poor patient survival (P = 0.004). Continuous downstream signal activation after KIT-SCF binding is accomplished through PKC-δ-mediated recycling of KIT. This sustained KIT activation may contribute to tumor progression in a subset of colon cancers with KIT expression and might provide the rationale for a therapeutic approach targeting KIT. ©2013 AACR.

Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS.

PubMed

Shubhakar, Archana; Kozak, Radoslaw P; Reiding, Karli R; Royle, Louise; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

2016-09-06

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.

21 CFR 864.1860 - Immunohistochemistry reagents and kits.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunohistochemistry reagents and kits. 864.1860 Section 864.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1860...

21 CFR 864.1860 - Immunohistochemistry reagents and kits.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunohistochemistry reagents and kits. 864.1860 Section 864.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1860...

21 CFR 864.1860 - Immunohistochemistry reagents and kits.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunohistochemistry reagents and kits. 864.1860 Section 864.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1860...

Evaluation of ELISA screening test for detecting aflatoxin in biogenic dust samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Durant, J.T.

Aflatoxin is a carcinogenic chemical that is sometimes produced when agricultural commodities are infested by the fungi Aspergillus flavus and A. Parasiticus. Aflatoxin has been found to be present in air samples taken around persons handling materials likely to be contaminated. The purpose of this investigation was to demonstrate the feasibility of using an Enzyme Linked Immunosorbent Assay (ELISA) test kit that was developed to screen for aflatoxin in bulk agricultural commodities, to an air sample. Samples were taken from two environments likely to be contaminated with aflatoxin, a dairy farm feed mixing operation and a peanut bagging operation. Themore » dust collected from these environments was considered to be biogenic, in that it originated primarily from biological materials.« less

Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

PubMed

Lim, Hazel Sin Yue; Zheng, Qianwang; Miks-Krajnik, Marta; Turner, Matthew; Yuk, Hyun-Gyun

2015-06-01

The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in less than 26 h.

Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach.

PubMed

Percy, Andrew J; Yang, Juncong; Chambers, Andrew G; Mohammed, Yassene; Miliotis, Tasso; Borchers, Christoph H

2016-01-01

Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

PubMed

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Comparison of Quantifiler(®) Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints.

PubMed

Goecker, Zachary C; Swiontek, Stephen E; Lakhtakia, Akhlesh; Roy, Reena

2016-06-01

The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside

PubMed Central

2012-01-01

Background Vulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection. Methods c-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes–Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients. Results c-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009). Conclusions Our findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women. PMID:22839358

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-04-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed Central

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-01-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation. Images Figure 4 Figure 6 Figure 7 Figure 8 PMID:9176104

Optimising the quantification of cytokines present at low concentrations in small human mucosal tissue samples using Luminex assays☆

PubMed Central

Staples, Emily; Ingram, Richard James Michael; Atherton, John Christopher; Robinson, Karen

2013-01-01

Sensitive measurement of multiple cytokine profiles from small mucosal tissue biopsies, for example human gastric biopsies obtained through an endoscope, is technically challenging. Multiplex methods such as Luminex assays offer an attractive solution but standard protocols are not available for tissue samples. We assessed the utility of three commercial Luminex kits (VersaMAP, Bio-Plex and MILLIPLEX) to measure interleukin-17A (IL-17) and interferon-gamma (IFNγ) concentrations in human gastric biopsies and we optimised preparation of mucosal samples for this application. First, we assessed the technical performance, limits of sensitivity and linear dynamic ranges for each kit. Next we spiked human gastric biopsies with recombinant IL-17 and IFNγ at a range of concentrations (1.5 to 1000 pg/mL) and assessed kit accuracy for spiked cytokine recovery and intra-assay precision. We also evaluated the impact of different tissue processing methods and extraction buffers on our results. Finally we assessed recovery of endogenous cytokines in unspiked samples. In terms of sensitivity, all of the kits performed well within the manufacturers' recommended standard curve ranges but the MILLIPLEX kit provided most consistent sensitivity for low cytokine concentrations. In the spiking experiments, the MILLIPLEX kit performed most consistently over the widest range of concentrations. For tissue processing, manual disruption provided significantly improved cytokine recovery over automated methods. Our selected kit and optimised protocol were further validated by measurement of relative cytokine levels in inflamed and uninflamed gastric mucosa using Luminex and real-time polymerase chain reaction. In summary, with proper optimisation Luminex kits (and for IL-17 and IFNγ the MILLIPLEX kit in particular) can be used for the sensitive detection of cytokines in mucosal biopsies. Our results should help other researchers seeking to quantify multiple low concentration cytokines in small tissue samples. PMID:23644159

Colorimetric measurement of carbohydrates in biological wastewater treatment systems: A critical evaluation.

PubMed

Le, Chencheng; Stuckey, David C

2016-05-01

Four laboratory preparations and three commercially available assay kits were tested on the same carbohydrate samples with the addition of 14 different interfering solutes typically found in wastewater treatment plants. This work shows that a wide variety of solutes can interfere with these assays. In addition, a comparative study on the use of these assays with different carbohydrate samples was also carried out, and the metachromatic response was clearly influenced by variation in sample composition. The carbohydrate content in the supernatant of a submerged anaerobic membrane bioreactor (SAMBR) was also measured using these assays, and the amount in the different supernatant samples, with and without a standard addition of glucose to the samples, showed substantial differences. We concluded that the carbohydrates present in wastewater measured using these colorimetric methods could be seriously under- or over-estimated. A new analytical method needs to be developed in order to better understand the biological transformations occurring in anaerobic digestion that leads to the production of soluble microbial products (SMPs) and extracellular polymeric substance (EPS). Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of a sensitive enzyme-linked immunosorbent assay for the measurement of biologically active etanercept in patients with ankylosing spondylitis.

PubMed

Wang, Lei; Wang, Xiaoxia; Li, Ying; Cheng, Zeneng

2016-01-01

Etanercept is the first tumor necrosis factor inhibitor to be approved for rheumatic disease treatment. Its in vivo concentration is usually detected with commercial enzyme-linked immunosorbent assay (ELISA) kits; specifically, previous researchers have mostly used double-antibody sandwich ELISA technology. Double-antibody sandwich ELISA is employed to detect the total etanercept rather than biologically active etanercept, which is more relevant in terms of therapeutic drug monitoring. In this work, a sensitive ELISA that employed its antigen TNF-α to capture biologically active etanercept for concentration detection was established and validated for etanercept pharmacokinetic (PK) study in patients with ankylosing spondylitis (AS). The proposed assay was demonstrated to be precise and accurate over the linear range of 12.5-400pg/mL. The intra- and inter-assay relative standard deviation ranged from 3.9 to 12.2% and 6.2 to 11.1%, respectively, and recovery varied between 90.1 and 99.7%, confirming the assay's reliability. The effectiveness and accuracy of the assay was also validated according to quality samples containing etanercept with different TNF-α concentrations, and with plasma samples from patients with AS. To complete the study, both the proposed assay and double-antibody sandwich ELISA were applied to the PK study of etanercept in patients and compared. The multiple-dose results of both analytical methods were consistent, while the drug exposure of the first dose as-detected by the proposed assay was lower than that detected by double-antibody sandwich ELISA. In conclusion, the proposed ELISA was shown to provide more accurate concentration data for therapeutic drug monitoring in comparison to commercial ELISA kits. Copyright © 2015 Elsevier B.V. All rights reserved.

Radioassay kit for method of determining methotrexate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Charm, S.E.; Blair, H.E.

1978-07-25

A radioassay system for the determination of methotrexate in biological fluids based on the competitive binding of labeled and unlabeled methotrexate to the enzyme dihydrofolate reductase. Samples of unknown methotrexate level are mixed with I/sup 125/ labeled methotrexate. A portion of the total methotrexate present is bound by the addition of enzyme, and the unbound methotrexate is removed with charcoal. The level of bound I/sup 125/ labeled methotrexate is measured in a gamma counter. To calculate the methotrexate level of the unknown samples, the displacement of bound labeled methotrexate caused by the unknowns is compared to the displacement caused bymore » known methotrexate standards.« less

Simultaneous extraction of proteins and metabolites from cells in culture

PubMed Central

Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

2014-01-01

Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938

10 CFR 429.33 - Ceiling fan light kits.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 3 2013-01-01 2013-01-01 false Ceiling fan light kits. 429.33 Section 429.33 Energy... COMMERCIAL AND INDUSTRIAL EQUIPMENT Certification § 429.33 Ceiling fan light kits. (a) Sampling plan for selection of units for testing. (1) The requirements of § 429.11 are applicable to ceiling fan light kits...

10 CFR 429.33 - Ceiling fan light kits.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 3 2014-01-01 2014-01-01 false Ceiling fan light kits. 429.33 Section 429.33 Energy... COMMERCIAL AND INDUSTRIAL EQUIPMENT Certification § 429.33 Ceiling fan light kits. (a) Sampling plan for selection of units for testing. (1) The requirements of § 429.11 are applicable to ceiling fan light kits...

10 CFR 429.33 - Ceiling fan light kits.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 3 2012-01-01 2012-01-01 false Ceiling fan light kits. 429.33 Section 429.33 Energy... COMMERCIAL AND INDUSTRIAL EQUIPMENT Certification § 429.33 Ceiling fan light kits. (a) Sampling plan for selection of units for testing. (1) The requirements of § 429.11 are applicable to ceiling fan light kits...

Loss of c-KIT expression in thyroid cancer cells.

PubMed

Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

Loss of c-KIT expression in thyroid cancer cells

PubMed Central

Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression. PMID:28301608

Factors for C-Kit Expression in Cardiac Outgrowth Cells and Human Heart Tissue.

PubMed

Matsushita, Satoshi; Minematsu, Kazuo; Yamamoto, Taira; Inaba, Hirotaka; Kuwaki, Kenji; Shimada, Akie; Yokoyama, Yasutaka; Amano, Atsushi

2017-12-12

We determined the factors associated with the expression of c-kit in the heart and the proliferation of c-kit-positive (c-kit pos ) cardiac stem cells among the outgrowth cells cultured from human cardiac explants.Samples of the right atrium (RA), left atrium (LA), and left ventricle obtained from patients during open-heart surgery were processed for cell culture of outgrowth cells and tissue analysis. The total number of growing cells and the population of c-kit pos cells were measured and compared with c-kit expression in native tissues and characteristics of the patients according to the region of the heart.We analyzed 452 samples from 334 patients. Atrial fibrillation (AF) in the patients reduced the number of outgrowth cells from the RA and LA, and aging was a co-factor for the LA. The c-kit pos population from the RA was associated with serum brain natriuretic peptide (BNP). C-kit expression in native tissue was also associated with BNP expression. However, we observed no relationship in expression between outgrowth cells and native tissue. In addition, the RA tissue provided the highest number of c-kit pos cells, and the left ventricle provided the lowest.C-kit was weakly expressed in response to damage. In addition, no correlation between outgrowth cells and native tissue was found for c-kit expression.

Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR

PubMed Central

Schorling, Stefan; Schalasta, Gunnar; Enders, Gisela; Zauke, Michael

2004-01-01

The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 104-1010 IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA. PMID:14736825

Student-Designed Enzyme-Linked Metabolite Assay Kits

ERIC Educational Resources Information Center

Hancock, D.; Johnston, J.; Dimauro, J.; Denyer, G.

2004-01-01

The extensive use of commercial kits in molecular biology and biochemistry has prompted us to design a series of practical sessions to help students become familiar with the uses and limitations of pre-packaged assay systems. To facilitate an understanding of these assay systems and to promote reflection on their appropriate use, students…

Qualitative assessment of study materials and communication strategies used in studies that include DNA collection.

PubMed

Jenkins, Mary M; Reed-Gross, Erika; Barfield, Wanda D; Prue, Christine E; Gallagher, Margaret L; Rasmussen, Sonja A; Honein, Margaret A

2011-11-01

To understand motivations and barriers to participation in studies that include DNA collection, focus group discussions were held with mothers who had participated in a case-control study of birth defects. Recruited mothers had completed an interview and had received a mailed kit containing cytobrushes to collect buccal cells for DNA from herself, her infant, and her infant's father. Six moderator-led focus groups were attended by a total of 38 women residing in Atlanta, Georgia. Focus groups were segmented by DNA collection status (biologics participants or nonparticipants), infant case-control status, infant birthweight, and maternal race and ethnicity. This report assesses maternal attitudes toward study materials and communication strategies. Across groups, respondents expressed concern about how their contact information was obtained. Study materials were described as clear and professional by most women, although some respondents reported confusion about disclosure of individual genetic results. Respondents generally reported that monetary incentives were not a motivation to participate, but increased perceived study legitimacy. Biologics nonparticipants expressed concerns about kit component sterility; government involvement; and DNA sample use, storage, and disposal. Respondents suggested that investigators provide feedback on whether sample collection was performed correctly and provide materials targeted to fathers to help alleviate paternal skepticism. Participation in DNA collection might be improved by strengthening study materials and communication strategies. Published 2011 Wiley Periodicals, Inc. This article is a U.S. Government work and is in the public domain in the USA.

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

PubMed

Lai, Hung-En; Moore, Simon; Polizzi, Karen; Freemont, Paul

2018-01-01

Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

PubMed

Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

2016-08-01

Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

Improved technical success and radiation safety of adrenal vein sampling using rapid, semi-quantitative point-of-care cortisol measurement.

PubMed

Page, Michael M; Taranto, Mario; Ramsay, Duncan; van Schie, Greg; Glendenning, Paul; Gillett, Melissa J; Vasikaran, Samuel D

2018-01-01

Objective Primary aldosteronism is a curable cause of hypertension which can be treated surgically or medically depending on the findings of adrenal vein sampling studies. Adrenal vein sampling studies are technically demanding with a high failure rate in many centres. The use of intraprocedural cortisol measurement could improve the success rates of adrenal vein sampling but may be impracticable due to cost and effects on procedural duration. Design Retrospective review of the results of adrenal vein sampling procedures since commencement of point-of-care cortisol measurement using a novel single-use semi-quantitative measuring device for cortisol, the adrenal vein sampling Accuracy Kit. Success rate and complications of adrenal vein sampling procedures before and after use of the adrenal vein sampling Accuracy Kit. Routine use of the adrenal vein sampling Accuracy Kit device for intraprocedural measurement of cortisol commenced in 2016. Results Technical success rate of adrenal vein sampling increased from 63% of 99 procedures to 90% of 48 procedures ( P = 0.0007) after implementation of the adrenal vein sampling Accuracy Kit. Failure of right adrenal vein cannulation was the main reason for an unsuccessful study. Radiation dose decreased from 34.2 Gy.cm 2 (interquartile range, 15.8-85.9) to 15.7 Gy.cm 2 (6.9-47.3) ( P = 0.009). No complications were noted, and implementation costs were minimal. Conclusions Point-of-care cortisol measurement during adrenal vein sampling improved cannulation success rates and reduced radiation exposure. The use of the adrenal vein sampling Accuracy Kit is now standard practice at our centre.

c-Kit modifies the inflammatory status of smooth muscle cells

PubMed Central

Song, Lei; Martinez, Laisel; Zigmond, Zachary M.; Hernandez, Diana R.; Lassance-Soares, Roberta M.; Selman, Guillermo

2017-01-01

Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation. PMID:28626608

c-Kit modifies the inflammatory status of smooth muscle cells.

PubMed

Song, Lei; Martinez, Laisel; Zigmond, Zachary M; Hernandez, Diana R; Lassance-Soares, Roberta M; Selman, Guillermo; Vazquez-Padron, Roberto I

2017-01-01

c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (Kit W/W-v ) and control (Kit +/+ ) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.

Validation of spot-testing kits to determine iodine content in salt.

PubMed Central

Pandav, C. S.; Arora, N. K.; Krishnan, A.; Sankar, R.; Pandav, S.; Karmarkar, M. G.

2000-01-01

Iodine deficiency disorders are a major public health problem, and salt iodization is the most widely practised intervention for their elimination. For the intervention to be successful and sustainable, it is vital to monitor the iodine content of salt regularly. Iodometric titration, the traditional method for measuring iodine content, has problems related to accessibility and cost. The newer spot-testing kits are inexpensive, require minimal training, and provide immediate results. Using data from surveys to assess the availability of iodized salt in two states in India, Madhya Pradesh and the National Capital Territory of Delhi, we tested the suitability of such a kit in field situations. Salt samples from Delhi were collected from 30 schools, chosen using the Expanded Programme on Immunization (EPI) cluster sampling technique. A single observer made the measurement for iodine content using the kit. Salt samples from Madhya Pradesh were from 30 rural and 30 urban clusters, identified by using census data and the EPI cluster sampling technique. In each cluster, salt samples were collected from 10 randomly selected households and all retailers. The 15 investigators performing the survey estimated the iodine content of salt samples in the field using the kit. All the samples were brought to the central laboratory in Delhi, where iodine content was estimated using iodometric titration as a reference method. The agreement between the kit and titration values decreased as the number of observers increased. Although sensitivity was not much affected by the increase in the number of observers (93.3% for a single observer and 93.9% for multiple observers), specificity decreased sharply (90.4% for a single observer and 40.4% for multiple observers). Due to the low specificity and resulting high numbers of false-positives for the kit when used by multiple observers ("real-life situations"), kits were likely to consistently overestimate the availability of iodized salt. This overestimation could result in complacency. Therefore, we conclude that until a valid alternative is available, the titration method should be used for monitoring the iodine content of salt at all levels, from producer to consumer, to ensure effectiveness of the programme. PMID:10994281

Evaluation of the Seeplex® Meningitis ACE Detection kit for the detection of 12 common bacterial and viral pathogens of acute meningitis.

PubMed

Shin, So Youn; Kwon, Kye Chul; Park, Jong Woo; Kim, Ji Myung; Shin, So Young; Koo, Sun Hoe

2012-01-01

Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. The lower detection limits ranged from 10(1) copies/µL to 5×10(1) copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

Evaluation of the Seeplex® Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis

PubMed Central

Shin, So Youn; Kwon, Kye Chul; Park, Jong Woo; Kim, Ji Myung; Shin, So Young

2012-01-01

Background Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. Methods Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. Results The lower detection limits ranged from 101 copies/µL to 5×101 copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. Conclusions The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens. PMID:22259778

Detection of Cytomegalovirus (CMV) DNA in EDTA Whole-Blood Samples: Evaluation of the Quantitative artus CMV LightCycler PCR Kit in Conjunction with Automated Sample Preparation▿

PubMed Central

Michelin, Birgit D. A.; Hadžisejdić, Ita; Bozic, Michael; Grahovac, Maja; Hess, Markus; Grahovac, Blaženka; Marth, Egon; Kessler, Harald H.

2008-01-01

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within ±0.5 log10 unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay. PMID:18272703

Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation.

PubMed

Michelin, Birgit D A; Hadzisejdic, Ita; Bozic, Michael; Grahovac, Maja; Hess, Markus; Grahovac, Blazenka; Marth, Egon; Kessler, Harald H

2008-04-01

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.

Can we use postal surveys with anonymous testing to monitor chlamydia prevalence in young women in England? Pilot study incorporating randomised controlled trial of recruitment methods.

PubMed

Woodhall, Sarah C; Nichols, Tom; Alexander, Sarah; da Silva, Filomeno Coelho; Mercer, Catherine H; Ison, Catherine; Gill, O Noel; Soldan, Kate

2015-09-01

Chlamydia prevalence in the general population is a potential outcome measure for the evaluation of chlamydia control programmes. We carried out a pilot study to determine the feasibility of using a postal survey for population-based chlamydia prevalence monitoring. Postal invitations were sent to a random sample of 2000 17-year-old to 18-year-old women registered with a general practitioner in two pilot areas in England. Recipients were randomised to receive either a self-sampling kit (n=1000), a self-sampling kit and offer of £5 voucher on return of sample (n=500) or a self-sampling kit on request (n=500). Participants returned a questionnaire and self-taken vulvovaginal swab sample for unlinked anonymous Chlamydia trachomatis testing. Non-responders were sent a reminder letter 3 weeks after initial invitation. We calculated the participation rate (number of samples returned/number of invitations sent) and cost per sample returned (including cost of consumables and postage) in each group. A total of 155/2000 (7.8%) samples were returned with consent for testing. Participation rates varied by invitation group: 7.8% in the group who were provided with a self-sampling kit, 14% in the group who were also offered a voucher and 1.0% in the group who were not sent a kit. The cost per sample received was lowest (£36) in the group who were offered both a kit and a voucher. The piloted survey methodology achieved low participation rates. This approach is not suitable for population-based monitoring of chlamydia prevalence among young women in England. (UKCRN ID 10913). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.

PubMed

Flores, Shahida; Sun, Jie; King, Jonathan; Budowle, Bruce

2014-05-01

The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A First Application of Enzyme-Linked Immunosorbent Assay for Screening Cyclodiene Insecticides in Ground Water

USGS Publications Warehouse

Dombrowski, T.R.; Thurman, E.M.; Mohrman, G.B.

1996-01-01

A commercially available enzyme-linked immunosorbent assay (ELISA) plate kit for screening of cyclodiene insecticides (aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor) was evaluated for sensitivity, cross reactivity, and overall performance using groundwater samples from a contaminated site. Ground-water contaminants included several pesticide compounds and their manufacturing byproducts, as well as many other organic and inorganic compounds. Cross-reactivity studies were carried out for the cyclodiene compounds, and results were compared to those listed by the manufacturer. Data obtained were used to evaluate the sensitivity of the ELISA kit to the cyclodiene compounds in ground water samples with a contaminated matrix. The method quantitation limit for the ELISA kit was 15 ??g/L (as chlordane). Of the 56 ground-water samples analyzed using the ELISA plate kits, more than 85% showed cyclodiene insecticide contamination. The ELISA kit showed excellent potential as a screening tool for sites with suspected groundwater contamination by insecticides.

DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.

PubMed

Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana

2018-04-01

Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

Total 25-Hydroxyvitamin D Determination by an Entry Level Triple Quadrupole Instrument: Comparison between Two Commercial Kits

PubMed Central

Cocci, Andrea; Zuppi, Cecilia; Persichilli, Silvia

2013-01-01

Objective. 25-hydroxyvitamin D2/D3 (25-OHD2/D3) determination is a reliable biomarker for vitamin D status. Liquid chromatography-tandem mass spectrometry was recently proposed as a reference method for vitamin D status evaluation. The aim of this work is to compare two commercial kits (Chromsystems and PerkinElmer) for 25-OHD2/D3 determination by our entry level LC-MS/MS. Design and Methods. Chromsystems kit adds an online trap column to an HPLC column and provides atmospheric pressure chemical ionization, isotopically labeled internal standard, and 4 calibrator points. PerkinElmer kit uses a solvent extraction and protein precipitation method. This kit can be used with or without derivatization with, respectively, electrospray and atmospheric pressure chemical ionization. For each analyte, there are isotopically labeled internal standards and 7 deuterated calibrator points. Results. Performance characteristics are acceptable for both methods. Mean bias between methods calculated on 70 samples was 1.9 ng/mL. Linear regression analysis gave an R 2 of 0.94. 25-OHD2 is detectable only with PerkinElmer kit in derivatized assay option. Conclusion. Both methods are suitable for routine. Chromsystems kit minimizes manual sample preparation, requiring only protein precipitation, but, with our system, 25-OHD2 is not detectable. PerkinElmer kit without derivatization does not guarantee acceptable performance with our LC-MS/MS system, as sample is not purified online. Derivatization provides sufficient sensitivity for 25-OHD2 detection. PMID:23555079

Factors Associated with Returning At-Home Specimen Collection Kits for HIV Testing among Internet-Using Men Who Have Sex with Men.

PubMed

Ricca, Alexandra V; Hall, Eric W; Khosropour, Christine M; Sullivan, Patrick S

2016-11-01

In the United States, men who have sex with men (MSM) are known to disproportionately have HIV. The authors sought to describe the acceptability of providing at-home dried blood spot specimen collection kits for HIV testing among MSM. Between August 2010 and December 2010, the authors recruited Internet-using, HIV-negative or -unknown MSM to participate in a 12-month study of behavioral risks. Eligible participants were mailed an at-home HIV test. Of the 896 men who were sent a test kit, 735 (82%) returned the kit. Returning a test kit was significantly associated with race (P = .002), highest level of education (P = .012), and annual income (P = .026). The adjusted odds of black, non-Hispanic men returning a test kit were about half of the odds of white, non-Hispanic men returning a test kit (adjusted odds ratios: 0.49; 95% confidence intervals: 0.31-0.78). Men who have sex with men are willing to provide biological specimens as part of an Internet-based HIV prevention study. © The Author(s) 2016.

Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

PubMed

Ravez, Séverine; Arsenlis, Stéphane; Barczyk, Amélie; Dupont, Anthony; Frédérick, Raphaël; Hesse, Stéphanie; Kirsch, Gilbert; Depreux, Patrick; Goossens, Laurence

2015-11-15

Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of a newly developed quantitative determination kit for tumor marker CA15-3 with chemiluminescent assay.

PubMed

Li, Peihua; Ye, Huiming; Liu, Jiangwu; Jin, Hongwei; Lin, Yongzhi; Yan, Shuidi; Yu, Yang; Gao, Lei; Xu, Feihai; Zhang, Zhongying

2018-01-01

Tumor marker carbohydrate antigen 15-3 (CA15-3) is used as a biomarker to aid to diagnose and monitor the prognosis of breast cancer patients. A new quantitative determination kit for CA15-3 with chemiluminescent assay was developed by Xiamen InnoDx Biotech Co., Ltd, China. Therefore, we conducted the report to evaluate the performance of the kit. According to the "Guiding principles on performance analysis of diagnostic reagents in vitro", the calibration curve, limit of detection, reportable range, accuracy, precision, anti-interference capability, cross-reaction and comparison by measuring EDTA plasma and serum were carried out. In addition, the kit was performed in parallel to electrochemiluminescence immunoassay kit (Roche) to analyze the correlation between the two kits. Regression equation of calibration curve of the kit was Y=0.7914X+4.1032 (R 2 =.990). Limit of detection was 0.0347 U/mL. The reportable range was 0.5-2400 U/mL. Recovery ratio was 100.0%-104.8%. Coefficient of variations (CVs) of within-run and between-run were 4.8%-7.6% and 5.8%-7.4% respectively. No remarkable interferences (all Bias% were less than ±10%) were detected when samples contained hemoglobin ≤183.8 μmol/L, bilirubin ≤340 μmol/L, triglyceride ≤18.1 mmol/L, or rheumatoid factor ≤400 U/mL. No cross-reaction was present in the kit. Moreover, compared with the results from electrochemiluminescence immunoassay kit (Roche) in 345 serum samples, there was a satisfied correlation coefficient of 0.977 (P<.01), and the kit was simultaneously fit for the detection of EDTA plasma and serum samples. The new kit validated satisfactorily, and it can be used for detecting CA15-3 in clinical practice. © 2017 The Authors Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.

Repeatability and validity of a field kit for estimation of cholinesterase in whole blood.

PubMed Central

London, L; Thompson, M L; Sacks, S; Fuller, B; Bachmann, O M; Myers, J E

1995-01-01

OBJECTIVES--To evaluate a spectrophotometric field kit (Test-Mate-OP) for repeatability and validity in comparison with reference laboratory methods and to model its anticipated sensitivity and specificity based on these findings. METHODS--76 farm workers between the age of 20 and 55, of whom 30 were pesticide applicators exposed to a range of organophosphates in the preceding 10 days, had blood taken for plasma cholinesterase (PCE) and erythrocyte cholinesterase (ECE) measurement by field kit or laboratory methods. Paired blinded duplicate samples were taken from subgroups in the sample to assess repeatability of laboratory and field kit methods. Field kits were also used to test venous blood in one subgroup. The variance obtained for the field kit tests was then applied to two hypothetical scenarios that used published action guidelines to model the kit's sensitivity and specificity. RESULTS--Repeatability for PCE was much poorer and for ECE slightly poorer than that of laboratory measures. A substantial upward bias for field kit ECE relative to laboratory measurements was found. Sensitivity of the kit to a 40% drop in PCE was 67%, whereas that for ECE was 89%. Specificity of the kit with no change in mean of the population was 100% for ECE and 91% for PCE. CONCLUSION--Field kit ECE estimation seems to be sufficiently repeatable for surveillance activities, whereas PCE does not. Repeatability of both tests seems to be too low for use in epidemiological dose-response investigations. Further research is indicated to characterise the upward bias in ECE estimation on the kit. PMID:7697143

The stem cell factor (SCF)/c-KIT system in carcinogenesis of reproductive tissues: What does the hormonal regulation tell us?

PubMed

Figueira, Marília I; Cardoso, Henrique J; Correia, Sara; Maia, Cláudio J; Socorro, Sílvia

2017-10-01

The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF) are expressed in several tissues of male and female reproductive tract, playing an important role in the regulation of basic biological processes. The activation of c-KIT by SCF controls, cell survival and death, cell differentiation and migration. Also, the SCF/c-KIT system has been implicated in carcinogenesis of reproductive tissues due to its altered expression pattern or overactivation in consequence of gain-of-functions mutations. Over the years, it has also been shown that hormones, the primary regulators of reproductive function and causative agents in the case of hormone-dependent cancers, are also able to control the SCF/c-KIT tissue levels. Therefore, it is liable to suppose that disturbed SCF/c-KIT expression driven by (de)regulated hormone actions can be a relevant step towards carcinogenesis. The present review describes the SCF and c-KIT expression in cancers of reproductive tissues, discussing the implications of the hormonal regulation of the SCF/c-KIT system in cancer development. Understanding the relationship between hormonal imbalance and the SCF/c-KIT expression and activity would be relevant in the context of novel therapeutic approaches in reproductive cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

PubMed

Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

2012-09-01

Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Evaluation of the PrioCHECK™ Trichinella AAD Kit for the digestion and recovery of larvae in pork, horse meat and wild meat.

PubMed

Konecsni, Kelly; Scheller, Cheryl; Scandrett, Brad; Buholzer, Patrik; Gajadhar, Alvin

2017-08-30

The artificial digestion magnetic stirrer method using pepsin protease and hydrochloric acid is the standard assay for the detection of Trichinella larvae in muscle of infected animals. Recently, an alternative enzyme, serine protease, was employed in the development of a commercially available digestion kit (PrioCHECK™ Trichinella AAD Kit). This assay requires a higher digestion temperature of 60°C which kills the larvae during the digestion process, mitigating the risk of environmental contamination from the parasite. The present study was conducted to determine the performance of the PrioCHECK™ Trichinella AAD Kit compared to the conventional pepsin/HCl digestion. Replicate paired 115g samples of Trichinella-negative pork diaphragm and masseter, and of horse tongue and masseter, were used to compare the two methods for tissue digestibility. Similarly, paired 100g samples of pork diaphragm and horse tongue were spiked with proficiency samples containing known numbers of Trichinella spiralis first stage larvae to compare larval recoveries for the two methods. Masseter samples from wild bears and wolves naturally infected with Trichinella nativa or T6 were also used to compare the performance of the methods. The results of the study showed that the PrioCHECK™ Trichinella AAD Kit, when used according to the manufacturer's instructions, was effective in detecting Trichinella infection in all samples that contained 0.05 or more larvae per gram of tissue. Although there was no significant difference between the Kit method and the standard pepsin/HCl digestion procedure in the average number of larvae recovered from spiked pork diaphragm, 38% fewer larvae were recovered from similarly spiked samples of horse tongue by digestion using serine protease (one way ANOVA, P value <0.001). Additional clarification was also more often required for both horse meat and pork when using the Kit compared to the pepsin/HCl method. The results of testing wildlife samples were similar for the two methods. Overall, the performance of the Kit method was suitable for the digestion of muscle samples and recovery of Trichinella larvae, according to international standards. It also provides advantages of faster digestion, safer reagents and recovered parasites that are non-hazardous for analysts and the environment. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Sample rotating turntable kit for infrared spectrometers

DOEpatents

Eckels, Joel Del [Livermore, CA; Klunder, Gregory L [Oakland, CA

2008-03-04

An infrared spectrometer sample rotating turntable kit has a rotatable sample cup containing the sample. The infrared spectrometer has an infrared spectrometer probe for analyzing the sample and the rotatable sample cup is adapted to receive the infrared spectrometer probe. A reflectance standard is located in the rotatable sample cup. A sleeve is positioned proximate the sample cup and adapted to receive the probe. A rotator rotates the rotatable sample cup. A battery is connected to the rotator.

Mutation scanning of BRAF, NRAS, KIT, and GNAQ/GNA11 in oral mucosal melanoma: a study of 57 cases.

PubMed

Lyu, Jiong; Wu, Yunteng; Li, Chaojun; Wang, Runxiang; Song, Hao; Ren, Guoxin; Guo, Wei

2016-04-01

Recent advances in novel targeted therapies have created the need for molecular characterization of cancer to allow accurate personalized treatments. In this study, our aim was to investigate the incidence of activating mutations of oncogenes BRAF, NRAS, KIT, and GNAQ/GNA11 in oral mucosal melanoma. We analyzed a cohort of 57 oral mucosal melanoma samples, including 27 frozen samples and 30 formalin-fixed paraffin-embedded samples. The tumors were screened for hotspot mutations of BRAF (exon 15), NRAS (exons 2 and 3), KIT (exons 9, 11, 13, and 17), and GNAQ/GNA11 (exon 5) by high-resolution melting and direct sequencing. In oral mucosal melanoma, 7.0% of tumors harbored KIT mutations and 3.5% harbored BRAF mutations, while classic BRAF V600E mutation was not detected. We found no mutations of NRAS or GNAQ/GNA11 in oral mucosal melanoma. We demonstrated that driver mutations are rare in mutational hotspots of BRAF, NRAS, KIT, and GNAQ/GNA11 in oral mucosal melanoma. The majority of patients will not benefit from KIT and BRAF inhibitors. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

c-KIT receptor expression is strictly associated with the biological behaviour of thyroid nodules

PubMed Central

2012-01-01

Background A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively. Methods In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene. Results We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%. Conclusion We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first, then the use of the c-KIT expression to ultimately assess the diagnosis of the nodules that otherwise would remain suspicious. The c-KIT expression-based classification is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of thyroid suspicious malignancies. PMID:22233760

Evaluation of Scat Deposition Transects versus Radio Telemetry for Developing a Species Distribution Model for a Rare Desert Carnivore, the Kit Fox.

PubMed

Dempsey, Steven J; Gese, Eric M; Kluever, Bryan M; Lonsinger, Robert C; Waits, Lisette P

2015-01-01

Development and evaluation of noninvasive methods for monitoring species distribution and abundance is a growing area of ecological research. While noninvasive methods have the advantage of reduced risk of negative factors associated with capture, comparisons to methods using more traditional invasive sampling is lacking. Historically kit foxes (Vulpes macrotis) occupied the desert and semi-arid regions of southwestern North America. Once the most abundant carnivore in the Great Basin Desert of Utah, the species is now considered rare. In recent decades, attempts have been made to model the environmental variables influencing kit fox distribution. Using noninvasive scat deposition surveys for determination of kit fox presence, we modeled resource selection functions to predict kit fox distribution using three popular techniques (Maxent, fixed-effects, and mixed-effects generalized linear models) and compared these with similar models developed from invasive sampling (telemetry locations from radio-collared foxes). Resource selection functions were developed using a combination of landscape variables including elevation, slope, aspect, vegetation height, and soil type. All models were tested against subsequent scat collections as a method of model validation. We demonstrate the importance of comparing multiple model types for development of resource selection functions used to predict a species distribution, and evaluating the importance of environmental variables on species distribution. All models we examined showed a large effect of elevation on kit fox presence, followed by slope and vegetation height. However, the invasive sampling method (i.e., radio-telemetry) appeared to be better at determining resource selection, and therefore may be more robust in predicting kit fox distribution. In contrast, the distribution maps created from the noninvasive sampling (i.e., scat transects) were significantly different than the invasive method, thus scat transects may be appropriate when used in an occupancy framework to predict species distribution. We concluded that while scat deposition transects may be useful for monitoring kit fox abundance and possibly occupancy, they do not appear to be appropriate for determining resource selection. On our study area, scat transects were biased to roadways, while data collected using radio-telemetry was dictated by movements of the kit foxes themselves. We recommend that future studies applying noninvasive scat sampling should consider a more robust random sampling design across the landscape (e.g., random transects or more complete road coverage) that would then provide a more accurate and unbiased depiction of resource selection useful to predict kit fox distribution.

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia.

PubMed

Hernandez, Diana R; Artiles, Adriana; Duque, Juan C; Martinez, Laisel; Pinto, Mariana T; Webster, Keith A; Velazquez, Omaida C; Vazquez-Padron, Roberto I; Lassance-Soares, Roberta M

2018-04-01

Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. Mutant mice with loss of c-Kit function (Kit W/W-v ), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. Kit W/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. Kit W/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. Kit W/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted Kit W/W-v mice. This study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of Kit W/W-v mouse hindlimbs. Copyright © 2017 Elsevier Inc. All rights reserved.

A cost effective hydrogel test kit for pre and post blast trinitrotoluene.

PubMed

Choodum, Aree; Malathong, Khanitta; NicDaeid, Niamh; Limsakul, Wadcharawadee; Wongniramaikul, Worawit

2016-09-01

A cost effective hydrogel test kit was successfully developed for the detection of pre- and post-blast trinitrotoluene (TNT). A polyvinyl alcohol (PVA) hydrogel matrix was used to entrap the potassium hydroxide (KOH) colourimetric reagent. The easily portable test kit was fabricated in situ in a small tube to which the sample could be added directly. The test kit was used in conjunction with digital image colourimetry (DIC) to demonstrate the rapid quantitative analysis of TNT in a test soil sample. The built-in digital camera of an iPhone was used to capture digital images of the colourimetric products from the test kit. Red-Green-Blue (RGB) colour data from the digital images of TNT standard solutions were used to establish a calibration graph. The validation of the DIC method indicated excellent inter day precision (0.12-3.60%RSD) and accuracy (93-108% relative accuracy). Post-blast soil samples containing TNT were analysed using the test kit and were in good agreement with spectrophotometric analysis. The intensity of the RGB data from the TNT complex deviated by +6.3%, +5.1%, and -4.9% after storage of the test kits in a freezer for 3 months. The test kit was also reusable for up to 12 times with only -5.4%, +0.3%, and +4.0% deviations. The hydrogel test kit was applied in the detection of trace explosive residues at the scene of the recent Bangkok bombing at the Ratchaprasong intersection and produced positive results for TNT demonstrating its operational field application as a rapid and cost effective quantitative tool for explosive residue analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparison of the sensitivity of the Legionella urinary antigen EIA kits from Binax and Biotest with urine from patients with infections caused by less common serogroups and subgroups of Legionella.

PubMed

Olsen, C W; Elverdal, P; Jørgensen, C S; Uldum, S A

2009-07-01

The detection of urinary antigen is the most widely used method to diagnose Legionnaires' disease (LD), so it is important that these assays have a high sensitivity for the disease. In this study, we compare two kits for their ability to detect urinary antigen in urine samples from patients infected with Legionella species and L. pneumophila sero- and subgroups not considered as the most common causes of LD. Urine samples (n = 33) from 30 culture-proven cases of L. pneumophila serogroup (sg) 1, subgroup non-Pontiac infection, and urine samples (n = 35) from 32 cases of non-L. pneumophila species or non-sg 1 infection were examined using the Binax EIA and Biotest EIA kits. For both groups, the overall diagnostic sensitivity of the Binax kit was significantly better than the sensitivity of the Biotest kits (P < 0.0001). For the non-Pontiac group, the sensitivity was 81.8 and 42.4%, respectively, and for the non-sg1 group, it was 51.4 and 28.6%, respectively. It was concluded that the Binax kit was more suitable for the general diagnosis of LD than the Biotest kit, but we still need urinary antigen detection methods with higher sensitivity for non-sg1 LD.

Development and evaluation of the RT-PCR kit for the rabies virus diagnosis.

PubMed

Dedkov, Vladimir G; Deviatkin, A A; Poleschuk, E M; Safonova, M V; Markelov, M L; Shipulin, G A

To improve the diagnosis, surveillance, and control for the rabies virus, a kit for hybridization-triggered fluorescence detection of rabies virus DNA by the RT-PCR technique was developed and evaluated. The analytical sensitivity of the kit was 4*10 GE per ml. High specificity of the kit was shown using representative sampling of viral, bacterial, and human nucleic acids.

Ancient DNA analyses of museum specimens from selected Presbytis (primate: Colobinae) based on partial Cyt b sequences

NASA Astrophysics Data System (ADS)

Aifat, N. R.; Yaakop, S.; Md-Zain, B. M.

2016-11-01

The IUCN Red List of Threatened Species has categorized Malaysian primates from being data deficient to critically endanger. Thus, ancient DNA analyses hold great potential to understand phylogeny, phylogeography and population history of extinct and extant species. Museum samples are one of the alternatives to provide important sources of biological materials for a large proportion of ancient DNA studies. In this study, a total of six museum skin samples from species Presbytis hosei (4 samples) and Presbytis frontata (2 samples), aged between 43 and 124 years old were extracted to obtain the DNA. Extraction was done by using QIAGEN QIAamp DNA Investigator Kit and the ability of this kit to extract museum skin samples was tested by amplification of partial Cyt b sequence using species-specific designed primer. Two primer pairs were designed specifically for P. hosei and P. frontata, respectively. These primer pairs proved to be efficient in amplifying 200bp of the targeted species in the optimized PCR conditions. The performance of the sequences were tested to determine genetic distance of genus Presbytis in Malaysia. From the analyses, P. hosei is closely related to P. chrysomelas and P. frontata with the value of 0.095 and 0.106, respectively. Cyt b gave a clear data in determining relationships among Bornean species. Thus, with the optimized condition, museum specimens can be used for molecular systematic studies of the Malaysian primates.

[The Quality Analysis of National Supervising Sampling for Rubella Virus IgM Diagnostic Kits in 2014].

PubMed

Yu, Tina; Qu, Shoufang; Zhang, Xiaoyan; Sun, Nan; Gao, Shangxian; Li, Haining; Huang, Jie

2015-07-01

To evaluate the quality status of rubella virus IgM diagnostic kits by national supervising sampling. Using legal inspection combining with exploratory study, the positive and negative coincidence rate, detection limit and repeatability of kits were verified. The results showed that 15 of 16 batches of kits were qualified using legal inspection, and the passing rate was 93.8%. The unqualified item was negative coincidence rate. In exploratory study, only 11 batches (68.8%) complied with industry standard. The unqualified items were negative coincidence rate, detection limit and repeatability. At present, rubella virus IgM diagnostic Kits have some quality problems in the market. It is recommended that we adopt industry standard and national reference panel in the registration inspection for the future, which will prompt enterprises to improve quality.

Evaluation of Cariogenic Bacteria

PubMed Central

Nishikawara, Fusao; Nomura, Yoshiaki; Imai, Susumu; Senda, Akira; Hanada, Nobuhiro

2007-01-01

Objectives The evaluation of Mutans streptococci (MS) is one of the index for caries risk. DentocultTM and CRTTM are commercial kits to detect and evaluate MS, conveniently. However, the evaluation of MS has also been carried out simply using an instruction manual. But the instruction manual is not easy to use for evaluation of MS. The aim of this study was to examine the utility of modified Mitis-Salivalius Bacitracin (MSB) agar medium compared with MSB agar medium and commercial kits, and to establish a convenient kit (mMSB-kit) using modified MSB agar. Methods The MS in stimulated saliva from 27 subjects were detected by MSB, modified MSB agar medium and commercial kits. Laboratory and clinically isolated strains of MS were similarly evaluated. The ratios of MS in detected bacteria were compared by ELISA. Results The scores using an mMSB-kit on the basis of modified MSB agar medium were tabulated. Saliva samples showed different levels of MS between culture methods and the commercial kit. Some samples which were full of MS were not detected by the commercial kit. The detection of MS by modified MSB agar medium and mMSB-kit were significantly higher when compared with MSB agar medium,CRTTM, (P< .01) and Dentocult SMTM (P<.05). Conclusion The sensitivity for detection of MS is higher for modified MSB agar medium when compared with MSB agar medium. The mMSB-kit can be used simply, and can be an important contributor for the evaluation of MS as a caries risk factor. PMID:19212495

Immunomagnetic Separation of Cryptosporidium parvum from Source Water Samples of Various Turbidities

PubMed Central

Bukhari, Z.; McCuin, R. M.; Fricker, C. R.; Clancy, J. L.

1998-01-01

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts. PMID:9797313

Coxiellosis in domestic livestock of Puducherry and Tamil Nadu: Detection of Coxiella burnetii DNA by polymerase chain reaction in slaughtered ruminants

PubMed Central

Pradeep, Jothimani; Stephen, Selvaraj; Pooja, Pratheesh; Akshayavardhini, Anbalagan; Sangeetha, Balakrishnan; Antony, Prabakar Xavier

2017-01-01

Background and Aim:: In the course of our Indian Council of Medical Research project on coxiellosis in Puducherry and Tamil Nadu, 5.64% goat, 1.85% sheep, 1.06% buffaloes, and 0.97% cattle were positive for Coxiella burnetii antibodies by enzyme linked immunosorbent assay kit (IDEXX, Liebefeld, Switzerland). In this preliminary study, we have proceeded to look for C. burnetii DNA in those antibody positive specimens employing an imported commercial C. burnetii polymerase chain reaction (PCR) kit. Materials and Methods:: Blood samples were collected during slaughtering. All 15 blood samples of antibody positive ruminants and three antibody negative samples were subjected to conventional Trans-PCR assay with a commercial PCR kit (Genekam Biotechnology AG, Duisburg, Germany). An in-house Trans-PCR was included in the study for comparison. Results:: A total of 15 antibody positive and three antibody-negative serum samples belonging to 11 goat, 4 sheep, 1 cattle, and 2 buffaloes were tested in duplicate for the presence of C. burnetii DNA by the commercial agar gel PCR kit and an in-house Trans-PCR. Only one buffalo serum sample was positive for C. burnetii with a band at 243 bp in in-house Trans-PCR. Discussion:: Seropositivity for C. burnetii need not necessarily translate into infectivity status of the animal. Conversely, seronegative ruminants can shed C. burnetii. Rapid disintegration of C. burnetii DNA during the storage period is an important impediment in QF-PCR research. This is the first time the performance of this commercial PCR kit is being validated in India. Conclusion:: Commercial PCR kit, Genekam did not identify any positive sample, probably because it targeted a larger amplicon of 687 bp. PMID:28717320

Comparison of a fluorometric assay kit with high-performance liquid chromatography for the assessment of serum retinol concentration.

PubMed

Elom, Aglago Kouassivi; Imane, El Menchawy; Kaoutar, Benjeddou; Khalid, El Kari; Asmaa, El Hamdouchi; Mehdi, Azlaf; Noureddine, El Haloui; Hassan, Aguenaou

2015-06-01

Although high-performance liquid chromatography (HPLC) is the commonly used method for the analysis of retinol in biological samples, simple and rapid test kits are available. This study compared a rapid test kit (ICHECK Fluoro®) to HPLC for the assessment of serum retinol concentrations. For the analysis by HPLC, sample preparation included standard deproteinization and extraction phases. The analysis by ICHECK was performed by injecting serum into IEX reagent vials (n=89) and mixing manually for separation. After precipitation of the proteins, the vial was introduced into the chamber of the ICHECK Fluoro and analysed at 0 min (ICHECK0min) and 15 min later (ICHECK15min). Bland and Altman approach was applied to test the agreement between HPLC and ICHECK. Mean HPLC, ICHECK0min and ICHECK15min values were 421.2±106.0 µg/L, 423.1±118.3 µg/L and 413.2±107.6 µg/L, respectively. Retinol concentrations significantly decreased in the IEX solution over time (p<0.001). No significant proportional bias was observed between HPLC and ICHECK0min (r-0.038, p=0.73) and ICHECK15min (r=-0.024, p=0.82). Fixed biases (HPLC minus ICHECK) for ICHECK0min and ICHECK15min were respectively -1.9±23.1 µg/l (p=0.45) and 8.0±22.7 µg/l (p=0.002). ICHECK Fluoro may offer a reliable mean for assessing serum retinol for measurements performed with no significant time delay.

Board Policies on Student Records. Educational Policies Development Kit.

ERIC Educational Resources Information Center

National School Boards Association, Waterford, CT. Educational Policies Service.

This report of policy samples is the 17th in a continuing series of kit-booklets issued to help school boards develop written policies in key subject areas. The intent in providing samples is to encourage thinking in policy terms; and to provide working papers to be edited, modified, or adapted to meet local requirements. Policy samples herein…

A framework for measurement and harmonization of pediatric multiple sclerosis etiologic research studies: The Pediatric MS Tool-Kit.

PubMed

Magalhaes, Sandra; Banwell, Brenda; Bar-Or, Amit; Fortier, Isabel; Hanwell, Heather E; Lim, Ming; Matt, Georg E; Neuteboom, Rinze F; O'Riordan, David L; Schneider, Paul K; Pugliatti, Maura; Shatenstein, Bryna; Tansey, Catherine M; Wassmer, Evangeline; Wolfson, Christina

2018-06-01

While studying the etiology of multiple sclerosis (MS) in children has several methodological advantages over studying etiology in adults, studies are limited by small sample sizes. Using a rigorous methodological process, we developed the Pediatric MS Tool-Kit, a measurement framework that includes a minimal set of core variables to assess etiological risk factors. We solicited input from the International Pediatric MS Study Group to select three risk factors: environmental tobacco smoke (ETS) exposure, sun exposure, and vitamin D intake. To develop the Tool-Kit, we used a Delphi study involving a working group of epidemiologists, neurologists, and content experts from North America and Europe. The Tool-Kit includes six core variables to measure ETS, six to measure sun exposure, and six to measure vitamin D intake. The Tool-Kit can be accessed online ( www.maelstrom-research.org/mica/network/tool-kit ). The goals of the Tool-Kit are to enhance exposure measurement in newly designed pediatric MS studies and comparability of results across studies, and in the longer term to facilitate harmonization of studies, a methodological approach that can be used to circumvent issues of small sample sizes. We believe the Tool-Kit will prove to be a valuable resource to guide pediatric MS researchers in developing study-specific questionnaire.

Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits.

PubMed

Mohammadi, M; Talebkhan, Y; Khalili, G; Mahboudi, F; Massarrat, S; Zamaninia, L; Oghalaei, A

2008-01-01

To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.

Arsenic exposure in drinking water: an unrecognized health threat in Peru.

PubMed

George, Christine Marie; Sima, Laura; Arias, M Helena Jahuira; Mihalic, Jana; Cabrera, Lilia Z; Danz, David; Checkley, William; Gilman, Robert H

2014-08-01

To assess the extent of arsenic contamination of groundwater and surface water in Peru and, to evaluate the accuracy of the Arsenic Econo-Quick(™) (EQ) kit for measuring water arsenic concentrations in the field. Water samples were collected from 151 water sources in 12 districts of Peru, and arsenic concentrations were measured in the laboratory using inductively-coupled plasma mass spectrometry. The EQ field kit was validated by comparing a subset of 139 water samples analysed by laboratory measurements and the EQ kit. In 86% (96/111) of the groundwater samples, arsenic exceeded the 10 µg/l arsenic concentration guideline given by the World Health Organization (WHO) for drinking water. In 56% (62/111) of the samples, it exceeded the Bangladeshi threshold of 50 µg/l; the mean concentration being 54.5 µg/l (range: 0.1-93.1). In the Juliaca and Caracoto districts, in 96% (27/28) of groundwater samples arsenic was above the WHO guideline; and in water samples collected from the section of the Rímac river running through Lima, all had arsenic concentrations exceeding the WHO limit. When validated against laboratory values, the EQ kit correctly identified arsenic contamination relative to the guideline in 95% (106/111) of groundwater and in 68% (19/28) of surface water samples. In several districts of Peru, drinking water shows widespread arsenic contamination, exceeding the WHO arsenic guideline. This poses a public health threat requiring further investigation and action. For groundwater samples, the EQ kit performed well relative to the WHO arsenic limit and therefore could provide a vital tool for water arsenic surveillance.

Determining Indirect Cost Rates in Research Libraries. SPEC Kit 34.

ERIC Educational Resources Information Center

Association of Research Libraries, Washington, DC. Office of Management Studies.

This kit prepared by the Association of Research Libraries (ARL) contains 15 primary source documents on determining indirect cost rates in research libraries. The kit comprises: (1) six library cost studies and surveys, "Allocation of Library Expenditures to Research and Instruction" (University of Pennsylvania), "Sampling of Current Monograph…

Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience.

PubMed

Bogdanovska-Todorovska, Magdalena; Petrushevska, Gordana; Janevska, Vesna; Spasevska, Liljana; Kostadinova-Kunovska, Slavica

2018-05-20

Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001). The greatest discordance (82%) between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

Detection of novel polymorphisms in the ckit gene of canine patients with lymphoma, melanoma, haemangiosarcoma, and osteosarcoma.

PubMed

Gramer, Irina; Kessler, Martin; Geyer, Joachim

2016-06-01

Tyrosine kinase inhibitors (TKIs) that specifically target cKIT represent a therapeutic approach for non-resectable canine mast cell tumours (MCTs) grade II/III. The therapeutic benefit of TKIs has been investigated in other tumours based on clinical response rates and identification of gain-of-function mutations. In the present study, cKIT expression in 14 dogs with osteosarcoma, melanoma, haemangiosarcoma, lymphoma, and fibrosarcoma was analysed. Tissue samples were used for cKIT sequencing to (I) detect the cKIT transcript and to (II) identify gain-of-function mutations. The cKIT transcript was detected in ten patients. Four novel amino acid substitutions and five silent polymorphisms were identified. Furthermore, an insertion mutation (GNSK) was discovered in the tissue, but not in the blood sample of one dog. CKIT expression was identified in a variety of canine tumours and, therefore, TKIs might have a broader therapeutic indication apart from treatment of MCTs. Further investigations will be necessary to localize the cKIT protein in the respective tumours and to evaluate the functional consequence of the cKIT variants identified in the present study.

Evaluation of Two Commercial Enzyme-Linked Immunosorbent Assay Kits for the Detection of Human Circulating Metrnl.

PubMed

Zheng, Si-Li; Li, Zhi-Yong; Zhang, Zheng; Wang, Dong-Sheng; Xu, Jian; Miao, Chao-Yu

2018-04-01

Metrnl is a newly discovered secreted protein with neurotrophic activity and metabolic effect, while in earlier studies its circulating level in human was not explored. We evaluated two commercial enzyme-linked immunosorbent assay kits (DY7867-05, R&D Systems and SK00478-02, Aviscera Bioscience) for the detection of human circulating Metrnl. The DY7867-05 kit showed superiority over the SK00478-02 kit since it generated better curve fitting degree, smaller variation among tests, higher inter-assay reproducibility and better specificity, and could effectively detect human Metrnl in six types of blood samples. Subsequent analysis was performed using the DY7867-05 kit. Sample storage conditions were investigated. No gender difference in circulating Metrnl levels was found, while people with newly diagnosed type 2 diabetes mellitus (T2DM) had significantly lower Metrnl levels compared to the healthy controls.

C-MORE Science Kits: Putting Technology in the Hands of K-12 Teachers and Students

NASA Astrophysics Data System (ADS)

Achilles, K.; Weersing, K.; Daniels, C.; Puniwai, N.; Matsuzaki, J.; Bruno, B. C.

2008-12-01

The Center for Microbial Oceanography: Research and Education (C-MORE) is a NSF Science and Technology Center based at the University of Hawaii. The C-MORE education and outreach program offers a variety of resources and professional development opportunities for science educators, including online resources, participation in oceanography research cruises, teacher-training workshops, mini-grants to incorporate microbial oceanography-related content and activities into their classroom and, most recently, C- MORE science kits. C-MORE science kits provide hands-on classroom, field, and laboratory activities related to microbial oceanography for K-12 students. Each kit comes with complete materials and instructions, and is available free of charge to Hawaii's public school teachers. Several kits are available nationwide. C-MORE science kits cover a range of topics and technologies and are targeted at various grade levels. Here is a sampling of some available kits: 1) Marine Murder Mystery: The Case of the Missing Zooxanthellae. Students learn about the effect of climate change and other environmental threats on coral reef destruction through a murder-mystery experience. Participants also learn how to use DNA to identify a suspect. Grades levels: 3-8. 2) Statistical sampling. Students learn basic statistics through an exercise in random sampling, with applications to microbial oceanography. The laptops provided with this kit enable students to enter, analyze, and graph their data using EXCEL. Grades levels: 6-12. 3) Chlorophyll Lab. A research-quality fluorometer is used to measure the chlorophyll content in marine and freshwater systems. This enables students to compare biomass concentrations in samples collected from various locations. Grades levels: 9-12. 4) Conductivity-Temperature-Depth (CTD). Students predict how certain variables (e.g., temperature, pressure, chlorophyll, oxygen) vary with depth. A CTD, attached to a laptop computer, is deployed into deep water off a dock or a ship to collect real-time data and test their hypotheses. Grades levels: 9-12.

c-kit Positive Cardiac Outgrowth Cells Demonstrate Better Ability for Cardiac Recovery Against Ischemic Myopathy.

PubMed

Li, Chuan; Matsushita, Satoshi; Li, Zhengqing; Guan, Jianjun; Amano, Atsushi

2017-10-01

Resident cardiac stem cells are expected to be a therapeutic option for patients who suffer from severe heart failure. However, uncertainty remains over whether sorting cells for c-kit, a stem cell marker, improves therapeutic outcomes. Cardiac outgrowth cells cultured from explants of rat heart atrium were sorted according to their positivity (+) or negativity (-) for c-kit. These cells were exposed to hypoxia for 3 d, and subsequently harvested for mRNA expression measurement. The cell medium was also collected to assess cytokine secretion. To test for a functional benefit in animals, myocardial infarction (MI) was induced in rats, and c-kit+ or c-kit- cells were injected. The left ventricular ejection fraction (LVEF) was measured for up to 4 weeks, after which the heart was harvested for biological and histological analyses. Expression of the angiogenesis-related genes, VEGF and ANGPTL2, was significantly higher in c-kit+ cells after 3 d of hypoxic culture, although we found no such difference prior to hypoxia. Secretion of VEGF and ANGPTL2 was greater in the c-kit+ group than in the c-kit- group, while hypoxia tended to increase cytokine expression in both groups. In addition, IGF-1 was significantly increased in the c-kit+ group, consistent with the relatively low expression of cleaved-caspase 3 revealed by western blot assay, and the relatively low count of apoptotic cells revealed by histochemical analysis. Administration of c-kit+cells into the MI heart improved the LVEF and increased neovascularization. These results indicate that c-kit+cells may be useful in cardiac stem cell therapy.

Protein crystallography prescreen kit

DOEpatents

Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

2007-10-02

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

Protein crystallography prescreen kit

DOEpatents

Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

2005-07-12

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

Post-Mortem Identification of a Fire Carbonized Body by STR Genotyping.

PubMed

Dumache, Raluca; Muresan, Camelia; Ciocan, Veronica; Rogobete, Alexandru F; Enache, Alexandra

2016-10-01

Identification of bodies of unknown identity that are victims of exposure to very high temperatures, resulting from fires, plane crashes, and terrorist attacks, represents one of the most difficult sides of forensic genetics, because of the advanced state of decomposition. The aim of this study was the identification of the carbonized cadaver of a fire victim through STR genotyping. We used blood samples obtained from the iliac artery during the autopsy examination as biological samples from the unidentified victim. After DNA isolation and quantification, we proceeded to its amplification using the multiplex PCR kit AmpFlSTR Identifiler. The DNA products were separated using an ABI 3500 genetic analyzer. Further analysis of the data was done using Gene Mapper ID-X version 1.4 software. In this case, it was possible to obtain a complete DNA profile from the biological samples. Due to the fact that the amelogenin gene presented two alleles, X and Y, we concluded that the victim was a man. We conclude that STR profiling of unidentified bodies (carbonized, decomposed) represents a powerful method of human identification in forensic medicine.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

PubMed Central

Schulte, Simone Laura; Waha, Andreas; Steiger, Barbara; Denkhaus, Dorota; Dörner, Evelyn; Calaminus, Gabriele; Leuschner, Ivo; Pietsch, Torsten

2016-01-01

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID:27391150

Survey of undeclared egg allergen levels in the most frequently recalled food types (including products bearing precautionary labelling).

PubMed

Khuda, Sefat E; Sharma, Girdhari M; Gaines, Dennis; Do, Andrew B; Pereira, Marion; Chang, Michael; Ferguson, Martine; Williams, Kristina M

2016-08-01

Since the number of recalls involving undeclared allergens is commonly associated with bakery and snack foods, we aimed to determine the frequency of egg allergens in a large number of these products using two commercial enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no egg identified on the product label or which had an egg precautionary statement. Among all samples, egg protein was detected in 5% of products using a Morinaga (MO) kit and 1% of products using a R-Biopharm (RB) kit. For bakery samples, egg protein was detected in 6% of 363 samples with no precautionary labelling (6% by MO and 1% by RB kit) and 12% of 80 samples which had precautionary labelling. For snack samples, egg protein was detected in 2% of 371 samples with no precautionary labelling (2% by MO and < 1% by RB kit) and 5% of 21 samples which had precautionary labelling. The disagreement rates between two methods were 5.2% for bakery products and 2.6% for snack products. The sample repeatability was at an acceptable level for bakery (< 12.5%) and snack foods (< 7.5%) for each method. The relative standard deviation between test kits was high (103.1%) for bakery foods. Four bakery products without precautionary labelling had a higher level of egg protein per serving compared with the eliciting dose (ED10 of 3.7 mg protein) for egg allergic patients. These results highlight the fact that detection methodology plays a vital role for accurate labelling control and mitigation of risk for egg allergic consumers.

Evaluation of a new APTIMA specimen collection and transportation kit for high-risk human papillomavirus E6/E7 messenger RNA in cervical and vaginal samples.

PubMed

Chernesky, Max; Jang, Dan; Gilchrist, Jodi; Elit, Laurie; Lytwyn, Alice; Smieja, Marek; Dockter, Janel; Getman, Damon; Reid, Jennifer; Hill, Craig

2014-06-01

An APTIMA specimen collection and transportation (SCT) kit was developed by Hologic/Gen-Probe. To compare cervical SCT samples to PreservCyt and SurePath samples and self-collected vaginal samples to physician-collected vaginal and cervical SCT samples. To determine ease and comfort of self-collection with the kit. Each woman (n = 580) self-collected a vaginal SCT, then filled out a questionnaire (n = 563) to determine ease and comfort of self-collection. Colposcopy physicians collected a vaginal SCT and cervical PreservCyt, SCT, and SurePath samples. Samples were tested by APTIMA HPV (AHPV) assay. Agreement between testing of cervical SCT and PreservCyt was 91.1% (κ = 0.82), and that of SurePath samples was 86.7% (κ = 0.72). Agreement of self-collected vaginal SCT to physician-collected SCT was 84.7% (κ = 0.68), and that of self-collected vaginal to cervical SCT was 82.0% (κ = 0.63). For 30 patients with CIN2+, AHPV testing of cervical SCT was 100% sensitive and 59.8% specific compared with PreservCyt (96.6% and 66.2%) and SurePath (93.3% and 70.9%). Vaginal SCT sensitivity was 86.7% for self-collection and 80.0% for physician collection. Most patients found that vaginal self-collection was easy, 5.3% reported some difficulty, and 87.6% expressed no discomfort. Cervical samples collected with the new SCT kit compared well to traditional liquid-based samples tested by AHPV. Although there was good agreement between self-collected and physician-collected samples with the SCT, in a limited number of 30 women, vaginal sampling identified fewer with CIN2+ precancerous cervical lesions than cervical SCT sampling. Comfort, ease of use, and detection of high-risk HPV demonstrated that the kit could be used for cervical and vaginal sampling.

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

PubMed

Herbert, Zachary T; Kershner, Jamie P; Butty, Vincent L; Thimmapuram, Jyothi; Choudhari, Sulbha; Alekseyev, Yuriy O; Fan, Jun; Podnar, Jessica W; Wilcox, Edward; Gipson, Jenny; Gillaspy, Allison; Jepsen, Kristen; BonDurant, Sandra Splinter; Morris, Krystalynne; Berkeley, Maura; LeClerc, Ashley; Simpson, Stephen D; Sommerville, Gary; Grimmett, Leslie; Adams, Marie; Levine, Stuart S

2018-03-15

Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

Rapid Detection of the Varicella Zoster Virus

NASA Technical Reports Server (NTRS)

Lewis, Michelle P.; Harding, Robert

2011-01-01

1.Technology Description-Researchers discovered that when the Varicella Zoster Virus (VZV) reactivates from latency in the body, the virus is consistently present in saliva before the appearance of skin lesions. A small saliva sample is mixed with a specialized reagent in a test kit. If the virus is present in the saliva sample, the mixture turns a red color. The sensitivity and specificity emanates from an antibody-antigen reaction. This technology is a rapid, non-invasive, point of-of-care testing kit for detecting the virus from a saliva sample. The device is easy to use and can be used in clinics and in remote locations to quickly detect VZV and begin treatment with antiviral drugs. 2.Market Opportunity- RST Bioscience will be the first and only company to market a rapid, same day test kit for the detection of VZV in saliva. The RST detection test kit will have several advantages over existing, competitive technology. The test kit is self contained and laboratory equipment is not required for analysis of the sample. Only a single saliva sample is required to be taken instead of blood or cerebral spinal fluid. The test kit is portable, sterile and disposable after use. RST detection test kits require no electrical power or expensive storage equipment and can be used in remote locations. 3.Market Analysis- According to the CDC, it is estimated that 1 million cases of shingles occur each year in the U.S. with more than half over the age of sixty. There is a high demand for rapid diagnostics by the public. The point-of-care testing (POCT) market is growing faster than other segments of in vitro diagnostics. According to a July 2007 InteLab Corporation industry report the overall market for POCT was forecast to increase from $10.3 billion in 2005 to $18.7 billion by 2011. The market value of this test kit has not been determined. 4.Competition- The VZV vaccine prevents 50% of cases and reduces neuralgia by 66%. The most popular test detects VZV-specific IgM antibody in blood. Other tests include running a sample in a polymerase chain reaction analyzer, enzyme immunoassay, latex agglutination, indirect fluorescent antibody and fluorescent antibody to membrane antigen assay. These existing tests require laboratory analysis by trained personnel, expensive equipment, invasive procedures and a longer period of time to obtain test results.

Evaluation of two platelet-rich plasma processing methods and two platelet-activation techniques for use in llamas and alpacas.

PubMed

Semevolos, Stacy A; Youngblood, Cori D; Grissom, Stephanie K; Gorman, M Elena; Larson, Maureen K

2016-11-01

OBJECTIVE To evaluate 2 processing methods (commercial kit vs conical tube centrifugation) for preparing platelet rich plasma (PRP) for use in llamas and alpacas. SAMPLES Blood samples (30 mL each) aseptically collected from 6 healthy llamas and 6 healthy alpacas. PROCEDURES PRP was prepared from blood samples by use of a commercial kit and by double-step conical tube centrifugation. A CBC was performed for blood and PRP samples. Platelets in PRP samples were activated by means of a freeze-thaw method with or without 23mM CaCl 2 , and concentrations of platelet-derived growth factor-BB and transforming growth factor-β 1 were measured. Values were compared between processing methods and camelid species. RESULTS Blood CBC values for llamas and alpacas were similar. The commercial kit yielded a significantly greater degree of platelet enrichment (mean increase, 8.5 fold vs 2.8 fold) and WBC enrichment (mean increase, 3.7 fold vs 1.9 fold) than did conical tube centrifugation. Llamas had a significantly greater degree of platelet enrichment than alpacas by either processing method. No difference in WBC enrichment was identified between species. Concentrations of both growth factors were significantly greater in PRP samples obtained by use of the commercial kit versus those obtained by conical tube centrifugation. CONCLUSIONS AND CLINICAL RELEVANCE For blood samples from camelids, the commercial kit yielded a PRP product with a higher platelet and WBC concentration than achieved by conical tube centrifugation. Optimal PRP platelet and WBC concentrations for various applications need to be determined for llamas and alpacas.

Blood collection kit for Space Lab 1

NASA Image and Video Library

1981-02-02

S81-26158 (Feb 1981) --- A close-up view of a training version of a STS-40/SLS-1 blood kit. Blood samples from crewmembers are critical to a number of Space Life Sciences-1 (SLS-1) investigations. One day's collection equipment, color coded for each crewmember, is neatly organized in the kit.

46 CFR 160.054-7 - Procedure for approval.

Code of Federal Regulations, 2014 CFR

2014-10-01

...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-7 Procedure... the first-aid kit. At the time of selection of the pre-approval sample, the manufacturer shall furnish... § 160.054-5 to determine the suitability of the first-aid kit for use in conjunction with lifesaving...

46 CFR 160.054-7 - Procedure for approval.

Code of Federal Regulations, 2012 CFR

2012-10-01

...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-7 Procedure... the first-aid kit. At the time of selection of the pre-approval sample, the manufacturer shall furnish... § 160.054-5 to determine the suitability of the first-aid kit for use in conjunction with lifesaving...

46 CFR 160.054-7 - Procedure for approval.

Code of Federal Regulations, 2011 CFR

2011-10-01

...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-7 Procedure... the first-aid kit. At the time of selection of the pre-approval sample, the manufacturer shall furnish... § 160.054-5 to determine the suitability of the first-aid kit for use in conjunction with lifesaving...

46 CFR 160.054-7 - Procedure for approval.

Code of Federal Regulations, 2013 CFR

2013-10-01

...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-7 Procedure... the first-aid kit. At the time of selection of the pre-approval sample, the manufacturer shall furnish... § 160.054-5 to determine the suitability of the first-aid kit for use in conjunction with lifesaving...

Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid:
a systematic review

PubMed Central

Nunes, Lazaro Alessandro Soares; Mussavira, Sayeeda

2015-01-01

This systematic review presents the latest trends in salivary research and its applications in health and disease. Among the large number of analytes present in saliva, many are affected by diverse physiological and pathological conditions. Further, the non-invasive, easy and cost-effective collection methods prompt an interest in evaluating its diagnostic or prognostic utility. Accumulating data over the past two decades indicates towards the possible utility of saliva to monitor overall health, diagnose and treat various oral or systemic disorders and drug monitoring. Advances in saliva based systems biology has also contributed towards identification of several biomarkers, development of diverse salivary diagnostic kits and other sensitive analytical techniques. However, its utilization should be carefully evaluated in relation to standardization of pre-analytical and analytical variables, such as collection and storage methods, analyte circadian variation, sample recovery, prevention of sample contamination and analytical procedures. In spite of all these challenges, there is an escalating evolution of knowledge with the use of this biological matrix. PMID:26110030

Kit for the rapid preparation of .sup.99m Tc red blood cells

DOEpatents

Richards, Powell; Smith, Terry D.

1976-01-01

A method and sample kit for the preparation of .sup.99m Tc-labeled red blood cells in a closed, sterile system. A partially evacuated tube, containing a freeze-dried stannous citrate formulation with heparin as an anticoagulant, allows whole blood to be automatically drawn from the patient. The radioisotope is added at the end of the labeling sequence to minimize operator exposure. Consistent 97% yields in 20 minutes are obtained with small blood samples. Freeze-dried kits have remained stable after five months.

Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx™ Forensic Genomics System.

PubMed

Guo, Fei; Yu, Jiao; Zhang, Lu; Li, Jun

2017-11-01

The ForenSeq™ DNA Signature Prep Kit (ForenSeq Kit) is designed to detect more than 200 forensically relevant markers in a single reaction on the MiSeq FGx™ Forensic Genomics System (MiSeq FGx System), including Amelogenin, 27 autosomal short tandem repeats (A-STRs), 7 X chromosomal STRs (X-STRs), 24 Y chromosomal STRs (Y-STRs) and 94 identity-informative single nucleotide polymorphisms (iSNPs) with the option to contain 22 phenotypic-informative SNPs (pSNPs) and 56 ancestry-informative SNPs (aSNPs). In this study, we evaluated the MiSeq FGx System on three major parts: methodological optimization (DNA extraction, sample quantification, library normalization, diluted libraries concentration, and sample-to-cell arrangement), massively parallel sequencing (MPS) performance (depth of coverage, sequence coverage ratio, and allele coverage ratio), and ForenSeq Kit characteristics (repeatability and concordance, sensitivity, mixture, stability and case-type samples). Results showed that quantitative polymerase chain reaction (qPCR)-based sample quantification and library normalization and the appropriate number of pooled libraries and concentration of diluted libraries provided a greater level of MPS performance and repeatability. Repeatable and concordant genotypes were obtained by the ForenSeq Kit. Full profiles were obtained from ≥100pg input DNA for STRs and ≥200pg for SNPs. A sample with ≥5% minor contributors was considered as a mixture by imbalanced allele coverage ratio distribution, and full profiles from minor contributors were easily detected between 9:1 and 1:9 mixtures with known reference profiles. The ForenSeq Kit tolerated considerable concentrations of inhibitors like ≤200μM hematin and ≤50μg/ml humic acid, and >56% STR profiles and >88% SNP profiles were obtained from ≥200-bp degraded samples. Also, it was adapted to case-type samples. As a whole, the ForenSeq Kit is a well-performed, robust, reliable, reproducible and highly informative assay, and it can fully meet requirements for human identification. Further, sensitive QC indicator and automated sample comparison function in the ForenSeq™ Universal Analysis Software are quite helpful, so that we can concentrate on questionable genotypes and avoid tedious and time-consuming labor to maximum the time spent in data analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Emergence of CTNNB1 mutation at acquired resistance to KIT inhibitor in metastatic melanoma.

PubMed

Cho, J; Kim, S Y; Kim, Y J; Sim, M H; Kim, S T; Kim, N K D; Kim, K; Park, W; Kim, J H; Jang, K-T; Lee, J

2017-10-01

The KIT inhibitor, imatinib, has shown promising efficacy in patients with KIT-mutated melanoma; however, acquisition of resistance to imatinib occurs rapidly in the majority of patients. The mechanisms of acquired resistance to imatinib in melanoma remain unclear. We analyzed biopsy samples from paired baseline and post-treatment tumor lesions in one patient with KIT-mutated melanoma who had had an initial objective tumor regression in response to imatinib treatment followed by disease progression 8 months later. Targeted deep sequencing from post-treatment biopsy samples detected an additional mutation in CTNNB1 (S33C) with original KIT L576P mutation. We examined the functional role of the additional CTNNB1 S33C mutation in resistance to imatinib indirectly using the Ba/F3 cell model. Ba/F3 cell lines transfected with both the L576P KIT mutation and the CTNNB1 S33C mutation demonstrated no growth inhibition despite imatinib treatment, whereas growth inhibition was observed in the Ba/F3 cell line transfected with the L576 KIT mutation alone. We report the first identification of the emergence of a CTNNB1 mutation that can confer acquired resistance to imatinib. Further investigation into the causes of acquired resistance to imatinib will be essential to improve the prognosis for patients with KIT-mutated melanoma.

Arsenic exposure in drinking water: an unrecognized health threat in Peru

PubMed Central

Sima, Laura; Arias, M Helena Jahuira; Mihalic, Jana; Cabrera, Lilia Z; Danz, David; Checkley, William; Gilman, Robert H

2014-01-01

Abstract Objective To assess the extent of arsenic contamination of groundwater and surface water in Peru and, to evaluate the accuracy of the Arsenic Econo-Quick™ (EQ) kit for measuring water arsenic concentrations in the field. Methods Water samples were collected from 151 water sources in 12 districts of Peru, and arsenic concentrations were measured in the laboratory using inductively-coupled plasma mass spectrometry. The EQ field kit was validated by comparing a subset of 139 water samples analysed by laboratory measurements and the EQ kit. Findings In 86% (96/111) of the groundwater samples, arsenic exceeded the 10 µg/l arsenic concentration guideline given by the World Health Organization (WHO) for drinking water. In 56% (62/111) of the samples, it exceeded the Bangladeshi threshold of 50 µg/l; the mean concentration being 54.5 µg/l (range: 0.1–93.1). In the Juliaca and Caracoto districts, in 96% (27/28) of groundwater samples arsenic was above the WHO guideline; and in water samples collected from the section of the Rímac river running through Lima, all had arsenic concentrations exceeding the WHO limit. When validated against laboratory values, the EQ kit correctly identified arsenic contamination relative to the guideline in 95% (106/111) of groundwater and in 68% (19/28) of surface water samples. Conclusion In several districts of Peru, drinking water shows widespread arsenic contamination, exceeding the WHO arsenic guideline. This poses a public health threat requiring further investigation and action. For groundwater samples, the EQ kit performed well relative to the WHO arsenic limit and therefore could provide a vital tool for water arsenic surveillance. PMID:25177071

Pure versus combined Merkel cell carcinomas: immunohistochemical evaluation of cellular proteins (p53, Bcl-2, and c-kit) reveals significant overexpression of p53 in combined tumors.

PubMed

Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M

2015-09-01

Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Novel somatic KIT exon 8 mutation with dramatic response to imatinib in a patient with mucosal melanoma: a case report.

PubMed

Rapisuwon, Suthee; Parks, Kellie; Al-Refaie, Waddah; Atkins, Michael B

2014-10-01

Primary mucosal melanomas represent ∼1.3% of all cases of melanoma diagnosed in the USA. The sinonasal location is the most common primary site. Mutations in the KIT gene occur in 10-22% of mucosal melanomas. Tumor response to imatinib mesylate has been reported in about half of the patients with tumors harboring KIT mutations. Responses are almost exclusively restricted to tumors with mutations in KIT exon 9 or 11. We report a case of a patient with a sinonasal mucosal melanoma with a novel exon 8 mutation (C443S) who had marked initial response to imatinib. Somatic exon 8 KIT mutations have not been previously reported in mucosal melanoma or in other human solid tumors; however, such mutations have been reported in canine and feline mast cell tumors. Protein transcripts from exon 8 play an important role in the structural and functional integrity of the extracellular domain of KIT. In preclinical studies, a mutation in exon 8 led to autophosphorylation, independent of KIT ligand, and constitutive activation of the tyrosine kinase. This biology may explain the successful application of imatinib in animals with tumors harboring exon 8 KIT mutations and in our patient with mucosal melanoma. This report expands the population of patients with melanoma who might benefit from imatinib to those with somatic exon 8 KIT mutations. Such mutations should be looked for in patients with mucosal melanoma.

Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus.

PubMed

Le Dimna, M; Vrancken, R; Koenen, F; Bougeard, S; Mesplède, A; Hutet, E; Kuntz-Simon, G; Le Potier, M F

2008-01-01

Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.

Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation

PubMed Central

Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi

2015-01-01

Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. PMID:26722383

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B.

PubMed

Ahn, Sang Hoon; Chun, Ji-Yong; Shin, Soo-Kyung; Park, Jun Yong; Yoo, Wangdon; Hong, Sun Pyo; Kim, Soo-Ok; Han, Kwang-Hyub

2013-12-01

Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B.

Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples

USDA-ARS?s Scientific Manuscript database

Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their effectiveness in detecting and differentiating between Shiga toxin 1 and 2 (Stx1 and Stx2) produced by Shiga toxin-producing E. coli (STEC) inoculated into food and water samples. Each kit incorporated monoclonal antibodies ...

Vitamin B12 absorption judged by measurement of holotranscobalamin, active vitamin B12: evaluation of a commercially available EIA kit.

PubMed

Greibe, Eva; Nexo, Ebba

2011-11-01

Active vitamin B12 absorption is followed by an increase in holotranscobalamin (holoTC) upon loading with a high physiological dose of the vitamin (the CobaSorb test). This study evaluates the use of a newly launched EIA kit for measurement of holoTC (active B12) in relation to the CobaSorb test. Intra-assay imprecision and linearity of the EIA kit was examined, employing serum pools of increasing holoTC concentrations. For the CobaSorb test, holoTC was measured before and after loading with 3-times 9 μg of vitamin B12 employing both the in-house ELISA and the EIA kit (n=25). The EIA kit showed an intra-assay CV between 2.2% and 5.8% for holoTC values ranging from 21 to 80 pmol/L. Employing diluted serum samples resulted in spurious high values of holoTC. The EIA kit performed well in relation to the CobaSorb test and classified the patients studied as capable of absorbing vitamin B12 (n=10) or not (n=15), as did the in-house ELISA. The Active B12 (holoTC) EIA kit proved suitable for use with the CobaSorb test, but not for analysis of diluted serum samples.

SEABEE Combat Handbook. Revision

DTIC Science & Technology

1989-02-01

early stages of any biological disease, the general symptoms are fever , malaise, and * Warning, reporting, detection, and inflammation, identification...be thoroughly scrubbed . Contaminated clothing i ilgclwraeee cus fyusolu scontract a disease from biological warfare in spite must be washed in hot...protective masks, skin decontamination caused by thermal radiation and injuries caused kits, and atropine. 19-16 Chapter 19-CHEMICAL, BIOLOGICAL , AND

Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics

PubMed Central

2011-01-01

Background High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. Results The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. Conclusions The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers. PMID:21554704

Automated diagnostic kiosk for diagnosing diseases

DOEpatents

Regan, John Frederick; Birch, James Michael

2014-02-11

An automated and autonomous diagnostic apparatus that is capable of dispensing collection vials and collections kits to users interesting in collecting a biological sample and submitting their collected sample contained within a collection vial into the apparatus for automated diagnostic services. The user communicates with the apparatus through a touch-screen monitor. A user is able to enter personnel information into the apparatus including medical history, insurance information, co-payment, and answer a series of questions regarding their illness, which is used to determine the assay most likely to yield a positive result. Remotely-located physicians can communicate with users of the apparatus using video tele-medicine and request specific assays to be performed. The apparatus archives submitted samples for additional testing. Users may receive their assay results electronically. Users may allow the uploading of their diagnoses into a central databank for disease surveillance purposes.

Shuttle Kit Freezer Refrigeration Unit Conceptual Design

NASA Technical Reports Server (NTRS)

Copeland, R. J.

1975-01-01

The refrigerated food/medical sample storage compartment as a kit to the space shuttle orbiter is examined. To maintain the -10 F in the freezer kit, an active refrigeration unit is required, and an air cooled Stirling Cycle refrigerator was selected. The freezer kit contains two subsystems, the refrigeration unit, and the storage volume. The freezer must provide two basic capabilities in one unit. One requirement is to store 215 lbs of food which is consumed in a 30-day period by 7 people. The other requirement is to store 128.3 lbs of medical samples consisting of both urine and feces. The unit can be mounted on the lower deck of the shuttle cabin, and will occupy four standard payload module compartments on the forward bulkhead. The freezer contains four storage compartments.

Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

PubMed

Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

2017-04-21

It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Design of Indigenous ELISA Using Tachyzoites from the RH Strain of Toxoplasma gondii and Comparison with Commercial Kits in Ahvaz, Southwest of Iran, 2015.

PubMed

Mohammadpour, Niloofar; Saki, Jasem; Rafiei, Abdollah; Khodadadi, Ali; Tavalla, Mehdi; Cheraghian, Bahman

2016-10-01

Toxoplasma gondii is one of the most common causes of latent infections in humans worldwide. Detecting anti- Toxoplasma antibodies in serum using serological tests is a common method to diagnose toxoplasmosis. In the present study, an indigenous ELISA kit was prepared using tachyzoites from the RH strain of T. gondii , and its sensitivity and specificity were compared with those of commercial kits. To produce antigens, 0.02 mL of locally isolated T. gondii RH strain parasites along with 10 9 tachyzoites were injected into the peritoneal cavities of 50 laboratory mice (BALB/C). Parasites were collected after 4 days. After filtering and washing, the concentration of protein in sonicated tachyzoites was calculated using the Lowry protein assay. The dilution of antigen, serum and alkaline phosphatase conjugate was assessed in designing an indigenous ELISA method; then ELISA was performed based on these dilutions, and its sensitivity was determined using 200 serum samples. In addition, the specificity of the assay was evaluated using 40 serum samples from patients with tuberculosis, leukemia or hydatid cyst. Indigenous ELISA was used to examine 100 serum samples containing anti- T. gondii IgG, with a sensitivity of 98% (commercial kits: 100%). Another 100 serum samples containing anti- T. gondii IgM were also tested, with a sensitivity of 99% (commercial kits: 100%). When 40 serum samples from patients with leukemia, hydatid cyst or tuberculosis were examined using anti- T. gondii IgG, the specificity was 100%, identical to commercial kits. However, the specificity of a similar test with anti- T. gondii IgM was just 28.6% for serum samples from leukemia patients, 21.4% for hydatid cyst and 16.7% for tuberculosis. We found that purified locally isolated soluble crude antigens of the RH strain of T. gondii from the peritoneal cavity of mice may be one of the most promising antigens for detection of human toxoplasmosis in routine screening.

Two simple techniques for the safe Sarcoptes collection and individual mite DNA extraction.

PubMed

Soglia, Dominga; Rambozzi, Luisa; Maione, Sandra; Spalenza, Veronica; Sartore, Stefano; Alasaad, Samer; Sacchi, Paola; Rossi, Luca

2009-10-01

Availability of mites is a recognized limiting factor of biological and genetic investigations of the genus Sarcoptes. Current methods of deoxyribonucleic acid (DNA) extraction from individual mites also need substantial improvement in efficiency and operator friendliness. We have first developed a technique for efficient and safe extraction of living mites from scabietic skin samples (crusts or deep skin scrapings). Its core device is a large plastic syringe connected with a 1.5-ml Eppendorf tube. The source material is introduced in the syringe and the device in a shoe box with the tip half of the tube emerging. Mites migrate towards a heat source during a minimum of 36 h. Then, the tube is detached and the mites utilized without risks for the operators. A second technique allows operator-friendly manipulation of individual mites for DNA extraction. Fixed mites are isolated by adhesion to a small strip of polyvinyl chloride (PVC) adhesive tape operated with tweezers. Then, mite and strip are plunged in the lyses buffer and the sample twice submitted to thermal shock for disruption of the chitinous exoskeleton. Data show that the tape does not interfere with successive DNA extraction with a commercial kit. The corresponding protocol, that we briefly name "PVC adhesive tape + thermal shock + kit DNA extraction," compares favorably with the available ones.

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

PubMed Central

Murayama, Asako; Sugiyama, Nao; Watashi, Koichi; Masaki, Takahiro; Suzuki, Ryosuke; Aizaki, Hideki; Mizuochi, Toshiaki; Wakita, Takaji

2012-01-01

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits. PMID:22495557

Efficacy of a lead based paint XRF analyzer and a commercially available colorimetric lead test kit as qualitative field tools for determining presence of lead in religious powders.

PubMed

Shah, Manthan P; Shendell, Derek G; Meng, Qingyu; Ohman-Strickland, Pamela; Halperin, William

2018-04-23

The performances of a portable X-Ray Fluorescence (XRF) lead paint analyzer (RMD LPA-1, Protec Instrument Corp., Waltham, MA) and a commercially available colorimetric lead test kit (First Alert Lead Test Kit, eAccess Solutions, Inc., Palatine, IL) were evaluated for use by local or state health departments as potential cost-effective rapid analysis or "spot test" field techniques for tentative identification of lead content in sindoor powders. For both field-sampling methods, sensitivity, specificity and predictive values varied widely for samples containing <300,000 μg/g lead. For samples containing ≥300,000 μg/g lead, the aforementioned metrics were 100% (however, the CIs had a wide range). In addition, both field sampling methods showed clear, consistent positive readings only for samples containing ≥300,000 μg/g lead. Even samples with lead content as high as 5,110 μg/g were not positively identified by either field analysis technique. The results of this study suggest the XRF analyzer and colorimetric lead test kit cannot be used as a rapid field test for sindoor by health department inspectors.

Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

PubMed

Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John

2016-11-01

Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

[Effect evaluation of three ELISA kits in detection of fasciolasis].

PubMed

Ai, Lin; Chen, Mu-Xin; Chen, Shao-Hong; Chu, Yan-Hong; Cai, Yu-Chun; Zhou, Xiao-Nong; Chen, Jia-Xu

2013-04-01

To evaluate the effect of 3 ELISA kits on detection of human fasciolasis. Twenty-six serum samples from patients with fasciolasis, 180 serum samples from patients with other parasitic diseases as well as 26 serum samples from healthy people were detected by ELISA kits which using soluble antigen of Fasciola gigantica, Fasciola hepatica (Fg-ELISA and Fh-ELISA) as well as IgG antigen ELISA detection kits made by DRG company in Germany. The effects of the 3 kits were evaluated. The sensitivities of Fg-ELISA, Fh-ELISA, and DRG-ELISA were 100.0%, 80.8% (95% CI: 65.7%-95.9%) and 100.0%, respectively; the specificities of the three were 87.9% (95% CI: 83.5%-92.4%), 85.0%(95% CI: 80.1%-89.9%) and 83.5% (95% CI: 78.4%-88.6%), respectively, and Youden indexes of them were 0.88, 0.66 and 0.84, respectively. The detection rate of Fg-ELISA (100%) was significantly higher than that of Fh-ELISA (80.8%) (P < 0.05). The A absolute value (A/CO) of Fg-ELISA was 1.70, which was also significantly higher than the value of Fh-ELISA (1.18) (P < 0.000 1). Fg-ELISA has a good detection effect and low cost, and is more suitable than Fh-ELISA and DRG-ELISA for clinical sample tests as well as massive screening in fasciolasis endemic areas in southwest China.

Identification of Differentially Methylated Sites with Weak Methylation Effects

PubMed Central

Tran, Hong; Zhu, Hongxiao; Wu, Xiaowei; Kim, Gunjune; Clarke, Christopher R.; Larose, Hailey; Haak, David C.; Westwood, James H.; Zhang, Liqing

2018-01-01

Deoxyribonucleic acid (DNA) methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs) among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM) was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ) twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs) are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same concepts, with the only difference being how methylation information across the genome is summarized. If methylation levels are determined by grouping neighboring cytosine sites, then they are DMRs; if methylation levels are calculated based on single cytosines, they are DMCs. PMID:29419727

A Field Evaluation of the Hardy TB MODS Kit™ for the Rapid Phenotypic Diagnosis of Tuberculosis and Multi-Drug Resistant Tuberculosis

PubMed Central

Martin, Laura; Coronel, Jorge; Faulx, Dunia; Valdez, Melissa; Metzler, Mutsumi; Crudder, Chris; Castillo, Edith; Caviedes, Luz; Grandjean, Louis; Rodriguez, Mitzi; Friedland, Jon S.; Gilman, Robert H.; Moore, David A. J.

2014-01-01

Background Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory. Methods & Findings 2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3–99.8%), 98.3% (97.5–98.8%), 95.8% (94.0–97.1%), and 99.7% (99.3–99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9–13) days for conventional MODS and 8.5 (7–11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples). Conclusions MODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest. PMID:25225802

A field evaluation of the Hardy TB MODS Kit™ for the rapid phenotypic diagnosis of tuberculosis and multi-drug resistant tuberculosis.

PubMed

Martin, Laura; Coronel, Jorge; Faulx, Dunia; Valdez, Melissa; Metzler, Mutsumi; Crudder, Chris; Castillo, Edith; Caviedes, Luz; Grandjean, Louis; Rodriguez, Mitzi; Friedland, Jon S; Gilman, Robert H; Moore, David A J

2014-01-01

Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory. 2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3-99.8%), 98.3% (97.5-98.8%), 95.8% (94.0-97.1%), and 99.7% (99.3-99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9-13) days for conventional MODS and 8.5 (7-11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples). MODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest.

[Comparison of Dengue viral nonstructural protein 1 antigen testing kits].

PubMed

Wu, D; Zhao, L Z; Wu, Y H; Zhang, H; Zhang, M; Tan, Q Q; Zhou, H Q; Zhang, F C; He, J F

2018-02-06

Objective: To investigate the sensitivity and specificity of commercial nonstructural protein 1 (NS1) testing kits for Dengue fever diagnose, and provide the evidence for diagnostic criteria revision. Methods: 300 PCR or virus isolation positive blood samples for dengue virus were collected from sentinel hospitals for dengue surveillance in Guangzhou, Dongguang and Zhongshang from May 2015 to Nov. 2016. At the same time, 308 PCR negative samples for Dengue virus were collected as control group. The information of the sample was collected using questionnaires. These samples were tested using imported and domestic ELISA and the colloidal gold-labeled kits that were widely used for detecting dengue NS1. Sensitivity, specificity and coincidence were calculated and analyzed, and Z hongshan's result was regarded as the reslut of the third part. Results: The positive group includes 133 males and 167 females, average ages are 47.2±13.3, 179, 110 and 11 of them is Dengue Ⅰ, Ⅱ and Ⅲ respectively. The negative group includes 154 males and 154 females, average ages are (40.1±11.6) years old. The sensitivity of domestic ELISA Kits (94.5%) is less than imported (99.5%), and the result has statistical significance (χ(2)=8.59, P= 0.030), the specificity is 99.7% and 97.7% respectively; The sensitivity of imported and domestic the colloidal gold-labeled Kits is 97.5% and 96.5% respectively, both of specificities are 100%. The sensitivity and specificity of Dengue Ⅰ for NS1 test are more than 97.0%. The sensitivity of domestic ELISA and gold-labeled Kits is 90.0% and 95.0%, and the specificity is 96.8% and 100% respectively for Dengue Ⅱ test. The sensitivity of imported ELISA and gold-labeled Kits is 100% and 98.0%, and the specificity is 99.4% and 100% respectively for Dengue Ⅱ test. The result of the third party show the sensitivity and specificity of domestic ELISA and gold-labeled Kits are 90.0% and 98.0%, the differences has statistical significance (χ(2)=5.67, P= 0.020). Conclusion: NS1 testing can be used as early dengue fever diagnose for higher sensitivity and specificity.

Study and evaluation of Wondfo rapid diagnostic kit based on nano-gold immunochromatography assay for diagnosis of Plasmodium falciparum.

PubMed

Peng, Yunping; Wu, Junlin; Wang, Jihua; Li, Wenmei; Yu, Shujuan

2012-04-01

Malaria has been recognized as a human disease for thousands of years and remains one of the most common diseases affecting humans worldwide. Therefore, a method for rapidly detecting Plasmodium falciparum is necessary and useful. We have developed Wondfo rapid diagnostic kit based on nano-gold immunochromatography assay for the detection of P. falciparum in patient specimen. In the present study, we demonstrated the sensitivity and specificity of the rapid diagnostic kit in which nano-gold labeling techniques and the monoclonal antibodies against histidine-rich protein-2 of P. falciparum were used to establish two-antibody sandwich immunochromatographic assay for detecting P. falciparum. By using microscopic examination of blood smears as control, the sensitivity, specificity, and feasibility of Wondfo rapid diagnostic kit was determined in the prompt and accurate diagnosis of malaria. In this study, 1,558 blood samples were collected from outpatient clinics in China and detected by both Wondfo kit and microscopic examination. The Wondfo kit did not show cross-reaction with microfilaria, Toxoplasma gondii, and other parasites in the blood. The patient samples positive for rheumatoid factor, HIV, tuberculosis, and syphilis did not show false positivity when testing with Wondfo kit. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 95.49% and 99.53%, respectively. These results indicate that our rapid diagnostic assay may be useful for detecting P. falciparum in patient specimen.

[Development of ELISA-kit of quantitative analysis for Zearalenone].

PubMed

Wang, Yu-ping; Ji, Rong; Jiang, Tao; Kang, Wei-jun

2006-03-01

To develope a rapid, sensitive, quantitative ELISA-kit for Zearalenone and determine zearalenone in cereals. On the base of monoclonal antibodies against ZEN, apply indirect ELISA to study the performance parameter of the kit. The limited concentration of detection of the ELISA-kit was 1ng/ml, linear range was 1-200 ng/ml, the linear equation was Y = 0.99 - 0.40 x (R2 = 0.99). The inhibition concentration of 50% against ZEN was 16.3 ng/ml. The average recovery rate of spiked corn and wheat was 96.5% and 95.5%, respectively, the coefficient of variant was 13.2% and 10.9%, respectively. The kit can be stored at 4 degrees C over 6 months. The cross reaction rate with the other mycotoxins was less than 1%, and coefficient of variant within-laboratory and between-laboratory was less than 15% and less than 20%, respectively. Detecting the VICAM sample with ELISA method and HPLC method, the results were within the range of the sample, and there was no statistic difference between the two methods. This ELISA-kit was quick, sensitive, stable and specific and can be used to determine ZEN in cereals.

Immunochemical analytical methods for the determination of peanut proteins in foods.

PubMed

Whitaker, Thomas B; Williams, Kristina M; Trucksess, Mary W; Slate, Andrew B

2005-01-01

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.

KIT amplification and gene mutations in acral/mucosal melanoma in Korea.

PubMed

Yun, Jina; Lee, Jeeyun; Jang, Jiryeon; Lee, Eui Jin; Jang, Kee Taek; Kim, Jung Han; Kim, Kyoung-Mee

2011-06-01

Mucosal and acral melanomas have demonstrated different genetic alterations and biological behavior compared with more common cutaneous melanomas. It was recently reported that gain-of-function KIT mutations and/or copy number increases are more common in mucosal and acral melanomas. Thus, we studied the frequency and pattern of KIT aberrations in mucosal and acral melanomas in Korea. We analyzed 97 patients who were pathologically confirmed with mucosal or acral melanoma between 1997 and 2010 at Samsung Medical Center. Of the 97 melanoma patients, 92 were screened for mutations in KIT exons 11, 13, 17, and 18, BRAF and NRAS genes. KIT copy number was assessed by quantitative, real-time PCR. Of the 97 patients, 55 (56.7%) were mucosal, 40 (41.2%) were acral melanoma, and two were of unknown primary origin. Among seven cases with KIT mutation, five (60.0%) occurred in exon 11, one (20.0%) in exon 17, and one (20.0%) in exon 13. Point mutations were the most common, resulting in substitutions in exon 11 (K558R, T574A, L576P, and V559A), exon 13 (N655K), and exon 17 (N822K). A novel Thr574Ala (c.1720A>G) KIT mutation, which has not been reported in melanoma or other tumor types, was identified in one genital melanoma case. Of the 97 mucosal or acral melanoma specimens, 49 were tested for KIT gene copy number changes using quantitative PCR. Increased KIT copy number was identified in 15 patients: seven (40%) of 20 acral melanomas and eight (31%) of 26 mucosal melanomas. Our study implicates that a significant proportion of acral and mucosal melanomas have KIT mutations in Asian population. © 2011 The Authors. APMIS © 2011 APMIS.

[Consistency study of PowerPlex 21 kit and Goldeneye 20A kit and forensic application].

PubMed

Ren, He; Liu, Ying; Zhang, Qing-Xia; Jiao, Zhang-Ping

2014-06-01

To ensure the consistency of genotype results for PowerPlex 21 kit and Goldeneye 20A kit. The STR loci were amplified in DNA samples from 205 unrelated individuals in Beijing Han population. And consistency of 19 overlap STR loci typing were observed. The genetic polymorphism of D1S1656 locus was obtained. All 19 overlap loci typing showed consistent. The proportion of peak height of heterozygous loci in two kits showed no statistical difference (P > 0.05). The observed heterozygosis of D1S1656 was 0.878. The discrimination power was 0.949. The excluding probability of paternity of triplet was 0.751. The excluding probability of paternity of diploid was 0.506. The polymorphism information content was 0.810. PowerPlex 21 kit and Goldeneye 20A kit present a good consistency. The primer design is reasonable. The polymorphism of D1S1656 is good. The two kits can be used for human genetic analysis, paternity test, and individual identification in forensic practice.

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B

PubMed Central

Ahn, Sang Hoon; Chun, Ji-Yong; Shin, Soo-Kyung; Park, Jun Yong; Yoo, Wangdon; Hong, Sun Pyo; Han, Kwang-Hyub

2013-01-01

Background/Aims Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. Methods The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. Results Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. Conclusions The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B. PMID:24459645

A "three-in-one" sample preparation method for simultaneous determination of B-group water-soluble vitamins in infant formula using VitaFast(®) kits.

PubMed

Zhang, Heng; Lan, Fang; Shi, Yupeng; Wan, Zhi-Gang; Yue, Zhen-Feng; Fan, Fang; Lin, Yan-Kui; Tang, Mu-Jin; Lv, Jing-Zhang; Xiao, Tan; Yi, Changqing

2014-06-15

VitaFast(®) test kits designed for the microbiological assay in microtiter plate format can be applied to quantitative determination of B-group water-soluble vitamins such as vitamin B12, folic acid and biotin, et al. Compared to traditional microbiological methods, VitaFast(®) kits significantly reduce sample processing time and provide greater reliability, higher productivity and better accuracy. Recently, simultaneous determination of vitamin B12, folic acid and biotin in one sample is urgently required when evaluating the quality of infant formulae in our practical work. However, the present sample preparation protocols which are developed for individual test systems, are incompatible with simultaneous determination of several analytes. To solve this problem, a novel "three-in-one" sample preparation method is herein developed for simultaneous determination of B-group water-soluble vitamins using VitaFast(®) kits. The performance of this novel "three-in-one" sample preparation method was systematically evaluated through comparing with individual sample preparation protocols. The experimental results of the assays which employed "three-in-one" sample preparation method were in good agreement with those obtained from conventional VitaFast(®) extraction methods, indicating that the proposed "three-in-one" sample preparation method is applicable to the present three VitaFast(®) vitamin test systems, thus offering a promising alternative for the three independent sample preparation methods. The proposed new sample preparation method will significantly improve the efficiency of infant formulae inspection. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Differentiation of influenza (Flu) type A, type B, and respiratory syncytial virus (RSV) by QuickNavi™-Flu+RSV].

PubMed

Kohiyama, Risa; Miyazawa, Takashi; Shibano, Nobuko; Inano, Koichi

2014-01-01

Because it is not easy to differentiate Influenza virus (Flu) from RS virus (RSV) just by clinical symptoms, to accurately diagnose those viruses in conjunction with patient's clinical symptoms, rapid diagnostic kits has been used separately for each of those viruses. In our new study, we have developed a new rapid diagnostic kit, QuickNavi™-Flu+RSV. The kit can detect Flu A, Flu B, and RSV antigens with a single sample collection and an assay. Total of 2,873 cases (including nasopharyngeal swabs and nasopharyngeal aspirates specimens) in 2010/2011 and 2011/2012 seasons were evaluated with QuickNavi™-Flu+RSV and a commercially available kit. Sensitivity, specificity, and accuracy of Flu type A, type B, and RSV were above 95% when compared to commercially available kits (QuickNavi™-Flu and QuickNavi™-RSV) and considered to be equivalent to the commercially available kits. In 2011/2012 season, RSV infections increased prior to Flu season and continued during the peak of the Flu season. The kit can contribute to accurate diagnosis of Flu and RSV infections since co-infection cases have also been reported during the 2011/2012 season. QuickNavi™-Flu+RSV is useful for differential diagnosis of respiratory infectious diseases since it can detect Flu type A, type B, and RSV virus antigens with a single sample collection.

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples.

PubMed

Thomas, M C; Shields, M J; Hahn, K R; Janzen, T W; Goji, N; Amoako, K K

2013-07-01

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10(1) and 1·3 × 10(2)  CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples. © Her Majesty the Queen in Right of Canada [2013]. Reproduced with the permission of the Canadian Food Inspection Agency.

Performances of Four Helicobacter pylori Serological Detection Kits Using Stool Antigen Test as Gold Standard.

PubMed

Biranjia-Hurdoyal, Susheela D; Seetulsingh-Goorah, Sharmila P

2016-01-01

The aim was to determine the performances of four Helicobacter pylori serological detection kits in different target groups, using Amplified IDEIA™ Hp StAR™ as gold standard. Kits studied were Rapid Immunochromatoghraphic Hexagon, Helicoblot 2.1, an EIA IgG kit and EIA IgA kit. Stool and blood samples were collected from 162 apparently healthy participants (control) and 60 Type 2 diabetes mellitus (T2DM) patients. The performances of the four serological detection kits were found to be affected by gender, age, health status and ethnicity of the participants. In the control group, the Helicoblot 2.1 kit had the best performance (AUC = 0.85; p<0.05, accuracy = 86.4%), followed by EIA IgG (AUC = 0.75; p<0.05, accuracy = 75.2%). The Rapid Hexagon and EIA IgA kits had relatively poor performances. In the T2DM subgroup, the kits H2.1 and EIA IgG had best performances, with accuracies of 96.5% and 93.1% respectively. The performance of EIA IgG improved with adjustment of its cut-off value. The performances of the detection kits were affected by various factors which should be taken into consideration.

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species

PubMed Central

Eriksson, Per; Mourkas, Evangelos; González-Acuna, Daniel; Olsen, Björn; Ellström, Patrik

2017-01-01

ABSTRACT Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host–microbial interactions. Fecal DNA analysis can provide information about the host’s microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers’ instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples. PMID:29152162

Portable field kit for determining uranium in water

USGS Publications Warehouse

McHugh, John B.

1979-01-01

The pressing need for on-site field analyses of the uranium content of surface and ground waters has promoted the development of a simple, light-weight, relatively cheap, portable kit to make such determinations in the field. Forty to sixty water samples per day can be analyzed for uranium to less than 0.2 parts per billion. The kit was tested in the field with excellent results.

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

PubMed Central

2010-01-01

Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST. PMID:21171987

Development of an On-Line Biological Detector

DTIC Science & Technology

1976-07-31

ib . Pert...chemical (Kit 0510). This kit uses the enzymes, glucose oxidase ( Aspergillus niger) and peroxidase (horseradish). In the presence of glucose oxidase, "D...tt -h l o)w the(m to( peteltl rate bt v 1tn t ;w I ib r .I "Flow i i | ;t t r ,I h--Ut cii I r’. 2 Iit I ii I iii t icý, ind M - ) i n i I.t r, i

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

PubMed

Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R T; Rodrigues, Sueli G; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

2006-11-01

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid.

PubMed

Sun, Ning; Deng, Congliang; Zhao, Xiaoli; Zhou, Qi; Ge, Guanglu; Liu, Yi; Yan, Wenlong; Xia, Qiang

2014-02-01

In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection. Copyright © 2013 Elsevier B.V. All rights reserved.

C-kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential

PubMed Central

Rangel, Erika B; Gomes, Samirah A; Dulce, Raul A; Premer, Courtney; Rodrigues, Claudia O; Kanashiro-Takeuchi, Rosemeire M; Oskouei, Behzad; Carvalho, Decio A; Ruiz, Phillip; Reiser, Jochen; Hare, Joshua M

2013-01-01

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit+ cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit+ cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex-vivo expanded c-kit+ cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together these findings document a novel neonatal rat kidney c-kit+ stem cell population that can be isolated, expanded, cloned, differentiated, and employed for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. PMID:23733311

Engineering CHO cells with an oncogenic KIT improves cells growth, resilience to stress, and productivity.

PubMed

Mahameed, Mohamed; Tirosh, Boaz

2017-11-01

An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Comparison of three commercial IgG and IgM ELISA kits for the detection of tick-borne encephalitis virus antibodies.

PubMed

Ackermann-Gäumann, Rahel; Tritten, Marie-Lise; Hassan, Mona; Lienhard, Reto

2018-05-01

Tick-borne encephalitis (TBE) is endemic in many parts of Europe and Asia. The diagnosis of this disease is essentially based on the demonstration of specific antibodies. For reasons of simplicity, automatization and quick availability of test results, enzyme-linked immunosorbent assays (ELISAs) are the method of choice for serological diagnosis of TBE. Here, we evaluated three commercially available anti-TBEV IgG and IgM ELISAs using 251 serum samples: the SERION ELISA classic FSME Virus/TBE Virus IgG and IgM kit (Virion\\Serion), the RIDASCREEN ® FSME/TBE IgG and IgM kit (R-Biopharm), and the anti-FSME/TBE virus ELISA "Vienna" IgG/anti-FSME/TBE virus ELISA IgM kit (Euroimmun). In total, discrepant test results for IgG and/or IgM were observed for 37/251 (14.7 %) of tested samples; differences were statistically significant. Reference values defined by serum neutralization test (SNT, n = 25) or results provided by EQA organizers (n = 2) were established for a subset of samples. In relation to these values, false-positive results were observed mainly for Euroimmun Vienna IgG and RIDASCREEN IgG, whereas false-negative results were primarily observed for Virion\\Serion IgG and RIDASCREEN IgM kits. In routine diagnostics, specificity problems are of major relevance and may be addressed by analyzing the respective samples using SNT. Copyright © 2018 Elsevier GmbH. All rights reserved.

Investigating the isolation and amplification of microRNAs for forensic body fluid identification.

PubMed

Glynn, Claire L; O Leary, Kelsie R

2018-04-30

The discovery of forensic DNA typing evolved molecular biology far beyond what could have been expected in terms of its forensic application, and now there exists other developments in molecular biology which are ready for application to forensic challenges. One such challenge is the identification of the body fluid source of stains recovered from evidence items and crime scenes. Currently there are significant efforts in the research field to develop novel methods for the molecular identification of body fluids, with microRNAs (miRNAs) revealing great potential. MiRNAs have been shown to have high tissue specificity and are less susceptible to degradation as a result of their small size, which infers great advantages to their potential role for identifying forensically relevant body fluids. This study investigated the isolation and amplification of miRNAs from forensically relevant body fluids. Venous blood, menstrual blood, semen, saliva, and vaginal material samples were extracted using; miRNeasy® mini kit (Qiagen), mirVana™ miRNA isolation kit (Ambion), and a modified mirVana™ method, and the quality/quantity of isolated miRNA was determined. miRNAs previously identified to show specificity for particular forensically relevant body fluids were examined. Real Time-Quantitative PCR (RT-qPCR) was performed targeting 5 miRNAs of interest, miR-451, miR-412, miR-891a, miR-205 and miR-124a. This study identified the miRNeasy® mini kit as the optimal method of the three methods investigated for the extraction of miRNAs from body fluids and further validates a selection of miRNAs previously suggested as potential biomarkers. This research highlights the potential of miRNAs as novel markers for the identification of forensically relevant body fluids. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study

PubMed Central

Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I.; Dwyer, Karen M.; Saffery, Richard

2018-01-01

Aim To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. PMID:29462136

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study.

PubMed

Lecamwasam, Ashani; Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I; Dwyer, Karen M; Saffery, Richard

2018-01-01

To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0-0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0-9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0-17.7μg/mL and 0-1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.

Anticipation of Personal Genomics Data Enhances Interest and Learning Environment in Genomics and Molecular Biology Undergraduate Courses

PubMed Central

Weber, K. Scott; Jensen, Jamie L.; Johnson, Steven M.

2015-01-01

An important discussion at colleges is centered on determining more effective models for teaching undergraduates. As personalized genomics has become more common, we hypothesized it could be a valuable tool to make science education more hands on, personal, and engaging for college undergraduates. We hypothesized that providing students with personal genome testing kits would enhance the learning experience of students in two undergraduate courses at Brigham Young University: Advanced Molecular Biology and Genomics. These courses have an emphasis on personal genomics the last two weeks of the semester. Students taking these courses were given the option to receive personal genomics kits in 2014, whereas in 2015 they were not. Students sent their personal genomics samples in on their own and received the data after the course ended. We surveyed students in these courses before and after the two-week emphasis on personal genomics to collect data on whether anticipation of obtaining their own personal genomic data impacted undergraduate student learning. We also tested to see if specific personal genomic assignments improved the learning experience by analyzing the data from the undergraduate students who completed both the pre- and post-course surveys. Anticipation of personal genomic data significantly enhanced student interest and the learning environment based on the time students spent researching personal genomic material and their self-reported attitudes compared to those who did not anticipate getting their own data. Personal genomics homework assignments significantly enhanced the undergraduate student interest and learning based on the same criteria and a personal genomics quiz. We found that for the undergraduate students in both molecular biology and genomics courses, incorporation of personal genomic testing can be an effective educational tool in undergraduate science education. PMID:26241308

Anticipation of Personal Genomics Data Enhances Interest and Learning Environment in Genomics and Molecular Biology Undergraduate Courses.

PubMed

Weber, K Scott; Jensen, Jamie L; Johnson, Steven M

2015-01-01

An important discussion at colleges is centered on determining more effective models for teaching undergraduates. As personalized genomics has become more common, we hypothesized it could be a valuable tool to make science education more hands on, personal, and engaging for college undergraduates. We hypothesized that providing students with personal genome testing kits would enhance the learning experience of students in two undergraduate courses at Brigham Young University: Advanced Molecular Biology and Genomics. These courses have an emphasis on personal genomics the last two weeks of the semester. Students taking these courses were given the option to receive personal genomics kits in 2014, whereas in 2015 they were not. Students sent their personal genomics samples in on their own and received the data after the course ended. We surveyed students in these courses before and after the two-week emphasis on personal genomics to collect data on whether anticipation of obtaining their own personal genomic data impacted undergraduate student learning. We also tested to see if specific personal genomic assignments improved the learning experience by analyzing the data from the undergraduate students who completed both the pre- and post-course surveys. Anticipation of personal genomic data significantly enhanced student interest and the learning environment based on the time students spent researching personal genomic material and their self-reported attitudes compared to those who did not anticipate getting their own data. Personal genomics homework assignments significantly enhanced the undergraduate student interest and learning based on the same criteria and a personal genomics quiz. We found that for the undergraduate students in both molecular biology and genomics courses, incorporation of personal genomic testing can be an effective educational tool in undergraduate science education.

Direct Determination of ECD in ECD Kit: A Solid Sample Quantitation Method for Active Pharmaceutical Ingredient in Drug Product

PubMed Central

Chao, Ming-Yu; Liu, Kung-Tien; Hsia, Yi-Chih; Liao, Mei-Hsiu; Shen, Lie-Hang

2011-01-01

Technetium-99m ethyl cysteinate dimer (Tc-99m-ECD) is an essential imaging agent used in evaluating the regional cerebral blood flow in patients with cerebrovascular diseases. Determination of active pharmaceutical ingredient, that is, L-Cysteine, N, N′-1,2-ethanediylbis-, diethyl ester, dihydrochloride (ECD) in ECD Kit is a relevant requirement for the pharmaceutical quality control in processes of mass fabrication. We here presented a direct solid sample determination method of ECD in ECD Kit without sample dissolution to avoid the rapid degradation of ECD. An elemental analyzer equipped with a nondispersive infrared detector and a calibration curve of coal standard was used for the quantitation of sulfur in ECD Kit. No significant matrix effect was found. The peak area of coal standard against the amount of sulfur was linear over the range of 0.03–0.10 mg, with a correlation coefficient (r) of 0.9993. Method validation parameters were achieved to demonstrate the potential of this method. PMID:21687539

Comparison of the CT OligoGen kit with cobas 4800 assay for detection of Chlamydia trachomatis.

PubMed

Parra-Sánchez, Manuel; Marcuello-López, Ana; García-Rey, Silvia; Zakariya-Yousef, Ismail; Sivianes-Valdecantos, Nieves; Sierra-Atienza, Celestina; Bernal-Martínez, Samuel; Pueyo-Rodrígez, Isabel; Martín-Mazuelos, Estrella; Palomares-Folía, José Carlos

2015-12-01

A prospective study was designed to assess the performance of the new CT OligoGen kit and the cobas 4800 assay for detection of Chlamydia trachomatis. A set of samples that included urine samples (n=212), endocervical (n=167), rectal (n=53), pharyngeal (n=7) and urethral swabs (n=3). The samples were sent from a regional sexually transmitted diseases (STD) clinic in Seville, Spain, and were collected from 261 men and 181 women. Discordant results were re-analyzed and clinical data and other tests were reviewed in order to resolve them. Sensitivity, specificity, positive predicative value (PPV), negative predictive value (NPV) and kappa value for C. trachomatis detection using the CT OligoGen kit were 98.5%, 100%, 100%, 95.4% and 0.97, respectively. This new kit had a high sensitivity, specificity, PPV and NPV for C. trachomatis, therefore the performance profile confirms the usefulness and reliable results of this new assay. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Mobile chemical detector (AP2C+SP4E) as an aid for medical decision making in the battlefield.

PubMed

Eisenkraft, Arik; Markel, Gal; Simovich, Shirley; Layish, Ido; Hoffman, Azik; Finkelstein, Arseny; Rotman, Eran; Dushnitsky, Tsvika; Krivoy, Amir

2007-09-01

The combination of the AP2C unit with the SP4E kit composes a lightweight mobile detector of chemical warfare agents (CWA), such as nerve and mustard agents, with both vapor- and liquid-sampling capabilities. This apparatus was recently introduced into our military medical units as an aid for detection of CWA on casualties. Importantly, critical information regarding the applicability in the battlefield was absent. In view of the serious consequences that might follow a proclamation of CWA recognition in battlefield, a high false-positive rate positions the utilization of this apparatus as a medical decision tool in question. We have therefore conducted a field experiment to test the false-positive rate as well as analyze possible factors leading to false-positive readings with this device. The experiment was carried out before and after a 4-day army field exercise, using a standard AP2C device, a SP4E surface sampling kit, and a specially designed medical sampling kit for casualties, intended for medical teams. Soldiers were examined at rest, after mild exercise, and after 4 days in the field. The readings with AP2C alone were compared to the combination of AP2C and SP4E and to the medical sampling kit. Various body fluids served as negative controls. Remarkably, we found a false-positive rate of 57% at rest and after mild exercise, and an even higher rate of 64% after the 4-day field exercise with the AP2C detector alone, as compared to almost no false-positive readings with the combination of AP2C and SP4E. Strikingly, the medical sampling kit has yielded numerous false-positive readings, even in normal body fluids such as blood, urine, and saliva. We therefore see no place for using the medical sampling kit due to an unaccepted high rate of false-positive readings. Finally, we have designed an algorithm that uses the entire apparatus of AP2C and SP4E as a reliable validation tool for medical triage in the setting of exposure to nerve agents in the battlefield.

Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System.

PubMed

Berlyne, Sigal; Oz, Carla; Einot, Naftaly; Avraham, Shlomit; Ram, Tanya; Goldberg, Miri D; Gafny, Ron

2017-07-01

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex ® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex ® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex ® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex ® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR.

PubMed

Dalla-Costa, Libera M; Morello, Luis G; Conte, Danieli; Pereira, Luciane A; Palmeiro, Jussara K; Ambrosio, Altair; Cardozo, Dayane; Krieger, Marco A; Raboni, Sonia M

2017-09-01

Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and its diagnosis remains a challenge. Blood culturing is the gold standard technique for blood stream infection (BSI) identification. Molecular tests to detect pathogens in whole blood enable early use of antimicrobials and affect clinical outcomes. Here, using real-time PCR, we evaluated DNA extraction using seven manual and three automated commercially available systems with whole blood samples artificially contaminated with Escherichia coli, Staphylococcus aureus, and Candida albicans, microorganisms commonly associated with BSI. Overall, the commercial kits evaluated presented several technical limitations including long turnaround time and low DNA yield and purity. The performance of the kits was comparable for detection of high microorganism loads (10 6 CFU/mL). However, the detection of lower concentrations was variable, despite the addition of pre-processing treatment to kits without such steps. Of the evaluated kits, the UMD-Universal CE IVD kit generated a higher quantity of DNA with greater nucleic acid purity and afforded the detection of the lowest microbial load in the samples. The inclusion of pre-processing steps with the kit seems to be critical for the detection of microorganism DNA directly from whole blood. In conclusion, future application of molecular techniques will require overcoming major challenges such as the detection of low levels of microorganism nucleic acids amidst the large quantity of human DNA present in samples or differences in the cellular structures of etiological agents that can also prevent high-quality DNA yields. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis on structure and properties of zinc nanocrystal in high ordered 3D nanostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sathyaseelan, B., E-mail: bsseelan03@gmail.com; Manigandan, A.; Anbarasu, V.

2015-06-24

The wet impregnation method was employed to prepare ZnO encapsulated in mesoporous silica (ZnO/KIT-6). The prepared ZnO/KIT-6 samples have been studied by X-ray diffraction, transmission electron microscope, and nitrogen adsorption–desorption isotherm. The low angle powder XRD patterns of Calcined ZnO/KIT-6 materials showed a phase that can be indexed to cubic Ia3d. Tem images revealed well ordered cubic 3D nanoporous chennels. The ZnO encapsulated in KIT-6 can be used as light-emitting diodes and ultraviolet nanolasers.

Statistical modeling of dental unit water bacterial test kit performance.

PubMed

Cohen, Mark E; Harte, Jennifer A; Stone, Mark E; O'Connor, Karen H; Coen, Michael L; Cullum, Malford E

2007-01-01

While it is important to monitor dental water quality, it is unclear whether in-office test kits provide bacterial counts comparable to the gold standard method (R2A). Studies were conducted on specimens with known bacterial concentrations, and from dental units, to evaluate test kit accuracy across a range of bacterial types and loads. Colony forming units (CFU) were counted for samples from each source, using R2A and two types of test kits, and conformity to Poisson distribution expectations was evaluated. Poisson regression was used to test for effects of source and device, and to estimate rate ratios for kits relative to R2A. For all devices, distributions were Poisson for low CFU/mL when only beige-pigmented bacteria were considered. For higher counts, R2A remained Poisson, but kits exhibited over-dispersion. Both kits undercounted relative to R2A, but the degree of undercounting was reasonably stable. Kits did not grow pink-pigmented bacteria from dental-unit water identified as Methylobacterium rhodesianum. Only one of the test kits provided results with adequate reliability at higher bacterial concentrations. Undercount bias could be estimated for this device and used to adjust test kit results. Insensitivity to methylobacteria spp. is problematic.

A cluster randomized trial of strategies to increase uptake amongst young women invited for their first cervical screen: The STRATEGIC trial.

PubMed

Kitchener, H; Gittins, M; Cruickshank, M; Moseley, C; Fletcher, S; Albrow, R; Gray, A; Brabin, L; Torgerson, D; Crosbie, E J; Sargent, A; Roberts, C

2018-06-01

Objectives To measure the feasibility and effectiveness of interventions to increase cervical screening uptake amongst young women. Methods A two-phase cluster randomized trial conducted in general practices in the NHS Cervical Screening Programme. In Phase 1, women in practices randomized to intervention due for their first invitation to cervical screening received a pre-invitation leaflet and, separately, access to online booking. In Phase 2, non-attenders at six months were randomized to one of: vaginal self-sample kits sent unrequested or offered; timed appointments; nurse navigator; or the choice between nurse navigator or self-sample kits. Primary outcome was uplift in intervention vs. control practices, at 3 and 12 months post invitation. Results Phase 1 randomized 20,879 women. Neither pre-invitation leaflet nor online booking increased screening uptake by three months (18.8% pre-invitation leaflet vs. 19.2% control and 17.8% online booking vs. 17.2% control). Uptake was higher amongst human papillomavirus vaccinees at three months (OR 2.07, 95% CI 1.69-2.53, p < 0.001). Phase 2 randomized 10,126 non-attenders, with 32-34 clusters for each intervention and 100 clusters as controls. Sending self-sample kits increased uptake at 12 months (OR 1.51, 95% CI 1.20-1.91, p = 0.001), as did timed appointments (OR 1.41, 95% CI 1.14-1.74, p = 0.001). The offer of a nurse navigator, a self-sample kits on request, and choice between timed appointments and nurse navigator were ineffective. Conclusions Amongst non-attenders, self-sample kits sent and timed appointments achieved an uplift in screening over the short term; longer term impact is less certain. Prior human papillomavirus vaccination was associated with increased screening uptake.

Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods.

PubMed

Kaneko, Ikuko; Miyamoto, Kazuaki; Mimura, Kanako; Yumine, Natsuko; Utsunomiya, Hirotoshi; Akimoto, Shigeru; McClane, Bruce A

2011-11-01

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ∼5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10³ cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.

Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling.

PubMed

Ding, Meng; Wang, Cheng; Lu, Xiaolan; Zhang, Cuiping; Zhou, Zhen; Chen, Xi; Zhang, Chen-Yu; Zen, Ke; Zhang, Chunni

2018-06-01

Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal miRNAsextraction and analyzed the advantages and weaknesses of each kit and their application to the sample ofserum and/or plasma.

Current state of biology and diagnosis of clonal mast cell diseases in adults.

PubMed

Alvarez-Twose, I; Morgado, J M; Sánchez-Muñoz, L; García-Montero, A; Mollejo, M; Orfao, A; Escribano, L

2012-10-01

Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of clonal mast cells (MC) in organs such as skin, bone marrow (BM), and gastrointestinal tract, among other tissues. The clonal nature of the disease can be established in most adult patients by the demonstration of activating KIT mutations in their BM MC. When highly sensitive techniques capable of identifying cells present at very low frequencies in a sample are applied, BM MC from virtually all systemic mastocytosis patients display unique immunophenotypical features, particularly the aberrant expression of CD25. By contrast, large, multifocal BM MC aggregates (the only World Health Organization major criterion for systemic mastocytosis) are absent in a significant proportion of patients fulfilling at least three minor criteria for systemic mastocytosis, particularly in subjects studied at early stages of the disease with very low MC burden. Moreover, recent molecular and immunophenotypical investigations of BM MC from patients with indolent systemic mastocytosis have revealed a close association of some biological features (e.g., multilineage involvement of hematopoiesis by the KIT mutation and an immature mast cell immunophenotype) with an increased risk for disease progression. These observations support the fact that, although the current consensus diagnostic criteria for systemic mastocytosis have been a major advance for the diagnosis and classification of the disease, rationale usage of the most sensitive diagnostic techniques available nowadays is needed to improve the diagnosis, refine the classification, and reach objective prognostic stratification of adult mastocytosis. © 2012 Blackwell Publishing Ltd.

An evaluation of direct PCR amplification

PubMed Central

Hall, Daniel E.; Roy, Reena

2014-01-01

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

Comparison of Three Different Commercial Kits for the Human Papilloma Virus Genotyping.

PubMed

Lim, Yong Kwan; Choi, Jee-Hye; Park, Serah; Kweon, Oh Joo; Park, Ae Ja

2016-11-01

High-risk type human papilloma virus (HPV) is the most important cause of cervical cancer. Recently, real-time polymerase chain reaction and reverse blot hybridization assay-based HPV DNA genotyping kits are developed. So, we compared the performances of different three HPV genotyping kits using different analytical principles and methods. Two hundred positive and 100 negative cervical swab specimens were used. DNA was extracted and all samples were tested by the MolecuTech REBA HPV-ID, Anyplex II HPV28 Detection, and HPVDNAChip. Direct sequencing was performed as a reference method for confirming high-risk HPV genotypes 16, 18, 45, 52, and 58. Although high-level agreement results were observed in negative samples, three kits showed decreased interassay agreement as screening setting in positive samples. Comparing the genotyping results, three assays showed acceptable sensitivity and specificity for the detection of HPV 16 and 18. Otherwise, various sensitivities showed in the detection of HPV 45, 52, and 58. The three assays had dissimilar performance of HPV screening capacity and exhibited moderate level of concordance in HPV genotyping. These discrepant results were unavoidable due to difference in type-specific analytical sensitivity and lack of standardization; therefore, we suggested that the efforts to standardization of HPV genotyping kits and adjusting analytical sensitivity would be important for the best clinical performance. © 2016 Wiley Periodicals, Inc.

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

PubMed

Kang, Keren; Wu, Peidian; Li, Wenmei; Tang, Shixing; Wang, Jihua; Luo, Xiaochun; Xie, Mingquan

2015-01-01

To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

PubMed

Peeters, M; Huang, C L; Vonk, L A; Lu, Z F; Bank, R A; Helder, M N; Doulabi, B Zandieh

2016-11-01

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. © 2016 Peeters et al.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis

PubMed Central

Peeters, M.; Huang, C. L.; Vonk, L. A.; Lu, Z. F.; Bank, R. A.; Doulabi, B. Zandieh

2016-01-01

Objectives Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. PMID:27881439

Development and performance evaluation of a novel immunofluorescence chromatographic assay for histidine-rich protein 2 of Plasmodium falciparum.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Huang, Yongping; Xie, Mingquan; Luo, Xiaochun; Li, Wenmei; Wang, Jihua

2015-05-30

The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. The cut-off level of detection of the kit was 25 parasites/μl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.

Food and medical sample freezer kit concept for Shuttle

NASA Technical Reports Server (NTRS)

Copeland, R. J.; Jaax, J. R.; Proctor, B. W.

1977-01-01

A variety of food and storage of samples can be provided by a Space Shuttle Orbiter Freezer Kit. The proposed concept is an integrated package consisting of four -23 C (-10 F) storage compartments and a Stirling cycle refrigeration unit. The Stirling cycle mechanical refrigeration was selected over alternative systems for superior efficiency and safety. The trade-offs and a conceptual design of the system are presented.

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

PubMed

Pereira, Valeriana Valadares; Conceição, Abiqueila da Silva; Maximiano, Leandro Henrique Silva; Belligoli, Leonardo de Queiroz Gomes; Silva, Eduardo Sergio da

2014-01-01

Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

PubMed

Brett, Sabine I; Lucien, Fabrice; Guo, Charles; Williams, Karla C; Kim, Yohan; Durfee, Paul N; Brinker, C J; Chin, Joseph I; Yang, Jun; Leong, Hon S

2017-05-01

The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches. © 2017 Wiley Periodicals, Inc.

Kit systems for granulated decontamination formulations

DOEpatents

Tucker, Mark D.

2010-07-06

A decontamination formulation and method of making that neutralizes the adverse health effects of both chemical and biological compounds, especially chemical warfare (CW) and biological warfare (BW) agents, and toxic industrial chemicals. The formulation provides solubilizing compounds that serve to effectively render the chemical and biological compounds, particularly CW and BW compounds, susceptible to attack, and at least one reactive compound that serves to attack (and detoxify or kill) the compound. The formulation includes at least one solubilizing agent, a reactive compound, a sorbent additive, and water. A highly adsorbent sorbent additive (e.g., amorphous silica, sorbitol, mannitol, etc.) is used to "dry out" one or more liquid ingredients into a dry, free-flowing powder that has an extended shelf life, and is more convenient to handle and mix in the field. The formulation can be pre-mixed and pre-packaged as a multi-part kit system, where one or more of the parts are packaged in a powdered, granulated form for ease of handling and mixing in the field.

A Self-Assisting Protein Folding Model for Teaching Structural Molecular Biology.

PubMed

Davenport, Jodi; Pique, Michael; Getzoff, Elizabeth; Huntoon, Jon; Gardner, Adam; Olson, Arthur

2017-04-04

Structural molecular biology is now becoming part of high school science curriculum thus posing a challenge for teachers who need to convey three-dimensional (3D) structures with conventional text and pictures. In many cases even interactive computer graphics does not go far enough to address these challenges. We have developed a flexible model of the polypeptide backbone using 3D printing technology. With this model we have produced a polypeptide assembly kit to create an idealized model of the Triosephosphate isomerase mutase enzyme (TIM), which forms a structure known as TIM barrel. This kit has been used in a laboratory practical where students perform a step-by-step investigation into the nature of protein folding, starting with the handedness of amino acids to the formation of secondary and tertiary structure. Based on the classroom evidence we collected, we conclude that these models are valuable and inexpensive resource for teaching structural molecular biology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preparation of therapeutic dose of 177Lu-DOTA-TATE using a novel single vial freeze-dried kit: a comparison with 'in-situ' preparation at hospital radiopharmacy.

PubMed

Das, Tapas; Banerjee, Sharmila; Shinto, Ajit; Kamaleshwaran, K K; Sarma, H D

2014-01-01

Patient dose of (177)Lu-DOTA-TATE, used for providing radiotherapeutic treatment to the patients suffering from cancers of neuroendocrine origin, could be prepared at the hospital radiopharmacy either 'in-situ' or by using freezedried kits. The objective of the present work is to formulate and evaluate a single vial freeze-dried DOTA-TATE kit, which is capable of producing up to 7.4 GBq (200 mCi) dose of (177)Lu-DOTA-TATE and to compare the two methodologies presently used for the preparation of the agent. Freeze-dried DOTA-TATE kits, comprising a lyophilized mixture of DOTA-TATE, gentisic acid and ammonium acetate, were prepared and used for the formulation of patient doses of (177)Lu-DOTA-TATE. The kits were subjected to detailed radiochemical evaluation and the shelf-life of the kits was determined. The pharmacokinetic behavior of the agent was studied in normal Wistar rats. These kits were utilized for treating the patients suffering from various types of neuroendocrine cancers. The freeze-dried kits were used for the preparation of up to 7.4 GBq (200 mCi) therapeutic doses of (177)Lu- DOTA-TATE with a radiochemical purity of >99% and were found to have sufficiently long shelf-life. Biological studies carried out in normal Wistar rats exhibited no significant accumulation of activity in any of the vital organs/tissue except in kidneys and non-accumulated activity showed major renal clearance. Clinical studies carried out in cancer patients exhibited accumulation of activity in the cancerous lesions and metastatic sites. The kit was useful for the convenient preparation of therapeutic dose of (177)Lu-DOTA-TATE, suitable for human administration. The use of kit is expected to reduce the batch failure and radiation exposure to the working personnel.

DEMONSTRATION BULLETIN: ENVIROGARD™ PCB TEST KIT - MILLIPORE, INC.

EPA Science Inventory

The EnviroGard™ polychlorinated biphenyl (PCB) immunoassay test kit rapidly analyzes for PCB concentrations in soils. Soil sample extracts are added to test tubes coated with antibodies that bind PCB molecules. The excess soil extracts are washed out of the tubes after incubat...

Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

PubMed

Aigrain, Louise; Gu, Yong; Quail, Michael A

2016-06-13

The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.

San Joaquin kit fox Vulpes macrotis mutica program, Camp Roberts, California

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Camp Roberts is a California Army National Guard Training Site located in central California. The San Joaquin kit fox, an endangered subspecies of kit fox, has been known to occur at Camp Roberts since 1960. The population of foxes began to increase in the early 1970's when use of rodenticides decreased. In 1987 the California Army National Guard contracted EG G Energy Measurements to conduct a 3-year study to assess the effects of Camp Roberts activities on the kit fox population. The major objective of the Camp Roberts Environmental Studies Program is to prepare a comprehensive Biological Assessment of themore » effects of all NGB-authorized activities (includes military training, anticipated construction projects, repair and maintenance activities, and all NGB-authorized non-military activities such as hunting and fishing programs, grazing leases, etc.) on San Joaquin kit fox. The program also provides NGB with the scientific expertise necessary to insure compliance with the Endangered Species Act. The specific objective of this report is to summarize progress and results of the Environmental Studies Program made during Fiscal Years 1989 and 1990 (FY89/90). 32 refs., 9 figs., 14 tabs.« less

DNA-based taxonomic identification of basidiospores in hallucinogenic mushrooms cultivated in "grow-kits" seized by the police: LC-UV quali-quantitative determination of psilocybin and psilocin.

PubMed

Gambaro, Veniero; Roda, Gabriella; Visconti, Giacomo Luca; Arnoldi, Sebastiano; Casagni, Eleonora; Dell'Acqua, Lucia; Farè, Fiorenza; Paladino, Eleonora; Rusconi, Chiara; Arioli, Stefania; Mora, Diego

2016-06-05

The taxonomic identification of the biological material contained in the hallucinogenic mushrooms culture media, was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are present as basidiospores mixed in culture media and spore-bearing fruiting body are not present. This approach is very useful as it allows the unequivocal identification of potentially illicit material before the cultivation and it enables to stop the material to the Customs and to destroy it due to its dangerousness without cultivating the "grow-kits" and without instructing a criminal case. In fact, even if psilocin and psilocybin and the whole mushrooms are illegal in many countries, there is no specific indication in the law about the so called "grow-kits", containing the spores. To confirm the data obtained by the taxonomic identification, a simple, reliable, efficient LC-UV method, using tryptamine as internal standard, suitable for the forensic quali-quantitative determination of psilocin and psilocybin in hallucinogenic mushroom was optimized, validated and applied to the mushrooms grown after the cultivation of the grow-kits seized by the judicial authority, with the authorization of the Ministry of Health. A cation exchange column was used in a gradient elution mode (Phase A: 50mMK2HPO4; 100mM NaCl pH=3 Phase B: methanol). The developed method was linear over the calibration range with a R(2)>0.9992 for both the analytes. The detection and quantification limits were respectively 0.01 and 0.1μg/mL for psilocybin and 0.05μg/mL and 0.1μg/mL for psilocin and the intra- and inter-day precision was satisfactory (coefficients of variation <2.0% for both the analytes). The content of psilocybin in the mushrooms grown from the seized "grow-kits" ranged from 1.02 to 7.60mg/g of dry vegetable material, while the content of psilocin from 0.415 to 8.36mg/g. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of acute ecotoxicity removal from industrial wastewater using a battery of rapid bioassays.

PubMed

Dries, Jan; Daens, Dominique; Geuens, Luc; Blust, Ronny

2014-01-01

The present study compares conventional wastewater treatment technologies (coagulation-flocculation and activated sludge) and powdered activated carbon (PAC) treatment for the removal of acute ecotoxicity from wastewater generated by tank truck cleaning (TTC) processes. Ecotoxicity was assessed with a battery of four commercially available rapid biological toxicity testing systems, verified by the US Environmental Protection Agency. Chemical coagulation-flocculation of raw TTC wastewater had no impact on the inhibition of the bioluminescence by Vibrio fischeri (BioTox assay). Subsequent biological treatment with activated sludge without PAC resulted in BioTox inhibition-free effluent (<10% inhibition). In contrast, activated sludge treatment without PAC produced an effluent that significantly inhibited (>50%) (i) the bioluminescence by Photobacterium leiognathi (ToxScreen³ test kit), (ii) the photosynthesis by the green algae Chlorella vulgaris (LuminoTox SAPS test kit), and (iii) the particle ingestion by the crustacean Thamnocephalus platyurus (Rapidtoxkit test kit). The lowest inhibition was measured after activated sludge treatment with the highest PAC dose (400 mg/L), demonstrating the effectiveness of PAC treatment for ecotoxicity removal from TTC wastewater. In conclusion, the combination of bioassays applied in the present study represents a promising test battery for rapid ecotoxicty assessment in wastewater treatment.

Evaluation of a culture-based pathogen identification kit for bacterial causes of bovine mastitis.

PubMed

Viora, L; Graham, E M; Mellor, D J; Reynolds, K; Simoes, P B A; Geraghty, T E

2014-07-26

Accurate identification of mastitis-causing bacteria supports effective management and can be used to implement selective use of antimicrobials for treatment. The objectives of this study were to compare the results from a culture-based mastitis pathogen detection test kit ('VetoRapid', Vétoquinol) with standard laboratory culture and to evaluate the potential suitability of the test kit to inform a selective treatment programme. Overall 231 quarter milk samples from five UK dairy farms were collected. The sensitivity and specificity of the test kit for the identification of Escherichia coli, Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis and Enterococcus spp. ranged from 17 per cent to 84 per cent and 92 per cent to 98 per cent, respectively. In total, 23 of 68 clinical samples were assigned as meeting the requirement for antimicrobial treatment (Gram-positive organism cultured) according to standard culture results, with the test kit results having sensitivity and specificity of 91 per cent and 78 per cent, respectively. Several occurrences of misidentification are reported, including S. aureus being misidentified as coagulase-negative staphylococci and vice versa. The test kit provides rapid preliminary identification of five common causes of bovine mastitis under UK field conditions and is likely to be suitable for informing selective treatment of clinical mastitis caused by Gram-positive organisms. British Veterinary Association.

Advanced forensic validation for human spermatozoa identification using SPERM HY-LITER™ Express with quantitative image analysis.

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2017-07-01

Identification of human semen is indispensable for the investigation of sexual assaults. Fluorescence staining methods using commercial kits, such as the series of SPERM HY-LITER™ kits, have been useful to detect human sperm via strong fluorescence. These kits have been examined from various forensic aspects. However, because of a lack of evaluation methods, these studies did not provide objective, or quantitative, descriptions of the results nor clear criteria for the decisions reached. In addition, the variety of validations was considerably limited. In this study, we conducted more advanced validations of SPERM HY-LITER™ Express using our established image analysis method. Use of this method enabled objective and specific identification of fluorescent sperm's spots and quantitative comparisons of the sperm detection performance under complex experimental conditions. For body fluid mixtures, we examined interference with the fluorescence staining from other body fluid components. Effects of sample decomposition were simulated in high humidity and high temperature conditions. Semen with quite low sperm concentrations, such as azoospermia and oligospermia samples, represented the most challenging cases in application of the kit. Finally, the tolerance of the kit against various acidic and basic environments was analyzed. The validations herein provide useful information for the practical applications of the SPERM HY-LITER™ Express kit, which were previously unobtainable. Moreover, the versatility of our image analysis method toward various complex cases was demonstrated.

Identifying Affordances of 3D Printed Tangible Models for Understanding Core Biological Concepts

ERIC Educational Resources Information Center

Davenport, Jodi L.; Silberglitt, Matt; Boxerman, Jonathan; Olson, Arthur

2014-01-01

3D models derived from actual molecular structures have the potential to transform student learning in biology. We share findings related to our research questions: 1) what types of interactions with a protein folding kit promote specific learning objectives?, and 2) what features of the instructional environment (e.g., peer interactions, teacher…

LAMP kit for diagnosis of non-falciparum malaria in Plasmodium ovale infected patients.

PubMed

Cuadros, Juan; Martin Ramírez, Alexandra; González, Iveth J; Ding, Xavier C; Perez Tanoira, Ramon; Rojo-Marcos, Gerardo; Gómez-Herruz, Peña; Rubio, Jose Miguel

2017-01-07

Microscopy and rapid diagnosis tests have a limited sensitivity in diagnosis of malaria by Plasmodium ovale. The LAMP kit (LoopAMP®) can be used in the field without special equipment and could have an important role in malaria control programmes in endemic areas and for malaria diagnosis in returned travellers. The performance of the Pan primer of the kit in detecting malaria by P. ovale was compared with the results of standard nPCR in samples of patients returning from P. ovale endemic areas. Plasmodium ovale positive samples (29, tested by PCR and/or microscopy) and malaria negative specimens (398, tested by microscopy and PCR) were collected in different hospitals of Europe from June 2014 to March 2016 and frozen at -20 °C. Boil and spin method was used to extract DNA from all samples and amplification was performed with LoopAMP® MALARIA kit (Eiken Chemical, Japan) in an automated turbidimeter (Eiken 500). The results of LAMP read by turbidimetry and with the naked eye were compared. The kit showed a sensitivity of 100% and a specificity of 97.24% with positive and negative predictive values of 72.5 and 100%, respectively. Naked eyed readings were in accordance with turbidimetry readings (sensitivity, 92.5%, specificity, 98.96% and positive and negative predictive values, respectively, 90.24 and 99.22%). The limit of detection of LAMP assay for P. ovale was between 0.8 and 2 parasites/µl. The Pan primer of the Malaria kit LoopAMP® can detect P. ovale at very low-levels and showed a predictive negative value of 100%. This tool can be useful in malaria control and elimination programmes and in returned travellers from P. ovale endemic areas. Naked eye readings are equivalent to automated turbidimeter readings in specimens obtained with EDTA.

Lack of reliability of nanotechnology in the of free plasma DNA in samples of patients with prostate cancer

PubMed Central

2013-01-01

Background Several studies seek biological markers that give diagnostic and degree of tumor development. The aim of this study was to validate the determination of plasma DNA using nanotechnology (Nanovue™-NV) in samples of 80 patients with prostate cancer. Methods Blood samples of 80 patients of the Urology Ambulatory of Faculdade de Medicina do ABC with prostate cancer confirmed by anatomical-pathology criteria were analyzed. DNA extraction was performed using a GFX TM kit (Amersham Pharmacia Biotech, Inc, USA) following the adapted protocol. Plasma was subjected to centrifugation. Results There was a big difference between the first and the second value obtained by NanoVue Only two samples had no differences between duplicates. Maximum difference between duplicates was 38 μg/mL. Average variation between 51 samples was 10.29 μg/mL, although 21 samples had differences above this average. No correlation was observed between pDNA obtained by traditional spectrophotometry and by nanotechnology. Conclusion Determination of plasma DNA by nanotechnology was not reproducible. PMID:23311763

Developing Save Your Food Kit (Sayofu Kit) to Support Inquiry, Improve Student Learning Outcomes at SMP Plus Hidayatul Mubtadiin and Public Awareness on Food Additives

NASA Astrophysics Data System (ADS)

Astutik, J.

2017-02-01

Food additives are materials that can not be separated from the lives of students and the community. Based on the preliminary questionnaire, it indicates the lack of kit supporting material additives in some schools and communities. The research objectives of this development are (1) to develop Kit experiment (SAYOFU KIT) and supplementary books to improve student learning outcomes in the classroom and public awareness on food additives (2) to describe the feasibility and potential effectiveness of SAYOFU KIT developed (3) to analyze the practice of SAYOFU KIT and benefits for students and the community. This development study uses 4-D models Thiagarajan, et al (1974). Through some stages, they are: defining, designing, developing and disseminating which involes the students and community. The developed SAYOFU KIT includes additives sample kit, borax test kit, curcumin test kit, formaldehyde test kit, modification heater to the identification of dyes and dye test paper. The study is conducted at SMP Plus Hidayatul Mubtadiin, and TKIT Al Uswah. The products are validated by experts and education practitioners. Qualitative data processing uses descriptive method, whereas quantitative data by using the N-gain. The average yield of expert validation of SAYOFU KIT with supplementary books 76.50% teacher’s book and 76.30% student’s book are eligible. The average yield of 96.81% validation of educational practitioners criteria, piloting a small group of 83.15%, and 82.89% field trials are very decent. The average yield on the student questionnaire responses SAYOFU kit and supplementary book is 87.6% with the criteria very well worth it. N-Gain 0:56 cognitive achievement with the criteria enough. The results of the public poll showed 95% feel the benefits SAYOFU kits for testing food. Based from description indicates that SAYOFU Kit developed feasible, practical, useful to support inquiry learning and improve student learning outcomes as well as public awareness of food additives.

Mast cells and matrix metalloproteinase 9 expression in actinic cheilitis and lip squamous cell carcinoma.

PubMed

Souza Freitas, Valéria; de Andrade Santos, Pedro Paulo; de Almeida Freitas, Roseana; Pereira Pinto, Leão; de Souza, Lélia Batista

2011-09-01

The aim of this study was to evaluate mast cell (MC) density and migration and their association with matrix metalloproteinase (MMP) 9 expression in squamous cell carcinoma (SCC) and actinic cheilitis (AC). Tryptase, c-Kit, and MMP-9 expression was evaluated in 20 cases of SCC, 20 cases of AC, and 7 cases of normal lip (control samples) by immunohistochemistry techniques. Tryptase(+) and c-Kit(+) MC densities were significantly higher in SCCs than in ACs and control samples (P < .001). However, no significant difference was found when comparing tryptase(+) and c-Kit(+) MC densities between ACs and control samples (P values .185 and .516, respectively). MMP-9 was strongly expressed in SCCs and moderately expressed in ACs and control samples. A highly significant association was found between tryptase(+) MC density and the expression of MMP-9 (P < .001). The increase in MC density associated with the strong expression of MMP-9 may favor SCC progression. Copyright © 2011 Mosby, Inc. All rights reserved.

[Development of a SPA-ELISA method for detecting anti-coronavirus IgG antibodies in serum samples from fulvous fruit bats].

PubMed

Zhou, Jie; Liao, Yu-xue; Chen, Zhong; Li, Yu-chun; Gao, Lu-Lu; Chen, Yi-xiong; Cai, Lian-gong; Chen, Qing; Yu, Shou-yi

2008-05-01

To develop an simple and sensitive method for detecting anti-coronavirus IgG antibodies in bat sera based on enzyme-linked immunosorbent assay (ELISA). A commercial ELISA kit for detecting SARS-CoV antibody was modified for detecting coronavirus antibodies in bat serum samples. The second antibody in the kit was replaced with horseradish peroxidase-conjugated protein-A (HRP-SPA) based on the characteristics of binding between Staphylococcus aureus protein A (SPA) and mammal IgG Fc fragment. The sera of 55 fulvous fruit bats (Rousettus dasymallus) were tested using the SPA-ELISA. The test results of the positive and negative controls in the kit and the serum samples from convalescent ;patient were consistent with expectation. Coronavirus antibody was detected in 2 out of the 55 bat serum samples. Serum neutralization test confirmed the validity of the SPA-ELISA method. This SPA-ELISA method is applicable for detecting coronavirus antibody in bat sera.

Development of a quantitative validation method for forensic investigation of human spermatozoa using a commercial fluorescence staining kit (SPERM HY-LITER™ Express).

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2016-11-01

In investigations of sexual assaults, as well as in identifying a suspect, the detection of human sperm is important. Recently, a kit for fluorescent staining of human spermatozoa, SPERM HY-LITER™, has become available. This kit allows for microscopic observation of the heads of human sperm using an antibody tagged with a fluorescent dye. This kit is specific to human sperm and provides easy detection by luminescence. However, criteria need to be established to objectively evaluate the fluorescent signals and to evaluate the staining efficiency of this kit. These criteria will be indispensable for investigation of forensic samples. In the present study, the SPERM HY-LITER™ Express kit, which is an improved version of SPERM HY-LITER™, was evaluated using an image analysis procedure using Laplacian and Gaussian methods. This method could be used to automatically select important regions of fluorescence produced by sperm. The fluorescence staining performance was evaluated and compared under various experimental conditions, such as for aged traces and in combination with other chemical staining methods. The morphological characteristics of human sperm were incorporated into the criteria for objective identification of sperm, based on quantified features of the fluorescent spots. Using the criteria, non-specific or insignificant fluorescent spots were excluded, and the specificity of the kit for human sperm was confirmed. The image analysis method and criteria established in this study are universal and could be applied under any experimental conditions. These criteria will increase the reliability of operator judgment in the analysis of human sperm samples in forensics.

Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

PubMed

Tarasova, Irina A; Lobas, Anna A; Černigoj, Urh; Solovyeva, Elizaveta M; Mahlberg, Barbara; Ivanov, Mark V; Panić-Janković, Tanja; Nagy, Zoltan; Pridatchenko, Marina L; Pungor, Andras; Nemec, Blaž; Vidic, Urška; Gašperšič, Jernej; Krajnc, Nika Lendero; Vidič, Jana; Gorshkov, Mikhail V; Mitulović, Goran

2016-09-01

Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mutational status of EGFR and KIT in thymoma and thymic carcinoma.

PubMed

Yoh, Kiyotaka; Nishiwaki, Yutaka; Ishii, Genichiro; Goto, Koichi; Kubota, Kaoru; Ohmatsu, Hironobu; Niho, Seiji; Nagai, Kanji; Saijo, Nagahiro

2008-12-01

This study was conducted to evaluate the prevalence of EGFR and KIT mutations in thymomas and thymic carcinomas as a means of exploring the potential for molecularly targeted therapy with tyrosine kinase inhibitors. Genomic DNA was isolated from 41 paraffin-embedded tumor samples obtained from 24 thymomas and 17 thymic carcinomas. EGFR exons 18, 19, and 21, and KIT exons 9, 11, 13, and 17, were analyzed for mutations by PCR and direct sequencing. Protein expression of EGFR and KIT was evaluated immunohistochemically. EGFR mutations were detected in 2 of 20 thymomas, but not in any of the thymic carcinomas. All of the EGFR mutations detected were missense mutations (L858R and G863D) in exon 21. EGFR protein was expressed in 71% of the thymomas and 53% of the thymic carcinomas. The mutational analysis of KIT revealed only a missense mutation (L576P) in exon 11 of one thymic carcinoma. KIT protein was expressed in 88% of the thymic carcinomas and 0% of the thymomas. The results of this study indicate that EGFR and KIT mutations in thymomas and thymic carcinomas are rare, but that many of the tumors express EGFR or KIT protein.

Direct detection of methicillin-resistant Staphylococcus aureus from blood cultures using an immunochromatographic immunoassay-based MRSA rapid kit for the detection of penicillin-binding protein 2a.

PubMed

Shin, Kyeong Seob; Song, Hyung Geun; Kim, Haejung; Yoon, Sangsun; Hong, Seung Bok; Koo, Sun Hoe; Kim, Jimyung; Kim, Jongwan; Roh, Kyoung Ho

2010-07-01

Using an EZ-Step MRSA rapid kit, a novel screening test for methicillin-resistant Staphylococcus aureus (MRSA) that detects penicillin-binding protein 2a, 34 of 36 MRSA-positive clinical blood culture samples were positive on direct testing (sensitivity, 94.4%), whereas 21 of 21 methicillin-susceptible S. aureus-positive samples were negative (specificity, 100%).

Human Papilloma Virus (HPV) self-sampling: do women accept it?

PubMed

Abdullah, Nik Nairan; Daud, Suzanna; Wang, Seok Mui; Mahmud, Zamalia; Mohd Kornain, Noor Kaslina; Al-Kubaisy, Waqar

2018-04-01

This study aims to determine the acceptability of Human Papilloma Virus (HPV) self-sampling and the factors associated with willingness to buy HPV self-sampling kit in the future. A total of 164 women aged 28-60 years old from Obstetrics & Gynaecology clinics at a teaching hospital performed HPV self-sampling using the Digene HC2 DNA collection kit. After samples were taken, the participants were given self-administered questionnaires. The majority of the participants were Malay (93.9%), had attained tertiary education (65.2%) and were employed (70.1%). The acceptability was good. More than half of the participants felt that self-sampling was easy. Only 1.2% felt that the procedure was difficult to perform. Most reported no pain at all during the procedure (66.9%). The commonest concern was getting a good sample (90.1%). A number of Pap smears were found to be significantly associated with the willingness to buy the HPV self-sampling kit. HPV self-sampling has the potential to be included in the cervical cancer screening programme. Impact Statement What is already known on this subject: HPV self-sampling is acceptable in some developed and developing countries. It is acceptable because it was easy to perform with very minimal pain or discomfort. Studies on the acceptance of self-screening are needed to plan a policy on self-sampling in the future. What the results of this study add: Our study adds new findings to the body of knowledge on self-sampling in the local population. We found that more women are willing to do the self-sampling at the clinic rather than at home. Although more than 90% expressed willingness to do self-sampling in the future, only 70% of them were willing to purchase the kit. Cost is a potential barrier to women who have the interest to perform the self-sampling. Given the global economic challenges, cost is inevitably an important predictor that we have to consider. What the implications are of these findings for clinical practice and/or further research: Future research should examine women from the rural areas and those who are resilient to Pap smear screening. In clinical practice, clinicians should acknowledge that cost is a potential barrier for women who are willing to do self-sampling. Self-sampling could be an option for women with no financial constraint to buy the kit. However, clinicians should counsel women so that they can make an informed choice in determining their screening method.

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

PubMed Central

Harder, Melanie; Renneberg, Rebecca; Meyer, Patrick; Krause-Kyora, Ben; von Wurmb-Schwark, Nicole

2012-01-01

Aim To comparatively test nine commercially available short tandem repeat (STR)-multiplex kits (PowerPlex 16, 16HS, ES, ESI17, ESX17, S5 [all Promega]; AmpFiSTR Identifiler, NGM and SEfiler [all Applied Biosystems]) for their efficiency and applicability to analyze ancient and thus highly degraded DNA samples. Methods Fifteen human skeletal remains from the late medieval age were obtained and analyzed using the nine polymerase chain reaction assays with slightly modified protocols. Data were systematically compared to find the most meaningful and sensitive assay. Results The ESI, ESX, and NGM kits showed the best overall results regarding amplification success, detection rate, identification of heterozygous alleles, sex determination, and reproducibility of the obtained data. Conclusion Since application of these three kits enables the employment of different primer sequences for all the investigated amplicons, a combined application is recommended for best possible and – most importantly – reliable genetic analysis of ancient skeletal material or otherwise highly degraded samples, eg, from forensic cases. PMID:23100203

[Preparation of freeze - drying control materials of IgG antibody against Schistosoma japonicum for immunodetection kits].

PubMed

Jin, Huang; Chun-Lian, Tang; Zu-Wu, Tu; Li, Tang; Ke-Hui, Zhang; Qian, Li; Jun, Ye

2018-04-18

To prepare freeze-drying control materials of IgG antibody against Schistosoma japonicum for detection kits. The serum samples of schistosomiasis patients from endemic areas and normal people without history of schistosome infection or contact with infested water in Hubei Province were collected. All the sera were detected by the method approved by China Food and Drug Administration and selected for preparation of quality control samples. Totally twelve positive quality control materials, ten negative quality control materials, and one sensitive and one precision quality control materials were screened. According to the positive serum level, the positive degrees of quality control materials were divided into strong, medium and weak levels. The stability could be valid for one year. The freeze-drying quality control materials of IgG antibody against S. japonicum for detection kits are prepared. They are easy to use and have good stability, and therefore, they may meet the requirement of quality control for the detection of schistosomiasis diagnostics kits.

Tyrosine Kinase Inhibition in HPV-related Squamous Cell Carcinoma Reveals Beneficial Expression of cKIT and Src.

PubMed

Kramer, Benedikt; Kneissle, Marcel; Birk, Richard; Rotter, Nicole; Aderhold, Christoph

2018-05-01

Therapeutic options of locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) are limited. Src and cKIT are key protein regulators for local tumor progression. The aim of the study was to investigate the therapeutic potential of targeted therapies in human squamous cell carcinoma (HNSCC) in vitro. Therefore, the influence of the selective tyrosine kinase inhibitors niotinib, dasatinib, erlotinib, gefitinib and afatinib on Src and cKIT expression in Human papilloma virus (HPV)-positive and HPV-negative squamous cancer cells (SCC) was analyzed in vitro. ELISA was performed to evaluate the expression of Src and cKIT under the influence of nilotinib, dasatinib, erlotinib, gefitinib and afatinib (10 μmol/l) in HPV-negative and HPV-positive SCC (24-96 h of incubation). Gefitinib significantly increased cKIT expression in HPV-positive and HPV-negative cells whereas nilotinib and afatinib decreased cKIT expression in HPV-positive SCC. The influence of tyrosine kinase inhibitors in HPV-negative SCC was marginal. Surprisingly, Src expression was significantly increased by all tested tyrosine kinase inhibitors in HPV-positive SCC. The results revealed beneficial and unexpected information concerning the interaction of selective tyrosine kinase inhibitors and the tumor biology of HNSCC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples.

PubMed

Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Oldroyd, Nicola J; Hennessy, Lori K

2011-07-01

The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping. © 2011 American Academy of Forensic Sciences.

Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

PubMed

Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

2015-12-29

Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.

RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation.

PubMed

Chatterjee, Anirban; Mirer, Paul L; Zaldivar Santamaria, Elvira; Klapperich, Catherine; Sharon, Andre; Sauer-Budge, Alexis F

2010-06-01

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.

Human autosomal DNA and X chromosome STR profiles obtained from Chrysomya albiceps (Diptera: Calliphoridae) larvae used as a biological trace.

PubMed

Oliveira, T C; Santos, A B R; Rabelo, K C N; Souza, C A; Santos, S M; Crovella, S

2016-11-03

The use of insects to answer questions in criminal investigations, as well as a combination of forensic genetic techniques to obtain human DNA from the organisms, especially necrophagous dipterians, have gained ground in recent decades among researchers and professionals in this area. The objective of our study was to evaluate and compare two methods of human DNA extraction, commonly used for forensic samples, to obtain human autosomal DNA and X chromosome short tandem repeat profiles from the digestive tract of Chrysomya albiceps (Diptera: Calliphoridae) larvae. Immature specimens were collected from corpses at the Institute of Forensic Medicine of Pernambuco and raised in bovine ground meat to allow stabilization of the colony. Groups of larvae in the third instar were provided with bovine ground meat plus human blood for 48 h, dissected, and then subjected to DNA extraction. DNA was extracted using two methods: a DNA IQ™ kit and a phenol-chloroform method. Genomic DNA was amplified using AmpFℓSTR ® Identifiler ® Plus PCR and Argus-X-12 ® kits, and samples were sequenced to determine if the two extraction techniques generated reliable profiles that were compatible with a reference sample. The existence of comparable profiles from both techniques demonstrates the usefulness of dipteran larvae for obtaining human DNA from corpses, which can be further used to correlate genetic profiles in a crime scene when other traces are not available. However, several variables still require revision; thus, the technique should be further investigated for its validity, security, and, in particular, its reproducibility.

Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.

PubMed

Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

2015-01-01

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

NASA Technical Reports Server (NTRS)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Novel Therapeutic and Prophylactic Modalities to Protect U.S. Armed Forces Against Major Biological Threat Agents

DTIC Science & Technology

2004-10-01

using flow cytometry after staining with CBA kit produced by BD-Pharmingen. The CBA kit can simultaneously test 5 inflammatory cytokines that include...or TLR4 transfected cells using flow cytometry . CHOK1 and CHOR1.1 cells were plated out in a 24-well dish and transfected 24 h later with either TLR2...with PE-labeled anti-hTLR2 antibody or PE-isotype control antibody (eBiosciences, CA), and cells were analyzed by flow cytometry . 39 The expression

DogMATIC--A Remote Biospecimen Collection Kit for Biobanking.

PubMed

Milley, Kristi M; Nimmo, Judith S; Bacci, Barbara; Ryan, Stewart D; Richardson, Samantha J; Danks, Janine A

2015-08-01

Canine tumors are valuable comparative oncology models. This research was designed to create a sustainable biobank of canine mammary tumors for breast cancer research. The aim was to provide a well-characterized sample cohort for specimen sharing, data mining, and long-term research aims. Canine mammary tumors are most frequently managed at a local veterinary clinic or hospital. We adopted a biobank framework based on a large number of participating veterinary hospitals and clinics acting as collection centers that were serviced by a centralized storage facility. Recruitment was targeted at rural veterinary clinics. A tailored, stable collection kit (DogMATIC) was designed that was used by veterinarians in remote or rural locations to collect both fresh and fixed tissue for submission to the biobank. To validate this methodology the kit design, collection rate, and sample quality were analyzed. The Australian Veterinary Cancer Biobank was established as a network of 47 veterinary clinics and three veterinary pathology laboratories spanning over 200,000 km(2). In the first 12 months, 30 canine mammary tumor cases were submitted via the DogMATIC kit. Pure intact RNA was isolated in over 80% of samples with an average yield of 14.49 μg. A large network biobank, utilizing off-site collection with the DogMATIC kit, was successfully coordinated. The creation of the Australian Veterinary Cancer Biobank has established a long-term, sustainable, comparative oncology research resource in Australia. There are broader implications for biobanking with this very different form of collection and banking.

A simple field kit for the determination of drug susceptibility in Plasmodium falciparum.

PubMed

Nguyen-Dinh, P; Magloire, R; Chin, W

1983-05-01

A field kit has been developed which greatly simplifies the performance of the 48-hour in vitro test for drug resistance in Plasmodium falciparum. The kit uses an easily reconstituted lyophilized culture medium, and requires only a fingerprick blood sample. In parallel tests with 13 isolates of P. falciparum in Haiti, the new technique had a success rate equal to that of the previously described method, with comparable results in terms of parasite susceptibility in vitro to chloroquine and pyrimethamine.

Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences.

PubMed

Wagner Mackenzie, Brett; Waite, David W; Taylor, Michael W

2015-01-01

The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.

Single and multiple streamer DBD micro-discharges for testing inactivation of biologically contaminated surfaces

NASA Astrophysics Data System (ADS)

Prukner, Vaclav; Dolezalova, Eva; Simek, Milan

2014-10-01

Highly reactive environment produced by atmospheric-pressure, non-equilibrium plasmas generated by surface dielectric barrier discharges (SDBDs) may be used for inactivation of biologically contaminated surfaces. We investigated decontamination efficiency of reactive environment produced by single/multiple surface streamer micro-discharge driven by amplitude-modulated AC power in coplanar electrode geometry on biologically contaminated surface by Escherichia coli. The discharges were fed by synthetic air with water vapor admixtures at atmospheric pressure, time of treatment was set from 10 second to 10 minutes, diameters of used SDBD electrodes (single and multiple streamer) and homogeneously contaminated disc samples were equal (25 mm), the distance between the electrode and contaminated surface was 2 mm. Both a conventional cultivation and fluorescent method LIVE/DEAD Bacterial Viability kit were applied to estimate counts of bacteria after the plasma treatment. Inactivation was effective and bacteria partly lost ability to grow and became injured and viable/active but non-cultivable (VBNC/ABNC). Work was supported by the MEYS under Project LD13010, VES13 COST CZ (COST Action MP 1101).

Computerized Online Bibliographic Searching. SPEC Kit #154.

ERIC Educational Resources Information Center

Hocker, Susan

For this kit, 106 Association of Research Libraries (ARL) academic libraries were surveyed concerning: (1) current administration/organization; (2) evaluation; (3) patron relations; (4) services; and (5) the impact of online searching on collections. Responses were received from 83 libraries, many of which contributed sample materials. Analyses of…

Low sensitivity and frequent cross-reactions in commercially available antibody detection ELISA assays for Taenia solium cysticercosis.

PubMed

Garcia, Hector H; Castillo, Yesenia; Gonzales, Isidro; Bustos, Javier A; Saavedra, Herbert; Jacob, Louis; Del Brutto, Oscar H; Wilkins, Patricia P; Gonzalez, Armando E; Gilman, Robert H

2018-01-01

To evaluate the diagnostic performance of two commercially available ELISA kits, Novalisa ® and Ridascreen ® , for the detection of antibodies to Taenia solium, compared to serological diagnosis of neurocysticercosis (NCC) by LLGP-EITB (electro-immunotransfer blot assay using lentil-lectin purified glycoprotein antigens). Archive serum samples from patients with viable NCC (n = 45) or resolved, calcified NCC (n = 45), as well as sera from patients with other cestode parasites (hymenolepiasis, n = 45 and cystic hydatid disease, n = 45), were evaluated for cysticercosis antibody detection using two ELISA kits, Novalisa ® and Ridascreen ® . All NCC samples had previously tested positive, and all samples from heterologous infections were negative on LLGP-EITB for cysticercosis. Positive rates were calculated by kit and sample group and compared between the two kits. Compared to LLGP-EITB, the sensitivity of both ELISA assays to detect specific antibodies in patients with viable NCC was low (44.4% and 22.2%), and for calcified NCC, it was only 6.7% and 4.5%. Sera from patients with cystic hydatid disease were highly cross-reactive in both ELISA assays (38/45, 84.4%; and 25/45, 55.6%). Sera from patients with hymenolepiasis cross-reacted in five cases in one of the assays (11.1%) and in only one sample with the second assay (2.2%). The performance of Novalisa ® and Ridascreen ® was poor. Antibody ELISA detection cannot be recommended for the diagnosis of neurocysticercosis. © 2017 John Wiley & Sons Ltd.

Performance Evaluation of a Novel Chemiluminescence Assay Detecting Treponema Pallidum Antibody as a Syphilis Screening Method.

PubMed

Chen, Qixia; An, Jingna; Rao, Chenli; Wang, Tingting; Li, Dongdong; Feng, Shu; Tao, Chuanmin

2016-01-01

Syphilis is a major concern to global public health with increasing incidence. So its screening test should have sufficient sensitivity and specificity. We evaluated the performance of the Lumipulse G TP-N assay detection for syphilis screening and compared it with the InTec ELISA test kit for TP, which is widely used. Samples of several patient groups including 133 clinical and serologically characterized syphilitic sera, 175 samples containing potentially interfering agents, and 2290 unselected samples submitted for routine screening were detected by both the Lumipulse G TP-N assay and the InTec ELISA test kit for TP. Inconsistent samples were confirmed by RecomLine Treponema IgG, IgM immunoblot. Coefficient of variations of the Lumipulseo G TP-N assay at both levels were below 5% and of the InTec ELISA test kit for TP both over 5%. The sensitivity of the Lumipulse G TP-N assay and the InTec ELISA test kit for TP were 100% for all stages of syphilis. The two methods had consistent analytical specificity of 100% (95% CI: 97.21 - 100.00), while the clinical specificity was 100% (95% CI: 99.79 - 100.00) and 99.82% (95% CI: 99.51 - 99.94), respectively. Between them, Spearman's correlation coefficient was 0.455 and kappa value was 0.986. The overall sensitivity and specificity of the Lumipulse G TP-N assay was higher than the InTec ELISA test kit for TP (sensitivity: 100.0 versus 99.5, specificity: 100.0 versus 99.8). The automated Lumipulse G TP-N assay demonstrated excellent diagnostic sensitivity and specificity when evaluated as a screening test for syphilis. Thus, it can be an alternative to the treponemal screening test.

Evaluation of Two Commercial Real-Time PCR Kits for Aspergillus DNA Detection in Bronchoalveolar Lavage Fluid in Patients with Invasive Pulmonary Aspergillosis.

PubMed

Denis, Julie; Forouzanfar, Faezeh; Herbrecht, Raoul; Toussaint, Elise; Kessler, Romain; Sabou, Marcela; Candolfi, Ermanno; Letsher-Bru, Valérie

2018-05-01

Invasive pulmonary aspergillosis (IPA) is a common complication of immunosuppression. Rapid diagnosis using molecular techniques is essential to improve patient survival. PCR techniques are promising in enhancing Aspergillus detection in blood and respiratory samples. We evaluate for the first time the performances of two commercial real-time PCR kits, the A. fumigatus Bio-Evolution and the MycoGENIE A. fumigatus for the detection of A. fumigatus DNA in bronchoalveolar lavage (BAL) from patients with and without IPA. Seventy-three BAL samples were included. Thirty-one of them corresponded to patients with probable IPA, 11 to patients with possible IPA, and 31 to patients without aspergillosis, according to the 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. In the probable IPA group, A. fumigatus Bio-Evolution and the MycoGENIE A. fumigatus real-time PCR kits showed a specificity of 100% and a sensitivity of 81% and 71%, respectively. The A. fumigatus Bio-Evolution detected Aspergillus DNA in the 14 BAL samples with a positive Aspergillus culture result, whereas the MycoGENIE A. fumigatus PCR result was positive only for 12. In the possible IPA group, there were no positive real-time PCR or positive Aspergillus culture results. For the patients without aspergillosis, no positive result was observed for real-time PCR kit, despite the presence of various other non-Aspergillus pathogens in this group. Our study demonstrates an excellent specificity and a good sensitivity of A. fumigatus DNA detection in BAL samples with both kits. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Buffer substitution in malaria rapid diagnostic tests causes false-positive results

PubMed Central

2010-01-01

Background Malaria rapid diagnostic tests (RDTs) are kits that generally include 20 to 25 test strips or cassettes, but only a single buffer vial. In field settings, laboratory staff occasionally uses saline, distilled water (liquids for parenteral drugs dilution) or tap water as substitutes for the RDT kit's buffer to compensate for the loss of a diluent bottle. The present study assessed the effect of buffer substitution on the RDT results. Methods Twenty-seven RDT brands were run with EDTA-blood samples of five malaria-free subjects, who were negative for rheumatoid factor and antinuclear antibodies. Saline, distilled water and tap water were used as substitute liquids. RDTs were also run with distilled water, without adding blood. Results were compared to those obtained with the RDT kit's buffer and Plasmodium positive samples. Results Only eight cassettes (in four RDT brands) showed no control line and were considered invalid. Visible test lines occurred for at least one malaria-free sample and one of the substitutes in 20/27 (74%) RDT brands (saline: n = 16; distilled water: n = 17; and tap water: n = 20), and in 15 RDTs which were run with distilled water only. They occurred for all Plasmodium antigens and RDT formats (two-, three- and four-band RDTs). Clearance of the background of the strip was excellent except for saline. The aspects (colour, intensity and crispness) of the control and the false-positive test lines were similar to those obtained with the RDT kits' buffer and Plasmodium positive samples. Conclusion Replacement of the RDT kit's dedicated buffer by saline, distilled water and tap water can cause false-positive test results. PMID:20650003

NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

PubMed Central

Malerba, Francesca; Paoletti, Francesca; Cattaneo, Antonino

2016-01-01

The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, toward the development of a selective assay. In particular, the use of the well validated anti-NGF αD11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological samples, and propose concrete solutions toward the solution of this problem. PMID:27536217

Unreliability of three commercial Coxiella burnetii phase II IgM ELISA kits for the seroscreening of acute Q fever in human cases.

PubMed

Stephen, Selvaraj; Ambroise, Stanley; Pradeep, Jothimani; Gunasekaran, Dhandapany; Sangeetha, Balakrishnan; Sarangapani, Kengamuthu

2017-09-01

Seroprevalence of Q fever (QF) caused by Coxiella burnetii has been reported from different parts of India. Usually serological/molecular tests are employed for detection of infection. The present study was undertaken to verify the validity of three different QF phase II IgM ELISA kits for acute QF diagnosis by comparing with the gold standard indirect fluorescent antibody assay (IFA). Fifty eight serum samples collected from 42 patients (26 patients provided acute sample only and 16 both acute and convalescent samples) which were examined by all three commercial kits, were cross-checked with QF Phase II IgM IFA for confirmation. Eleven patients were positive for C. burnetii antibodies by IFA in acute and/or convalescent serum samples. Taking IFA as a reference, percentages of sensitivity, specificity, positive predictive value and negative predictive value for Virion-Serion/Vircell/NovaTec were 36.36, 61.29, 25.00, 73.08; 81.82, 35.48, 31.03, 84.62 and 100, 25.81, 32.35, 100 per cent, respectively. The three different ELISA kits exhibited poor agreement amongst them and unacceptable level of false positivity. IFA remains to be the only option for diagnosing acute QF. Discrepancy between the clinical findings and IFA/ELISA results needs confirmation by C. burnetii DNA detection in real-time polymerase chain reaction.

Unreliability of three commercial Coxiella burnetii phase II IgM ELISA kits for the seroscreening of acute Q fever in human cases

PubMed Central

Stephen, Selvaraj; Ambroise, Stanley; Pradeep, Jothimani; Gunasekaran, Dhandapany; Sangeetha, Balakrishnan; Sarangapani, Kengamuthu

2017-01-01

Background & objectives: Seroprevalence of Q fever (QF) caused by Coxiella burnetii has been reported from different parts of India. Usually serological/molecular tests are employed for detection of infection. The present study was undertaken to verify the validity of three different QF phase II IgM ELISA kits for acute QF diagnosis by comparing with the gold standard indirect fluorescent antibody assay (IFA). Methods: Fifty eight serum samples collected from 42 patients (26 patients provided acute sample only and 16 both acute and convalescent samples) which were examined by all three commercial kits, were cross-checked with QF Phase II IgM IFA for confirmation. Results: Eleven patients were positive for C. burnetii antibodies by IFA in acute and/or convalescent serum samples. Taking IFA as a reference, percentages of sensitivity, specificity, positive predictive value and negative predictive value for Virion-Serion/Vircell/NovaTec were 36.36, 61.29, 25.00, 73.08; 81.82, 35.48, 31.03, 84.62 and 100, 25.81, 32.35, 100 per cent, respectively. Interpretation & conclusions: The three different ELISA kits exhibited poor agreement amongst them and unacceptable level of false positivity. IFA remains to be the only option for diagnosing acute QF. Discrepancy between the clinical findings and IFA/ELISA results needs confirmation by C. burnetii DNA detection in real-time polymerase chain reaction. PMID:29355147

[Multisite validation of CDT measurement by the %CDT TIA and the Tina Quant %CDT kits].

PubMed

Boehrer, J L; Cano, Y; Capolaghi, B; Desch, G; Dosbaa, I; Estepa, L; Hennache, B; Schellenberg, F

2007-01-01

The measurement of CDT (Carbohydrate Deficient Transferrin) is an essential biological tool in the diagnosis and follow-up of alcohol abuse. It is also employed as a marker of abstinence for the restitution of driving licences. However, the precision of measurement, and the between laboratory homogeneity of the results are still discussed. The ion exchange followed by immunodetermination of CDT is available in two products, the Tina Quant %CDT (Roche, Mannheim, Germany) and the %CDT TIA (Bio-Rad, Hercules, United States). This multicentre study was undertaken: 1) to evaluate the analytical characteristics of these kits and the homogeneity of the results from one laboratory to another, independently of the method used, 2) to validate the differences between the proposed normal values of both kits, 3) to study the possibility of using commercial control sera as external quality control. Four analytical systems were included in the study (Roche Modular/Hitachi 717, Beckman Coulter Immage and LX20, Dade Behring BNII). Determinations were carried out on pools of sera, commercial control sera, kit controls, and 30 serums of patients. These latter were also analyzed in capillary electrophoresis in order to establish correlations between the techniques. The calibrations were stable over one 2 weeks period. The repeatability of measurements spread out from 3,1% to 24,7%, for a mean value lower than 10%. The commercial control sera provided reliable results, with values adapted to a routine quality control use. The results of the Bio-Rad applications were lower by approximately 20% than those of the Roche application, which justifies the difference of the normal values (2,6% versus 3%), and an identical classification of the patients in at least 27 of the 30 samples. We conclude that the analytical quality of the compared techniques, even if it could be improved, is sufficient to guarantee a good reliability of the results. An external quality control could be proposed by using the control sera that we tested.

An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints.

PubMed

Solomon, April D; Hytinen, Madison E; McClain, Aryn M; Miller, Marilyn T; Dawson Cruz, Tracey

2018-01-01

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases. © 2017 American Academy of Forensic Sciences.

Reiter works with SWAB ASD Filter Kit in the U.S. Laboratory during Expedition 13

NASA Image and Video Library

2006-09-10

ISS013-E-80066 (10 Sept. 2006) --- European Space Agency (ESA) astronaut Thomas Reiter, Expedition 13 flight engineer, works with the surface, water and air biocharacterization (SWAB) air sampling device (ASD) filter kit in the Destiny laboratory of the International Space Station.

Low-cost field test kits for arsenic detection in water.

PubMed

Das, Joyati; Sarkar, Priyabrata; Panda, Jigisha; Pal, Priyabrata

2014-01-01

Arsenic, a common contaminant of groundwater, affects human health adversely. According to the World Health Organization (WHO), the maximum recommended contamination level of arsenic in drinking water is 10 μg/L. The purpose of this research was to develop user-friendly kits for detection of arsenic to measure at least up to 10 μg/L in drinking water, so that a preventive measure could be taken. Two different kits for detection of total arsenic in water are reported here. First, the arsenic in drinking water was converted to arsine gas by a strong reducing agent. The arsine produced was then detected by paper strips via generation of color due to reaction with either mercuric bromide (KIT-1) or silver nitrate (KIT-2). These were previously immobilized on the detector strip. The first one gave a yellow color and the second one grey. Both of these kits could detect arsenic contamination within a range of 10 μg/L-250 μg/L. The detection time for both the kits was only 7 min. The kits exhibited excellent performance compared to other kits available in the market with respect to detection time, ease of operation, cost and could be easily handled by a layman. The field trials with these kits gave very satisfactory results. A study on interference revealed that these kits could be used in the presence of 24 common ions present in the arsenic contaminated water. Though the kits were meant for qualitative assay, the results with unknown concentrations of real samples, when compared with atomic absorption spectrophotometer (AAS) were in good agreement as revealed by the t-test.

The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder.

PubMed

Gevaert, Thomas; Ridder, Dirk De; Vanstreels, Els; Daelemans, Dirk; Everaerts, Wouter; Aa, Frank Van Der; Pintelon, Isabel; Timmermans, Jean-Pierre; Roskams, Tania; Steiner, Clara; Neuhaus, Jochen

2017-06-01

The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT + ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody-related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin-1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT + cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT + ICC are present in bladder. In this perspective, functional concepts of KIT + ICC being involved in sensory and/or motor aspects of bladder physiology should be revised. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Chlamyweb Study I: rationale, design and acceptability of an internet-based chlamydia testing intervention.

PubMed

Lydié, Nathalie; de Barbeyrac, Bertille; Bluzat, Lucile; Le Roy, Chloé; Kersaudy-Rahib, Delphine

2017-05-01

In recent years, the internet has widely facilitated Chlamydia trachomatis home-sampling. In France (2012), the Chlamyweb Study evaluated an intervention (Chlamyweb) involving home-based self-sampling via the internet. One element of the study consisted of a randomised controlled trial (RCT), which is reported in detail elsewhere. The focus of this paper, however, is on describing the Chlamyweb Intervention and reporting on the non-RCT element of the evaluation of that intervention by the Chlamyweb Study. This involves (1) describing the design and roll-out of the Chlamyweb Intervention, (2) comparing the socio-behavioural profiles of the participants in the intervention with a nationally representative general population sample and (3) examining the factors that influence the acceptance and return of a self-sampling kit supplied to participants in the course of the intervention. Self-sampling kits were offered to sexually active people aged 18-24 years living on the mainland French. Participants' characteristics were compared with the general population to describe recruited and participant populations. Multivariate analyses by conditional logistic regression were performed to determine factors that were predictors of kit acceptation and use. 7215 people aged 18-24 years were included. Compared with the general population, Chlamyweb reached larger proportions of women, younger people and people with several partners in the previous year. 3372 (46.7%) agreed to receive a self-sampling kit and 2084 (61.8%) returned it, with more women doing so than men. The participation rate was associated with age, place of birth, occupational status, number of partners and condom use, differently for men and women. The offer of easy-to-use, self-sampling kits free of charge appeared to be a logistically feasible strategy for testing in France and reached a large and diverse population including individuals who have limited access to the traditional healthcare system. AFFSAPS n° IDRCB 0211-A01000-41; pre-results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

PubMed

Domonova, É A; Shipulina, O Iu; Kuevda, D A; Larichev, V F; Safonova, A P; Burchik, M A; Butenko, A M; Shipulin, G A

2012-01-01

Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

PubMed

Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

2015-05-01

Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Development and validation of an ELISA kit (YF MAC-HD) to detect IgM to yellow fever virus.

PubMed

Basile, Alison Jane; Goodman, Christin; Horiuchi, Kalanthe; Laven, Janeen; Panella, Amanda J; Kosoy, Olga; Lanciotti, Robert S; Johnson, Barbara W

2015-12-01

Yellow fever virus (YFV) is endemic in tropical and sub-tropical regions of the world, with around 180,000 human infections a year occurring in Africa. Serologic testing is the chief laboratory diagnostic means of identifying an outbreak and to inform the decision to commence a vaccination campaign. The World Health Organization disseminates the reagents for YFV testing to African reference laboratories, and the US Centers for Disease Control and Prevention (CDC) is charged with producing and providing these reagents. The CDC M-antibody capture ELISA is a 2-day test, requiring titration of reagents when new lots are received, which leads to inconsistency in testing and wastage of material. Here we describe the development of a kit-based assay (YF MAC-HD) based upon the CDC method, that is completed in approximately 3.5h, with equivocal samples being reflexed to an overnight protocol. The kit exhibits >90% accuracy when compared to the 2-day test. The kits were designed for use with a minimum of equipment and are stored at 4°C, removing the need for freezing capacity. This kit is capable of tolerating temporary sub-optimal storage conditions which will ease shipping or power outage concerns, and a shelf life of >6 months was demonstrated with no deterioration in accuracy. All reagents necessary to run the YF MAC-HD are included in the kit and are single-use, with 8 or 24 sample options per kit. Field trials are envisioned for the near future, which will enable refinement of the method. The use of the YF MAC-HD is anticipated to reduce materials wastage, and improve the quality and consistency of YFV serologic testing in endemic areas. Published by Elsevier B.V.

Comparison of clinical application of the Abbott HBV PCR kit and the VERSANT HBV DNA 3.0 test to measure serum hepatitis B virus DNA in Taiwanese patients.

PubMed

Yang, Jeng-Fu; Lin, Ya-Yun; Huang, Jee-Fu; Liu, Shu-Fen; Chu, Pei-Yu; Hsieh, Ming-Yen; Lin, Zu-Yau; Chen, Shinn-Cherng; Wang, Liang-Yen; Dai, Chia-Yen; Chuang, Wan-Long; Yu, Ming-Lung

2009-08-01

With an estimated 350-400 million people worldwide chronically infected with hepatitis B virus (HBV), and the subsequent serious complications caused by liver damage including cirrhosis, liver failure, and hepatocellular carcinoma, HBV infection remains a global health issue, particularly in Taiwan, an HBV-hyperendemic area. Sensitive and accurate quantification of HBV DNA is necessary to monitor patients with chronic hepatitis B who are receiving antiviral therapy to determine treatment response and adapt therapy. We evaluated and compared the clinical performance of two HBV DNA assays based on different technologies: the RealArt HBV PCR Kit (Abbott HBV DNA PCR kit, real-time polymerase chain reaction assay, detection limit: 27 IU/mL) and the VERSANT bDNA 3.0 assay (Bayer, branched DNA signal amplification assay, detection limit: 357 IU/mL). Serum levels of HBV DNA in 173 chronic HBV carriers were determined using both the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 test. Of the 173 samples analyzed for baseline viral load detection, HBV DNA was quantifiable in 147 patients (82.1%) by the RealArt HBV PCR Kit, which was significantly higher than the 92 (53.2%) samples quantified by the VERSANT bDNA 3.0 assay. A total of 86 (49.7%) samples were quantifiable by both assays, whereas 25 (14.5%) were below the detection limit of both assays. The HBV DNA quantification values measured by the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 assay were positively correlated (Spearman's rank correlation coefficient r = 0.932, p < 0.001). On average, the results derived from the RealArt HBV PCR Kit were 0.67 log lower than those of the VERSANT bDNA 3.0 assay. HBV DNA concentrations were significantly higher in 63 HBV e antigen (HBeAg)-seropositive patients than in 110 HBeAg-seronegative patients (5.42 +/- 2.34 logs vs. 3.21 +/- 2.27 logs, p < 0.001). The RealArt HBV PCR Kit is more sensitive and has a wider dynamic range than the VERSANT bDNA 3.0 assay in the clinical setting of chronic hepatitis B patients. The sensitivity and wide dynamic range of the PCR assay allow optimal monitoring and timely adaptation of antiviral therapy. Nevertheless, the HBV DNA values measured by the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 assay were significantly correlated.

Solid phase sequencing of biopolymers

DOEpatents

Cantor, Charles; Koster, Hubert

2010-09-28

This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

An immunohistochemical analysis of canine haemangioma and haemangiosarcoma.

PubMed

Sabattini, S; Bettini, G

2009-01-01

The aim of the present study was to investigate immunohistochemically aspects of the biology of canine endothelial neoplasia. Forty samples of canine cutaneous and visceral haemangiosarcoma (HSA), 29 samples of cutaneous and visceral haemangioma (HA) and 10 control samples of granulation tissue (GT) were labelled with antisera specific for vimentin, smooth muscle actin, von Willebrand factor (vWF), CD117 (KIT), vascular endothelial growth factor receptor-3 (VEGFR-3), vascular endothelial growth factor-C (VEGFC) and CD44. Further antisera were employed to determine the level of cellular proliferation (MIB-1 index) and toluidine blue staining was used to detect populations of tumour-infiltrating mast cells (MCs). There was greater expression of CD117, VEGFR-3 and CD44 in HSA than in HA, suggesting that these proteins might be suitable targets for the future development of novel therapeutic approaches to canine HSA. Marked infiltration of MC was detected in HA, suggesting a possible role for these cells in the pathogenesis of benign vascular neoplasia in the dog.

An indigenously developed nitrite kit to aid in the diagnosis of urinary tract infection.

PubMed

Sood, S; Upadhyaya, P; Kapil, A; Lodha, R; Jain, Y; Bagga, A

1999-09-01

To evaluate the utility of an indigenously developed nitrite kit for the rapid diagnosis of urinary tract infection (UTI) METHODS: 1018 urine specimens were collected from all cases where there was clinical suspicion of UTI. Samples were cultured as per standard microbiological protocol. Presence of nitrites was indicated by the development of purple color on addition of color developing solution and compared with the set of graded positive and negative controls also provided in the Kit. The results of the nitrite kit were compared with the semi-quantitative urine culture as the gold standard. The sensitivity, specificity, positive predictive and negative predictive values were 47%, 87%, 31% and 93%, respectively. Nitrite kit as a screening test can decrease the work load in the clinical bacteriology laboratory. More importantly in a field set up that is devoid of culture facilities, it can be used to correctly predict the absence of UTI.

Huntingtin-interacting protein 1: a Merkel cell carcinoma marker that interacts with c-Kit.

PubMed

Ames, Heather M; Bichakjian, Christopher K; Liu, Grace Y; Oravecz-Wilson, Katherine I; Fullen, Douglas R; Verhaegen, Monique E; Johnson, Timothy M; Dlugosz, Andrzej A; Ross, Theodora S

2011-10-01

Merkel cell carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. Although the prevalence of MCC has been increasing, treatments for this disease remain limited because of a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin-interacting protein 1 (HIP1) is expressed at high levels in ∼90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20. Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working toward an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF-activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit.

TECHNOLOGIES FOR MONITORING AND MEASUREMENT ...

EPA Pesticide Factsheets

A demonstration of technologies for determining the presence of dioxin and dioxin-like compounds in soil and sediment was conducted under EPA's Superfund Innovative Technology Evaluation Program in Saginaw, Michigan in April 2004. This report describes the performance evaluation of CAPE Technologies DF-1 Dioxin/Furan and PCB TEQ Immunoassay Kits. The kits are immunoassay techniques that report the total toxicity equivalents (TEQ) of dioxin/furans and polychlorinated biphenyls (PCBs. The technology results were compared to high resolution mass spectrometry TEQ results generated using EPA Methods 1613B and 1668A.The CAPE Technologies kits generally reported data higher than the certified PE and reference laboratory values. The technologys estimated MDL was 12 to 33 pg/g TEQ. Results from this demonstration suggest that the CAPE Technologies kits could be an effective screening tool for determining sample results above and below 20 pg/g TEQ and even more effective as a screen for sample above and below 50 pg/g TEQ, particularly considering that both the cost ($59,234 vs. $398,029) and the time (3 weeks vs. 8 months) to analyze the 209 demonstration samples were significantly less than those of the reference laboratory. The objective of this program is to promote the acceptance and use of innovative field technologies by providing well-documented performance and cost data obtained from field demonstrations.

77 FR 1396 - Procurement of Commodities and Services Financed by USAID Federal Program Funds.

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-10

... Agriculture Commodities, has been revised to state that USAID provides a list of restricted agricultural... salts, biologicals, and some in-vitro diagnostic reagents/test kits; but does not include devices or...

Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections.

PubMed

Zhao, Meng-Chuan; Li, Gui-Xia; Zhang, Dan; Zhou, Hang-Yu; Wang, Hao; Yang, Shuo; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

2017-06-01

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.

Performance evaluation of a chemiluminescence microparticle immunoassay for CK-MB.

PubMed

Lin, Zhi-Yuan; Fang, Yi-Zhen; Jin, Hong-Wei; Lin, Hua-Yue; Dai, Zhang; Luo, Qing; Li, Hong-Wei; Lin, Yan-Ling; Huang, Shui-Zhen; Gao, Lei; Xu, Fei-Hai; Zhang, Zhong-Ying

2018-03-31

To verify and evaluate the performance characteristics of a creatine kinase phosphokinase isoenzymes MB (CK-MB) assay kit, which produced by Xiamen Innodx Biotech Co. Ltd. Evaluation was carried out according to "Guidelines for principle of analysis performance evaluation of in vitro diagnostic reagent." The performance parameters included detection limit, linearity range, reportable range, recovery test, precision verification, interference test, cross-reactivity, matrix effect, and method comparison. The detection limit was 0.1 ng/mL. The assay had clinical linearity over range of 0.1 ng/mL-500 ng/mL. Reportable range was from 0.1 ng/mL to 1000 ng/mL. The average percent of recovery was 99.66%. The coefficient of variation (CV) for within-run and between-run of low CK-MB sample was 5.55% and 6.16%, respectively. As for high-level sample, it was 7.88% and 7.80%. In medical decision level, the relative deviation (Bias) of all interference tests was lower than 15%. When the sample had mild-hemolysis; hemoglobin ≤15 g/L; triglyceride ≤17 mmol/L; bilirubin ≤427.5 μmol/L; rheumatoid factor ≤206U/mL, there was no significant interference to be found. Moreover, assay kit had no cross-reaction with CK-MM and CK-BB. At last, total diagnostic accuracy of kit was 93.24%, when compared with refer kit. Overall the results of the verification study indicated the performance of kit is met the requirements of the clinical test. © 2018 Wiley Periodicals, Inc.

Therapeutic drug monitoring of infliximab: performance evaluation of three commercial ELISA kits.

PubMed

Schmitz, Ellen M H; van de Kerkhof, Daan; Hamann, Dörte; van Dongen, Joost L J; Kuijper, Philip H M; Brunsveld, Luc; Scharnhorst, Volkher; Broeren, Maarten A C

2016-07-01

Therapeutic drug monitoring (TDM) of infliximab (IFX, Remicade®) can aid to optimize therapy efficacy. Many assays are available for this purpose. However, a reference standard is lacking. Therefore, we evaluated the analytical performance, agreement and clinically relevant differences of three commercially available IFX ELISA kits on an automated processing system. The kits of Theradiag (Lisa Tracker Infliximab), Progenika (Promonitor IFX) and apDia (Infliximab ELISA) were implemented on an automated processing system. Imprecision was determined by triplicate measurements of patient samples on five days. Agreement was evaluated by analysis of 30 patient samples and four spiked samples by the selected ELISA kits and the in-house IFX ELISA of Sanquin Diagnostics (Amsterdam, The Netherlands). Therapeutic consequences were evaluated by dividing patients into four treatment groups using cut-off levels of 1, 3 and 7 μg/mL and determining assay concordance. Within-run and between-run imprecision were acceptable (≤12% and ≤17%, respectively) within the quantification range of the selected ELISA kits. The apDia assay had the best precision and agreement to target values. Statistically significant differences were found between all assays except between Sanquin Diagnostics and the Lisa Tracker assay. The Promonitor assay measured the lowest IFX concentrations, the apDia assay the highest. When patients were classified in four treatment categories, 70% concordance was achieved. Although all assays are suitable for TDM, significant differences were observed in both imprecision and agreement. Therapeutic consequences were acceptable when patients were divided in treatment categories, but this could be improved by assay standardization.

How To Sample in Surveys. The Survey Kit, Volume 6.

ERIC Educational Resources Information Center

Fink, Arlene

The nine-volume Survey Kit is designed to help readers prepare and conduct surveys and become better users of survey results. All the books in the series contain instructional objectives, exercises and answers, examples of surveys in use, illustrations of survey questions, guidelines for action, checklists of "dos and don'ts," and…

Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness

PubMed Central

Siemens, Helge; Jackstadt, Rene; Kaller, Markus; Hermeking, Heiko

2013-01-01

The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches. PMID:24009080

Audit of lymphadenectomy in lung cancer resections using a specimen collection kit and checklist

PubMed Central

Osarogiagbon, Raymond U.; Sareen, Srishti; Eke, Ransome; Yu, Xinhua; McHugh, Laura M.; Kernstine, Kemp H.; Putnam, Joe B.; Robbins, Edward T.

2014-01-01

Background Audits of operative summaries and pathology reports reveal wide discordance in identifying the extent of lymphadenectomy performed (the communication gap). We tested the ability of a pre-labeled lymph node specimen collection kit and checklist to narrow the communication gap between operating surgeons, pathologists, and auditors of surgeons’ operation notes. Methods We conducted a prospective single cohort study of lung cancer resections performed with a lymph node collection kit from November 2010 to January 2013. We used the kappa statistic to compare surgeon claims on a checklist of lymph node stations harvested intraoperatively, to pathology reports, and an independent audit of surgeons’ operative summaries. Lymph node collection procedures were classified into 4 groups based on the anatomic origin of resected lymph nodes: mediastinal lymph node dissection, systematic sampling, random sampling and no sampling. Results From the pathology report, 73% of 160 resections had a mediastinal lymph node dissection or systematic sampling procedure, 27% had random sampling. The concordance with surgeon claims was 80% (kappa statistic 0.69 [CI 0.60 – 0.79]). Concordance between independent audits of the operation notes and either the pathology report (kappa 0.14 [0.04 – 0.23]), or surgeon claims (kappa 0.09 [0.03 – 0.22]), was poor. Conclusion A pre-labeled specimen collection kit and checklist significantly narrowed the communication gap between surgeons and pathologists in identifying the extent of lymphadenectomy. Audit of surgeons’ operation notes did not accurately reflect the procedure performed, bringing its value for quality improvement work into question. PMID:25530090

Survey of undeclared soy allergen levels in the most frequently recalled food categories with or without precautionary labelling.

PubMed

Khuda, Sefat E; Sharma, Girdhari M; Gaines, Dennis; Do, Andrew B; Pereira, Marion; Chang, Michael; Ferguson, Martine; Williams, Kristina M

2016-08-01

A comprehensive study was designed to determine the frequency and levels of soy allergen in packaged bakery and snack food products. A representative sample of products with no soy allergen disclosed on the label was analysed using two widely used enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no soy identified on the product label or which had a soy precautionary statement. Among 558 bakery and snack products, soy protein was detected in 17% of the products using the Neogen (NE) kit and 11% of the products using the Elisa Systems (ES) kit. The disagreement rates between kits were 8.8% for bakery products and 3.3% for snack products. Overall soy protein was detected at higher frequency in bakery products than in snack foods. Among 284 bakery samples, soy protein was detected in 25% of the samples with no precautionary statement and 19% of the samples which had a precautionary statement. Among 274 snack samples, soy protein was detected in 11% of the samples with no precautionary statement and 9% of the samples which had a precautionary statement. The sample repeatability was at an acceptable level (< 9%) for each method and food commodity. The reproducibility between kits was 23% for bakery foods and 36% for snack foods. None of the bakery (21) and snack (6) products without precautionary labelling (measured level > 5 ppm) had a higher level of soy protein per serving compared with the eliciting dose10 (ED10) of 10.6 mg for soy allergic patients. But the level of soy protein per serving may be clinically relevant to a subpopulation of soy allergic patients if a more stringent eliciting dose is applied. These findings emphasise that suitable detection methodologies and references doses are crucial for labelling accuracy and the safety of soy allergic consumers.

Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis.

PubMed

Greiner, Georg; Gurbisz, Michael; Ratzinger, Franz; Witzeneder, Nadine; Simonitsch-Klupp, Ingrid; Mitterbauer-Hohendanner, Gerlinde; Mayerhofer, Matthias; Müllauer, Leonhard; Sperr, Wolfgang R; Valent, Peter; Hoermann, Gregor

2018-03-01

The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR ( P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival ( P < 0.001). dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis. © 2017 American Association for Clinical Chemistry.

Gene expression in gastrointestinal stromal tumors is distinguished by KIT genotype and anatomic site.

PubMed

Antonescu, Cristina R; Viale, Agnes; Sarran, Lisa; Tschernyavsky, Sylvia J; Gonen, Mithat; Segal, Neil H; Maki, Robert G; Socci, Nicholas D; DeMatteo, Ronald P; Besmer, Peter

2004-05-15

Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

PubMed

Jara-Acevedo, Maria; Teodosio, Cristina; Sanchez-Muñoz, Laura; Álvarez-Twose, Ivan; Mayado, Andrea; Caldas, Carolina; Matito, Almudena; Morgado, José M; Muñoz-González, Javier I; Escribano, Luis; Garcia-Montero, Andrés C; Orfao, Alberto

2015-08-01

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

PubMed

Vojkovska, H; Kubikova, I; Kralik, P

2015-03-01

Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014 The Society for Applied Microbiology.

On-farm biosecurity practices and causes of preweaning mortality in Canadian commercial mink kits.

PubMed

Compo, Nicole; Pearl, David L; Tapscott, Brian; Storer, Amanda; Hammermueller, Jutta; Brash, Marina; Turner, Patricia V

2017-09-08

Mink are an important animal commodity group in Canada and excessive kit mortality represents a significant loss to production. National biosecurity standards have been developed for Canadian mink farms, but it is unclear how well these standards have been implemented as there are no studies correlating management practices of mink producers with causes of death in mink kits. To that end, we surveyed Ontario mink producers on their biosecurity and management practices and conducted almost 5660 post mortem examinations on found-dead, preweaned kits to characterize mink farm biosecurity practices and causes of death in preweaned kits. We found that very few biosecurity and management practices were uniformly used by producers, despite good awareness of appropriate practices. Use of personal protective equipment was implemented by fewer than 50% of respondents, while control of mink shed access, disinfection of feed containers after use, and use of a rodent control program were the only practices implemented by greater than 70% of respondents. Only 18% of producers reported regular use of antimicrobials in feed or water, although 91% stated they used antimicrobials for treatment of bacterial diseases on a regular basis. On post mortem examination, no gross abnormalities were noted in 71% of the kits, 45% were thought to be stillborn or aborted, 27% had some form of abnormal fluid distribution in the body, and 2% had a congenital malformation. A subset of 69 gastrointestinal tract samples was submitted for bacterial culture, of which 45 samples yielded sufficient growth. Most interesting was the identification of Salmonella enterica serovar Heidelberg in 11% of samples. The results of this study will provide a benchmark for Canadian mink producers and their veterinarians, defining the areas to which greater attention should be given to ensure more rigorous biosecurity practices are in place. Ultimately, these improvements in practices may contribute to increased mink production and animal well-being.

Attitudes towards HIV testing via home-sampling kits ordered online (RUClear pilots 2011-12).

PubMed

Ahmed-Little, Y; Bothra, V; Cordwell, D; Freeman Powell, D; Ellis, D; Klapper, P; Scanlon, S; Higgins, S; Vivancos, R

2016-09-01

The burden of disease relating to undiagnosed HIV infection is significant in the UK. BHIVA (British HIV Association) recommends population screening in high prevalence areas, expanding outside traditional antenatal/GUM settings. RUClear 2011-12 piloted expanding HIV testing outside traditional settings using home-sampling kits (dry-blood-spot testing) ordered online. Greater Manchester residents (≥age 16) could request testing via an established, online chlamydia testing service (www.ruclear.co.uk). Participant attitudes towards this new service were assessed. Qualitative methods (thematic analysis) were used to analyse free-text data submitted by participants via hard copy questionnaires issued in all testing kits. 79.9% (2447/3062) participants completed questionnaires, of which 30.9% (756/2447) provided free-text data. Participants overwhelmingly supported the service, valuing particularly accessibility and convenience, allowing individuals to order tests any time of day and self-sample comfortably at home; avoiding the invasive nature of venipuncture and avoiding the need for face-to-face interaction with health services. The pilot was also clinically and cost-effective. Testing via home-sampling kits ordered online (dry-blood-spot testing) was felt to be an acceptable and convenient method for accessing a HIV test. Many individuals undertook HIV testing where they would otherwise not have been tested at all. Expansion of similar services may increase the uptake of HIV testing. © The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

NASA Astrophysics Data System (ADS)

Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

1994-03-01

Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

Evaluation of Commercially Available Chikungunya Virus Immunoglobulin M Detection Assays

PubMed Central

Johnson, Barbara W.; Goodman, Christin H.; Holloway, Kimberly; de Salazar, P. Martinez; Valadere, Anne M.; Drebot, Michael A.

2016-01-01

Commercial chikungunya virus (CHIKV)–specific IgM detection kits were evaluated at the Centers for Disease Control and Prevention (CDC), the Public Health Agency of Canada National Microbiology Laboratory, and the Caribbean Public Health Agency (CARPHA). The Euroimmun Anti-CHIKV IgM ELISA kit had ≥ 95% concordance with all three reference laboratory results. The limit of detection for low CHIK IgM+ samples, as measured by serial dilution of seven sera up to 1:12,800 ranged from 1:800 to 1:3,200. The Euroimmun IIFT kit evaluated at CDC and CARPHA performed well, but required more retesting of equivocal results. The InBios CHIKjj Detect MAC-ELISA had 100% and 98% concordance with CDC and CARPHA results, respectively, and had equal sensitivity to the CDC MAC-ELISA to 1:12,800 dilution in serially diluted samples. The Abcam Anti-CHIKV IgM ELISA had high performance at CARPHA, but at CDC, performance was inconsistent between lots. After replacement of the biotinylated IgM antibody controls with serum containing CHIKV-specific IgM and additional quality assurance/control measures, the Abcam kit was rereleased and reevaluated at CDC. The reformatted Abcam kit had 97% concordance with CDC results and limit of detection of 1:800 to 1:3,200. Two rapid tests and three other CHIKV MAC-ELISAs evaluated at CDC had low sensitivity, as the CDC CHIKV IgM in-house positive controls were below the level of detection. In conclusion, laboratories have options for CHIKV serological diagnosis using validated commercial kits. PMID:26976887

Comparison of Multiplex Suspension Array Large-Panel Kits for Profiling Cytokines and Chemokines in Rheumatoid Arthritis Patients

PubMed Central

Khan, Imran H.; Krishnan, V.V.; Ziman, Melanie; Janatpour, Kim; Wun, Ted; Luciw, Paul A.; Tuscano, Joseph

2015-01-01

Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients. PMID:18823005

NextGen Home Sperm Banking Kit: Outcomes of Offsite vs Onsite Collection--Preliminary Findings.

PubMed

Agarwal, Ashok; Sharma, Reecha; Gupta, Sajal; Sharma, Rakesh

2015-06-01

To compare cryosurvival rates between remote collections with NextGen kit (offsite) and onsite collection of semen samples from infertile men and those with cancer. Prefreeze and post-thaw sperm motility, total motile sperm, and percent cryosurvival rates were compared between samples collected from infertile men onsite at the Andrology Center (n = 10) and samples collected from infertile patients at home (offsite; n = 9), which were shipped by NextGen to our laboratory. A second group (n = 17) consisted of 10 semen samples from cancer patients collected onsite, which were compared with 7 semen samples from cancer patients shipped by the NextGen. All semen samples were assessed within 18 hours of collection. In the infertile men, percent cryosurvival rates were similar with NextGen compared with those of onsite collection (53.14 ± 28.9% vs 61.90 ± 20.46%; P = .51). Similarly, in the cancer patients, all 4 parameters were comparable between the onsite and NextGen. Cryosurvival rates were also similar between NextGen compared with those of onsite collection (52.71 ± 20.37% vs 58.90 ± 22.68%; P = .46). Cancer patients can bank sperm as effectively as men banking for infertility reasons using the NextGen kit. Copyright © 2015 Elsevier Inc. All rights reserved.

The Development of a Portable SPR Bioanalyzer for Sensitive Detection of Escherichia coli O157:H7.

PubMed

Wang, Shun; Xie, Jiufeng; Jiang, Min; Chang, Keke; Chen, Ruipeng; Ma, Liuzheng; Zhu, Juanhua; Guo, Qingqian; Sun, Haifeng; Hu, Jiandong

2016-11-04

The purpose of this study was to develop a portable surface plasmon resonance (SPR) bioanalyzer for the sensitive detection of Escherichia coli O157:H7 in comparison with an enzyme-linked immunosorbent assay (ELISA). The experimental setup mainly consisted of an integrated biosensor and a homemade microfluidic cell with a three-way solenoid valve. In order to detect Escherichia coli O157:H7 using the SPR immunoassay, 3-mercaptopropionic acid (3-MPA) was chemisorbed onto a gold surface via covalent bond for the immobilization of biological species. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used as crosslinker reagents to enable the reaction between 3-MPA and Escherichia coli O157:H7 antibodies by covalent -CO-NH- amide bonding. The experimental results were obtained from the Escherichia coli O157:H7 positive samples prepared by 10-, 20-, 40-, 80-, and 160-fold dilution respectively, which show that a good linear relationship with the correlation coefficient R of 0.982 existed between the response units from the portable SPR bioanalyzer and the concentration of Escherichia coli O157:H7 positive samples. Moreover, the theoretical detection limit of 1.87 × 10³ cfu/mL was calculated from the positive control samples. Compared with the Escherichia coli O157:H7 ELISA kit, the sensitivity of this portable SPR bioanalyzer is four orders of magnitude higher than the ELISA kit. The results demonstrate that the portable SPR bioanalyzer could provide an alternative method for the quantitative and sensitive determination of Escherichia coli O157:H7 in field.

The Development of a Portable SPR Bioanalyzer for Sensitive Detection of Escherichia coli O157:H7

PubMed Central

Wang, Shun; Xie, Jiufeng; Jiang, Min; Chang, Keke; Chen, Ruipeng; Ma, Liuzheng; Zhu, Juanhua; Guo, Qingqian; Sun, Haifeng; Hu, Jiandong

2016-01-01

The purpose of this study was to develop a portable surface plasmon resonance (SPR) bioanalyzer for the sensitive detection of Escherichia coli O157:H7 in comparison with an enzyme-linked immunosorbent assay (ELISA). The experimental setup mainly consisted of an integrated biosensor and a homemade microfluidic cell with a three-way solenoid valve. In order to detect Escherichia coli O157:H7 using the SPR immunoassay, 3-mercaptopropionic acid (3-MPA) was chemisorbed onto a gold surface via covalent bond for the immobilization of biological species. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used as crosslinker reagents to enable the reaction between 3-MPA and Escherichia coli O157:H7 antibodies by covalent –CO–NH– amide bonding. The experimental results were obtained from the Escherichia coli O157:H7 positive samples prepared by 10-, 20-, 40-, 80-, and 160-fold dilution respectively, which show that a good linear relationship with the correlation coefficient R of 0.982 existed between the response units from the portable SPR bioanalyzer and the concentration of Escherichia coli O157:H7 positive samples. Moreover, the theoretical detection limit of 1.87 × 103 cfu/mL was calculated from the positive control samples. Compared with the Escherichia coli O157:H7 ELISA kit, the sensitivity of this portable SPR bioanalyzer is four orders of magnitude higher than the ELISA kit. The results demonstrate that the portable SPR bioanalyzer could provide an alternative method for the quantitative and sensitive determination of Escherichia coli O157:H7 in field. PMID:27827923

[Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

PubMed

Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

2012-01-01

Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

Clinical, biological, and molecular characteristics of clonal mast cell disorders presenting with systemic mast cell activation symptoms.

PubMed

Alvarez-Twose, Iván; González de Olano, David; Sánchez-Muñoz, Laura; Matito, Almudena; Esteban-López, Maria I; Vega, Arantza; Mateo, Maria Belén; Alonso Díaz de Durana, Maria D; de la Hoz, Belén; Del Pozo Gil, Maria D; Caballero, Teresa; Rosado, Ana; Sánchez Matas, Isabel; Teodósio, Cristina; Jara-Acevedo, María; Mollejo, Manuela; García-Montero, Andrés; Orfao, Alberto; Escribano, Luis

2010-06-01

Systemic mast cell activation disorders (MCADs) are characterized by severe and systemic mast cell (MC) mediators-related symptoms frequently associated with increased serum baseline tryptase (sBt). To analyze the clinical, biological, and molecular characteristics of adult patients presenting with systemic MC activation symptoms/anaphylaxis in the absence of skin mastocytosis who showed clonal (c) versus nonclonal (nc) MCs and to provide indication criteria for bone marrow (BM) studies. Eighty-three patients were studied. Patients showing clonal BM MCs were grouped into indolent systemic mastocytosis without skin lesions (ISMs(-); n = 48) and other c-MCADs (n = 3)-both with CD25(++) BM MCs and either positive mast/stem cell growth factor receptor gene (KIT) mutation or clonal human androgen receptor assay (HUMARA) tests-and nc-MCAD (CD25-negative BM MCs in the absence of KIT mutation; n = 32) and compared for their clinical, biological, and molecular characteristics. Most clonal patients (48/51; 94%) met the World Health Organization criteria for systemic mastocytosis and were classified as ISMs(-), whereas the other 3 c-MCAD and all nc-MCAD patients did not. In addition, although both patients with ISMs(-) and patients with nc-MCAD presented with idiopathic and allergen-induced anaphylaxis, the former showed a higher frequency of men, cardiovascular symptoms, and insect bite as a trigger, together with greater sBt. Based on a multivariate analysis, a highly efficient model to predict clonality before BM sampling was built that includes male sex (P = .01), presyncopal and/or syncopal episodes (P = .009) in the absence of urticaria and angioedema (P = .003), and sBt >25 microg/L (P = .006) as independent predictive factors. Patients with c-MCAD and ISMs(-) display unique clinical and laboratory features different from nc-MCAD patients. A significant percentage of c-MCAD patients can be considered as true ISMs(-) diagnosed at early phases of the disease. Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Finding a fox: an evaluation of survey methods to estimate abundance of a small desert carnivore.

PubMed

Dempsey, Steven J; Gese, Eric M; Kluever, Bryan M

2014-01-01

The status of many carnivore species is a growing concern for wildlife agencies, conservation organizations, and the general public. Historically, kit foxes (Vulpes macrotis) were classified as abundant and distributed in the desert and semi-arid regions of southwestern North America, but is now considered rare throughout its range. Survey methods have been evaluated for kit foxes, but often in populations where abundance is high and there is little consensus on which technique is best to monitor abundance. We conducted a 2-year study to evaluate four survey methods (scat deposition surveys, scent station surveys, spotlight survey, and trapping) for detecting kit foxes and measuring fox abundance. We determined the probability of detection for each method, and examined the correlation between the relative abundance as estimated by each survey method and the known minimum kit fox abundance as determined by radio-collared animals. All surveys were conducted on 15 5-km transects during the 3 biological seasons of the kit fox. Scat deposition surveys had both the highest detection probabilities (p = 0.88) and were most closely related to minimum known fox abundance (r2 = 0.50, P = 0.001). The next best method for kit fox detection was the scent station survey (p = 0.73), which had the second highest correlation to fox abundance (r2 = 0.46, P<0.001). For detecting kit foxes in a low density population we suggest using scat deposition transects during the breeding season. Scat deposition surveys have low costs, resilience to weather, low labor requirements, and pose no risk to the study animals. The breeding season was ideal for monitoring kit fox population size, as detections consisted of the resident population and had the highest detection probabilities. Using appropriate monitoring techniques will be critical for future conservation actions for this rare desert carnivore.

Finding a Fox: An Evaluation of Survey Methods to Estimate Abundance of a Small Desert Carnivore

PubMed Central

Dempsey, Steven J.; Gese, Eric M.; Kluever, Bryan M.

2014-01-01

The status of many carnivore species is a growing concern for wildlife agencies, conservation organizations, and the general public. Historically, kit foxes (Vulpes macrotis) were classified as abundant and distributed in the desert and semi-arid regions of southwestern North America, but is now considered rare throughout its range. Survey methods have been evaluated for kit foxes, but often in populations where abundance is high and there is little consensus on which technique is best to monitor abundance. We conducted a 2-year study to evaluate four survey methods (scat deposition surveys, scent station surveys, spotlight survey, and trapping) for detecting kit foxes and measuring fox abundance. We determined the probability of detection for each method, and examined the correlation between the relative abundance as estimated by each survey method and the known minimum kit fox abundance as determined by radio-collared animals. All surveys were conducted on 15 5-km transects during the 3 biological seasons of the kit fox. Scat deposition surveys had both the highest detection probabilities (p = 0.88) and were most closely related to minimum known fox abundance (r2 = 0.50, P = 0.001). The next best method for kit fox detection was the scent station survey (p = 0.73), which had the second highest correlation to fox abundance (r2 = 0.46, P<0.001). For detecting kit foxes in a low density population we suggest using scat deposition transects during the breeding season. Scat deposition surveys have low costs, resilience to weather, low labor requirements, and pose no risk to the study animals. The breeding season was ideal for monitoring kit fox population size, as detections consisted of the resident population and had the highest detection probabilities. Using appropriate monitoring techniques will be critical for future conservation actions for this rare desert carnivore. PMID:25148102

SURVEY OF GROUND, SURFACE, AND POTABLE WATERS FOR THE PRESENCE OF LEGIONELLA SPECIES BY ENVIROAMP PCR LEGIONELLA KIT, CULTURE, AND IMMUNOFLUORESCENT STAINING

EPA Science Inventory

A total of 116 samples from numerous aquatic sources including water from faucets, showerheads, dental units, fire sprinklers, and surface waters were examined for the presence of Legionella by the EnviroAmp Legionella PCR kit, culture on BCYEx, or direct fluorescent antibody (DF...

Acute Pancreatitis as a Model to Predict Transition of Systemic Inflammation to Organ Failure in Trauma and Critical Illiness

DTIC Science & Technology

2015-10-01

of the samples. Table 1. Characteristics of common filters suitable for use with the LIVE/DEAD® Viability Kit Omega Filters* Chroma Filters* Notes...www.omegafilters.com). Chroma filters are supplied by Chroma Technology Corp. (www.chroma.com). LIVE/DEAD® Viability/Cytotoxicity Kit 3 4.2 Incubate the cells

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

PubMed

Bidmon, Nicole; Kind, Sonja; Welters, Marij J P; Joseph-Pietras, Deborah; Laske, Karoline; Maurer, Dominik; Hadrup, Sine Reker; Schreibelt, Gerty; Rae, Richard; Sahin, Ugur; Gouttefangeas, Cécile; Britten, Cedrik M; van der Burg, Sjoerd H

2018-07-01

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification.

PubMed

Frégeau, Chantal J; Laurin, Nancy

2015-05-01

The Investigator® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049ng/μL and 0.0003ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR® Profiler® Plus or AmpFlSTR® Identifiler® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

Evaluation of Immunomagnetic Separation for Recovery of Infectious Cryptosporidium parvum Oocysts from Environmental Samples

PubMed Central

Rochelle, Paul A.; De Leon, Ricardo; Johnson, Anne; Stewart, Mic H.; Wolfe, Roy L.

1999-01-01

Two commercial immunomagnetic separation (IMS) kits for Cryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity. PMID:9925626

[Evaluation of Wondfo Rapid Diagnostic Kit for detecting Plasmodium ovale and analysis of influencing factors].

PubMed

Feng, Tang; Jian-Xia, Tang; Feng, Lu; Sui, Xu; Ya-Ping, Gu; De-Sheng, Tong; Guo-Ding, Zhu; Hai-Yong, Hua; Hua-Yun, Zhou; Jun, Cao

2016-03-21

To evaluate the Wondfo Rapid Diagnostic Kit (Pf-LDH/Pan -pLDH) for detecting Plasmodium ovale and analyze the influence of parasitaemia, concentration and polymorphism of pLDH on the performances. A total of 100 blood samples from P. ovale patients confirmed by PCR were detected with the Wondfo Rapid Diagnostic Kit according to the manufacturers'instructions. The parasitaemia was determined by the microscopic examination. The concentration of pLDH was measured by ELISA tests. The LDH gene of P. ovale was amplified by PCR and sequenced. The influence of these three factors on the positive rate was analyzed. The overall positive rate of Wondfo Rapid Diagnostic Kit was 70.0% (70/100). The positive rate was 27.3% for the samples with parasitaemia ≤ 500 parasites/μl and reached 75.0%-75.4% when parasitaemia > 500 parasites/μl. The positive rate was 6.7% for samples with a low pLDH concentration ( A values ≤ 0.100) and reached 95.1%-100% at a high pLDH concentration ( A values > 0.100). The results of sequence analysis indicated that all the samples could be divided into 2 types, P. o. curtisi and P. o. wallikeri . The gene homology of LDH between 2 types was 97%. There were 24 single nucleotide polymorphism (s) (SNPs) between 2 types, while only 3 SNPs were non-synonymous mutations. The homology of LDH amino acid sequences between 2 types was 99%; only 3 amino acids were different. The positive rates for P. o. curtisi and P. o. wallikeri were 73.1% (38/52) and 66.7% (32/48) respectively; there was no statistically significant difference ( P > 0.05). The Wondfo Rapid Diagnostic Kit (Pf-LDH/Pan-pLDH) performs better than most of the similar products for the detection of P. ovale , and the positive rates are closely related to the parasitaemia and concentration of pLDH, while no related to the polymorphism of pLDH gene.

Evaluation of the Coca-Cola company travel health kit.

PubMed

Harper, Lynne A; Bettinger, Julie; Dismukes, Roberta; Kozarsky, Phyllis E

2002-01-01

The Coca-Cola travel health kit has been used for about one decade for international travelers and required evaluation to see if the items contained were appropriate for the employees. Two hundred thirty-four travelers were sampled and filled out a voluntary survey including questions about demographic information, travel history, and usage and value of the contents of the travel health kit. One hundred eighty-one surveys were returned; 65% of the respondents were male, and the majority of travelers were between the ages of 36 and 45 years. The most useful items were analgesics and medications used for gastrointestinal problems. In general, the items identified as being the least useful were those requiring specialized use by a medical practitioner, such as needles and syringes. Suggestions of items to be added to the kit included vitamins, cough drops, sleep aids, and eye drops. A surprising result that Coca-Cola employees expressed the desire for brand name rather than generic items. Evaluation of the Coca-Cola Company travel health kit revealed it to be very useful to most corporate travelers. Suggestions that were made will be taken into consideration in designing a new kit, and consideration is being given to whether a basic travel health kit should be provided to which travelers can add other items depending on their personal needs.

[Comparison between the LightCycler CMV Quant Kit (Roche Diagnostics) with a standardized in-house Taqman assay for cytomegalovirus blood viral load quantification].

PubMed

Alain, S; Lachaise, V; Hantz, S; Denis, F

2010-04-01

The broad use of cytomegalovirus (CMV) viral load quantification in blood to follow immunosuppressed patients need standardized assays. Choice of whole blood allows follow-up for several viruses and simplifies pretreatment and storage of samples. We therefore evaluated the LightCycler CMV Quant Kit (Roche Diagnostics) assay on whole blood after a manual extraction (High Pure viral nucleic acid kit, Roche Diagnostics), using as a reference an in-house Taqman assay (LC1UL83) which has been validated in various clinical situations. A panel obtained by serial dilutions of a virion stock in CMV whole blood, a commercial plasma quality control (VQC, Argène, France) crude or diluted in whole blood, infected cells extracts and 46 clinical samples from transplanted patients were tested simultaneously by both techniques. For plasma quality controls, both PCR assays are correlated VQC (R(2)=0.93). On whole blood or infected cells dilutions, correlation shows an overestimation by the LC1UL83 assay (mean 1.2 log copies/ml) over 3 log though R(2)=0.94. Results with CMV Quant Kit are closer to expected values. Results on clinical samples are close to quality controls with a lower variation of quantification (0.76 log copies/ml). CMV Quant Kit performs well when compared with a clinically validated PCR. Quality control results showed discrepancies between plasma and whole blood, demonstrating the need for whole blood standardized panels to compare the methods. This underlines the need to follow a patient with the same technique during his follow-up. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.

PubMed

Filisetti, Denis; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Pelloux, Hervé; Touafek, Fériel; Varlet-Marie, Emmanuelle; Yera, Hélène; Candolfi, Ermano; Bastien, Patrick

2015-01-01

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

PubMed Central

Filisetti, Denis; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Pelloux, Hervé; Touafek, Fériel; Varlet-Marie, Emmanuelle; Yera, Hélène; Candolfi, Ermano

2014-01-01

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents. PMID:25339393

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Clinical, Cytological, Histological and Immunohistochemical Features of Cutaneous Mast Cell Tumours in Ferrets (Mustela putorius furo).

PubMed

Vilalta, L; Meléndez-Lazo, A; Doria, G; Ramis, A; Solano-Gallego, L; Pastor, J; Martorell, J

2016-11-01

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of Listeria monocytogenes in ready-to-eat food by Step One real-time polymerase chain reaction.

PubMed

Pochop, Jaroslav; Kačániová, Miroslava; Hleba, Lukáš; Lopasovský, L'ubomír; Bobková, Alica; Zeleňáková, Lucia; Stričík, Michal

2012-01-01

The aim of this study was to follow contamination of ready-to-eat food with Listeria monocytogenes by using the Step One real time polymerase chain reaction (PCR). We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Listeria monocytogenes Detection Kit for the real-time PCR performance. In 30 samples of ready-to-eat milk and meat products without incubation we detected strains of Listeria monocytogenes in five samples (swabs). Internal positive control (IPC) was positive in all samples. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in ready-to-eat food without incubation.

Effect of blanching treatments on antioxidant activity of frozen green capsicum (Capsicum annuum L. var bell pepper) using radical scavenging activity (DPPH) assay

NASA Astrophysics Data System (ADS)

Azizzuddin, Norafida; Abdullah, Aminah

2016-11-01

Blanching treatments are needed to deactivate enzymes in frozen vegetables. Antioxidant activity using DPPH radical scavenging activity assay were evaluated in steaming, boiling water, and microwave blanching at different temperature, time and microwave power level on frozen green capsicum. Green capsicum was chosen for frozen treatment compared to other capsicum with different maturity index because of the firm texture. The objective of this study was to compare the antioxidant activity of frozen green capsicum between conventional and Oxi Count Kit® assay for DPPH radical scavenging activity. Results showed frozen green capsicum blanched using microwave at high level/90 seconds (sample J) contained higher level of DPPH in both conventional method and Oxi Count Kit® compared to other treatments. However, there were no significant differences between sample J and fresh sample (sample A). Overall, the sequences from highest to lowest in blanching treatments for both DPPH conventional method, and DPPH Oxi Count Kit® were J (microwave high level/90 seconds) > A (Fresh) > H (Microwave Medium Level/120 seconds) > D (Boiling Water 80°C/150 seconds) > K (Microwave High Level/120 seconds) > I (Microwave Medium Level/150 seconds) > F (Microwave Low Level/150 seconds)> B (Steam 100°C/150 seconds) > E (Boiling Water 100°C /120 seconds) > G (Microwave Low Level /180 seconds)> C (Steam 100°C/180 seconds). Almost all frozen green capsicum samples showed no significant differences for comparison between test using DPPH conventional method and Oxi Count Kit®. Frozen storage for 0, and 3rd months showed no significant differences which indicate no changes on antioxidant activity during frozen storage at -18°C.

Audit of lymphadenectomy in lung cancer resections using a specimen collection kit and checklist.

PubMed

Osarogiagbon, Raymond U; Sareen, Srishti; Eke, Ransome; Yu, Xinhua; McHugh, Laura M; Kernstine, Kemp H; Putnam, Joe B; Robbins, Edward T

2015-02-01

Audits of operative summaries and pathology reports reveal wide discordance in identifying the extent of lymphadenectomy performed (the communication gap). We tested the ability of a prelabeled lymph node specimen collection kit and checklist to narrow the communication gap between operating surgeons, pathologists, and auditors of surgeons' operation notes. We conducted a prospective single cohort study of lung cancer resections performed with a lymph node collection kit from November 2010 to January 2013. We used the kappa statistic to compare surgeon claims on a checklist of lymph node stations harvested intraoperatively with pathology reports and an independent audit of surgeons' operative summaries. Lymph node collection procedures were classified into four groups based on the anatomic origin of resected lymph nodes: mediastinal lymph node dissection, systematic sampling, random sampling, and no sampling. From the pathology reports, 73% of 160 resections had a mediastinal lymph node dissection or systematic sampling procedure, 27% had random sampling. The concordance with surgeon claims was 80% (kappa statistic 0.69, 95% confidence interval: 0.60 to 0.79). Concordance between independent audits of the operation notes and either the pathology report (kappa 0.14, 95% confidence interval: 0.04 to 0.23) or surgeon claims (kappa 0.09, 95% confidence interval: 0.03 to 0.22) was poor. A prelabeled specimen collection kit and checklist significantly narrowed the communication gap between surgeons and pathologists in identifying the extent of lymphadenectomy. Audit of surgeons' operation notes did not accurately reflect the procedure performed, bringing its value for quality improvement work into question. Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.

PubMed

Balbi, Carolina; Piccoli, Martina; Barile, Lucio; Papait, Andrea; Armirotti, Andrea; Principi, Elisa; Reverberi, Daniele; Pascucci, Luisa; Becherini, Pamela; Varesio, Luigi; Mogni, Massimo; Coviello, Domenico; Bandiera, Tiziano; Pozzobon, Michela; Cancedda, Ranieri; Bollini, Sveva

2017-05-01

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT + hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O 2 versus 1% O 2 preconditioning). The capacity of the c-KIT + hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn F7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT + hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Wilms tumor gene 1 (WT1), TP53, RAS/BRAF and KIT aberrations in testicular germ cell tumors.

PubMed

Boublikova, L; Bakardjieva-Mihaylova, V; Skvarova Kramarzova, K; Kuzilkova, D; Dobiasova, A; Fiser, K; Stuchly, J; Kotrova, M; Buchler, T; Dusek, P; Grega, M; Rosova, B; Vernerova, Z; Klezl, P; Pesl, M; Zachoval, R; Krolupper, M; Kubecova, M; Stahalova, V; Abrahamova, J; Babjuk, M; Kodet, R; Trka, J

2016-07-01

Wilms tumor gene 1 (WT1), a zinc-finger transcription factor essential for testis development and function, along with other genes, was investigated for their role in the pathogenesis of testicular germ cell tumors (TGCT). In total, 284 TGCT and 100 control samples were investigated, including qPCR for WT1 expression and BRAF mutation, p53 immunohistochemistry detection, and massively parallel amplicon sequencing. WT1 was significantly (p < 0.0001) under-expressed in TGCT, with an increased ratio of exon 5-lacking isoforms, reaching low levels in chemo-naïve relapsed TGCT patients vs. high levels in chemotherapy-pretreated relapsed patients. BRAF V600E mutation was identified in 1% of patients only. p53 protein was lowly expressed in TGCT metastases compared to the matched primary tumors. Of 9 selected TGCT-linked genes, RAS/BRAF and WT1 mutations were frequent while significant TP53 and KIT variants were not detected (p = 0.0003). WT1 has been identified as a novel factor involved in TGCT pathogenesis, with a potential prognostic impact. Distinct biologic nature of the two types of relapses occurring in TGCT has been demonstrated. Differential mutation rate of the key TGCT-related genes has been documented. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Internet-based recruitment system for HIV and STI screening for men who have sex with men in Estonia, 2013: analysis of preliminary outcomes.

PubMed

Ruutel, K; Lohmus, L; Janes, J

2015-04-16

The aim of the current project was to develop an Internet-based recruitment system for HIV and sexually transmitted infection (STI) screening for men who have sex with men (MSM) in Estonia in order to collect biological samples during behavioural studies. In 2013, an Internet-based HIV risk-behaviour survey was conducted among MSM living in Estonia. After completing the questionnaire, all participants were offered anonymous and free-of-charge STI testing. They could either order a urine sample kit by post to screen for chlamydia infections (including lymphogranuloma venereum (LGV)), trichomoniasis, gonorrhoea and Mycoplasma genitalium infections, or visit a laboratory for HIV, hepatitis A virus, hepatitis B virus,hepatitis C virus and syphilis screening. Of 301 participants who completed the questionnaire, 265 (88%),reported that they were MSM. Of these 265 MSM,68 (26%) underwent various types of testing. In the multiple regression analysis, Russian as the first language,previous HIV testing and living in a city or town increased the odds of testing during the study. Linking Internet-based behavioural data collection with biological sample collection is a promising approach. As there are no specific STI services for MSM in Estonia,this system could also be used as an additional option for anonymous and free-of-charge STI screening.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

PubMed Central

Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

2015-01-01

Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

PubMed

Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G

2015-05-01

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

A method suitable for DNA extraction from humus-rich soil.

PubMed

Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

2014-11-01

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

C3-PRO: Connecting ResearchKit to the Health System Using i2b2 and FHIR.

PubMed

Pfiffner, Pascal B; Pinyol, Isaac; Natter, Marc D; Mandl, Kenneth D

2016-01-01

A renewed interest by consumer information technology giants in the healthcare domain is focused on transforming smartphones into personal health data storage devices. With the introduction of the open source ResearchKit, Apple provides a framework for researchers to inform and consent research subjects, and to readily collect personal health data and patient reported outcomes (PRO) from distributed populations. However, being research backend agnostic, ResearchKit does not provide data transmission facilities, leaving research apps disconnected from the health system. Personal health data and PROs are of the most value when presented in context along with health system data. Our aim was to build a toolchain that allows easy and secure integration of personal health and PRO data into an open source platform widely adopted across 140 academic medical centers. We present C3-PRO: the Consent, Contact, and Community framework for Patient Reported Outcomes. This open source toolchain connects, in a standards-compliant fashion, any ResearchKit app to the widely-used clinical research infrastructure Informatics for Integrating Biology and the Bedside (i2b2). C3-PRO leverages the emerging health data standard Fast Healthcare Interoperability Resources (FHIR).

C3-PRO: Connecting ResearchKit to the Health System Using i2b2 and FHIR

PubMed Central

Pfiffner, Pascal B.; Pinyol, Isaac; Natter, Marc D.; Mandl, Kenneth D.

2016-01-01

A renewed interest by consumer information technology giants in the healthcare domain is focused on transforming smartphones into personal health data storage devices. With the introduction of the open source ResearchKit, Apple provides a framework for researchers to inform and consent research subjects, and to readily collect personal health data and patient reported outcomes (PRO) from distributed populations. However, being research backend agnostic, ResearchKit does not provide data transmission facilities, leaving research apps disconnected from the health system. Personal health data and PROs are of the most value when presented in context along with health system data. Our aim was to build a toolchain that allows easy and secure integration of personal health and PRO data into an open source platform widely adopted across 140 academic medical centers. We present C3-PRO: the Consent, Contact, and Community framework for Patient Reported Outcomes. This open source toolchain connects, in a standards-compliant fashion, any ResearchKit app to the widely-used clinical research infrastructure Informatics for Integrating Biology and the Bedside (i2b2). C3-PRO leverages the emerging health data standard Fast Healthcare Interoperability Resources (FHIR). PMID:27031856

Huntingtin Interacting Protein 1: a Merkel Cell Carcinoma Marker That Interacts with c-Kit

PubMed Central

Ames, Heather M.; Bichakjian, Christopher K.; Liu, Grace Y.; Oravecz-Wilson, Katherine I.; Fullen, Douglas R.; Verhaegen, Monique; Johnson, Timothy M.; Dlugosz, Andrzej A.; Ross, Theodora S.

2011-01-01

Merkel Cell Carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. While the prevalence of MCC has been increasing, treatments for this disease remain limited due to a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin interacting protein 1 (HIP1) is expressed at high levels in close to 90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20 (CK20). Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working towards an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit. PMID:21697888

Standardisation of a novel sperm banking kit - NextGen(®) - to preserve sperm parameters during shipment.

PubMed

Agarwal, A; Sharma, R; Singh, A; Gupta, S; Sharma, R

2016-08-01

Many male patients diagnosed with cancer are within their reproductive years. These men are advised to freeze their spermatozoa prior to the start of cancer treatment. Very often, sperm banking facilities may not be readily available and patients may be required to travel to distant sperm bank centres. Our objective was to design and standardise a remote home shipping sperm kit that allows patients to collect a semen sample at home and ship it overnight to a sperm bank. A total of 21 semen samples and two transport media (refrigeration media and human tubal fluid) and five different combinations of ice packs were tested for maintaining desired shipping temperature. Ten semen samples were assessed for pre- and post-shipment changes in sperm motility, membrane integrity, total motile spermatozoa and recovery of motile spermatozoa. Even though motility, membrane integrity and total motile spermatozoa declined both in samples examined under simulated shipped conditions and in overnight-shipped samples, the observed motility and total motile spermatozoa were adequate for use with assisted reproductive techniques. Using refrigeration media, cooling sleeve and ice packs, adequate sperm motility can be maintained utilising NextGen(®) kit and these spermatozoa can be used for procreation utilising ART techniques such as intracytoplasmic sperm injection. © 2015 Blackwell Verlag GmbH.

A Lateral Flow Rapid Test for Human Toxocariasis Developed Using Three Toxocara canis Recombinant Antigens.

PubMed

Yunus, Muhammad Hafiznur; Tan Farrizam, Siti Naqiuyah; Abdul Karim, Izzati Zahidah; Noordin, Rahmah

2018-01-01

Laboratory diagnosis of toxocariasis is still a challenge especially in developing endemic countries with polyparasitism. In this study, three Toxocara canis recombinant antigens, rTES-26, rTES-30, and rTES-120, were expressed and used to prepare lateral flow immunoglobulin G4 (IgG4) dipsticks. The concordance of the results of the rapid test (comprising three dipsticks) with a commercial IgG-enzyme-linked immunosorbent assay (ELISA) (Cypress Diagnostics, Belgium) was compared against the concordance of two other commercial IgG-ELISA kits (Bordier, Switzerland and NovaTec, Germany) with the Cypress kit. Using Toxocara- positive samples, the concordance of the dipstick dotted with rTES-26, rTES-30, and rTES-120 was 41.4% (12/29), 51.7% (15/29), and 72.4% (21/29), respectively. When positivity with any dipstick was considered as an overall positive rapid test result, the concordance with the Cypress kit was 93% (27/29). Meanwhile, when compared with the results of the Cypress kit, the concordance of IgG-ELISA from NovaTec and Bordier was 100% (29/29) and 89.7% (26/29), respectively. Specific IgG4 has been recognized as a marker of active infection for several helminthic diseases; therefore, the two non-concordant results of the rapid test when compared with the NovaTec IgG-ELISA kit may be from samples of people with non-active infection. All the three dipsticks showed 100% (50/50) concordance with the Cypress kit when tested with serum from individuals who were healthy and with other infections. In conclusion, the lateral flow rapid test is potentially a good, fast, and easy test for toxocariasis. Next, further validation studies and development of a test with the three antigens in one dipstick will be performed.

Sitophilus granarius L. (Coleoptera) Toxicity and Biological Activities of the Essential Oils of Tanacetum macrophyllum (Waldst. & Kit.) Schultz Bip.

PubMed

Polatoğlu, Kaan; Karakoç, Ömer Cem; Demirci, Betül; Gören, Nezhun; Can Başer, Kemal Hüsnü

2015-01-01

Insecticides of the natural origin are an important alternative to the synthetic insecticides that are being employed for the preserving stored products. The volatiles obtained from T. cinerariifolium (=Pyrethrum cinerariifolium) is being used for many types of insecticidal applications; however there is a very little information on the insecticidal activity of the essential oils of other Tanacetum species. The main purpose of the present study is to determine the chemical composition of T. macrophyllum (Waldst. & Kit.) Schultz Bip. essential oils and evaluate their insecticidal activity against S. granarius as well as its other beneficial biological activities. Highest contact toxicity was observed in the leaf oil of (88.93%) against S. granarius. The flower oil showed considerable fumigant toxicity against L. minor at 10 mg/mL application concentration (61.86 %) when compared with other samples at the same concentration. The highest DPPH (2,2-Diphenyl-1-picrylhydrazyl) scavenging activity (47.7%) and phosphomolybdenum reducing activity was observed also for the flower oil of T. macrophyllum at 10 mg/mL concentration. The essential oils were analyzed by GC, GC/MS. The flower and leaf oils were characterized with γ-eudesmol 21.5%, (E)-sesquilavandulol 20.3%, copaborneol 8.5% and copaborneol 14.1%, 1,8-cineole 11%, bornyl acetate 9.6%, borneol 6.3% respectively. AHC analysis of the qualitative and quantitative data obtained from the essential oil composition of the T. macrophyllum essential oil from the present research and previous reports pointed out that two different chemotypes could be proposed with current findings which are p-methyl benzyl alcohol/ cadinene and eudesmane chemotypes.

Diagnosis of Acute Q Fever by Detection of Coxiella burnetii DNA using Real-Time PCR, Employing a Commercial Genesig Easy Kit

PubMed Central

Pradeep, Jothimani; Ambroise, Stanley; Gunasekaran, Dhandapany

2017-01-01

Introduction Query (Q) fever is an important zoonosis and a cause of concern for humans, due to the potential bioterrorism threat posed by the causative agent, Coxiella burnetii. Because of the danger of contracting the illness, isolation attempts are seldom made. Serological and molecular diagnostic tests are the main option. Aim To study the prevalence of acute Q fever in Puducherry and surrounding districts of Tamil Nadu, India, employing a new commercial Real-Time Polymerase Chain Reaction (RT-PCR) kit and confirming it by the gold standard Immunofluorescence Assay (IFA). Materials and Methods Acute phase blood samples from 72 consecutive febrile patients and 24 healthy individuals were included in this prospective study. DNA was extracted from the buffy coats and preserved at -80°C. Detection of C. burnetii was carried out employing a commercial Real-Time PCR kit. Serum samples were tested for IgM (Phase I+II) and IgG (Phase I+II) by QM-120 and QG-120, Coxiella burnetii IFA Fuller Laboratories, California, USA. Sensitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were calculated keeping IFA as the reference. Results Presumptive diagnosis of acute Q fever was made in two febrile patients by the Genesig Easy kit (2.78%). In addition to these two PCR positive cases, one more patient was positive for both Phase II IgM and Phase II IgG antibodies by the gold standard IFA. All 24 healthy controls were negative for Q fever by both PCR and IFA. The sensitivity, specificity, NPV and PPV for Genesig Easy kit PCR were: 66.67%, 100%, 100% and 98.57 % respectively against IFA as the reference. Conclusion The true prevalence of Q fever in India and other developing countries is poorly understood, owing to the difficulties in the diagnosis of this infection. Since molecular diagnostic tests have good specificity and are mandated for confirmation of single acute samples, validation of commercial Q fever PCR kits is the need of the hour. Genesig Easy kit in our hands was found to be reliable with the moderate sensitivity and high specificity. Performing both PCR (with acute specimens) and IFA (with paired sera) would be ideal for Q fever diagnosis. PMID:29207703

Highly efficient nuclear DNA typing of the World War II skeletal remains using three new autosomal short tandem repeat amplification kits with the extended European Standard Set of loci

PubMed Central

Zupanič Pajnič, Irena; Gornjak Pogorelc, Barbara; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut

2012-01-01

Aim To perform an efficiency study of three new amplification kits with the extended European Standard Set (ESS) of loci for autosomal short tandem repeat (STR) typing of skeletal remains excavated from the World War II mass graves in Slovenia. Methods In the beginning of the 2011, we analyzed 102 bones and teeth using the PowerPlex ESX 17 System (Promega), AmpFiSTR NGM PCR Amplification Kit (Applied Biosystems), and Investigator ESSplex Kit (Qiagen). We cleaned the bones and teeth, removed surface contamination, and ground them into a powder using liquid nitrogen. Prior to DNA isolation with Biorobot EZ1 (Qiagen), 0.5 g bone or tooth powder was decalcified. Nuclear DNA of the samples was quantified using real-time polymerase chain reaction. All three kits used the same extract with the amplification conditions recommended by the manufacturers. Results We extracted up to 131 ng DNA/g of powder from the bones and teeth. All three amplification kits showed very similar efficiency, since DNA typing was successful with all amplification kits in 101 out of 102 bones and teeth, which represents a 99% success rate. Conclusion The commercially available ESX 17, ESSplex, and NGM kits are highly reliable for STR typing of World War II skeletal remains with the DNA extraction method optimized in our laboratory. PMID:22351574

Imatinib mesylate inhibits Leydig cell tumor growth: evidence for in vitro and in vivo activity.

PubMed

Basciani, Sabrina; Brama, Marina; Mariani, Stefania; De Luca, Gabriele; Arizzi, Mario; Vesci, Loredana; Pisano, Claudio; Dolci, Susanna; Spera, Giovanni; Gnessi, Lucio

2005-03-01

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

The National Problem of Untested Sexual Assault Kits (SAKs): Scope, Causes, and Future Directions for Research, Policy, and Practice.

PubMed

Campbell, Rebecca; Feeney, Hannah; Fehler-Cabral, Giannina; Shaw, Jessica; Horsford, Sheena

2015-12-23

Victims of sexual assault are often advised to have a medical forensic exam and sexual assault kit (SAK; also termed a "rape kit") to preserve physical evidence (e.g., semen, blood, and/or saliva samples) to aid in the investigation and prosecution of the crime. Law enforcement are tasked with submitting the rape kit to a forensic laboratory for DNA (deoxyribonucleic acid) analysis, which can be instrumental in identifying offenders in previously unsolved crimes, confirming identify in known-offender assaults, discovering serial rapists, and exonerating individuals wrongly accused. However, a growing number of media stories, investigative advocacy projects, and social science studies indicate that police are not routinely submitting SAKs for forensic testing, and instead rape kits are placed in evidence storage, sometimes for decades. This review article examines the growing national problem of untested rape kits by summarizing current research on the number of untested SAKs in the United States and exploring the underlying reasons why police do not submit this evidence for DNA testing. Recommendations for future research that can guide policy and practice are discussed. © The Author(s) 2015.

Undergraduate virology exercises demonstrate conventional and real-time PCR using commercially available HIV primers and noninfectious target.

PubMed

Sulzinski, Michael A; Wasilewski, Melissa A; Farrell, James C; Glick, David L

2009-07-01

It is an extraordinary challenge to offer an undergraduate laboratory course in virology that teaches hands-on, relevant molecular biology techniques using nonpathogenic models of human virus detection. To our knowledge, there exists no inexpensive kits or reagent sets that are appropriate for demonstrating real-time PCR (RT-PCR) in an undergraduate laboratory course in virology. Here we describe simple procedures for student exercises that demonstrate the PCR detection of an HIV target nucleic acid. Our procedures combine a commercially available kit for conventional PCR with a modification for RT-PCR using the same reagents in the kit, making it possible for an instructor with access to a LightCycler® instrument to implement a relevant student exercise on RT-PCR detection of HIV nucleic acid targets. This combination of techniques is useful for demonstrating and comparing conventional PCR amplification and detection with agarose gel electrophoresis, with real-time PCR over a series of three laboratory periods. The series of laboratory periods also is used to provide the foundation for teaching the concept of PCR primer design, optimization of PCR detection systems, and introduction to nucleic acid queries using NCBI-BLAST to find and identify primers, amplicons, and other potential amplification targets within the HIV viral genome. The techniques were successfully implemented at the Biology 364 undergraduate virology course at the University of Scranton during the Fall 2008 semester. The techniques are particularly targeted to students who intend to pursue either postgraduate technical employment or graduate studies in the molecular life sciences. Copyright © 2009 International Union of Biochemistry and Molecular Biology, Inc.

Immunochemistry for high-throughput screening of human exhaled breath condensate (EBC) media: implementation of automated Quanterix SIMOA instrumentation.

PubMed

Pleil, Joachim D; Angrish, Michelle M; Madden, Michael C

2015-12-11

Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-α, IL-1β and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r(2)  >  0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg ml(-1). All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ~45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml(-1)). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels.

Evaluation of a monoclonal antibody-based latex agglutination test for diagnosis of cryptococcosis: comparison with two tests using polyclonal antibodies.

PubMed Central

Temstet, A; Roux, P; Poirot, J L; Ronin, O; Dromer, F

1992-01-01

Cryptococcal antigen detection has become a routine biological test performed for patients with AIDS. The poor prognosis of cryptococcosis explains the need for reliable tests. We evaluated the performances of a newly commercialized agglutination test that uses a monoclonal antibody specific for cryptococcal capsular polysaccharide (Pastorex Cryptococcus; Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) and compared them with those of tests that use polyclonal immune sera (Cryptococcal Antigen Latex Agglutination System, Meridian Diagnostics, Inc., Cincinnati, Ohio; and Crypto-LA, International Biological Labs Inc., Cranbury, N.J.). The sensitivities and specificities of the tests were compared by using purified polysaccharides and yeast suspensions. Clinical specimens (131 serum samples, 41 cerebrospinal fluid samples, 34 urine samples, and 19 bronchoalveolar lavage samples) from 87 human immunodeficiency virus-positive subjects with (40 patients) and without (47 patients) culture-proven cryptococcosis were retrospectively tested during a blinded study. The effect of pronase treatment of samples was assessed for Pastorex Cryptococcus and the Cryptococcal Antigen Latex Agglutination System, and the antigen titers were compared. Our results show that (i) during the screening, concordance among the three tests was 97%; (ii) the use of pronase enhanced both the sensitivities and specificities of the Pastorex Cryptococcus test; (iii) titers agreed for 67% of the cerebrospinal fluid samples and 60% of the serum samples; and (iv) cryptococcosis was detected equally well with Pastorex Cryptococcus and with the other tests, whatever the infecting serotype (A, B, or D). The meaning of in vitro sensitivity and the relationship between titers and sensitivity are discussed. The results show that Pastorex Cryptococcus is a rapid and reliable test for the detection of cryptococcal antigen in body fluids and suggest that kits cannot be used interchangeably to monitor antigen titers in patients. PMID:1400951

Analysis of DNA from post-blast pipe bomb fragments for identification and determination of ancestry.

PubMed

Tasker, Esiri; LaRue, Bobby; Beherec, Charity; Gangitano, David; Hughes-Stamm, Sheree

2017-05-01

Improvised explosive devices (IEDs) such as pipe bombs are weapons used to detrimentally affect people and communities. A readily accessible brand of exploding targets called Tannerite® has been identified as a potential material for abuse as an explosive in pipe bombs. The ability to recover and genotype DNA from such weapons may be vital in the effort to identify suspects associated with these devices. While it is possible to recover DNA from post-blast fragments using short tandem repeat markers (STRs), genotyping success can be negatively affected by low quantities of DNA, degradation, and/or PCR inhibitors. Alternative markers such as insertion/null (INNULs) and single nucleotide polymorphisms (SNPs) are bi-allelic genetic markers that are shorter genomic targets than STRs for amplification, which are more likely to resist degradation. In this study, we constructed pipe bombs that were spiked with known amounts of biological material to: 1) recover "touch" DNA from the surface of the device, and 2) recover traces of blood from the ends of wires (simulated finger prick). The bombs were detonated with the binary explosive Tannerite® using double-base smokeless powder to initiate the reaction. DNA extracted from the post-blast fragments was quantified with the Quantifiler® Trio DNA Quantification Kit. STR analysis was conducted using the GlobalFiler® Amplification Kit, INNULs were amplified using an early-access version of the InnoTyper™ 21 Kit, and SNP analysis via massively parallel sequencing (MPS) was performed using the HID-Ion Ampliseq™ Identity and Ancestry panels using the Ion Chef and Ion PGM sequencing system. The results of this study showed that INNUL markers resulted in the most complete genetic profiles when compared to STR and SNP profiles. The random match probabilities calculated for samples using INNULs were lower than with STRs when less than 14 STR alleles were reported. These results suggest that INNUL analysis may be well suited for low-template and/or degraded DNA samples, and may be used to supplement incomplete or failed STR analysis. Human identification using SNP analysis via MPS showed variable success with low-level post-blast samples in this study (<150pg). While neat DNA samples (6μL input as recommended) resulted in <50% of SNP calls, samples that were concentrated from 15μL to 6μL (15μL was added for STR and INNUL typing) resulted in more complete SNP profiles. Five out of six blood samples recovered from the wires attached to the pipe-bombs resulted in the correct ancestry predictions. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of genetically modified soybean in crude soybean oil.

PubMed

Nikolić, Zorica; Vasiljević, Ivana; Zdjelar, Gordana; Ðorđević, Vuk; Ignjatov, Maja; Jovičić, Dušica; Milošević, Dragana

2014-02-15

In order to detect presence and quantity of Roundup Ready (RR) soybean in crude oil extracted from soybean seed with a different percentage of GMO seed two extraction methods were used, CTAB and DNeasy Plant Mini Kit. The amplifications of lectin gene, used to check the presence of soybean DNA, were not achieved in all CTAB extracts of DNA, while commercial kit gave satisfactory results. Comparing actual and estimated GMO content between two extraction methods, root mean square deviation for kit is 0.208 and for CTAB is 2.127, clearly demonstrated superiority of kit over CTAB extraction. The results of quantification evidently showed that if the oil samples originate from soybean seed with varying percentage of RR, it is possible to monitor the GMO content at the first stage of processing crude oil. Copyright © 2013 Elsevier Ltd. All rights reserved.

Qualitative and quantitative assessment of Illumina's forensic STR and SNP kits on MiSeq FGx™.

PubMed

Sharma, Vishakha; Chow, Hoi Yan; Siegel, Donald; Wurmbach, Elisa

2017-01-01

Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina's MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina's FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

PubMed Central

2013-01-01

Background Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis. All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort. Results Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples. Conclusions gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies. PMID:24168095

miR-148b-3p functions as a tumor suppressor in GISTs by directly targeting KIT.

PubMed

Wang, Yu; Li, Jun; Kuang, Dong; Wang, Xiaoyan; Zhu, Yuanli; Xu, Sanpeng; Chen, Yaobing; Cheng, Henghui; Zhao, Qiu; Duan, Yaqi; Wang, Guoping

2018-04-16

Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis. miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117 IHC+ /KIT mutation GISTs and 19 CD117 IHC- /wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3. Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117 IHC+ /KIT mutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3'-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients. miR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs.

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

PubMed Central

Li, Linlin; Deng, Xutao; Mee, Edward T.; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D.; Delwart, Eric

2014-01-01

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces. PMID:25497414

Study protocol of the CHOiCE trial: a three-armed, randomized, controlled trial of home-based HPV self-sampling for non-participants in an organized cervical cancer screening program.

PubMed

Tranberg, Mette; Bech, Bodil Hammer; Blaakær, Jan; Jensen, Jørgen Skov; Svanholm, Hans; Andersen, Berit

2016-11-03

The effectiveness of cervical cancer screening programs is challenged by suboptimal participation and coverage. Offering cervico-vaginal self-sampling for human papillomavirus testing (HPV self-sampling) to non-participants can increase screening participation. However, the effect varies substantially among studies, especially depending on the approach used to offer HPV self-sampling. The present trial evaluates the effect on participation in an organized screening program of a HPV self-sampling kit mailed directly to the home of the woman or mailed to the woman's home on demand only, compared with the standard second reminder for regular screening. The CHOiCE trial is a parallel, randomized, controlled, open-label trial. It will include 9327 women aged 30-64 years who are living in the Central Denmark Region and who have not participated in cervical cancer screening after an invitation and one reminder. The women will be equally randomized into three arms: 1) Directly mailed a second reminder including a HPV self-sampling kit; 2) Mailed a second reminder offering a HPV self-sampling kit, to be ordered by e-mail, text message, phone, or through a webpage; and 3) Mailed a second reminder for a practitioner-collected sample (control group). The primary outcome will be the proportion of women in the intervention groups who participate by returning their HPV self-sampling kit or have a practitioner-collected sample compared with the proportion of women who have a practitioner-collected sample in the control group at 90 and 180 days after mail out of the second reminders. Per-protocol and intention-to-treat analyses will be performed. The secondary outcome will be the proportion of women with a positive HPV self-collected sample who attend follow-up testing at 30, 60, or 90 days after mail out of the results. The CHOiCE trial will provide strong and important evidence allowing us to determine if and how HPV self-sampling can be used to increase participation in cervical cancer screening. This trial therefore has the potential to improve prevention and reduce the number of deaths caused by cervical cancer. Current Controlled Trials NCT02680262 . Registered 10 February 2016.

Monitoring of left ventricular ejection fraction with a miniature, nonimaging nuclear detector: accuracy and reliability over time with special reference to blood labeling.

PubMed

Lindhardt, T B; Hesse, B; Gadsbøll, N

1997-01-01

The purpose of this study was to determine the accuracy of determinations of left ventricular ejection fraction (LVEF) by a nonimaging miniature nuclear detector system (Cardioscint) and to evaluate the feasibility of long-term LVEF monitoring in patients admitted to the coronary care unit, with special reference to the blood-labeling technique. Cardioscint LVEF values were compared with measurements of LVEF by conventional gamma camera radionuclide ventriculography in 33 patients with a wide range of LVEF values. In 21 of the 33 patients, long-term monitoring was carried out for 1 to 4 hours (mean 186 minutes), with three different kits: one for in vivo and two for in vitro red blood cell labeling. The stability of the labeling was assessed by determination of the activity of blood samples taken during the first 24 hours after blood labeling. The agreement between Cardioscint LVEF and gamma camera LVEF was good with automatic background correction (r = 0.82; regression equation y = 1.04x + 3.88) but poor with manual background correction (r = 0.50; y = 0.88x - 0.55). The agreement was highest in patients without wall motion abnormalities. The long-term monitoring showed no difference between morning and afternoon Cardioscint LVEF values. Short-lasting fluctuations in LVEFs greater than 10 EF units were observed in the majority of the patients. After 24 hours, the mean reduction in the physical decay-corrected count rate of the blood samples was most pronounced for the two in vitro blood-labeling kits (57% +/- 9% and 41% +/- 3%) and less for the in vivo blood-labeling kit (32% +/- 26%). This "biologic decay" had a marked influence on the Cardioscint monitoring results, demanding frequent background correction. A fairly accurate estimate of LVEF can be obtained with the nonimaging Cardioscint system, and continuous bedside LVEF monitoring can proceed for hours with little inconvenience to the patients. Instability of the red blood cell labeling during long-term monitoring necessitates frequent background correction.

[Double-antigen sandwich ELISA for detecting Aspergillus fumigatus anti-Afmp1cr and Afmp2cr antibodies].

PubMed

Yang, Mei; Wang, Zhuoya; Hao, Wei; Wang, Yanfang; Huang, Li; Cai, Jianpiao; Jiang, Lingxiao; Che, Xiaoyan; Zhong, Xiaozhu; Yu, Nan

2014-05-01

To establish two double-antigen sandwich ELISA systems to detect anti-Afmp1cr and Afmp2cr antibodies of Aspergillus fumigatus. Recombinant Afmp1cr and Afmp2cr proteins of A.fumigatus expressed in Pichia pastoris were obtained. Double-antigen sandwich ELISA systems for detecting specific anti-Afmp1cr and anti-Afmp2cr antibodies were developed after chessboard titrating to determine the appropriate concentrations of the recombinant proteins and HRP-labeled proteins. The sensitivity of the assay was evaluated using serum samples of rabbits immunized with Afmp1cr and Afmp2cr. The specificity of the assay was evaluated by detecting serum samples from healthy donors and patients with other pathogenic fungal and baterial infections. The performance of the two ELISA kits was furthered evaluated using serum samples from patients with suspected Aspergillus infection. The established ELISA kits were capable of detecting anti-Afmp1cr and anti-Afmp2cr antibodies in immunized rabbit serum at the maximum dilutions of 800 and 3200, respectively. No cross-reactivity was observed in detecting serum from patients with other pathogenic fungal or bactetial infections. Both of the two kits yielded positive results in sera from two established Aspergillus-infected cases and a suspected case. Two antibody-capture ELISA kits were developed for the laboratory diagnosis of A.fumigatus infection and can be potentially useful in the clinical diagnosis of Aspergillosis infections.

Gastrointestinal stromal tumors: the histology report.

PubMed

Dei Tos, Angelo P; Laurino, Licia; Bearzi, Italo; Messerini, Luca; Farinati, Fabio

2011-03-01

Gastrointestinal stromal tumors (GISTs) represent a mesenchymal neoplasm occurring primarily in the gastrointestinal tract, and showing differentiation toward the interstitial cell of Cajal. Its incidence is approximately 15 case/100,000/year. Stomach and small bowel are the most frequently affected anatomic sites. GIST represents a morphological, immunophenotypical and molecular distinct entity, the recognition of which has profound therapeutic implications. In fact, they have shown an exquisite sensitivity to treatment with the tyrosine kinase inhibitor imatinib. Diagnosis relies upon morphology along with immunodetection of KIT and/or DOG1. When dealing with KIT negative cases, molecular analysis of KIT/PDGFRA genes may help in confirming diagnosis. Molecular evaluation of both genes are in any case recommended as mutational status provides key predictive information. Pathologists also play a key role in providing an estimation of the risk of biological aggressiveness, which is currently based on anatomic location of the tumor, size, and mitotic activity. Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd.. All rights reserved.

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401.

PubMed

Junge, Benjamin; Berghof-Jäger, Kornelia

2006-01-01

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.

Smartphone-based assessment of blood alteration severity

NASA Astrophysics Data System (ADS)

Li, Xianglin; Xue, Jiaxin; Li, Wei; Li, Ting

2018-02-01

Blood quality and safety management is a critical issue for cold chain transportation of blood or blood-based biological reagent. The conventional methods of blood alteration severity assessment mainly rely on kit test or blood-gas analysis required opening the blood package to get samples, which cause possible blood pollution and are complicate, timeconsuming, and expensive. Here we proposed to develop a portable, real-time, safety, easy-operated and low cost method aimed at assessing blood alteration severity. Color images of the blood in transparent blood bags were collected with a smartphone and the alteration severity of the blood was assessed by the smartphone app offered analysis of RGB color values of the blood. The algorithm is based on a large number sample of RGB values of blood at different alteration degree. The blood quality results evaluated by the smartphone are in accordance with the actual data. This study indicates the potential of smart phone in real time, convenient, and reliable blood quality assessment.

Solid phase sequencing of double-stranded nucleic acids

DOEpatents

Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

2002-01-01

This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

PubMed Central

Derouin, F.; Garin, Y. J.; Buffard, C.; Berthelot, F.; Petithory, J. C.

1994-01-01

A collaborative study conducted by the French National Agency for Quality Control in Parasitology (CNQP) and various manufacturers of ELISA kits, represented by the Association of Laboratory Reagent Manufacturers (SFRL) compared the toxoplasmosis IgG antibody titres obtained with different ELISA-IgG kits and determined the relationships between the titres obtained by these techniques and the titre defined in international units (IU). Fifty-one serum samples with toxoplasmosis antibody titres ranging from 0 to 900 IU were tested in two successive studies with 16 ELISA-IgG kits. For the negative sera, false-positive reactions were observed with one kit. For the positive sera, the titres observed in ELISA were generally higher than those expressed in IU. Above 250 IU, the very wide variability of the titres found with the different ELISA kits renders any comparative analysis impossible. For titres below 250 IU, the results are sufficiently homogeneous to permit the use of regression analysis to study how the results for each ELISA kit compare with the mean results for the other kits. The slope of the line of regression shows a tendency to over-titration or under-titration compared with the results of the other manufacturers; the ordinate at the origin reflects the positivity threshold of the reaction and can be used to assess the risk of a lack of sensitivity (high threshold) or of specificity (threshold too low). On the whole, the trends revealed for a given manufacturer are constant from one study to the other. Within this range of titres, regression analysis also reveals the general tendency of ELISA kits to overestimate the titres by comparison with immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8205645

Identification and measurement of beta-lactam antibiotic residues in milk: integration of screening kits with liquid chromatography.

PubMed

Harik-Khan, R; Moats, W A

1995-01-01

A procedure for identifying and quantitating violative beta-lactams in milk is described. This procedure integrates beta-lactam residue detection kits with the multiresidue automated liquid chromatographic (LC) cleanup method developed in our laboratory. Spiked milk was deproteinized, extracted, and subjected to reversed-phase LC using a gradient program that concentrated the beta-lactams. Amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillin G were, thus, separated into 5 fractions that were subsequently tested for activity by using 4 kits. beta-lactams in the positive fractions were quantitated by analytical LC methods developed in our laboratory. The LC cleanup method separated beta-lactam antibiotics from each other and from interferences in the matrix and also concentrated the antibiotics, thus increasing the sensitivity of the kits to the beta-lactam antibiotics. The procedure facilitated the task of identifying and measuring the beta-lactam antibiotics that may be present in milk samples.

A silent allele in the locus D5S818 contained within the PowerPlex®21 PCR Amplification Kit.

PubMed

Chen, Ling; Tai, Yunchun; Qiu, Pingming; Du, Weian; Liu, Chao

2015-11-01

Three paternity tests cases were found with a single locus mismatch at the locus D5S818 with PowerPlex®21 PCR Amplification Kit (Promega). Forward and reverse primers were redesigned to type the samples again and to evaluate if there were alleles dropped out. The results showed the existence of a silent allele 12 in all the three families, due to a point mutation that changed cytosine to adenine at 90 nucleotides upstream from the 5' end of the AGAT repeat sequences in all the six individuals. A single locus mismatch due to a silent allele may occur in any locus using any kit. Therefore, we recommend using multiple kits to confirm the results in paternity testing cases with mismatches, especially when there is a single locus mismatch with homozygote involved. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Reaching women who do not participate in the regular cervical cancer screening programme by offering self-sampling kits: a systematic review and meta-analysis of randomised trials.

PubMed

Verdoodt, F; Jentschke, M; Hillemanns, P; Racey, C S; Snijders, P J F; Arbyn, M

2015-11-01

Population coverage for cervical cancer screening is an important determinant explaining differences in the incidence of cervical cancer between countries. Offering devices for self-sampling has the potential to increase participation of hard-to-reach women. A systematic review and meta-analysis were performed to evaluate the participation after an invitation including a self-sampling device (self-sampling arm) versus an invitation to have a sample taken by a health professional (control arm), sent to under-screened women. Sixteen randomised studies were found eligible. In an intention-to-treat analysis, the pooled participation in the self-sampling arm was 23.6% (95% confidence interval (CI)=20.2-27.3%), when self-sampling kits were sent by mail to all women, versus 10.3% (95% CI=6.2-15.2%) in the control arm (participation difference: 12.6% [95% CI=9.3-15.9]). When women had to opt-in to receive the self-sampling device, as used in three studies, the pooled participation was not higher in the self-sampling compared to the control arm (participation difference: 0.2% [95% CI=-4.5-4.9%]). An increased participation was observed in the self-sampling arm compared to the control arm, if self-sampling kits were sent directly to women at their home address. However, the size of the effect varied substantially among studies. Since participation was similar in both arms when women had to opt-in, future studies are warranted to discern opt-in scenarios that are most acceptable to women. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preliminary characterization of digestive enzymes in freshwater mussels

USGS Publications Warehouse

Sauey, Blake W.; Amberg, Jon J.; Cooper, Scott T.; Grunwald, Sandra K.; Newton, Teresa J.; Haro, Roger J.

2015-01-01

Resource managers lack an effective chemical tool to control the invasive zebra mussel Dreissena polymorpha. Zebra mussels clog water intakes for hydroelectric companies, harm unionid mussel species, and are believed to be a reservoir of avian botulism. Little is known about the digestive physiology of zebra mussels and unionid mussels. The enzymatic profile of the digestive glands of zebra mussels and native threeridge (Amblema plicata) and plain pocketbook mussels (Lampsilis cardium) are characterized using a commercial enzyme kit, api ZYM, and validated the kit with reagent-grade enzymes. A linear correlation was shown for only one of nineteen enzymes, tested between the api ZYM kit and a specific enzyme kit. Thus, the api ZYM kit should only be used to make general comparisons of enzyme presence and to observe trends in enzyme activities. Enzymatic trends were seen in the unionid mussel species, but not in zebra mussels sampled 32 days apart from the same location. Enzymatic classes, based on substrate, showed different trends, with proteolytic and phospholytic enzymes having the most change in relative enzyme activity.

Analysis of direct-to-consumer marketed Chlamydia trachomatis diagnostic tests in Norway.

PubMed

Reinton, Nils; Hjelmevoll, Stig Ove; Håheim, Håkon; Garstad, Kjersti; Mørch-Reiersen, Lisa Therese; Moghaddam, Amir

2015-08-01

Background In 2014, and for the first time in Norway, a pharmacy chain started selling home sampling kits for Chlamydia trachomatis (C. trachomatis) detection. Direct-to-consumer diagnostic kits for C. trachomatis have been available in Norway from an Internet company since 2005. There has been little assessment of persons who purchase direct-to-consumer diagnostic tests for sexually transmissible infections (STIs) detection and if low-risk populations are being unnecessarily encouraged to buy these tests. The prevalence of C. trachomatis in customers who purchased home sampling kits from the pharmacy chain and from the commercial Internet Co. were compared to that of patients attending STI clinics and other free primary healthcare services. Prevalences of other STIs in pharmacy and Internet customers were also determined. The prevalence of C. trachomatis among pharmacy customers was 11%, almost identical to the prevalence among Internet customers (12%). In comparison, the prevalence among patients attending STI clinics in Oslo was 7.2%, which is similar to the prevalence among patients who have been tested through primary healthcare services. The prevalence of Mycoplasma genitalium was two-fold less than that of C. trachomatis in the STI and primary physician population, and significantly less in the Internet and the pharmacy population. Neisseria gonorrhoeae was not detected in urine samples from pharmacy customers or from Internet customers. Both pharmacy and Internet C. trachomatis home-sampling kits seem to be purchased by the right risk population. Marketing of direct-to-consumer N. gonorrhoeae tests and possibly M. genitalium tests cannot be justified in Norway. Direct-to-consumer diagnostic tests should be actively utilised as part of national programs in preventing the spread of C. trachomatis.

Modification of the BAX Salmonella test kit to include a hot start functionality (modification of AOAC Official Method 2003.09).

PubMed

Wallace, F Morgan; DiCosimo, Deana; Farnum, Andrew; Tice, George; Andaloro, Bridget; Davis, Eugene; Burns, Frank R

2011-01-01

In 2010, the BAX System PCR assay for Salmonella was modified to include a hot start functionality designed to keep the reaction enzyme inactive until PCR begins. To validate the assay's Official Methods of Analysis status to include this procedure modification, an evaluation was conducted on four food types that were simultaneously analyzed with the BAX System and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Identical performance between the BAX System method and the reference methods was observed. Additionally, lysates were analyzed using both the BAX System Classic and BAX System Q7 instruments with identical results using both platforms for all samples tested. Of the 100 samples analyzed, 34 samples were positive for both the BAX System and reference methods, and 66 samples were negative by both the BAX System and reference methods, demonstrating 100% correlation. No instrument platform variation was observed. Additional inclusivity and exclusivity testing using the modified test kit demonstrated the test kit to be 100% accurate in evaluation of test panels of 352 Salmonella strains and 46 non-Salmonella strains.

The Use of Laser Microdissection in Forensic Sexual Assault Casework: Pros and Cons Compared to Standard Methods.

PubMed

Costa, Sergio; Correia-de-Sá, Paulo; Porto, Maria J; Cainé, Laura

2017-07-01

Sexual assault samples are among the most frequently analyzed in a forensic laboratory. These account for almost half of all samples processed routinely, and a large portion of these cases remain unsolved. These samples often pose problems to traditional analytic methods of identification because they consist most frequently of cell mixtures from at least two contributors: the victim (usually female) and the perpetrator (usually male). In this study, we propose the use of current preliminary testing for sperm detection in order to determine the chances of success when faced with samples which can be good candidates to undergo analysis with the laser microdissection technology. Also, we used laser microdissection technology to capture fluorescently stained cells of interest differentiated by gender. Collected materials were then used for DNA genotyping with commercially available amplification kits such as Minifiler, Identifiler Plus, NGM, and Y-Filer. Both the methodology and the quality of the results were evaluated to assess the pros and cons of laser microdissection compared with standard methods. Overall, the combination of fluorescent staining combined with the Minifiler amplification kit provided the best results for autosomal markers, whereas the Y-Filer kit returned the expected results regardless of the used method. © 2017 American Academy of Forensic Sciences.

TECHNOLOGIES FORM MONITORING AND ...

EPA Pesticide Factsheets

A demonstration of technologies for determining the presence of dioxin and dioxin-like compounds in soil and sediment was conducted under EPA's Superfund Innovative Technology Evaluation Program in Saginaw, Michigan in April 2004. This report describes the evaluation of Wako Pure Chemical Industries's Dioxin ELISA Kit. The kit is an immunoassay technique that reports toxicity equivalents (TEQ) of dioxin/furans. The sample units are in pg/g 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (EQ). The technology results were compared to high resolution mass spectrometry (HRMS) TEQ results generated using EPA Method 1613B.The Wako results were biased both positively and negatively relative to HRMS results. The technologys estimated method detection limit was 83-201 pg/g 2,3,7,8-TCDD EQ, but this should be considered a rough estimate. Results from this demonstration suggest that the Wako kit could be an effective screening tool for determining sample results above and below 20 pg/g TEQ, and even more effective as a screen for samples above and below 50 pg/g TEQ, particularly considering the cost to analyze the 209 demonstration samples was significantly less than that of the reference laboratory ($150,294 vs. $213,580), and all samples were analyzed on-site in 9 days (in comparison to the reference laboratory which took 8 months). The objective of this program is to promote the acceptance and use of innovative field technologies by providing well-documented per

Thirsk with SSK in JPM

NASA Image and Video Library

2009-07-15

ISS020-E-020652 (15 July 2009) --- Canadian Space Agency astronaut Robert Thirsk, Expedition 20 flight engineer, uses the Surface Sample Kit (SSK) to collect microbiology samples from specific sampling locations in the Harmony node and other modules of the International Space Station.

Use of DNA sequencing to detect pathogenic, saprotrophic, and stain fungi in sapwood of declining red pine (Pinus resinosa) in the Upper Midwest

Treesearch

M.T. Banik; D.L. Lindner; J. Juzwik; J.A. Glaeser

2013-01-01

An inexpensive kit was developed to collect wood samples for molecular detection of pathogenic, saprotrophic and stain fungi in declining Pinus resinosa in the Upper Midwest. The kit contained materials for "clean" collection of sapwood drill shavings, which were then subjected to PCR of the rDNA ITS region with fungal-specific primers,...

Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples

PubMed Central

Dong, Chun-nan; Yang, Ya-dong; Li, Shu-jin; Yang, Ya-ran; Zhang, Xiao-jing; Fang, Xiang-dong; Yan, Jiang-wei; Cong, Bin

2016-01-01

In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and identified 2,462 single nucleotide variations (SNVs), 128 insertion-deletion polymorphisms (indels). After comparing the sequence reads with 44 STR loci commonly used in forensics, five STRs (TH01, TPOX, D18S51, DYS391, and D10S1248)were matched. We compared these “nucleosome protected STRs” (NPSTRs) with five other non-NPSTRs using mini-STR primer design, real-time PCR, and capillary gel electrophoresis on artificially degraded DNA. Moreover, genotyping performance of the five NPSTRs and five non-NPSTRs was also tested with real casework samples. All results show that loci located in nucleosomes are more likely to be successfully genotyped in degraded samples. In conclusion, after further strict validation, these markers could be incorporated into future forensic and paleontology identification kits, resulting in higher discriminatory power for certain degraded sample types. PMID:27189082

Oncogenic mutations in melanomas and benign melanocytic nevi of the female genital tract

PubMed Central

Tseng, Diane; Kim, Julie; Warrick, Andrea; Nelson, Dylan; Pukay, Marina; Beadling, Carol; Heinrich, Michael; Selim, Maria Angelica; Corless, Christopher L.; Nelson, Kelly

2015-01-01

Background The genetic heterogeneity of melanomas and melanocytic nevi of the female genital tract is poorly understood. Objective We aim to characterize the frequency of mutations of the following genes: BRAF, NRAS, KIT, GNA11, and GNAQ in female genital tract melanomas. We also characterize the frequency of BRAF mutations in female genital tract melanomas compared with melanocytic nevi. Methods Mutational screening was performed on the following female genital tract melanocytic neoplasms: 25 melanomas, 7 benign melanocytic nevi, and 4 atypical melanocytic nevi. Results Of the 25 female genital tract melanoma specimens queried, KIT mutations were detected in 4 (16.0%), NRAS mutations in 4 (16.0%), and BRAF mutations in 2 (8.0%) samples. Two of the tumors with KIT mutations harbored double mutations in the same exon. No GNAQ or GNA11 mutations were identified among 11 melanomas screened. BRAF V600E mutations were detected in 7 of 7 benign melanocytic genital nevi (100%) and 3 of 4 atypical genital nevi (75%). Limitations Our study is limited by the small sample size of this rare subset of melanomas. Conclusion KIT, NRAS, and BRAF mutations are found in a subset of female genital tract melanomas. Screening for oncogenic mutations is important for developing and applying clinical therapies for melanomas of the female genital tract. PMID:24842760

Fast and simple DNA extraction from saliva and sperm cells obtained from the skin or isolated from swabs.

PubMed

von Wurmb-Schwark, Nicole; Mályusz, Victoria; Fremdt, Heike; Koch, Christine; Simeoni, Eva; Schwark, Thorsten

2006-05-01

The forensic scientist often has to cope with problematic samples from the crime scene due to their minute size and thus the low amount of extractable DNA. The retrieval of DNA from swabs taken from the surface of the skin, for example, in cases of strangulation, can be especially difficult. We systematically investigated swabs taken from the skin (to obtain a genetic profile from the victim and also from a possible offender) and from sperm cell containing swabs using two extraction kits: the Invisorb forensic and the Invisorb spin swab kit (both Invitek, Germany). DNA quality and quantity were tested on ethidium bromide containing agarose gels and in a highly sensitive duplex-PCR, which amplifies fragments specific for mitochondrial and nuclear DNA. Absolute quantification was done using real time PCR. Samples, which were positive in the duplex-PCR, were also employed to genetic fingerprinting using the Powerplex ES and the AmpFlSTRIdentifiler(TM) kits. Our study shows that the easy-to-use Invisorb spin swab kit is very suitable for DNA isolation from swabs taken from the skin and also from sperm cells. Retrieval of cells from the skin with swabs moistened in extraction buffer, not in distilled water, led to a significant higher DNA yield.

Long-lived immunity to canine core vaccine antigens in UK dogs as assessed by an in-practice test kit.

PubMed

Killey, R; Mynors, C; Pearce, R; Nell, A; Prentis, A; Day, M J

2018-01-01

To determine the utility of an in-practice test kit to detect protective serum antibody against canine distemper virus, canine adenovirus and canine parvovirus type 2 in a sample of the UK dog population. Serum samples from 486 dogs, last vaccinated between less than 1 month and 124 months previously, were tested with the VacciCheck™ test kit for protective antibodies against distemper, adenovirus and parvovirus type 2. A high proportion of the dogs tested (93·6%) had protective antibody against all three of the core vaccine antigens: 95·7% of the dogs were seropositive against canine distemper virus, 97·3% against canine adenovirus and 98·5% against canine parvovirus type 2. The small number of dogs that were seronegative for one or more of the antigens (n = 31) may have had waning of previous serum antibody or may have been rare genetic non-responders to that specific antigen. UK veterinarians can be reassured that triennial revaccination of adult dogs with core vaccines provides long-lived protective immunity. In-practice serological test kits are a valuable tool for informing decision-making about canine core revaccination. © 2017 British Small Animal Veterinary Association.

Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in non-small-cell lung cancer.

PubMed

Lim, Sun Min; Chang, Hyun; Cha, Yoon Jin; Liang, Shile; Tai, Yan Chin; Li, Gu; Pestova, Ekaterina; Policht, Frank; Perez, Thomas; Soo, Ross A; Park, Won Young; Kim, Hye Ryun; Shim, Hyo Sup; Cho, Byoung Chul

2017-09-01

ALK and ROS1 gene rearrangements are distinct molecular subsets of non-small-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to develop an effective screening strategy to detect patients who will benefit from such treatment. In this study, we aimed to validate analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. Study population composed of three patient cohorts with histologically confirmed lung adenocarcinoma (patients with ALK rearrangement, patients with ROS1 rearrangement and patients with wild-type ALK and ROS1). Specimens consisted of 12 ALK-positive, 8 ROS1-positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement was previously assessed by Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbot Park, IL, USA) and ROS1 rearrangement was previously assessed by ZytoLight ® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). All specimens were re-evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit. FISH images were scanned on BioView AllegroPlus system and interpreted via BioView SoloWeb remotely. For a total of 41 patient samples, the concordance of the results by Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and compared to the known ALK and ROS1 rearrangement status of the specimen. Of the 12 ALK-positive cases, hybridization with Vysis ALK/ROS1 Dual Break Apart Probe Kit was successful in 10 cases (success rate 10/12, 83%) and of these 10 cases, all showed ALK rearrangement (100% concordance with the results of Vysis ALK Break Apart FISH Probe Kit). Two of the ALK+ cases were excluded due to weak ROS1 signals that could not be enumerated. Of the 8 ROS1-positive cases, 6 cases were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The success rate was 75% (6/8), and of these 6 cases, all showed ROS1 rearrangement, giving a 100% concordance with ZytoLight ® SPEC ROS1 Break Apart Probe. Two of the cases were excluded due to weak ROS1 gold signal or high background. In the cohort of 21 wild-type cases, the success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 85% (18/21) and the concordance with ALK and ROS1 probe kit was 100% (18/18). Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC. Copyright © 2017 Elsevier B.V. All rights reserved.

High levels of macrolide-resistant Mycoplasma genitalium in Queensland, Australia.

PubMed

Trembizki, Ella; Buckley, Cameron; Bletchly, Cheryl; Nimmo, Graeme R; Whiley, David M

2017-10-01

The macrolide azithromycin is recommended for treatment of Mycoplasma genitalium infection; however, M. genitalium strains possessing macrolide resistance-mediating mutations (MRMMs) are increasingly being reported. Here, we used the SpeeDx ResistancePlus MG kit, which provides simultaneous detection of M. genitalium and MRMMs, to assess MRMM carriage among M. genitalium infections in Queensland, Australia. Performance characteristics of the ResistancePlus MG kit for M. genitalium detection were compared to in-house PCR. Available M. genitalium PCR-positive (n=67) and negative (n=281) samples from the years 2011 to 2017 were tested using the SpeeDx ResistancePlus MG kit. In total, 63.6 % M. genitalium-positive samples were indicated to harbour MRMMs. The ResistancePlus MG method provided sensitivity and specificity of 97 and 99.6 % respectively compared to in-house PCR for M. genitalium detection. Such high levels of macrolide-resistant M. genitalium raise further concerns over future use of azithromycin for treatment of M. genitalium infection.

Assessment of DNA extracted from FTA® cards for use on the Illumina iSelect BeadChip

PubMed Central

McClure, Matthew C; McKay, Stephanie D; Schnabel, Robert D; Taylor, Jeremy F

2009-01-01

Background As FTA® cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes ≥ 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. Findings An ANOVA analysis indicated no significant difference (P > 0.72) in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. Conclusion We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform. PMID:19531223

Assessment of DNA extracted from FTA cards for use on the Illumina iSelect BeadChip.

PubMed

McClure, Matthew C; McKay, Stephanie D; Schnabel, Robert D; Taylor, Jeremy F

2009-06-16

As FTA cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes >or= 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. An ANOVA analysis indicated no significant difference (P > 0.72) in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform.

The multikinase inhibitor midostaurin (PKC412A) lacks activity in metastatic melanoma: a phase IIA clinical and biologic study.

PubMed

Millward, M J; House, C; Bowtell, D; Webster, L; Olver, I N; Gore, M; Copeman, M; Lynch, K; Yap, A; Wang, Y; Cohen, P S; Zalcberg, J

2006-10-09

Midostaurin (PKC412A), N-benzoyl-staurosporine, potently inhibits protein kinase C alpha (PKCalpha), VEGFR2, KIT, PDGFR and FLT3 tyrosine kinases. In mice, midostaurin slows growth and delays lung metastasis of melanoma cell lines. We aimed to test midostaurin's safety, efficacy and biologic activity in a Phase IIA clinical trial in patients with metastatic melanoma. Seventeen patients with advanced metastatic melanoma received midostaurin 75 mg p.o. t.i.d., unless toxicity or disease progression supervened. Patient safety was assessed weekly, and tumour response was assessed clinically or by CT. Tumour biopsies and plasma samples obtained at entry and after 4 weeks were analysed for midostaurin concentration, PKC activity and multidrug resistance. No tumour responses were seen. Two (12%) patients had stable disease for 50 and 85 days, with minor response in one. The median overall survival was 43 days. Seven (41%) discontinued treatment with potential toxicity, including nausea, vomiting, diarrhoea and/or fatigue. One patient had >50% reduction in PKC activity. Tumour biopsies showed two PKC isoforms relatively insensitive to midostaurin, out of three patients tested. No modulation of multidrug resistance was demonstrated. At this dose schedule, midostaurin did not show clinical or biologic activity against metastatic melanoma. This negative trial reinforces the importance of correlating biologic and clinical responses in early clinical trials of targeted therapies.

Detection of sex chromosome aneuploidies using quantitative fluorescent PCR in the Hungarian population.

PubMed

Nagy, Balint; Nagy, Richard Gyula; Lazar, Levente; Schonleber, Julianna; Papp, Csaba; Rigo, Janos

2015-05-20

Aneuploidies are the most frequent chromosomal abnormalities at birth. Autosomal aneuploidies cause serious malformations like trisomy 21, trisomy 18 and trisomy 13. However sex chromosome aneuploidies are causing less severe syndromes. For the detection of these aneuploidies, the "gold standard" method is the cytogenetic analysis of fetal cells, karyograms show all numerical and structural abnormalities, but it takes 2-4 weeks to get the reports. Molecular biological methods were developed to overcome the long culture time, thus, FISH and quantitative fluorescent PCR were introduced. In this work we show our experience with a commercial kit for the detection of sex chromosome aneuploidies. We analyzed 20.173 amniotic fluid samples for the period of 2006-2013 in our department. A conventional cytogenetic analysis was performed on the samples. We checked the reliability of quantitative fluorescent PCR and DNA fragment analysis on those samples where sex chromosomal aneuploidy was diagnosed. From the 20.173 amniotic fluid samples we found 50 samples with sex chromosome aneuploidy. There were 19 samples showing 46, XO, 17 samples with 46, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes. The applied quantitative fluorescent PCR and DNA fragment analyses method are suitable to detect all abnormal sex chromosome aneuploidies. Quantitative fluorescent PCR is a fast and reliable method for detection of sex chromosome aneuploidies. Copyright © 2015. Published by Elsevier B.V.

Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

PubMed

Frye, Chris W; Enders, Andrew; Brooks, Marjory B; Struble, Angela M; Wakshlag, Joseph J

2016-01-01

To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

Investigator® HDplex (Qiagen) reference population database for forensic use in Argentina.

PubMed

Martínez, Gustavo; Borosky, Alicia; Corach, Daniel; Llull, Cintia; Locarno, Laura; Lojo, Mercedes; Marino, Miguel; Miozzo, María Cecilia; Modesti, Nidia; Pacharoni, Carla; Pilili, Juan Pablo; Ramella, María Isabel; Sala, Andrea; Schaller, Cecilia; Vullo, Carlos; Toscanini, Ulises

2017-01-01

Currently, autosomal Short Tandem Repeat (STR) markers represent the method of election in forensic human identification. Commercial kits of most common use nowadays -e.g. PowerPlex ® Fusion, Promega Corp.; AmpFlSTR GlobalFiler, Thermofisher scientific; Investigator 24Plex QS,Qiagen-, allow the co-amplification of 23 highly polymorphic STR loci providing a high discrimination power in human identity testing. However, in complex kinship analysis and familial database searches involving distant relationships, additional DNA typing is often required in order to achieve well-founded conclusions. The recently developed kit Investigator ® HDplex (Qiagen) co-amplify twelve autosomal STRs markers (D7S1517, D3S1744, D12S391, D2S1360, D6S474, D4S2366, D8S1132, D5S2500, D18S51, D21S2055, D10S2325, SE33), nine of which are not present in the above mentioned kits, providing a set of efficient supplementary markers for human identification purposes. In this study we genotyped a sample of 980 individuals from urban areas of ten Argentinean provinces using the Investigator ® HDplex kit, aiming to provide forensic estimates for use in forensic casework and parentage testing in Argentina. We report reference allelic frequency databases for each of the provinces studied as well as for the combined samples. No deviation of Hardy-Weinberg equilibrium was observed. A reasonable discrimination capacity and power of exclusion was estimated which allowed predicting an acceptable forensic behavior of this kit, either to be used as the main STR panel for simple cases or as an auxiliary tool in complex cases. Additionally, population comparison tests showed that the studied samples are relatively homogeneous across the country for these STR set. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Investigating Associations Between Proliferation Indices, C-kit, and Lymph Node Stage in Canine Mast Cell Tumors.

PubMed

Krick, Erika Lauren; Kiupel, Matti; Durham, Amy C; Thaiwong, Tuddow; Brown, Dorothy C; Sorenmo, Karin U

Previous studies have evaluated cellular proliferation indices, KIT expression, and c-kit mutations to predict the clinical behavior of canine mast cell tumors (MCTs). The study purpose was to retrospectively compare mitotic index, argyrophilic nucleolar organizer regions (AgNORs)/nucleus, Ki-67 index, KIT labeling pattern, and internal tandem duplication mutations in c-KIT between stage I and stage II grade II MCTs. Medical records and tumor biopsy samples from dogs with Grade II MCTs with cytological or histopathological regional lymph node evaluation were included. Signalment, tumor location and stage, and presence of a recurrent versus de novo tumor were recorded. Mitotic index, AgNORs/nucleus, Ki-67, KIT staining pattern, and internal tandem duplication mutations in exon 11 of c-KIT were evaluated. Sixty-six tumors (51 stage I; 15 stage II) were included. Only AgNORs/nucleus and recurrent tumors were significantly associated with stage (odds ratio 2.8, 95% confidence interval [CI] 1.0-8.0, P = .049; odds ratio 8.8, 95% CI 1.1-69.5; P = .039). Receiver-operator characteristic analysis showed that the sensitivity and specificity of AgNORs/cell ≥ 1.87 were 93.3% and 27.4%, respectively, (area under the curve: 0.65) for predicting stage. Recurrent tumors and higher AgNORs/nucleus are associated with stage II grade II MCTs; however, an AgNOR cutoff value that reliably predicts lymph node metastasis was not determined.

Mkit: A cell migration assay based on microfluidic device and smartphone.

PubMed

Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

2018-01-15

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS 2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS 2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS 2 -based cell functional assay for testing cell migration (the M kit ). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the M kit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the M kit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the M kit . In addition to research applications, we demonstrated the effective use of the M kit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed M kit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Active and realistic passive marijuana exposure tested by three immunoassays and GC/MS in urine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mule, S.J.; Lomax, P.; Gross, S.J.

Human urine samples obtained before and after active and passive exposure to marijuana were analyzed by immune kits (Roche, Amersham, and Syva) and gas chromatography/mass spectrometry (GC/MS). Seven of eight subjects were positive for the entire five-day test period with one immune kit. The latter correlated with GC/MS in 98% of the samples. Passive inhalation experiments under conditions likely to reflect realistic exposure resulted consistently in less than 10 ng/mL of cannabinoids. The 10-100-ng/mL cannabinoid concentration range essential for detection of occasional and moderate marijuana users is thus unaffected by realistic passive inhalation.

In-Flight Water Quality Monitoring on the International Space Station (ISS): Measuring Biocide Concentrations with Colorimetric Solid Phase Extraction (CSPE)

NASA Technical Reports Server (NTRS)

Gazda, Daniel B.; Schultz, John R.; Siperko, Lorraine M.; Porter, Marc D.; Lipert, Robert J.; Flint, Stephanie M.; McCoy, J. Torin

2011-01-01

The colorimetric water quality monitoring kit (CWQMK) was delivered to the International Space Station (ISS) on STS-128/17A and was initially deployed in September 2009. The kit was flown as a station development test objective (SDTO) experiment to evaluate the acceptability of colorimetric solid phase extraction (CSPE) technology for routine water quality monitoring on the ISS. During the SDTO experiment, water samples from the U.S. water processor assembly (WPA), the U.S. potable water dispenser (PWD), and the Russian system for dispensing ground-supplied water (SVO-ZV) were collected and analyzed with the CWQMK. Samples from the U.S. segment of the ISS were analyzed for molecular iodine, which is the biocide added to water in the WPA. Samples from the SVOZV system were analyzed for ionic silver, the biocide used on the Russian segment of the ISS. In all, thirteen in-flight analysis sessions were completed as part of the SDTO experiment. This paper provides an overview of the experiment and reports the results obtained with the CWQMK. The forward plan for certifying the CWQMK as operational hardware and expanding the capabilities of the kit are also discussed.

Detection of human papillomavirus DNA in patients referred to a family practice colposcopy clinic.

PubMed

Holman, J R

1996-01-01

Human papillomavirus (HPV) is strongly implicated in the pathogenesis of cervical neoplasia. The ability of a commercially available kit (Virapap/Viratype) to detect evidence of HPV is compared with cervical cytology, colposcopy, and directed biopsies. During a period of 16 months, cervical samples from 241 consecutive new patients referred for a colposcopy examination were obtained for HPV-DNA hybridization typing according to the kit instructions. Samples were sent to a reference laboratory for testing. The results were compared with results of the colposcopy examination, cervical cytology, and directed cervical biopsy samples processed and evaluated by our hospital laboratory. HPV DNA was detected in 27 of 107 patients who had abnormal colposcopy findings for a sensitivity of 25 +/- 7.5 percent at the 90 percent confidence interval. One of 134 patients with normal findings was positive for a specificity of 99 +/- 5 percent at the 95 percent confidence interval. Based on a 75 percent probability of HPV in the population, the positive predictive value was 99 percent and the negative predictive value 30 percent. With the low negative predictive value and sensitivity, HPV-DNA testing by this commercial kit is not an adequate tool for screening HPV in this population.

Distribution and predominance of genotype 3 in hepatitis C virus carriers in the province of kahramanmaras, Turkey.

PubMed

Caliskan, Ahmet; Kirisci, Ozlem; Ozkaya, Esra; Ozden, Sevinc; Tumer, Seray; Caglar, Serkan; Guler, Selma Ates; Senol, Hande

2015-04-01

The hepatitis C virus (HCV) has six major genotypes and more than 100 subtypes, and the determination of the responsible genotype, collection of epidemiological data, tailoring antiviral therapy, and prediction of prognosis have an important place in disease management. The aim of the present study was to determine the distribution of HCV genotypes across geographic regions and compare these data with those obtained from other geographic locations. The HCV genotypes were identified in HCV RNA positive blood samples, obtained from different centers. The HCV genotype was determined using molecular methods [Real-Time Polymerase Chain Reaction (RT-PCR)] in 313 patients, who were found to be positive for HCV RNA. The presence of HCV RNA was investigated using the RT-PCR method in serum samples delivered to the Microbiology Laboratory at Kahramanmaras Necip Fazıl City Hospital, Kahramanmaras, Turkey, from the centers located in Kahramanmaras City center and peripheral districts of the province, between March 2010 and August 2014. The HCV genotype analysis was performed in HCV RNA positive samples, using RT-PCR reagents kit. Urine samples from the patients were tested for amphetamine with an Amphetamines II (AMPS2) kit, cocaine was tested with a Cocaine II (COC2) kit, opiates were tested with an Opiates II (OPI2) kit, and cannabinoids were tested with a Cannabinoids II (THC2) kit in Roche/Hitachi Cobas c501 device. The blood samples collected from 313 patients were included in the study. Of these patients, 212 (67.7%) were male and 101 (32.3%) were female. The mean age of the patients was 41.29 ± 20.32 years. In terms of HCV genotype distribution, 162 patients (51.7%) had genotype 1, 144 patients (46%) had genotype 3, four patients (1.3%) had genotype 2, and three patients (1%) had genotype 4. The results of urine drug tests were available in only 65 patients (20.2%). Of these, 61 (93.8%) patients had HCV genotype 3. In conclusion, the prevalence of HCV genotype 1 was 51.7%, which was lower than the rates reported in other studies in Turkey, while the prevalence of HCV genotype 3 was 46%, which was remarkably higher than the reported Turkish data. In addition, the prevalence rate for genotype 3 reported in the present study is the highest that has ever been reported in the literature.

Assessment of "grading" with Ki-67 and c-kit immunohistochemical expressions may be a helpful tool in management of patients with flat epithelial atypia (FEA) and columnar cell lesions (CCLs) on core breast biopsy.

PubMed

Tomasino, Rosa M; Morello, Vincenza; Gullo, Arianna; Pompei, Giancarlo; Agnese, Valentina; Russo, Antonio; Rinaldi, Gaetana

2009-11-01

It is essential to reach a better understanding of "flat epithelial atypia/columnar cell lesions" (FEA/CCLs) in breast core biopsies. Our aim was to explore their biological nature, in order to predict the likelihood of an upgrade to carcinoma. "Cytological grading" has been specially focused, in view of its possible utility in the choice of management. One hundred thirty of a total of 900 cases core needle (CN)/vacuum-assisted biopsies (VABs), with diagnoses of "hyperplasia" and "atypia" were retrospectively re-evaluated. Pathological findings of further excision biopsies (FEBs) performed in 40/75 patients with follow-up were compared with the previous diagnoses. In all cases, both Ki-67 and c-kit immunoreactivities were explored and compared with both normal breast tissues and subsequently documented cancers, with special reference to the hyperplastic FEA/CCLs, with "mild" atypia (FEA/CCHAm). Sixteen cases were re-diagnosed as "usual ductal hyperplasia" (UDH), 60 as "columnar cell hyperplasia" (CCH), and 54 as FEA/CCHA, 30 of which FEA/CCHAm and 24 FEA/CCHAh (with high atypia). Significantly, the Ki-67 index proved to be on the increase and c-kit expression on the decrease in FEA/CCHA lesions, mainly in the FEA/CCHAh group and in the subsequently observed cancers, compared with either benign tissues or the FEA/CCH cases. It was also significant that most of the carcinomas were found in FEBs within the FEA/CCHAh group. In this study cytological grading, together with Ki-67 and c-kit indices, proved to be helpful in FEA/CCLs evaluation. With regard to FEA/CCHAm lesions, an adequate surveillance appears to be a more appropriate management tool than FEB, as a result of their biological nature and behavior.

Prediction of autosomal STR typing success in ancient and Second World War bone samples.

PubMed

Zupanič Pajnič, Irena; Zupanc, Tomaž; Balažic, Jože; Geršak, Živa Miriam; Stojković, Oliver; Skadrić, Ivan; Črešnar, Matija

2017-03-01

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

PubMed

Muiños-Bühl, Anixa; González-Recio, Oscar; Muñoz, María; Óvilo, Cristina; García-Casco, Juan; Fernández, Ana I

2018-06-01

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N 2 , room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N 2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.

Field soil aggregate stability kit for soil quality and rangeland health evaluations

USGS Publications Warehouse

Herrick, J.E.; Whitford, W.G.; de Soyza, A. G.; Van Zee, J. W.; Havstad, K.M.; Seybold, C.A.; Walton, M.

2001-01-01

Soil aggregate stability is widely recognized as a key indicator of soil quality and rangeland health. However, few standard methods exist for quantifying soil stability in the field. A stability kit is described which can be inexpensively and easily assembled with minimal tools. It permits up to 18 samples to be evaluated in less than 10 min and eliminates the need for transportation, minimizing damage to soil structure. The kit consists of two 21??10.5??3.5 cm plastic boxes divided into eighteen 3.5??3.5 cm sections, eighteen 2.5-cm diameter sieves with 1.5-mm distance openings and a small spatula used for soil sampling. Soil samples are rated on a scale from one to six based on a combination of ocular observations of slaking during the first 5 min following immersion in distilled water, and the percent remaining on a 1.5-mm sieve after five dipping cycles at the end of the 5-min period. A laboratory comparison yielded a correlation between the stability class and percent aggregate stability based on oven dry weight remaining after treatment using a mechanical sieve. We have applied the method in a wide variety of agricultural and natural ecosystems throughout western North America, including northern Mexico, and have found that it is highly sensitive to differences in management and plant community composition. Although the field kit cannot replace the careful laboratory-based measurements of soil aggregate stability, it can clearly provide valuable information when these more intensive procedures are not possible.

Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

PubMed

Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David

2011-08-01

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

PubMed

Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

2017-08-01

HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; P<0.0001), with an average bias of -0.24 log 10 copies/ml. The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar ® HHV-6 PCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

Spacelab

NASA Image and Video Library

1995-07-01

While instruments on the pallets in the payload bay observed the universe, biological experiments were performed in the middeck of the Shuttle Orbiter Challenger. Studying life processes in a microgravity environment can shed new light on the functioning of biological systems on Earth. These investigations can also help us understand how living organisms react to prolonged weightlessness. One such experiment was the vitamin D metabolites and bone demineralization experiment. This investigation measured the vitamin d metabolite levels of crew members to gain information on the cause of bone demineralization and mineral imbalance that occur during prolonged spaceflight as well as on Earth. Research into the biochemical nature of vitamin D has shown that the D-metabolites play a major role in regulating the body's calcium and phosphorus levels. One major function of the most biologically active vitamin D metabolite is to regulate the amount of calcium absorbed from the diet and taken out of bones. This investigation had two phases. The first was the developmental phase, which included extensive testing before flight, and the second, or final phase, involved the postflight analysis of the crew's blood samples. This photograph shows a blood draw test kit and centrifuge used for the experiment aboard the Spacelab-2. Marshall Space Flight Center had management responsibilities of all Spacelab missions.

cl-dash: rapid configuration and deployment of Hadoop clusters for bioinformatics research in the cloud.

PubMed

Hodor, Paul; Chawla, Amandeep; Clark, Andrew; Neal, Lauren

2016-01-15

: One of the solutions proposed for addressing the challenge of the overwhelming abundance of genomic sequence and other biological data is the use of the Hadoop computing framework. Appropriate tools are needed to set up computational environments that facilitate research of novel bioinformatics methodology using Hadoop. Here, we present cl-dash, a complete starter kit for setting up such an environment. Configuring and deploying new Hadoop clusters can be done in minutes. Use of Amazon Web Services ensures no initial investment and minimal operation costs. Two sample bioinformatics applications help the researcher understand and learn the principles of implementing an algorithm using the MapReduce programming pattern. Source code is available at https://bitbucket.org/booz-allen-sci-comp-team/cl-dash.git. hodor_paul@bah.com. © The Author 2015. Published by Oxford University Press.

cl-dash: rapid configuration and deployment of Hadoop clusters for bioinformatics research in the cloud

PubMed Central

Hodor, Paul; Chawla, Amandeep; Clark, Andrew; Neal, Lauren

2016-01-01

Summary: One of the solutions proposed for addressing the challenge of the overwhelming abundance of genomic sequence and other biological data is the use of the Hadoop computing framework. Appropriate tools are needed to set up computational environments that facilitate research of novel bioinformatics methodology using Hadoop. Here, we present cl-dash, a complete starter kit for setting up such an environment. Configuring and deploying new Hadoop clusters can be done in minutes. Use of Amazon Web Services ensures no initial investment and minimal operation costs. Two sample bioinformatics applications help the researcher understand and learn the principles of implementing an algorithm using the MapReduce programming pattern. Availability and implementation: Source code is available at https://bitbucket.org/booz-allen-sci-comp-team/cl-dash.git. Contact: hodor_paul@bah.com PMID:26428290

[Individual Identification of Cartilage by Direct Amplification in Mass Disasters].

PubMed

Wang, C H; Xu, C; Li, X Q; Wu, Y; Du, Z

2017-06-01

To explore the effectiveness of direct amplification for the STR analysis of cartilage, and to accelerate the effectiveness of disaster victim identification. Eighty-eight cartilage samples were directly amplified by PowerPle® 21 kit, and the results of genotyping were compared with that obtained by the magnetic beads method. In 88 cartilage samples, the STR genotypes were successfully detected from 84 samples by direct amplification and magnetic beads method, and both the results of genotyping by two method were consistent. Direct amplification with PowerPlex® 21 kit can be used for STR genotyping of cartilages. This method is operated easily and promptly, which has a potential application in the individual identification of mass disasters. Copyright© by the Editorial Department of Journal of Forensic Medicine

Enhanced DNA Profiling of the Semen Donor in Late Reported Sexual Assaults: Use of Y-Chromosome-Targeted Pre-amplification and Next Generation Y-STR Amplification Systems.

PubMed

Hanson, Erin K; Ballantyne, Jack

2016-01-01

In some cases of sexual assault the victim may not report the assault for several days after the incident due to various factors. The ability to obtain an autosomal STR profile of the semen donor from a living victim rapidly diminishes as the post-coital interval is extended due to the presence of only a small amount of male DNA amidst an overwhelming amount of female DNA. Previously, we have utilized various technological tools to overcome the limitations of male DNA profiling in extended interval post-coital samples including the use of Y-chromosome STR profiling, cervical sample, and post-PCR purification permitting the recovery of Y-STR profiles of the male DNA from samples collected 5-6 days after intercourse. Despite this success, the reproductive biology literature reports the presence of spermatozoa in the human cervix up to 7-10 days post-coitus. Therefore, novel and improved methods for recovery of male profiles in extended interval post-coital samples were required. Here, we describe enhanced strategies, including Y-chromosome-targeted pre-amplification and next generation Y-STR amplification kits, that have resulted in the ability to obtain probative male profiles from samples collected 6-9 days after intercourse.

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

PubMed Central

Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

2015-01-01

Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions. PMID:26247357

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.

PubMed

Leontiou, Chrysanthia A; Hadjidaniel, Michael D; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C

2015-01-01

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

The Moon Beams on Westlake.

ERIC Educational Resources Information Center

Bishop, Jeanne E.

1979-01-01

Presented is a community's experience with a lunar sample education kit containing actual pieces of moon rocks and soil on loan from NASA. School and community activities including mini-labs, seminars, and lunar sample viewing sessions, are described. (SA)

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis

PubMed Central

Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick

2017-01-01

ABSTRACT Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal. PMID:28202794

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis.

PubMed

Robert-Gangneux, Florence; Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick

2017-05-01

Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations ( P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal. Copyright © 2017 American Society for Microbiology.

Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

PubMed Central

Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

2014-01-01

Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

Gel Electrophoresis on a Budget to Dye for

ERIC Educational Resources Information Center

Yu, Julie H.

2010-01-01

Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

With "Biobricks," Students Snap Together a New Science

ERIC Educational Resources Information Center

Trivedi, Bijal

2007-01-01

The underlying goal of the International Genetically Engineered Machine Competition, known as iGEM, is to figure out whether biological organisms and devices can be built from a collection of standard, off-the-shelf parts, just as someone might build a kit plane or car. For the undergraduates, it's an opportunity to construct whatever creature…

42 CFR 403.902 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-10-01

..., repackagers, relabelers, and kit assemblers) that do not hold title to any covered drug, device, biological or... sister corporations. Covered device means any device for which payment is available under Title XVIII of the Act or under a State plan under Title XIX or XXI of the Act (or a waiver of such plan), either...

42 CFR 403.902 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-10-01

..., repackagers, relabelers, and kit assemblers) that do not hold title to any covered drug, device, biological or... sister corporations. Covered device means any device for which payment is available under Title XVIII of the Act or under a State plan under Title XIX or XXI of the Act (or a waiver of such plan), either...

How great a thirst? Assembling a river restoration toolkit

Treesearch

Steve Harris

1999-01-01

The Rio Grande River's biologically troubled status is clearly linked to present and historic water management. To restore the river to pre-settlement conditions will take a "tool kit" that holds authorities, knowledge, and skills needed to correct historical neglect and abuse. Tools include awareness, planning, partnerships, engineering solutions, and a...

A New Three-Dimensional Educational Model Kit for Building DNA and RNA Molecules: Development and Evaluation

ERIC Educational Resources Information Center

Beltramini, Leila Maria; Araujo, Ana Paula Ulian; de Oliveira, Tales Henrique Goncalves; dos Santos Abel, Luciano Douglas; da Silva, Aparecido Rodrigues; dos Santos, Neusa Fernandes

2006-01-01

International specialized literature focused on research in biology education is sadly scarce, especially regarding biochemical and molecular aspects. In this light, researchers from this Centre for Structural Molecular Biotechnology developed and evaluated a three-dimensional educational model named "Building Life Molecules DNA and RNA." The…

[BIOLOGICALLY ACTIVE ALKALOIDS FROM RHIZOMES WITH ROOTS OF VINCA HERBACEA WALDST. ET KIT, GROWING IN GEORGIA].

PubMed

Vachnadze, V; Vachnadze, N; Gogitidze, N; Mushkiashvili, N; Mchedlidze, K

2017-10-01

Roots and rhizomes of Vinca herbacea Waldst. et Kit, were collected during early flowering and fruiting. Рhenophases biologically active substances I and II were obtained by liquid-liquid extraction. Dominant alkaloids: tabersonin, reserpine, maidine, norfluorocurarin and copsinin were obtained after the dispertion in citrare-phosfhate buffer and subsequent TLC. Accelerated restitution of granulocytopoiesis was observed in mice during both irradiation and myelotoxic drug-induced acute leucopenia. Increase in total WBC over 200% was observed after treatment by substance I in drug-induced leucopenia model (fivefold oral administration) and over 130% after treatment by substance I in irradiate mice (fivefold intraperitoneal administration). Morphological and anatomical structures of the underground organs of V. herbacea have been studied. The main microstructural characteristics are revealed - Rhizomes are characterized by coutinized epidermis, lamellar collenchyma, fibers and the texture of the vascular system of a monocyclic structure. The root system shows the whole cortex, the endoderm with Kaspar spots; the outer, radially continuous phloem tissue is located in the conducting system and distinguishes the cylindrical xylem tissue with annular and spiral-circular blood vessels.

Development of an incurred cornbread model for gluten detection by immunoassays.

PubMed

Sharma, Girdhari M; Khuda, Sefat E; Pereira, Marion; Slate, Andrew; Jackson, Lauren S; Pardo, Christopher; Williams, Kristina M; Whitaker, Thomas B

2013-12-11

Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision.

Evaluation of two strategies for heart health promotion by direct mail in a low-income urban community.

PubMed

O'Loughlin, J; Paradis, G; Meshefedjian, G

1997-01-01

The objective of this study was to evaluate the reach of mass mailings of heart health education print materials in a low-income, urban community. Materials included a monthly newsletter and a self-help behavior change kit, both distributed to all 12,789 households in the study community. Recall, use, and self-reported impact of the materials were measured in a cross-sectional survey of a random sample of 345 adults conducted 2 weeks after distribution of the kit and 18 months after delivery of the first newsletter. Over one-third of the subjects (38.6%) recalled the newsletter and 27.9% had read one or more newsletters; 21.7% recalled the kit and 10.8% had read it. Few subjects had read both materials. Female gender and older age were independent correlates of having seen and read the newsletters. Older age, being widowed/separated/divorced, and infrequent physical activity were correlates of having seen and read the kit. Although the newsletter and kit formats might appeal to different segments of the population, mass mailings of heart health education print materials in a low-income urban community can reach large numbers of individuals. The cost effectiveness of repeated mailings of short, simple newsletters might be higher than a single mailing of a more complex behavior change kit.

Optimizing direct amplification of forensic commercial kits for STR determination.

PubMed

Caputo, M; Bobillo, M C; Sala, A; Corach, D

2017-04-01

Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman ® 3 MM paper, FTA™ Classic cards, and Whatman ® Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Dual-Targeting of AR and Akt Pathways by Berberine in Castration-Resistant Prostate Cancer

DTIC Science & Technology

2015-08-01

BCA Protein Assay kit (Pierce). The samples were separated on 10% SDS- polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF...Microtubule/Tubulin In Vivo Assay Kit (Cytoskeleton Inc., Cat.# BK038) following the manufacturer’s instructions. Briefly, 3 × 106 cells were lysed in 4...reporter gene assay . Although the protein fusion affected 193 the relative activities of the fusion proteins (Figs. 1D and S1), all the fusion

Comparing Standard and Selective Degradation DNA Extraction Methods: Results from a Field Experiment with Sexual Assault Kits^.

PubMed

Campbell, Rebecca; Pierce, Steven J; Sharma, Dhruv B; Shaw, Jessica; Feeney, Hannah; Nye, Jeffrey; Schelling, Kristin; Fehler-Cabral, Giannina

2017-01-01

A growing number of U.S. cities have large numbers of untested sexual assault kits (SAKs) in police property facilities. Testing older kits and maintaining current case work will be challenging for forensic laboratories, creating a need for more efficient testing methods. We evaluated selective degradation methods for DNA extraction using actual case work from a sample of previously unsubmitted SAKs in Detroit, Michigan. We randomly assigned 350 kits to either standard or selective degradation testing methods and then compared DNA testing rates and CODIS entry rates between the two groups. Continuation-ratio modeling showed no significant differences, indicating that the selective degradation method had no decrement in performance relative to customary methods. Follow-up equivalence tests indicated that CODIS entry rates for the two methods could differ by more than ±5%. Selective degradation methods required less personnel time for testing and scientific review than standard testing. © 2016 American Academy of Forensic Sciences.

Oncogenic mutations in melanomas and benign melanocytic nevi of the female genital tract.

PubMed

Tseng, Diane; Kim, Julie; Warrick, Andrea; Nelson, Dylan; Pukay, Marina; Beadling, Carol; Heinrich, Michael; Selim, Maria Angelica; Corless, Christopher L; Nelson, Kelly

2014-08-01

The genetic heterogeneity of melanomas and melanocytic nevi of the female genital tract is poorly understood. We aim to characterize the frequency of mutations of the following genes: BRAF, NRAS, KIT, GNA11, and GNAQ in female genital tract melanomas. We also characterize the frequency of BRAF mutations in female genital tract melanomas compared with melanocytic nevi. Mutational screening was performed on the following female genital tract melanocytic neoplasms: 25 melanomas, 7 benign melanocytic nevi, and 4 atypical melanocytic nevi. Of the 25 female genital tract melanoma specimens queried, KIT mutations were detected in 4 (16.0%), NRAS mutations in 4 (16.0%), and BRAF mutations in 2 (8.0%) samples. Two of the tumors with KIT mutations harbored double mutations in the same exon. No GNAQ or GNA11 mutations were identified among 11 melanomas screened. BRAF V600E mutations were detected in 7 of 7 benign melanocytic genital nevi (100%) and 3 of 4 atypical genital nevi (75%). Our study is limited by the small sample size of this rare subset of melanomas. KIT, NRAS, and BRAF mutations are found in a subset of female genital tract melanomas. Screening for oncogenic mutations is important for developing and applying clinical therapies for melanomas of the female genital tract. Copyright © 2014 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Women's views on human papillomavirus self-sampling: focus groups to assess acceptability, invitation letters and a test kit in the Australian setting.

PubMed

Sultana, Farhana; Mullins, Robyn; Murphy, Michael; English, Dallas R; Simpson, Julie A; Drennan, Kelly T; Heley, Stella; Wrede, C David; Brotherton, Julia M L; Saville, Marion; Gertig, Dorota M

2015-08-01

Background The study evaluated acceptability, invitation letters and the test kit for a trial of human papillomavirus (HPV) self-sampling among never- and under-screened women in Australia. Victorian women, 30-69 years, who had never had a Pap test or were overdue for one, participated. Four focus groups including eight to nine participants segmented by age (30-49 and 50-69 years) and screening history (never- and under-screened) were conducted in August 2013. Discussions were recorded and transcribed verbatim and data analysed using thematic content analysis. The response to the concept of HPV self-sampling was positive. Decision-making was largely influenced by the content of a pre-invitation letter. Appealing features of self-sampling were cost (free), convenience (home-based) and anticipated less discomfort (with a swab) than a Pap test. Small kits that fit in mailboxes were preferred over post office parcel collection. The perceived barriers include concerns about test accuracy and lack of confidence that a home-based test would give the same results as a physician administered test. Women wanted information on the timing of receipt of the results and information about the organisation providing the test. HPV self-sampling is a possible alternative for Australian women who are reluctant to have a Pap test and may increase the likelihood of participation in cervical cancer screening if women's concerns about it can be addressed. The findings of this study are relevant for researchers, policymakers and practitioners implementing self-sampling for under-screened women as part of cervical screening programs.

Human Papillomavirus Infection in Women Who Submit Self-collected Vaginal Swabs After Internet Recruitment.

PubMed

Nelson, Erik J; Hughes, John; Oakes, J Michael; Thyagarajan, Bharat; Pankow, James S; Kulasingam, Shalini L

2015-06-01

Submission of vaginal samples collected at home could remove barriers that women face in getting screened for cervical cancer. From December 2013 to January 2014, women aged 21-30 years were recruited online to participate in either (1) self-collected testing for human papillomavirus (HPV) infection and an online survey, or (2) an online survey regarding their perceptions of self-collected testing for HPV infection. Demographics, risk factors, testing perceptions, and satisfaction with self-collected testing were assessed with online questionnaires. Women who performed self-collection were sent a home sampling kit by US mail, which was returned via US mail for HPV testing. A total of 197 women were enrolled, with 130 completing the online survey and 67 participating in both the survey and self-collection. Of the 67 women who were sent kits, 62 (92.5%) were returned for testing. Sixty kits contained a sample sufficient for testing. The overall prevalence of HPV infection was 17.8%, however 6 women (9.7%) were infected with >1 type of HPV. Women who self-collected a sample reported more favorable attributes of self-collection compared to women who only participated in the online survey, including ease of sampling (87.1 vs. 18.9%), no pain during sampling (72.6 vs. 5.6%), and lack of embarrassment (67.7 vs. 12.9%). A high prevalence of HPV infection was demonstrated among women recruited via the internet. Online recruitment and at home screening methods have the potential to engage women in screening by offering an approach that might be more acceptable to women of different backgrounds.

Comparing rapid methods for detecting Listeria in seafood and environmental samples using the most probably number (MPN) technique.

PubMed

Cruz, Cristina D; Win, Jessicah K; Chantarachoti, Jiraporn; Mutukumira, Anthony N; Fletcher, Graham C

2012-02-15

The standard Bacteriological Analytical Manual (BAM) protocol for detecting Listeria in food and on environmental surfaces takes about 96 h. Some studies indicate that rapid methods, which produce results within 48 h, may be as sensitive and accurate as the culture protocol. As they only give presence/absence results, it can be difficult to compare the accuracy of results generated. We used the Most Probable Number (MPN) technique to evaluate the performance and detection limits of six rapid kits for detecting Listeria in seafood and on an environmental surface compared with the standard protocol. Three seafood products and an environmental surface were inoculated with similar known cell concentrations of Listeria and analyzed according to the manufacturers' instructions. The MPN was estimated using the MPN-BAM spreadsheet. For the seafood products no differences were observed among the rapid kits and efficiency was similar to the BAM method. On the environmental surface the BAM protocol had a higher recovery rate (sensitivity) than any of the rapid kits tested. Clearview™, Reveal®, TECRA® and VIDAS® LDUO detected the cells but only at high concentrations (>10(2) CFU/10 cm(2)). Two kits (VIP™ and Petrifilm™) failed to detect 10(4) CFU/10 cm(2). The MPN method was a useful tool for comparing the results generated by these presence/absence test kits. There remains a need to develop a rapid and sensitive method for detecting Listeria in environmental samples that performs as well as the BAM protocol, since none of the rapid tests used in this study achieved a satisfactory result. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative analysis of fungicide azoxystrobin in agricultural samples with rapid, simple and reliable monoclonal immunoassay.

PubMed

Watanabe, Eiki; Miyake, Shiro

2013-01-15

This work presents analytical performance of a kit-based direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for azoxystrobin detection in agricultural products. The dc-ELISA was sufficiently sensitive for analysis of residue levels close to the maximum residue limits. The dc-ELISA did not show cross-reactivity to other strobilurin analogues. Absorbance decreased with the increase of methanol concentration in sample solution from 2% to 40%, while the standard curve became most linear when the sample solution contained 10% methanol. Agricultural samples were extracted with methanol, and the extracts were diluted with water to 10% methanol adequate. No significant matrix interference was observed. Satisfying recovery was found for all of spiked samples and the results were well agreed with the analysis with liquid chromatography. These results clearly indicate that the kit-based dc-ELISA is suitable for the rapid, simple, quantitative and reliable determination of the fungicide. Copyright © 2012 Elsevier Ltd. All rights reserved.

Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using the Bio-Quant direct ELISA presumptive assays for amphetamine and methamphetamine.

PubMed

Apollonio, Luigino G; Whittall, Ian R; Pianca, Dennis J; Kyd, Jennelle M; Maher, William A

2007-05-01

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.

Functional TRPV2 and TRPV4 channels in human cardiac c-kit(+) progenitor cells.

PubMed

Che, Hui; Xiao, Guo-Sheng; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2016-06-01

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

PubMed Central

Gabriel, Frédéric; Gaboyard, Manuel; Lagardere, Gaëlle; Audebert, Lucile; Quesne, Gilles; Godichaud, Sandrine; Verweij, Paul E.; Accoceberry, Isabelle

2017-01-01

ABSTRACT Aspergillus fumigatus is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in A. fumigatus is worrisome. The aim of this study was to validate the new MycoGENIE A. fumigatus real-time PCR kit and to evaluate its performance on clinical samples for the detection of A. fumigatus and its azole resistance. This multiplex assay detects DNA from the A. fumigatus species complex by targeting the multicopy 28S rRNA gene and specific TR34 and L98H mutations in the single-copy-number cyp51A gene of A. fumigatus. The specificity of cyp51A mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical A. fumigatus isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for the Aspergillus 28S rRNA gene and 6 copies for the cyp51A gene harboring the TR34 and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34 and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of A. fumigatus DNA and azole resistance due to TR34 and L98H mutations in clinical samples. PMID:28814586

TECHNOLOGIES FOR MONITORING AND MEASUREMENT ...

EPA Pesticide Factsheets

A demonstration of technologies for determining the presence of dioxin and dioxin-like compounds in soil and sediment was conducted under EPA's Superfund Innovative Technology Evaluation Program in Saginaw, Michigan in April 2004. This report describes the performance evaluation of the Abraxis LLC Coplanar PCB Enzyme-Linked Immunosorbent Assay (ELISA) kit. The kit is an immunoassay technique that reports the total toxicity equivalents (TEQ) of polychlorinated biphenyls (PCBs). The technology results were compared to high resolution mass spectrometry TEQ results generated using EPA Method 1668A.Abraxis generally reported data that were higher than the reference laboratory TEQPCB values, with the exception of ultra-high level PCB samples [> 10,000 picogram/gram (pg/g) TEQ] where Abraxis reported values lower than the reference method. The technologys estimated MDL was 6 to 31 pg/g TEQPCB. Results from this demonstration suggest that the Abraxis kit could be an effective screening tool for screening sample concentrations above and below 50 pg/g TEQPCB, particularly considering that the cost ($22,668 vs. $184,449) and the time to analyze the 209 demonstration samples were significantly less than those of the reference laboratory. The objective of this program is to promote the acceptance and use of innovative field technologies by providing well-documented performance and cost data obtained from field demonstrations.

Platelet collection efficiencies of three different platelet-rich plasma preparation systems.

PubMed

Aydin, Fatma; Pancar Yuksel, Esra; Albayrak, Davut

2015-06-01

Different systems have been used for the preparation of platelet-rich plasma (PRP), but platelet collection efficiencies of these systems are not clear. To evaluate the platelet collection efficiencies of three different PRP preparation systems. Blood samples were obtained from the same 16 volunteers for each system. The samples were centrifuged and PRP was prepared by three systems. The ratio of the total number of platelets in PRP to the total number of platelets of the venous blood sample of the patient expressed in percentage was named as platelet collection efficiency and calculated for each system. Mean platelet collection efficiencies were 66.6 (min: 56.9, max: 76.9), 58.3 (min: 27.3, max: 102.8), 50.8 (min: 27.2, max: 73) for top and bottom bag system, system using citrated tube, and the system using tube with Ficoll and cell extraction kit, respectively. Statistically significant difference was found only between the platelet collection efficiencies of systems using the tube with ficoll and cell extraction kit and the top and bottom bag system (p = 0.002). All three systems could be used for PRP preparation, but top and bottom bag system offers a slight advantage over the system using Ficoll and cell extraction kit regarding the platelet collection efficiency.

HIV-1 and HCV viral load cost models for bDNA: 440 Molecular System versus real-time PCR AmpliPrep/TaqMan test.

PubMed

Elbeik, Tarek; Dalessandro, Ralph; Loftus, Richard A; Beringer, Scott

2007-11-01

Comparative cost models were developed to assess cost-per-reportable result and annual costs for HIV-1 and HCV bDNA and AmpliPrep/TaqMan Test (PCR). Model cost components included kit, disposables, platform and related equipment, equipment service plan, equipment maintenance, equipment footprint, waste and labor. Model assessment was most cost-effective when run by bDNA with 36 or more clinical samples and PCR with 30 or fewer clinical samples. Lower costs are attained with maximum samples (84-168) run daily. Highest cost contributors include kit, platform and PCR proprietary disposables. Understanding component costs and the most economic use of HIV-1 and HCV viral load will aid in attaining lowest costs through selection of the appropriate assay and effective negotiations.

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices.

PubMed

Hoople, Gordon D; Richards, Andrew; Wu, Yan; Pisano, Albert P; Zhang, Kun

2018-03-26

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

PubMed Central

Lim, Natalie Y. N.; Roco, Constance A.; Frostegård, Åsa

2016-01-01

Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. PMID:27803690

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

PubMed Central

El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

2014-01-01

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

Evaluation of the Aspergillus Western Blot IgG Kit for Diagnosis of Chronic Aspergillosis

PubMed Central

Oliva, A.; Flori, P.; Hennequin, C.; Dubus, J.-C.; Reynaud-Gaubert, M.; Charpin, D.; Vergnon, J. M.; Gay, P.; Colly, A.; Piarroux, R.; Pelloux, H.

2014-01-01

Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. PMID:25392351

Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

PubMed

Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

2015-01-01

Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform.

PubMed

Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

2016-01-01

Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.

Performance Evaluation of the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit: Comparison with the Roche COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test Ver.2.0 for Quantification of HIV-1 Viral Load in Indonesia.

PubMed

Kosasih, Agus Susanto; Sugiarto, Christine; Hayuanta, Hubertus Hosti; Juhaendi, Runingsih; Setiawan, Lyana

2017-08-08

Measurement of viral load in human immunodeficiency virus type 1 (HIV-1) infected patients is essential for the establishment of a therapeutic strategy. Several assays based on qPCR are available for the measurement of viral load; they differ in sample volume, technology applied, target gene, sensitivity and dynamic range. The Bioneer AccuPower® HIV-1 Quantitative RT-PCR is a novel commercial kit that has not been evaluated for its performance. This study aimed to evaluate the performance of the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit. In total, 288 EDTA plasma samples from the Dharmais Cancer Hospital were analyzed with the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit and the Roche COBAS? AmpliPrep/COBAS® TaqMan® HIV-1 version 2.0 (CAP/CTM v2.0). The performance of the Bioneer assay was then evaluated against the Roche CAP/CTM v2.0. Overall, there was good agreement between the two assays. The Bioneer assay showed significant linear correlation with CAP/CTM v2.0 (R2=0.963, p<0.001) for all samples (N=118) which were quantified by both assays, with high agreement (94.9%, 112/118) according to the Bland-Altman model. The mean difference between the quantitative values measured by Bioneer assay and CAP/CTM v2.0 was 0.11 Log10 IU/mL (SD=0.26). Based on these results, the Bioneer assay can be used to quantify HIV-1 RNA in clinical laboratories.

Evaluation of the performance of the IQ-check kits and the USDA microbiology laboratory guidebook methods for detection of Shiga Toxin-Producing E. coli (STEC) and STEC and Salmonella simultaneously in ground beef

USDA-ARS?s Scientific Manuscript database

Aims: To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top 7 Shiga toxin-producing E. coli (STEC) (O157:H7, O26, O45, O103, O111, O121, and O145) in ground beef and both STEC and Salmonella in co-inoculated samples. M...

Evaluation of Presto(plus) assay and LightMix kit Trichomonas vaginalis assay for detection of Trichomonas vaginalis in dry vaginal swabs.

PubMed

de Waaij, Dewi J; Ouburg, Sander; Dubbink, Jan Henk; Peters, Remco P H; Morré, Servaas A

2016-08-01

This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05). Copyright © 2016 Elsevier B.V. All rights reserved.

Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

NASA Technical Reports Server (NTRS)

Fan, Wenhong (Inventor); Han, Jie (Inventor); Cassell, Alan M. (Inventor)

2006-01-01

Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

Commercial test kits for detection of Lyme borreliosis: a meta-analysis of test accuracy

PubMed Central

Cook, Michael J; Puri, Basant K

2016-01-01

The clinical diagnosis of Lyme borreliosis can be supported by various test methodologies; test kits are available from many manufacturers. Literature searches were carried out to identify studies that reported characteristics of the test kits. Of 50 searched studies, 18 were included where the tests were commercially available and samples were proven to be positive using serology testing, evidence of an erythema migrans rash, and/or culture. Additional requirements were a test specificity of ≥85% and publication in the last 20 years. The weighted mean sensitivity for all tests and for all samples was 59.5%. Individual study means varied from 30.6% to 86.2%. Sensitivity for each test technology varied from 62.4% for Western blot kits, and 62.3% for enzyme-linked immunosorbent assay tests, to 53.9% for synthetic C6 peptide ELISA tests and 53.7% when the two-tier methodology was used. Test sensitivity increased as dissemination of the pathogen affected different organs; however, the absence of data on the time from infection to serological testing and the lack of standard definitions for “early” and “late” disease prevented analysis of test sensitivity versus time of infection. The lack of standardization of the definitions of disease stage and the possibility of retrospective selection bias prevented clear evaluation of test sensitivity by “stage”. The sensitivity for samples classified as acute disease was 35.4%, with a corresponding sensitivity of 64.5% for samples from patients defined as convalescent. Regression analysis demonstrated an improvement of 4% in test sensitivity over the 20-year study period. The studies did not provide data to indicate the sensitivity of tests used in a clinical setting since the effect of recent use of antibiotics or steroids or other factors affecting antibody response was not factored in. The tests were developed for only specific Borrelia species; sensitivities for other species could not be calculated. PMID:27920571

Mendel Meets CSI: Forensic Genotyping as a Method to Teach Genetics & DNA Science

ERIC Educational Resources Information Center

Kurowski, Scotia; Reiss, Rebecca

2007-01-01

This article describes a forensic DNA science laboratory exercise for advanced high school and introductory college level biology courses. Students use a commercial genotyping kit and genetic analyzer or gene sequencer to analyze DNA recovered from a fictitious crime scene. DNA profiling and STR genotyping are outlined. DNA extraction, PCR, and…

Genetic profile of a multi-ethnic population from Guiné-Bissau (west African coast) using the new PowerPlex 16 System kit.

PubMed

Gonçalves, Rita; Jesus, José; Fernandes, Ana Teresa; Brehm, António

2002-09-10

Allele and haplotype frequencies of 15 chromosome STR loci included in the kit PowerPlex16 System from Promega, were determined in a sample of unrelated males from Guiné-Bissau, a country from the west African coast. All individuals were subjected to an interview in order to make sure that their ancestors belonged to the same ethnic group. This way we intended to look for possible inter-ethnic differences. PowerPlex 16 includes STRs not studied before in any multi-ethnic population. The kit includes two new allele markers (Penta D and Penta E), which are very useful either in forensics or population genetic studies. The Guinean population presents significant differences when compared with other African populations.

Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq.

PubMed

Faherty, Sheena L; Campbell, C Ryan; Larsen, Peter A; Yoder, Anne D

2015-07-30

RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates. We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison. Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.

Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal.

PubMed

Hasan, J A; Huq, A; Nair, G B; Garg, S; Mukhopadhyay, A K; Loomis, L; Bernstein, D; Colwell, R R

1995-11-01

We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens. The BengalScreen test requires less than 5 min to complete and can be used in the field. Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V. cholerae O139 synonym Bengal. In tests for specificity, all 40 strains of V. cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study. A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods. BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94%. Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods. In a second evaluation, 93 stool specimens from Mexico that were negative for V. cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits. None of the 93 specimens were positive for V. cholerae O139 by both tests. A concentration method was optimized for screening of environmental water samples for V. cholerae O139 synonym Bengal with rapid test kits. BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml. When Bengal DFA was compared with conventional culture methods for enumeration of V. cholerae O139 synonym Bengal organisms, no difference was observed.

Effect of Temperature and Time on Fecal Hemoglobin Stability in 5 Fecal Immunochemical Test Methods and One Guaiac Method.

PubMed

Catomeris, Peter; Baxter, Nancy N; Boss, Sheila C; Paszat, Lawrence F; Rabeneck, Linda; Randell, Edward; Serenity, Mardie L; Sutradhar, Rinku; Tinmouth, Jill

2018-01-01

- Although promising for colorectal cancer screening, hemoglobin (Hb) stability remains a concern with fecal immunochemical tests. This study implemented a novel, standardized method to compare Hb stability across various fecal immunochemical tests. The method can be used to inform decisions when selecting a kit for use in colorectal cancer screening. In so doing, this work addressed a critical need for standardization in this field. - To compare the stability of Hb across 5 different immunochemical kits and one guaiac kit. - The stability of Hb was analyzed in collection devices inoculated with Hb-spiked feces and (1) stored at various temperatures (frozen, refrigerated, ambient, and elevated) for more than 60 days; (2) after undergoing 3 controlled, freeze-thaw cycles; and (3) after being transported by courier or postal services in uncontrolled temperature conditions from 3 locations in Ontario, Canada, to a central testing center. - The stability of Hb varied with time and temperature and by kit. Lower Hb recoveries occurred with increasing temperature and increasing time from sample collection to testing. Refrigeration provided the best stability, although results varied across kits (eg, from 4.2 days to >60 days before a prespecified threshold [<70% probability of the test results remaining positive] was reached). Freeze-thaw stability varied across kits and cycles (Hb recoveries: NS Plus [Alfresa Pharma, Chuo-ku, Osaka, Japan], 91.7% to 95.4%; OC Diana [Eiken Chemical, Taito-ku, Tokyo, Japan], 57.6% to 74.9%). Agreement regarding Hb levels before and after transportation varied across kits (from 57% to 100%). - Important differences in Hb stability were found across the included fecal immunochemical tests. These findings should inform practice-based and population-based colorectal cancer screening.

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm.

PubMed

Kucera, Aurelia C; Heidinger, Britt J

2018-02-05

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

Assessment of peracetic acid disinfected effluents by microbiotests.

PubMed

Antonelli, M; Mezzanotte, V; Panouillères, M

2009-09-01

Bioassays were performed by commercially available kits on peracetic acid (PAA) solutions, at different concentrations, and on secondary effluents (from two different wastewater treatment plants) after disinfection at bench-scale, considering both samples containing residual active PAA and the same samples where residual PAA was quenched. Four indicator organisms were used: Vibrio fischeri, Thamnocephalus platyurus, Daphnia magna, and Selenastrum capricornutum. The experiments lead to conclude that Thamnocephalus platyurus is a very sensitive organism, probably not adequate to perform a reliable toxicity assessment of effluents for monitoring purposes. The presence of specific organic compounds deriving from human metabolism and urban pollution, even at very low concentrations, can affect the results of bioassays, especially those performed on Vibrio fischeri. PAA is toxic for bacteria and crustaceans even at concentrations lower than the ones commonly used in wastewater disinfection (2-5 mg/L), while its effect on algae is smaller. The toxic effect on bacteria was expected, as PAA is used for disinfection, but its possible influence on biological processes in the receiving aquatic environment should be considered. Toxicity on crustaceans would confirm the fact that discharging disinfected effluents could raise some environmental problems.

Concordance study between the ParaDNA® Intelligence Test, a rapid DNA profiling assay, and a conventional STR typing kit (AmpFlSTR® SGM Plus®).

PubMed

Ball, G; Dawnay, N; Stafford-Allen, B; Panasiuk, M; Rendell, P; Blackman, S; Duxbury, N; Wells, S

2015-05-01

The ParaDNA® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR® SGM Plus® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Availability, Accessibility, and Price of Rapid HIV Self-Tests, New York City Pharmacies, Summer 2013.

PubMed

Myers, Julie E; El-Sadr Davis, Olivia Y; Weinstein, Elliott R; Remch, Molly; Edelstein, Amy; Khawja, Amina; Schillinger, Julia A

2017-02-01

We conducted an in-person survey of New York City (NYC) pharmacies to assess the availability, accessibility, and price of the over-the-counter, rapid HIV self-test kit. NYC pharmacies were stratified into high, moderate and low morbidity neighborhoods by the HIV diagnosis rate of the neighborhood in which the pharmacy was located. A random sample of 500 pharmacies was taken [250 from high morbidity neighborhoods (HighMN) and 250 from low morbidity neighborhoods (LowMN)]. Pharmacies were excluded if: closed during survey, non-retail, or >10 min walk from subway. Project staff visited pharmacies to determine kit availability (in pharmacy on day of survey), accessibility (not locked/behind counter), and price (marked on shelf/product). Of 361 pharmacies (161 LowMN; 200 HighMN), kits were available in 27 % and accessible in 10 %; there was no difference by neighborhood. Kits were most often kept behind the pharmacy counter; this was more common in HighMN than in LowMN. Kits were kept solely behind the pharmacy counter in 52 %. Median price was US $42.99 without variability across neighborhoods. The rapid HIV self-test had limited availability and access in retail pharmacies. The high median price measured suggests that cost remained a barrier.

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

PubMed Central

De Vita, Serena; Schneider, Rebekka K.; Garcia, Michael; Wood, Jenna; Gavillet, Mathilde; Ebert, Benjamin L.; Gerbaulet, Alexander; Roers, Axel; Levine, Ross L.; Mullally, Ann; Williams, David A.

2014-01-01

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM. PMID:24788138

Sample Collection for the Russian Biodegradatsiya Experiment

NASA Image and Video Library

2007-10-01

ISS015-E-32031 (October 2007) --- Cosmonaut Oleg V. Kotov, Expedition 15 flight engineer representing Russia's Federal Space Agency, uses a Bioproby Kit to collect surface samples for analysis for the Russian Biodegradation experiment in the Zvezda Service Module of the International Space Station.

mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

2008-12-01

Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

Optimization of a metatranscriptomic approach to study the lignocellulolytic potential of the higher termite gut microbiome.

PubMed

Marynowska, Martyna; Goux, Xavier; Sillam-Dussès, David; Rouland-Lefèvre, Corinne; Roisin, Yves; Delfosse, Philippe; Calusinska, Magdalena

2017-09-01

Thanks to specific adaptations developed over millions of years, the efficiency of lignin, cellulose and hemicellulose decomposition of higher termite symbiotic system exceeds that of many other lignocellulose utilizing environments. Especially, the examination of its symbiotic microbes should reveal interesting carbohydrate-active enzymes, which are of primary interest for the industry. Previous metatranscriptomic reports (high-throughput mRNA sequencing) highlight the high representation and overexpression of cellulose and hemicelluloses degrading genes in the termite hindgut digestomes, indicating the potential of this technology in search for new enzymes. Nevertheless, several factors associated with the material sampling and library preparation steps make the metatranscriptomic studies of termite gut prokaryotic symbionts challenging. In this study, we first examined the influence of the sampling strategy, including the whole termite gut and luminal fluid, on the diversity and the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. Secondly, we evaluated different commercially available kits combined in two library preparative pipelines for the best bacterial mRNA enrichment strategy. We showed that the sampling strategy did not significantly impact the generated results, both in terms of the representation of the microbes and their transcriptomic profiles. Nevertheless collecting luminal fluid reduces the co-amplification of unwanted RNA species of host origin. Furthermore, for the four studied higher termite species, the library preparative pipeline employing Ribo-Zero Gold rRNA Removal Kit "Epidemiology" in combination with Poly(A) Purist MAG kit resulted in a more efficient rRNA and poly-A-mRNAdepletion (up to 98.44% rRNA removed) than the pipeline utilizing MICROBExpress and MICROBEnrich kits. High correlation of both Ribo-Zero and MICROBExpresse depleted gene expression profiles with total non-depleted RNA-seq data has been shown for all studied samples, indicating no systematic skewing of the studied pipelines. We have extensively evaluated the impact of the sampling strategy and library preparation steps on the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. The presented methodological approach has great potential to enhance metatranscriptomic studies of the higher termite intestinal flora and to unravel novel carbohydrate-active enzymes.

InstantLabs Listeria monocytogenes food safety kit. Performance tested method 051304.

PubMed

Sharma, Neil; Bambusch, Lauren; Le, Thu; Morey, Amit

2014-01-01

The InstantLabs Listeria monocytogenes Food Safety Kit was validated against the International Organization for Standardization (ISO) reference method 11290-1 for the detection of Listeria monocytogenes and other Listeria species. The matrixes (stainless steel, sealed concrete, ice cream, whole milk, cheddar cheese, raw shrimp, hot dogs, deli turkey, and lettuce) were inoculated with approximately 1 CFU/test portion of L. monocytogenes to generate fractional positives (5-15) in 20 inoculated samples. Enrichments were also fractionally inoculated with L. grayii for side-by-side testing of the Listeria Species Food Safety Kit. Stainless steel and sealed concrete samples were validated using 4 x 4" and 1 x 1 " test areas, respectively, and enriched in Buffered Listeria Enrichment Broth (BLEB) at 35 +/- 1degreesC for 22-28 h. All food samples were tested at 25 g and enriched in BLEB at 35 +/- 1 degreesC for 24-28 h. All samples were confirmed using the ISO reference method, regardless of initial screen result. The InstantLabs test method performed as well as or better than the reference method for the detection of L. monocytogenes on stainless steel and sealed concrete and in ice cream, whole milk, cheddar cheese, raw shrimp, hot dogs, deli turkey, and lettuce. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 L. monocytogenes serovars and 30 non-L. monocytogenes species examined. The method was shown to be robust when the enrichment times, volumes for DNA extraction, and heat block times were varied.

Synthesis and application of mesoporous molecular sieve for miniaturized matrix solid-phase dispersion extraction of bioactive flavonoids from toothpaste, plant, and saliva.

PubMed

Cao, Wan; Cao, Jun; Ye, Li-Hong; Xu, Jing-Jing; Hu, Shuai-Shuai; Peng, Li-Qing

2015-12-01

This article describes the use of the mesoporous molecular sieve KIT-6 as a sorbent in miniaturized matrix solid-phase dispersion (MSPD) in combination with ultra-performance LC for the determination of bioactive flavonoids in toothpaste, Scutellariae Radix, and saliva. In this study, for the first time, KIT-6 was used as a sorbent material for this mode of extraction. Compared with common silica-based sorbents (C18 and activated silica gel), the proposed KIT-6 dispersant with a three-dimensional cubic Ia3d structure and highly ordered arrays of mesoporous channels exhibits excellent adsorption capability of the tested compounds. In addition, several experimental variables, such as the mass ratio of sample to dispersant, grinding time, and elution solvent, were optimized to maximize the extraction efficiency. The proposed analytical method is simple, fast, and entails low consumption of samples, dispersants and elution solvents, thereby meeting "green chemistry" requirements. Under the optimized conditions, the recoveries of three bioactive flavonoids obtained by analyzing the spiked samples were from 89.22 to 101.17%. Also, the LODs and LOQs for determining the analytes were in the range of 0.02-0.04 μg/mL and 0.07-0.13 μg/mL, respectively. Finally, the miniaturized matrix solid-phase dispersion method was successfully applied to the analysis of target solutes in real samples, and satisfactory results were obtained. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Science and Issues of Human DNA Polymorphisms: A Training Workshop for High School Biology Teachers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Micklos, David A.

2006-10-30

This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms â which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, andmore » computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrée to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nationâÂÂs oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human polymorphism kits by Carolina Biological rose from 700 units in 1999 to 1,132 in 2000 â a 62% increase. Competing kits using the Alu system, and based substantially on our earlier work, are also marketed by Biorad and Edvotek. In parallel with the lab experiments, we developed a suite of database/statistical applications and easy-to-use interfaces that allow students to use their own DNA data to explore human population genetics and to test theories of human evolution. Database searches and statistical analyses are launched from a centralized workspace. Workshop participants were introduced to these and other resources available at the DNALC WWW site (http://vector.cshl.org/bioserver/): 1) Allele Server tests Hardy-Weinberg equilibrium and statistically compares PV92 data from world populations. 2) Sequence Server uses DNA sequence data to search Genbank using BLASTN, compare sequences using CLUSTALW, and create phylogenetic trees using PHYLIP. 3) Simulation Server uses a Monte Carlo generator to model the long-term effects of drift, selection, and population bottlenecks. By targeting motivated and innovative biology faculty, we believe that this project offered a cost-effective means to bring high school biology education up-to-the-minute with genomic biology. The workshop reached a target audience of highly professional faculty who have already implemented hands-on labs in molecular genetics and many of whom offer laboratory electives in biotechnology. Many attend professional meetings, develop curriculum, collaborate with scientists, teach faculty workshops, and manage equipment-sharing programs. These individuals are life-long learners, anxious for deeper insight and additional training to further extend their leadership. This contention was supported by data from a mail survey, conducted in February-March 2000 and 2001, of 256 faculty who participated in workshops conducted during the current term of DOE support. Seventy percent of participants responded, providing direct reports on how their teaching behavior had changed since taking the DOE workshop. About nine of ten respondents said they had provided new classroom materials and first-hand accounts of DNA typing, sequencing, or PCR. Three-fourths had introduced new units on human molecular genetics. Most strikingly, half had students use PCR to amplify their own insertion polymorphisms (PV92), and better than one-fourth amplified a VNTR polymorphism and the mitochondrial control region. One in five had mitochondrial DNA sequenced by the DNALC Sequencing Service. A majority (58%) used online materials at the DNALC WWW site, and 28% analyzed student polymorphism data with Bioservers at the DNALC site. A majority (58%) assisted other faculty with student labs on polymorphisms, reaching an additional 786 teachers.« less

The Science and Issues of Human DNA Polymoprhisms: A Training Workshop for High School Biology Teachers

DOE Office of Scientific and Technical Information (OSTI.GOV)

David. A Micklos

2006-10-30

This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms – which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, andmore » computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrée to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nation’s oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human polymorphism kits by Carolina Biological rose from 700 units in 1999 to 1,132 in 2000 – a 62% increase. Competing kits using the Alu system, and based substantially on our earlier work, are also marketed by Biorad and Edvotek. In parallel with the lab experiments, we developed a suite of database/statistical applications and easy-to-use interfaces that allow students to use their own DNA data to explore human population genetics and to test theories of human evolution. Database searches and statistical analyses are launched from a centralized workspace. Workshop participants were introduced to these and other resources available at the DNALC WWW site (http://vector.cshl.org/bioserver/): 1) Allele Server tests Hardy-Weinberg equilibrium and statistically compares PV92 data from world populations. 2) Sequence Server uses DNA sequence data to search Genbank using BLASTN, compare sequences using CLUSTALW, and create phylogenetic trees using PHYLIP. 3) Simulation Server uses a Monte Carlo generator to model the long-term effects of drift, selection, and population bottlenecks. By targeting motivated and innovative biology faculty, we believe that this project offered a cost-effective means to bring high school biology education up-to-the-minute with genomic biology. The workshop reached a target audience of highly professional faculty who have already implemented hands-on labs in molecular genetics and many of whom offer laboratory electives in biotechnology. Many attend professional meetings, develop curriculum, collaborate with scientists, teach faculty workshops, and manage equipment-sharing programs. These individuals are life-long learners, anxious for deeper insight and additional training to further extend their leadership. This contention was supported by data from a mail survey, conducted in February-March 2000 and 2001, of 256 faculty who participated in workshops conducted during the current term of DOE support. Seventy percent of participants responded, providing direct reports on how their teaching behavior had changed since taking the DOE workshop. About nine of ten respondents said they had provided new classroom materials and first-hand accounts of DNA typing, sequencing, or PCR. Three-fourths had introduced new units on human molecular genetics. Most strikingly, half had students use PCR to amplify their own insertion polymorphisms (PV92), and better than one-fourth amplified a VNTR polymorphism and the mitochondrial control region. One in five had mitochondrial DNA sequenced by the DNALC Sequencing Service. A majority (58%) used online materials at the DNALC WWW site, and 28% analyzed student polymorphism data with Bioservers at the DNALC site. A majority (58%) assisted other faculty with student labs on polymorphisms, reaching an additional 786 teachers.« less

[Performance evaluation of Abbott RealTime HBV Quantification Kit for HBV viral load by real-time PCR].

PubMed

Kim, Myeong Hee; Cha, Choong Hwan; An, Dongheui; Choi, Sung Eun; Oh, Heung Bum

2008-04-01

Hepatitis B virus (HBV) DNA quantification is necessary for starting and monitoring of antiviral therapy in patients with chronic hepatitis B. This study was intended to assess the clinical performance of Abbott RealTime HBV Quantification kit (Abbott Laboratories, USA). The performance was evaluated in terms of precision, linearity, detection sensitivity, cross-reactivity, and carry-over. A correlation with the Real-Q HBV Quantification kit (BioSewoom Inc., Korea) was also examined using serum samples from 64 patients diagnosed with chronic hepatitis B and underwent lamivudine therapy in Asan Medical Center. We verified the trueness of the system by comparing the outputs with the assigned values of the BBI panel (BBI Diagnostics, USA). Within-run and between-run coefficients of variation (CV) were 3.56-4.71% and 3.03-4.98%, respectively. Linearity was manifested ranging from 53 to 10(9)copies/mL and the detection sensitivity was verified to be 51 copies/mL. None of hepatitis C virus showed cross-reactivity. No cross-contamination occurred when negative and positive samples were alternatively placed in a row. It showed a good correlation with the Real-Q HBV (r(2)=0.9609) and the test results for the BBI panel were also well agreed to the assigned values (r(2)=0.9933). The performance of Abbott RealTime HBV Quantification kit was excellent; thus, it should be widely used in starting and monitoring of antiviral therapy in Korean patients with chronic hepatitis B.

Immunodiagnosis of toxocarosis in humans: evaluation of a new enzyme-linked immunosorbent assay kit.

PubMed Central

Jacquier, P; Gottstein, B; Stingelin, Y; Eckert, J

1991-01-01

Excretory/secretory (E/S) antigen derived from second-stage larvae of Toxocara canis maintained in defined medium in vitro has been well established worldwide for the immunodiagnosis of human toxocarosis by enzyme-linked immunosorbent assay. Such an enzyme-linked immunosorbent assay, based on the detection of human anti-T. canis (E/S antigen) serum immunoglobulin G, has recently been commercialized by Biokema-Affinity Products (Crissier-Lausanne, Switzerland). This commercial test kit was evaluated with regard to its application in a routine diagnostic laboratory and the reliability of the results. Of 78 patients with suspected clinical toxocarosis, 71 had anti-T. canis antibodies (positive serological result) corresponding to a diagnostic sensitivity of 91%; 14% of serum samples (n = 199) from patients with protozoan or with helminthic infections also showed positive reactions mainly related to infections with Trichinella, Strongyloides, and Fasciola species. An epidemiological study with 1,000 serum samples from randomly selected healthy blood donors and children in Switzerland demonstrated a seroprevalence of 2.7%. The test kit under evaluation had an overall diagnostic sensitivity of 91% and a relative specificity of 86%, the latter being related to some protozoan and helminthic infections. Because of the scarcity of such infections, potential cross-reactivity does not play a major role under the conditions found in the middle part of Europe. In conclusion, the application of the test kit provided for use in this study can be recommended for routine diagnostic use. PMID:1774303

An efficient recovery method for enteric viral particles from agricultural soils.

PubMed

Brassard, Julie; Gagné, Marie-Josée

2018-06-24

Enteric viruses have been recognized as the leading cause of non-bacterial gastroenteritis and hepatitis outbreaks around the world. Understanding their prevalence and persistence in the environment is important for the effective control of these infections. The aim of this study was to develop an efficient recovery procedure for viral infectious particles from agricultural soils. Samples (25 g) of soil (black earth soil, loamy soil, and sandy soil) were spiked with murine norovirus (MNV) and feline calicivirus (FCV), mixed with five different buffers and viral genetic material was extracted by 3 commercial kits. The combination consisted by the modified Eagle's medium buffer followed by Dynabeads nucleic acid extraction kit, when the detection is conducted by molecular biology, has been identified as being the most effective procedure to preserve the viral particle infectivity and also to remove PCR inhibitors.The recovery percentages of infectious MNV for the 3 types of soils were 54.3%, 54.4%, and 56.9%. In contrast, the titres of the FCV varied depending on the type of soil, and the recovery percentages were 47.8% in the black soil, 15.6% in the loamy soil, and 17.7% in the sandy soil. Also, the results presented in this study highlight the importance of using an internal process control such as artificial inoculation with MNV at known concentrations during detection by molecular methods, in order to avoid the occurrence of false negative reactions. Copyright © 2018. Published by Elsevier B.V.

Aviation Career Awareness

ERIC Educational Resources Information Center

Journal of Aerospace Education, 1976

1976-01-01

Describes a kit containing seven units, each designed to increase the elementary school student's awareness of aviation and career possibilities in aviation. Includes a sample section from one unit. (MLH)

Sensitivity or artifact? -- IQ Toxicity Test -- effluent values

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayes, K.R.; Novotny, A.N.; Batista, N.

1995-12-31

Several complex effluents were DAPHNIA MAGNA IQ TOXICITY TESTED -- (1.25 hours) and conventionally tested with Daphnia magna (48 hours). In many samples the IQ Technology yielded low EC50 values while the 48 hour exposures yielded no acute toxicity. Possible explanations have been suggested for this occurrence such as: genotoxicity, mutagenicity, substrate interference, and enzyme satiation. To identify the causative agent(s) of this response a Toxicity Identification Evaluation was performed on one of the samples. To define the nature of the response, THE SOS-CHROMOTEST KIT and THE MUTA-CHROMOPLATE KIT were utilized to characterize genotoxicity and mutagenicity respectively. The sample didmore » not test positive for genotoxicity but tested positive for mutagenicity only after activation with S9 enzymes, suggesting the presence of promutagens. Additional work needs to be performed to correlate IQ TOXICITY TEST sensitivity with positive MUTA-CHROMOPLATE response.« less

Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents.

PubMed

Altshuler, Hallie; Roy, Reena

2015-11-01

DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates. Each of the six non-FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents. Amplification was performed using direct and nondirect autosomal and Y-STR kits. Autosomal and Y-STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents. The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes. © 2015 American Academy of Forensic Sciences.

Evaluation of InnoTyper® 21 in a sample of Rio de Janeiro population as an alternative forensic panel.

PubMed

Moura-Neto, R S; Mello, I C T; Silva, R; Maette, A P C; Bottino, C G; Woerner, A; King, J; Wendt, F; Budowle, B

2018-01-01

The use of bi-allelic markers such as retrotransposable element insertion polymorphisms or Innuls (for insertion/null) can overcome some limitations of short tandem repeat (STR) loci in typing forensic biological evidence. This study investigated the efficiency of the InnoTyper® 21 Innul markers in an urban admixed population sample in Rio de Janeiro (n = 40) and one highly compromised sample collected as evidence by the Rio de Janeiro police. No significant departures from Hardy-Weinberg equilibrium were detected after the Bonferroni correction (α' ≈ 0.05/20, p < 0.0025), and no significant linkage disequilibrium was observed between markers. Assuming loci independence, the cumulative random match probability (RMP) was 2.3 × 10 -8 . A lower mean Fis value was obtained for this sample population compared with those of three North American populations (African-American, Southwest Hispanic, US Caucasian). Principal component analysis with the three North American populations and one from 21 East Asian population showed that African Americans segregated as an independent group while US Caucasian, Southwest Hispanic, East Asian, and Rio de Janeiro populations are in a single large heterogeneous group. Also, a full Innuls profile was produced from an evidence sample, despite the DNA being highly degraded. In conclusion, this system is a useful complement to standard STR kits.

Reduced weight decontamination formulation for neutralization of chemical and biological warfare agents

DOEpatents

Tucker, Mark D.

2014-06-03

A reduced weight DF-200 decontamination formulation that is stable under high temperature storage conditions. The formulation can be pre-packed as an all-dry (i.e., no water) or nearly-dry (i.e., minimal water) three-part kit, with make-up water (the fourth part) being added later in the field at the point of use.

Undergraduate Virology Exercises Demonstrate Conventional and Real-Time PCR Using Commercially Available HIV Primers and Noninfectious Target

ERIC Educational Resources Information Center

Sulzinski, Michael A.; Wasilewski, Melissa A.; Farrell, James C.; Glick, David L.

2009-01-01

It is an extraordinary challenge to offer an undergraduate laboratory course in virology that teaches hands-on, relevant molecular biology techniques using nonpathogenic models of human virus detection. To our knowledge, there exists no inexpensive kits or reagent sets that are appropriate for demonstrating real-time PCR (RT-PCR) in an…

Methods of biological dosimetry employing chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2000-01-01

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

BRISK--research-oriented storage kit for biology-related data.

PubMed

Tan, Alan; Tripp, Ben; Daley, Denise

2011-09-01

In genetic science, large-scale international research collaborations represent a growing trend. These collaborations have demanding and challenging database, storage, retrieval and communication needs. These studies typically involve demographic and clinical data, in addition to the results from numerous genomic studies (omics studies) such as gene expression, eQTL, genome-wide association and methylation studies, which present numerous challenges, thus the need for data integration platforms that can handle these complex data structures. Inefficient methods of data transfer and access control still plague research collaboration. As science becomes more and more collaborative in nature, the need for a system that adequately manages data sharing becomes paramount. Biology-Related Information Storage Kit (BRISK) is a package of several web-based data management tools that provide a cohesive data integration and management platform. It was specifically designed to provide the architecture necessary to promote collaboration and expedite data sharing between scientists. The software, documentation, Java source code and demo are available at http://genapha.icapture.ubc.ca/brisk/index.jsp. BRISK was developed in Java, and tested on an Apache Tomcat 6 server with a MySQL database. denise.daley@hli.ubc.ca.

School Board Policies on Leaves and Absences. Educational Policies Development Kit.

ERIC Educational Resources Information Center

National School Boards Association, Waterford, CT. Educational Policies Service.

This report provides board policy samples and other policy resources on leaves and absences. The intent in providing policy samples is to encourage thinking in policy terms and to provide working papers that can be edited, modified, or adapted to meet local requirements. Topics covered in the samples include (1) sick leave, (2) maternity leave,…

Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria.

PubMed Central

Mills, C. D.; Burgess, D. C.; Taylor, H. J.; Kain, K. C.

1999-01-01

Rapid, accurate and affordable methods are needed for the diagnosis of malaria. Reported here is an evaluation of a new immunochromatographic strip, the PATH Falciparum Malaria IC Strip, which is impregnated with an immobilized IgM monoclonal antibody that binds to the HRP-II antigen of Plasmodium falciparum. In contrast to other commercially available kits marketed for the rapid diagnosis of falciparum malaria, this kit should be affordable in the malaria-endemic world. Using microscopy and polymerase chain reaction (PCR)-based methods as reference standards, we compared two versions of the PATH test for the detection of P. falciparum infection in 200 febrile travellers. As determined by PCR and microscopy, 148 travellers had malaria, 50 of whom (33.8%) were infected with P. falciparum. Compared with PCR, the two versions of the PATH test had initial sensitivities of 90% and 88% and specificities of 97% and 96%, respectively, for the detection of falciparum malaria. When discrepant samples were retested blindly with a modified procedure (increased sample volume and longer washing step) the sensitivity and specificity of both kits improved to 96% and 99%, respectively. The two remaining false negatives occurred in samples with < 100 parasites per microliter of blood. The accuracy, simplicity and predicted low cost may make this test a useful diagnostic tool in malaria-endemic areas. PMID:10444878

Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

PubMed Central

Petrova, Olga E.; Garcia-Alcalde, Fernando; Zampaloni, Claudia; Sauer, Karin

2017-01-01

Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested. PMID:28117413

Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria

PubMed Central

Polley, Spencer D.; González, Iveth J.; Mohamed, Deqa; Daly, Rosemarie; Bowers, Kathy; Watson, Julie; Mewse, Emma; Armstrong, Margaret; Gray, Christen; Perkins, Mark D.; Bell, David; Kanda, Hidetoshi; Tomita, Norihiro; Kubota, Yutaka; Mori, Yasuyoshi; Chiodini, Peter L.; Sutherland, Colin J.

2013-01-01

Background. Diagnosis of malaria relies on parasite detection by microscopy or antigen detection; both fail to detect low-density infections. New tests providing rapid, sensitive diagnosis with minimal need for training would enhance both malaria diagnosis and malaria control activities. We determined the diagnostic accuracy of a new loop-mediated amplification (LAMP) kit in febrile returned travelers. Methods. The kit was evaluated in sequential blood samples from returned travelers sent for pathogen testing to a specialist parasitology laboratory. Microscopy was performed, and then malaria LAMP was performed using Plasmodium genus and Plasmodium falciparum–specific tests in parallel. Nested polymerase chain reaction (PCR) was performed on all samples as the reference standard. Primary outcome measures for diagnostic accuracy were sensitivity and specificity of LAMP results, compared with those of nested PCR. Results. A total of 705 samples were tested in the primary analysis. Sensitivity and specificity were 98.4% and 98.1%, respectively, for the LAMP P. falciparum primers and 97.0% and 99.2%, respectively, for the Plasmodium genus primers. Post hoc repeat PCR analysis of all 15 tests with discrepant results resolved 4 results in favor of LAMP, suggesting that the primary analysis had underestimated diagnostic accuracy. Conclusions. Malaria LAMP had a diagnostic accuracy similar to that of nested PCR, with a greatly reduced time to result, and was superior to expert microscopy. PMID:23633403

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard.

PubMed

Tucker, Valerie C; Hopwood, Andrew J; Sprecher, Cynthia J; McLaren, Robert S; Rabbach, Dawn R; Ensenberger, Martin G; Thompson, Jonelle M; Storts, Douglas R

2011-11-01

In response to the ENFSI and EDNAP groups' call for new STR multiplexes for Europe, Promega(®) developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex(®) ESI 16 (European Standard Investigator 16) and the PowerPlex(®) ESI 17 Systems. The PowerPlex(®) ESI 16 System combines the 11 loci compatible with the UK National DNA Database(®), contained within the AmpFlSTR(®) SGM Plus(®) PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR(®) SGM Plus(®) kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex(®) ESI 17 System amplifies the same loci as the PowerPlex(®) ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR(®) SGM Plus(®) kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex(®) ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54-86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Haplotype data for 23 Y-chromosome markers in four U.S. population groups.

PubMed

Coble, Michael D; Hill, Carolyn R; Butler, John M

2013-05-01

The PowerPlex Y23 kit contains 23 Y-chromosomal loci including all 17 of the markers in the Yfiler Y-STR kit plus six additional markers: DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643. We have typed 1032 unrelated population samples from four self-declared US groups: African Americans, Asians, Hispanics, and Western European Caucasians. An analysis of the population genetic parameters and the improvement of adding additional Y-STR markers to the dataset are described. Published by Elsevier Ireland Ltd.

Advanced systemic mastocytosis: from molecular and genetic progress to clinical practice.

PubMed

Ustun, Celalettin; Arock, Michel; Kluin-Nelemans, Hanneke C; Reiter, Andreas; Sperr, Wolfgang R; George, Tracy; Horny, Hans-Peter; Hartmann, Karin; Sotlar, Karl; Damaj, Gandhi; Hermine, Olivier; Verstovsek, Srdan; Metcalfe, Dean D; Gotlib, Jason; Akin, Cem; Valent, Peter

2016-10-01

Systemic mastocytosis is a heterogeneous disease characterized by the accumulation of neoplastic mast cells in the bone marrow and other organ organs/tissues. Mutations in KIT, most frequently KIT D816V, are detected in over 80% of all systemic mastocytosis patients. While most systemic mastocytosis patients suffer from an indolent disease variant, some present with more aggressive variants, collectively called "advanced systemic mastocytosis", which include aggressive systemic mastocytosis, systemic mastocytosis with an associated hematologic, clonal non mast cell-lineage disease, and mast cell leukemia. Whereas patients with indolent systemic mastocytosis have a near normal life expectancy, patients with advanced systemic mastocytosis have a reduced life expectancy. Although cladribine and interferon-alpha are of benefit in a group of patients with advanced systemic mastocytosis, no curative therapy is available for these patients except possible allogeneic hematopoietic stem cell transplantation. Recent studies have also revealed additional somatic defects (apart from mutations in KIT) in a majority of patients with advanced systemic mastocytosis. These include TET2, SRSF2, ASXL1, RUNX1, JAK2, and/or RAS mutations, which may adversely impact prognosis and survival in particular systemic mastocytosis with an associated hematological neoplasm. In addition, several additional signaling molecules involved in the abnormal proliferation of mast cells in systemic mastocytosis have been identified. These advances have led to a better understanding of the biology of advanced systemic mastocytosis and to the development of new targeted treatment concepts. Herein, we review the biology and pathogenesis of advanced systemic mastocytosis, with a special focus on novel molecular findings as well as current and evolving therapeutic options. Copyright© Ferrata Storti Foundation.

Commander De Winne poses for a photo during Air Sampling

NASA Image and Video Library

2009-11-11

ISS021-E-024700 (11 Nov. 2009) --- European Space Agency astronaut Frank De Winne, Expedition 21 commander, uses the Microbial Air Sampler kit (floating freely near De Winne) to obtain microbiology (bacterial & fungal) air samples in the Kibo laboratory of the International Space Station.

Molecular biological characteristics of the recruitment of hematopoietic stem cells from bone marrow niche in chronic myeloid leukemia

PubMed Central

Zhu, Biao; Zhang, Jianbo; Chen, Jiao; Li, Chenglong; Wang, Xiaodong

2015-01-01

Chronic myeloid leukemia (CML) can be contextualized as a disease of unregulated self-renewal of stem cells which exist in a quiescent state and are instructed to differentiate and mobilize to circulation under pathologic circumstances leading to tumor invasion and metastasis. Here we found that matrix metalloproteinase-9 (MMP-9), induced by TGF-β1, upregulated s-KitL and s-ICAM-1, permitting the transfer of c-kit+ hematopoietic stem cells (HSCs) from the quiescent to proliferative niche in CML. Further study showed that this MMP-9 production was raised by CML specific BCR/ABL+ oncogene mediated TGF-β1. Besides, phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was evidenced to govern this stem cell recruitment in CML pathogenesis. Overall, our observations defined a novel critical role for TGF-β1 induced PI3K/Akt/NF-κB signaling pathway in the recruitment of the malignant cells in CML by releasing s-KitL and s-ICAM-1 and this was through a distinct PI3K/Akt/NF-κB signaling pathway. PMID:26722450

Detection of oral HPV infection - Comparison of two different specimen collection methods and two HPV detection methods.

PubMed

de Souza, Marjorie M A; Hartel, Gunter; Whiteman, David C; Antonsson, Annika

2018-04-01

Very little is known about the natural history of oral HPV infection. Several different methods exist to collect oral specimens and detect HPV, but their respective performance characteristics are unknown. We compared two different methods for oral specimen collection (oral saline rinse and commercial saliva kit) from 96 individuals and then analyzed the samples for HPV by two different PCR detection methods (single GP5+/6+ PCR and nested MY09/11 and GP5+/6+ PCR). For the oral rinse samples, the oral HPV prevalence was 10.4% (GP+ PCR; 10% repeatability) vs 11.5% (nested PCR method; 100% repeatability). For the commercial saliva kit samples, the prevalences were 3.1% vs 16.7% with the GP+ PCR vs the nested PCR method (repeatability 100% for both detection methods). Overall the agreement was fair or poor between samples and methods (kappa 0.06-0.36). Standardizing methods of oral sample collection and HPV detection would ensure comparability between future oral HPV studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Correlates of requesting home HIV self-testing kits on online social networks among African-American and Latino men who have sex with men.

PubMed

Chiu, ChingChe J; Young, Sean D

2016-01-01

High levels of HIV stigma are one of the main difficulties in engaging African-American and Latino men who have sex with men (MSM) in HIV testing. The availability of home HIV test and the possibility of self-testing in private may improve uptake and counteract stigma. This paper sought to determine the correlates of requesting home HIV test kits among a sample of MSM social media users. The odds of participants requesting a test kit were significantly associated with using social networks to seek sexual partners (aOR: 2.47, 95% CI: 1.07-6.06) and thinking it is easier to use social networks for seeking sexual partners (1.87, 1.2-3.12), uncertain HIV status (4.29, 1.37-14.4), and having sex under the influence of alcohol (2.46, 1.06-5.77). Participants who had not been tested for more than 6 months were more likely to request a test kit than those who were tested in the past 6 months (2.53, 1.02-6.37). Participants who frequently talked to others about having sex with men online were less likely to request a test kit (0.73, 0.56-0.92). By reaching people over social media and offering them access to test kits, we were able to reach at-risk individuals who were uncertain about their HIV status and had not been regularly tested. The findings of the study will help to inform future HIV testing interventions.

Evaluation of the SD BIOLINE Dengue Duo rapid test in the course of acute and convalescent dengue infections in a Mexican endemic region.

PubMed

Sánchez-Vargas, Luis A; Sánchez-Marce, Elvis E; Vivanco-Cid, Héctor

2014-04-01

In this study, we evaluated the performance of a rapid test, the SD BIOLINE Dengue Duo (SD BDD) kit, with a panel of serum samples from 310 Mexican patients with diagnosis of dengue infection previously confirmed by reference enzyme-linked immunosorbent assay tests. Eighty-seven negative samples from other febrile illnesses were included as controls. The SD BDD showed an overall sensitivity of 90.65% and specificity of 89.66%. No statistically significant differences were found in the sensitivity of the SD BDD kit compared between primary or secondary infections (87.05% versus 93.57%, respectively, P = 0.0761) and dengue fever or dengue hemorrhagic fever cases (90.77% versus 89.74%, respectively, P = 0.7716). However, a higher sensitivity in the acute phase of dengue infection was found compared with the convalescent phase (93.03% versus 81.82%, respectively, P = 0.0089). These results indicate that the SD BDD kit is a useful tool to diagnose dengue infections, both in primary or secondary infections and mainly during the acute phase. Copyright © 2014 Elsevier Inc. All rights reserved.

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

PubMed Central

Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate

2012-01-01

Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (blaKPC) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 105 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship. PMID:23035190

Recognition of Y Fragment Deletion by Genotyping Graphs after Amplified by PowerPlex® 21 Detection Kit.

PubMed

Wang, S C; Ding, M M; Wei, X L; Zhang, T; Yao, F

2016-06-01

To recognize the possibility of Y fragment deletion of Amelogenin gene intuitively and simply according to the genotyping graphs. By calculating the ratio of total peak height of genotyping graphs, the statistics of equilibrium distribution between Amelogenin and D3S1358 loci, Amelogenin X-gene and Amelogenin Y-gene, and different alleles of D3S1358 loci from 1 968 individuals was analyzed after amplified by PowerPlex ® 21 detection kit. Sum of peak height of Amelogenin X allele was not less than 60% that of D3S1358 loci alleles in 90.8% female samples, and sum of peak height of Amelogenin X allele was not higher than 70% that of D3S1358 loci alleles in 94.9% male samples. The result of genotyping after amplified by PowerPlex ® 21 detection kit shows that the possibility of Y fragment deletion should be considered when only Amelogenin X-gene of Amelogenin is detected and the peak height of Amelogenin X-gene is not higher than 70% of the total peak height of D3S1358 loci. Copyright© by the Editorial Department of Journal of Forensic Medicine

[Analysis of the dilution deviation in CA19-9 measurement].

PubMed

Hanada, Hiroyuki; Takeoka, Keiko; Nomura, Tomoko; Moriyama, Takanori; Kanakura, Yuzuru

2005-04-01

CA19-9 widely used as a tumor marker of the pancreas and a bile duct. There are a number of reports which describes the measured value discrepancies between RIA and non-RIA kits. RIA results also have shown lack of the linearity over 70 U/ml when the samples are diluted. The pH condition at assay reaction for RIA had been suggested as the major reason, it has been denied by the results from the same pH condition at assay reaction used by COBAS CORE CA19-9 EIA II. On the other hand, the lack of RIA antibody titer is indicated for the discordant results by changing the sample volume to reagent volume ratio in the reaction. Our further investigation also indicates that the specific Lewis blood type, i.e. Le (a-b+), shows the linearity issues by RIA. The discrepancies are not caused by the reaction pH, but the amount of the antibody used in the RIA kit is closely associated. Considering the CA19-9 antibody nature used in RIA kit, which covers broad molecular range, users need to pay more attention to setting up each laboratory's measuring range.