Direct Y-STR amplification of body fluids deposited on commonly found crime scene substrates.

PubMed

Dargay, Amanda; Roy, Reena

2016-04-01

Body fluids detected on commonly found crime scene substrates require extraction, purification and quantitation of DNA prior to amplification and generation of short tandem repeat (STR) DNA profiles. In this research Y-STR profiles were generated via direct amplification of blood and saliva deposited on 12 different substrates. These included cigarette butts, straws, grass, leaves, woodchips and seven different types of fabric. After depositing either 0.1 μL of blood or 0.5 μL of saliva, each substrate containing the dry body fluid stain was punched using a Harris 1.2 mm micro-punch. Each of these punched substrates, a total of 720 samples, containing minute amount of blood or saliva was either amplified directly without any pre-treatment, or was treated with one of the four washing reagents or buffer. In each of these five experimental groups the substrates containing the body fluid remained in the amplification reagent during the thermal cycling process. Each sample was amplified with the three direct Y-STR amplification kits; AmpFℓSTR(®) Yfiler(®) Direct, Yfiler(®) Plus Amplification Kits and the PowerPlex(®) Y23 System. Complete and concordant Y-STR profiles were successfully obtained from most of these 12 challenging crime scene objects when the stains were analyzed by at least one of the five experimental groups. The reagents and buffer were interchangeable among the three amplification kits, however, pre-treatment with these solutions did not appear to enhance the quality or the number of the full profiles generated with direct amplification. This study demonstrates that blood and saliva deposited on these simulated crime scene objects can be amplified directly. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. 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.

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.

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.

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.

Comparison of manual and semi-automatic DNA extraction protocols for the barcoding characterization of hematophagous louse flies (Diptera: Hippoboscidae).

PubMed

Gutiérrez-López, Rafael; Martínez-de la Puente, Josué; Gangoso, Laura; Soriguer, Ramón C; Figuerola, Jordi

2015-06-01

The barcoding of life initiative provides a universal molecular tool to distinguish animal species based on the amplification and sequencing of a fragment of the subunit 1 of the cytochrome oxidase (COI) gene. Obtaining good quality DNA for barcoding purposes is a limiting factor, especially in studies conducted on small-sized samples or those requiring the maintenance of the organism as a voucher. In this study, we compared the number of positive amplifications and the quality of the sequences obtained using DNA extraction methods that also differ in their economic costs and time requirements and we applied them for the genetic characterization of louse flies. Four DNA extraction methods were studied: chloroform/isoamyl alcohol, HotShot procedure, Qiagen DNeasy(®) Tissue and Blood Kit and DNA Kit Maxwell(®) 16LEV. All the louse flies were morphologically identified as Ornithophila gestroi and a single COI-based haplotype was identified. The number of positive amplifications did not differ significantly among DNA extraction procedures. However, the quality of the sequences was significantly lower for the case of the chloroform/isoamyl alcohol procedure with respect to the rest of methods tested here. These results may be useful for the genetic characterization of louse flies, leaving most of the remaining insect as a voucher. © 2015 The Society for Vector Ecology.

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.

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.

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

A Y-chromosome STR marker should be added to commercial multiplex STR kits.

PubMed

Oz, Carla; Zaken, Neomi; Amiel, Merav; Zamir, Ashira

2008-07-01

Autosomal short tandem repeat (STR) analysis has become highly relevant in the identification of victims from mass disasters and terrorist attacks. In such events, gender misidentification can be of grave consequences, yet the list reporting amelogenin amplification failure using STR multiplex kits continues to grow. Presented here are three such examples. In the first case, we present two male suspects who demonstrated amelogenin Y-deficient results using two commercial kit procedures. The presence of their Y chromosomes was proven by obtaining a Y-haplotype. The second case demonstrated a profile from a third male suspect where only the Y homolog of the XY pair was amplified. In events such as mass disasters or terrorist attacks, timely and reliable high throughput DNA typing results are essential. As the number of reported cases of amplification failure at the amelogenin gene continues to grow, we suggest that the incorporation of a better gender identification tool in commercial kits is crucial.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms.

PubMed

Chen, DaYang; Zhen, HeFu; Qiu, Yong; Liu, Ping; Zeng, Peng; Xia, Jun; Shi, QianYu; Xie, Lin; Zhu, Zhu; Gao, Ya; Huang, GuoDong; Wang, Jian; Yang, HuanMing; Chen, Fang

2018-03-21

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Increased Sensitivity of HIV-1 p24 ELISA Using a Photochemical Signal Amplification System.

PubMed

Bystryak, Simon; Santockyte, Rasa

2015-10-01

In this study we describe a photochemical signal amplification method (PSAM) for increasing of the sensitivity of enzyme-linked immunosorbent assay (ELISA) for determination of HIV-1 p24 antigen. The photochemical signal amplification method is based on an autocatalytic photochemical reaction of a horseradish peroxidase (HRP) substrate, orthophenylenediamine (OPD). To compare the performance of PSAM-boosted ELISA with a conventional colorimetric ELISA for determination of HIV-1 p24 antigen we employed a PerkinElmer HIV-1 p24 ELISA kit, using conventional ELISA alongside ELISA + PSAM. In the present study, we show that PSAM technology allows one to increase the analytical sensitivity and dynamic range of a commercial HIV-1 p24 ELISA kit, with and without immune-complex disruption, by a factor of approximately 40-fold. ELISA + PSAM is compatible with commercially available microtiter plate readers, requires only an inexpensive illumination device, and the PSAM amplification step takes no longer than 15 min. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods.

Forensic and population genetic analyses of Danes, Greenlanders and Somalis typed with the Yfiler® Plus PCR amplification kit.

PubMed

Olofsson, Jill Katharina; Mogensen, Helle Smidt; Buchard, Anders; Børsting, Claus; Morling, Niels

2015-05-01

Recently, the Yfiler® Plus PCR Amplification Kit (Yfiler® Plus, Thermo Fisher Scientific, Waltham, MA, USA) was introduced. Yfiler® Plus amplifies 27 Y-chromosomal short tandem repeat loci (Y-STRs) and adds ten new Y-STRs to those analysed with the commonly used AmpFlSTR® Yfiler® PCR Amplification Kit (Yfiler®, Thermo Fisher Scientific, Waltham, MA, USA). Seven of the new Y-STRs are rapidly mutating Y-STRs (RM Y-STRs). In this study, 551 male individuals from Denmark, Greenland and Somalia were typed with Yfiler® Plus. The results were compared to those obtained with Yfiler® in the same individuals. Forensic and population genetic parameters were estimated for Yfiler® Plus. Yfiler® Plus had a higher power of discrimination than Yfiler® in all three populations. Compared to Yfiler®, Yfiler® Plus offers increased power of discrimination, which is obviously an advantage in crime case investigations. However, the inclusion of seven RM Y-STRs in Yfiler® Plus makes it less attractive for relationship testing because of the relatively high combined mutation rate, approximately 15%. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification and Identification of Deletion Genetic Subtypes in Prader-Willi Syndrome

PubMed Central

Henkhaus, Rebecca S.; Kim, Soo-Jeong; Kimonis, Virginia E.; Gold, June-Anne; Dykens, Elisabeth M.; Driscoll, Daniel J.

2012-01-01

Purpose: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are complex neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region depending on the parent of origin. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) kits from MRC-Holland (Amsterdam, The Netherlands) were used to detect PWS and AS deletion subtypes. We report our experience with two versions of the MS-MLPA-PWS/AS kit (original A1 and newer B1) in determining methylation status and deletion subtypes in individuals with PWS. Methods: MS-MLPA analysis was performed on DNA isolated from a large cohort of PWS subjects with the MS-MLPA-PWS/AS-A1 and -B1 probe sets. Results: Both MS-MLPA kits will identify deletions in the 15q11-q13 region but the original MS-MLPA-A1 kit has a higher density of probes at the telomeric end of the 15q11-q13 region, which is more useful for identifying individuals with atypical deletions. The newer B1 kit contains more probes in the imprinting center (IC) and adjoining small noncoding RNAs useful in identifying small microdeletions. Conclusion: The A1 kit identified the typical deletions and smaller atypical deletions, whereas the B1 kit was more informative for identifying microdeletions including the IC and SNORD116 regions. Both kits should be made available for accurate characterization of PWS/AS deletion subtypes as well as evaluating for IC and SNORD116 microdeletions. PMID:21977908

Loop-mediated isothermal amplification with the Procedure for Ultra Rapid Extraction kit for the diagnosis of pneumocystis pneumonia.

PubMed

Kawano, Shuichi; Maeda, Takuya; Suzuki, Takefumi; Abe, Tatsuhiro; Mikita, Kei; Hamakawa, Yusuke; Ono, Takeshi; Sonehara, Wataru; Miyahira, Yasushi; Kawana, Akihiko

2015-03-01

Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies/tube, even with the use of massive blood-contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A comprehensive Y-STR portrait of Yousafzai's population.

PubMed

Tabassum, Sadia; Ilyas, Muhammad; Ullah, Inam; Israr, Muhammad; Ahmad, Habib

2017-09-01

In the current study, 17 Y-Chromosomal short tandem repeats (Y-STRs) included in theAmpFlSTR Y-Filer amplification kit (Applied Biosystems, Foster City, USA) were investigated in 146 unrelated Yousafzai males residing in the Khyber Pakhtunkhwa Province of Pakistan. A total of 94 (89.52%) unique haplotypes were observed. Discrimination capacity was 71.92%. Haplotype diversity ranged from 0.354 (DYS456) to 0.663 (DYS458). Both Rst pairwise analysis and multidimensional scaling plot showed that the genetic structure of the Yousafzais is significantly different from neighbouring populations.

Identification of Clinical Coryneform Bacterial Isolates: Comparison of Biochemical Methods and Sequence Analysis of 16S rRNA and rpoB Genes▿

PubMed Central

Adderson, Elisabeth E.; Boudreaux, Jan W.; Cummings, Jessica R.; Pounds, Stanley; Wilson, Deborah A.; Procop, Gary W.; Hayden, Randall T.

2008-01-01

We compared the relative levels of effectiveness of three commercial identification kits and three nucleic acid amplification tests for the identification of coryneform bacteria by testing 50 diverse isolates, including 12 well-characterized control strains and 38 organisms obtained from pediatric oncology patients at our institution. Between 33.3 and 75.0% of control strains were correctly identified to the species level by phenotypic systems or nucleic acid amplification assays. The most sensitive tests were the API Coryne system and amplification and sequencing of the 16S rRNA gene using primers optimized for coryneform bacteria, which correctly identified 9 of 12 control isolates to the species level, and all strains with a high-confidence call were correctly identified. Organisms not correctly identified were species not included in the test kit databases or not producing a pattern of reactions included in kit databases or which could not be differentiated among several genospecies based on reaction patterns. Nucleic acid amplification assays had limited abilities to identify some bacteria to the species level, and comparison of sequence homologies was complicated by the inclusion of allele sequences obtained from uncultivated and uncharacterized strains in databases. The utility of rpoB genotyping was limited by the small number of representative gene sequences that are currently available for comparison. The correlation between identifications produced by different classification systems was poor, particularly for clinical isolates. PMID:18160450

[DNA analysis of chromosome Y in the area of the azoospermia factor (AZF) in infertile men].

PubMed

Kolárová, J; Santavá, A; Vrtĕl, R

2001-09-01

Establishment of investigation of sterile male DNA in AZF region--choice of loci and primers for investigation, optimization of PCR conditions (polymerase chain reaction). For practice. Department of Medical Genetics and Foetal Medicine, Faculty of Medicine, Palacky University and Faculty Hospital Olomouc. PCR amplification of DNA isolated from blood of sterile men and consequential electrophoresis of synthesized DNA fragments to appoint microdeletions in AZF. From January to June 2000 were detected the microdeletions in AZF of 3 out of 79 sterile men (3.80%) by means of the Experteam firm kit. From July to December 2000 were tested and optimized conditions of amplification of 10 AZF loci to substitute the kit and they were used for examination of the first 20 sterile men of our collection. In our laboratory was established routine examination male sterility related to microdeletions in AZF. With our collection of loci was substituted the original Experteam firm kit and was widened spectrum of observed loci.

High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma.

PubMed

Bernheim, Alain; Toujani, Saloua; Saulnier, Patrick; Robert, Thomas; Casiraghi, Odile; Validire, Pierre; Temam, Stéphane; Menard, Philippe; Dessen, Philippe; Fouret, Pierre

2008-05-01

Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.

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.

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

PubMed

Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William

2011-06-01

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Typeability of DNA in Touch Traces Deposited on Paper and Optical Data Discs.

PubMed

Sołtyszewski, Ireneusz; Szeremeta, Michał; Skawrońska, Małgorzata; Niemcunowicz-Janica, Anna; Pepiński, Witold

2015-01-01

Nucleated epithelial cells that are transferred by casual touching and handling of objects are the primary source of biological evidence that is found in high-volume crimes. Cellular material associated with touch traces usually contains low levels of DNA template making it challenging to acquire an informative profile. The main purpose of this study was to examine the efficacy of DNA typing in fingerprints deposited on optical data discs and the office paper. Latent fingerprints were made by 60 subjects of both sexes (30 males and 30 females). A highly effective DNA extraction method with QIAamp DNA Mini Kit (Qiagen) and an increased sensitivity PCR by AmpFlSTR® NGM™ Amplification Kit (Applied Biosystems) carried out at standard 30 cycles and at increased 34 cycles were used. The mean value of total DNA recovery was 0.4 ng from CDs/DVDs and 0.3 ng from the office paper. Amplification of Low Template DNA (LT-DNA) resulted in improved analytical success by increasing the number of PCR cycles from standard 30 to 34. On the other hand, the increased PCR cycles resulted in allele drop-ins showing additional peaks, the majority of which were outside the stutter positions. Rigorous procedures and interpretation guidelines are required during LT-DNA for producing reliable and reproducible DNA profiles for forensic purposes.

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.

[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

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.

Development of a highly sensitive immunochromatographic detection kit for H5 influenza virus hemagglutinin using silver amplification.

PubMed

Wada, Atsuhiko; Sakoda, Yoshihiro; Oyamada, Takayoshi; Kida, Hiroshi

2011-12-01

H5N1, a highly pathogenic avian influenza virus (HPAIV), has become a serious epizootic threat to the poultry population in Asia. In addition, significant numbers of human cases of HPAIV infection have been reported to date. To prevent the spread of HPAIV among humans and to allow for timely medical intervention, a rapid and high sensitive method is needed to detect and subtype the causative HPAIVs. In the present study, a silver amplification technique used in photographic development was combined with immunochromatography technologies and a highly sensitive and rapid diagnostic test to detect the hemagglutinin of H5 influenza viruses was developed. The sensitivity of the test kit was increased 500 times by silver amplification. The sensitivity of the method was more than 10 times higher than those of conventional rapid influenza diagnostic tests, which detect viral nucleoproteins. The diagnostic system developed in the present study can therefore provide rapid and highly sensitive results and will be useful for diagnosis of H5 HPAIV infection in humans and animals. Copyright © 2011 Elsevier B.V. All rights reserved.

KIT gene mutations and patterns of protein expression in mucosal and acral melanoma.

PubMed

Abu-Abed, Suzan; Pennell, Nancy; Petrella, Teresa; Wright, Frances; Seth, Arun; Hanna, Wedad

2012-01-01

Recently characterized KIT (CD117) gene mutations have revealed new pathways involved in melanoma pathogenesis. In particular, certain subtypes harbor mutations similar to those observed in gastrointestinal stromal tumors, which are sensitive to treatment with tyrosine kinase inhibitors. The purpose of this study was to characterize KIT gene mutations and patterns of protein expression in mucosal and acral melanoma. Formalin-fixed, paraffin-embedded tissues were retrieved from our archives. Histologic assessment included routine hematoxylin-eosin stains and immunohistochemical staining for KIT. Genomic DNA was used for polymerase chain reaction-based amplification of exons 11 and 13. We identified 59 acral and mucosal melanoma cases, of which 78% showed variable levels of KIT expression. Sequencing of exons 11 and 13 was completed on all cases, and 4 (6.8%) mutant cases were isolated. We successfully optimized conditions for the detection of KIT mutations and showed that 8.6% of mucosal and 4.2% of acral melanoma cases at our institution harbor KIT mutations; all mutant cases showed strong, diffuse KIT protein expression. Our case series represents the first Canadian study to characterize KIT gene mutations and patterns of protein expression in acral and mucosal melanoma.

Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: endogenous streptavidin-binding sites lead to false positive staining.

PubMed

Horling, L; Neuhuber, W L; Raab, M

2012-02-15

Highly sensitive immunohistochemical detection systems such as tyramide signal amplification (TSA) are widely used, since they allow using two primary antibodies raised in the same species. Most of them are based on the streptavidin-biotin-peroxidase system and include streptavidin-coupled secondary antibodies. Using TSA in cryostat-sectioned tissues of mouse esophagus, we were puzzled by negative controls with unexpected staining mostly in the ganglionic areas. This prompted us to search for the causing agent and to include also other parts of the mouse gastrointestinal tract for comparison. Streptavidin-coupled antibodies bound to endogenous binding sites yet to be characterized, which are present throughout the mouse intestines. Staining was mainly localized around neuronal cell bodies of enteric ganglia. Thus, caution is warranted when applying streptavidin-coupled antibodies in the mouse gastrointestinal tract. The use of endogenous biotin-blocking kits combined with a prolonged post-fixation time could significantly reduce unintentional staining. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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

Validation of a fully automated HER2 staining kit in breast cancer.

PubMed

Moelans, Cathy B; Kibbelaar, Robby E; van den Heuvel, Marius C; Castigliego, Domenico; de Weger, Roel A; van Diest, Paul J

2010-01-01

Testing for HER2 amplification and/or overexpression is currently routine practice to guide Herceptin therapy in invasive breast cancer. At present, HER2 status is most commonly assessed by immunohistochemistry (IHC). Standardization of HER2 IHC assays is of utmost clinical and economical importance. At present, HER2 IHC is most commonly performed with the HercepTest which contains a polyclonal antibody and applies a manual staining procedure. Analytical variability in HER2 IHC testing could be diminished by a fully automatic staining system with a monoclonal antibody. 219 invasive breast cancers were fully automatically stained with the monoclonal antibody-based Oracle HER2 Bond IHC kit and manually with the HercepTest. All cases were tested for amplification with chromogenic in situ hybridization (CISH). HercepTest yielded an overall sharper membrane staining, with less cytoplasmic and stromal background than Oracle in 17% of cases. Overall concordance between both IHC techniques was 89% (195/219) with a kappa value of 0.776 (95% CI 0.698-0.854), indicating a substantial agreement. Most (22/24) discrepancies between HercepTest and Oracle showed a weaker staining for Oracle. Thirteen of the 24 discrepant cases were high-level HER2 amplified by CISH, and in 12 of these HercepTest IHC better reflected gene amplification status. All the 13 HER2 amplified discrepant cases were at least 2+ by HercepTest, while 10/13 of these were at least 2+ for Oracle. Considering CISH as gold standard, sensitivity of HercepTest and Oracle was 91% and 83%, and specificity was 94% and 98%, respectively. Positive and negative predictive values for HercepTest and Oracle were 90% and 95% for HercepTest and 96% and 91% for Oracle, respectively. Fully-automated HER2 staining with the monoclonal antibody in the Oracle kit shows a high level of agreement with manual staining by the polyclonal antibody in the HercepTest. Although Oracle shows in general some more cytoplasmic staining and may be slightly less sensitive in picking up HER2 amplified cases, it shows a higher specificity and may be considered as an alternative method to evaluate the HER2 expression in breast cancer with potentially less analytical variability.

Whole genome amplification of DNA extracted from FFPE tissues.

PubMed

Bosso, Mira; Al-Mulla, Fahd

2011-01-01

Whole genome amplification systems were developed to meet the increasing research demands on DNA resources and to avoid DNA shortage. The technology enables amplification of nanogram amounts of DNA into microgram quantities and is increasingly used in the amplification of DNA from multiple origins such as blood, fresh frozen tissue, formalin-fixed paraffin-embedded tissues, saliva, buccal swabs, bacteria, and plant and animal sources. This chapter focuses on the use of GenomePlex(®) tissue Whole Genome Amplification Kit, to amplify DNA directly from archived tissue. In addition, this chapter documents our unique experience with the utilization of GenomePlex(®) amplified DNA using several molecular techniques including metaphase Comparative Genomic Hybridization, array Comparative Genomic Hybridization, and real-time quantitative polymerase chain reaction assays. GenomePlex(®) is a registered trademark of Rubicon Genomics Incorporation.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

PubMed

Lodh, Nilanjan; Mikita, Kei; Bosompem, Kwabena M; Anyan, William K; Quartey, Joseph K; Otchere, Joseph; Shiff, Clive J

2017-09-01

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

E-Cadherin as a Chemotherapy Resistance Mechanism on Metastatic Breast Cancer

DTIC Science & Technology

2011-01-01

Gold Kit (Zymo, San Diego, CA) per the manufacturer’s specifications. MSP was performed in the way of Corn et al [62] or using the CpG WIZ E-cadherin...Amplification Kit per the manufacturer’s instructions (Millipore, Temecula, CA). Briefly, in the method of Corn , a nested PCR method was used, in...cadherin gene promoter methylation in prostatic adenocarcinomas. Cancer 92(11): 2786-95. 29. Corn , PG, BD Smith, ES Ruckdeschel et al (2000) E-cadherin

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.

Diagnostic testing for Giardia infections.

PubMed

Heyworth, Martin F

2014-03-01

The traditional method for diagnosing Giardia infections involves microscopic examination of faecal specimens for Giardia cysts. This method is subjective and relies on observer experience. From the 1980s onwards, objective techniques have been developed for diagnosing Giardia infections, and are superseding diagnostic techniques reliant on microscopy. Detection of Giardia antigen(s) by immunoassay is the basis of commercially available diagnostic kits. Various nucleic acid amplification techniques (NAATs) can demonstrate DNA of Giardia intestinalis, and have the potential to become standard approaches for diagnosing Giardia infections. Of such techniques, methods involving either fluorescent microspheres (Luminex) or isothermal amplification of DNA (loop-mediated isothermal amplification; LAMP) are especially promising.

[Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens].

PubMed

Drouillon, V; Houriez, F; Buze, M; Lagrange, P; Herrmann, J-L

2006-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

PubMed

Cornelissen, Marion; Gall, Astrid; Vink, Monique; Zorgdrager, Fokla; Binter, Špela; Edwards, Stephanie; Jurriaans, Suzanne; Bakker, Margreet; Ong, Swee Hoe; Gras, Luuk; van Sighem, Ard; Bezemer, Daniela; de Wolf, Frank; Reiss, Peter; Kellam, Paul; Berkhout, Ben; Fraser, Christophe; van der Kuyl, Antoinette C

2017-07-15

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml. Copyright © 2016 The Authors. Published by 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.

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms.

PubMed

Wong, Y-P; Othman, S; Lau, Y-L; Radu, S; Chee, H-Y

2018-03-01

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease. © 2017 The Society for Applied Microbiology.

The effects of variable sample biomass on comparative metagenomics.

PubMed

Chafee, Meghan; Maignien, Loïs; Simmons, Sheri L

2015-07-01

Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

PubMed Central

Dayton, Melody; Koskinen, Mikko T; Tom, Bradley K; Mattila, Anna-Maria; Johnston, Eric; Halverson, Joy; Fantin, Dennis; DeNise, Sue; Budowle, Bruce; Smith, David Glenn; Kanthaswamy, Sree

2009-01-01

Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species-specificity, case type samples analyses and population studies. Results The kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. Conclusion The kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study’s evaluation to comply with the quality standards expected for forensic casework. PMID:19480022

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.

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.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

PubMed

Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

2015-01-01

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

KIT pathway alterations in mucosal melanomas of the vulva and other sites.

PubMed

Omholt, Katarina; Grafström, Eva; Kanter-Lewensohn, Lena; Hansson, Johan; Ragnarsson-Olding, Boel K

2011-06-15

A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens. Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry. KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis. Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. ©2011 AACR.

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.

Validation of a commercial kit aimed to the detection of pathogenic anisakid nematodes in fish products.

PubMed

Cavallero, Serena; Bruno, Alessandro; Arletti, Enrico; Caffara, Monica; Fioravanti, Maria Letizia; Costa, Antonella; Cammilleri, Gaetano; Graci, Stefania; Ferrantelli, Vincenzo; D'Amelio, Stefano

2017-09-18

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005. Copyright © 2017 Elsevier B.V. All rights reserved.

The Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification

DTIC Science & Technology

2014-11-01

using the CHEF Genomic DNA Plug Kit (Biorad), following the manufacturer’s instructions. Briefly, after the labeling, cells were washed twice with...with BD-CellQuest software. What opportunities for training and professional development has the project provided? During the funded project period I

Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems.

PubMed

Yao, Yining; Yang, Qinrui; Shao, Chengchen; Liu, Baonian; Zhou, Yuxiang; Xu, Hongmei; Zhou, Yueqin; Tang, Qiqun; Xie, Jianhui

2018-01-01

Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex ® 21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR ® . Identifiler ® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex ® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex.

PubMed

Matray, Olivier; Mouhajir, Abdelmounaim; Giraud, Sandrine; Godon, Charlotte; Gargala, Gilles; Labbé, Franck; Rougeron, Amandine; Ballet, Jean-Jacques; Zouhair, Rachid; Bouchara, Jean-Philippe; Favennec, Loïc

2016-05-01

The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF). © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

PubMed Central

Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier

2013-01-01

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779

Selective gene amplification to detect the T790M mutation in plasma from patients with advanced non-small cell lung cancer (NSCLC) who have developed epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance.

PubMed

Nishikawa, Shingo; Kimura, Hideharu; Koba, Hayato; Yoneda, Taro; Watanabe, Satoshi; Sakai, Tamami; Hara, Johsuke; Sone, Takashi; Kasahara, Kazuo; Nakao, Shinji

2018-03-01

The epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, tissues for the genotyping of the EGFR T790M mutation can be difficult to obtain in a clinical setting. The aims of this study were to evaluate a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients using the PointMan™ EGFR DNA enrichment kit, which is a novel method for the selective amplification of specific genotype sequences. Blood samples were collected from NSCLC patients who had activating EGFR mutations and who were resistant to EGFR-TKI treatment. Using cell-free DNA (cfDNA) from plasma, EGFR T790M mutations were amplified using the PointMan™ enrichment kit, and all the reaction products were confirmed using direct sequencing. The concentrations of plasma DNA were then determined using quantitative real-time PCR. Nineteen patients were enrolled, and 12 patients (63.2%) were found to contain EGFR T790M mutations in their cfDNA, as detected by the kit. T790M mutations were detected in tumor tissues in 12 cases, and 11 of these cases (91.7%) also exhibited the T790M mutation in cfDNA samples. The concentrations of cfDNA were similar between patients with the T790M mutation and those without the mutation. The PointMan™ kit provides a useful method for determining the EGFR T790M mutation status in cfDNA.

Development and evaluation of a rapid recombinase polymerase amplification assay for detection of coxsackievirus A6.

PubMed

Wang, Kaifeng; Wu, Yue; Yin, Dan; Tang, Shixing; Hu, Guifang; He, Yaqing

2017-01-01

Coxsackievirus A6 (CV-A6) is an important pathogen causing hand, foot and mouth disease (HFMD). The aim of this study was to develop and evaluate a rapid real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of CV-A6. The sensitivity of this assay was 202 copies/reaction, with 100 % specificity. Furthermore, this assay yielded consistent results comparable with a commercial qRT-PCR diagnostic kit. This assay is therefore potentially useful for surveillance of CV-A6 infections and outbreak control.

Growth control of genetically modified cells using an antibody/c-Kit chimera.

PubMed

Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

2012-05-01

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Optimal Design of a Traveling-Wave Kinetic Inductance Amplifier Operated in Three-Wave Mixing Mode

NASA Astrophysics Data System (ADS)

Erickson, Robert; Bal, Mustafa; Ku, Ksiang-Sheng; Wu, Xian; Pappas, David

In the presence of a DC bias, an injected pump, of frequency fP, and a signal, of frequency fS, undergo parametric three-way mixing (3WM) within a traveling-wave kinetic inductance (KIT) amplifier, producing an idler product of frequency fI =fP -fS . Periodic frequency stops are engineered into the coplanar waveguide of the device to enhance signal amplification. With fP placed just above the first frequency stop gap, 3WM broadband signal gain is achieved with maximum gain at fS =fP / 2 . Within a theory of the dispersion of traveling waves in the presence of these engineered loadings, which accounts for this broadband signal gain, we show how an optimal frequency-stop design may be constructed to achieve maximum signal amplification. The optimization approach we describe can be applied to the design of other nonlinear traveling-wave parametric amplifiers. This work was supported by the Army Research Office and the Laboratory for Physical Sciences under EAO221146, EAO241777, and the NIST Quantum Initiative. RPE acknowledges Grant 60NANB14D024 from the US Department of Commerce, NIST.

40 CFR 1054.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 33 2014-07-01 2014-07-01 false What special provisions apply for installing and removing altitude kits? 1054.655 Section 1054.655 Protection of Environment ENVIRONMENTAL... installing and removing altitude kits? An action for the purpose of installing or modifying altitude kits and...

40 CFR 1054.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 34 2012-07-01 2012-07-01 false What special provisions apply for installing and removing altitude kits? 1054.655 Section 1054.655 Protection of Environment ENVIRONMENTAL... installing and removing altitude kits? An action for the purpose of installing or modifying altitude kits and...

40 CFR 1054.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 34 2013-07-01 2013-07-01 false What special provisions apply for installing and removing altitude kits? 1054.655 Section 1054.655 Protection of Environment ENVIRONMENTAL... installing and removing altitude kits? An action for the purpose of installing or modifying altitude kits and...

40 CFR 1054.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 32 2010-07-01 2010-07-01 false What special provisions apply for installing and removing altitude kits? 1054.655 Section 1054.655 Protection of Environment ENVIRONMENTAL... installing and removing altitude kits? An action for the purpose of installing or modifying altitude kits and...

40 CFR 1054.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 33 2011-07-01 2011-07-01 false What special provisions apply for installing and removing altitude kits? 1054.655 Section 1054.655 Protection of Environment ENVIRONMENTAL... installing and removing altitude kits? An action for the purpose of installing or modifying altitude kits and...

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.

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors.

PubMed

Chen, Weicai; Kuang, Ye; Qiu, Hai-Bo; Cao, Zhifa; Tu, Yuqing; Sheng, Qing; Eilers, Grant; He, Quan; Li, Hai-Long; Zhu, Meijun; Wang, Yuexiang; Zhang, Rongqing; Wu, Yeqing; Meng, Fanguo; Fletcher, Jonathan A; Ou, Wen-Bin

2017-09-15

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR . ©2017 American Association for Cancer Research.

DNA analysis of molluscs from a museum wet collection: a comparison of different extraction methods.

PubMed

Jaksch, Katharina; Eschner, Anita; Rintelen, Thomas V; Haring, Elisabeth

2016-07-18

DNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S). Overall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did. Museum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The difference between extraction methods was minimal and we recommend using an established kit for a first attempt because experience and routine in handling might be more important than slight performance differences of the various kits. Finally, we identify fixation, storage, sample conservation and documentation of the specimens' history rather than the DNA extraction method to be the most crucial factors for PCR success.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

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.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

PubMed

Xu, Ruoyang; Shieh, Y Carol; Stewart, Diana S

2017-01-01

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization. Published by Elsevier B.V.

An Ultra-High Discrimination Y Chromosome Short Tandem Repeat Multiplex DNA Typing System

PubMed Central

Hanson, Erin K.; Ballantyne, Jack

2007-01-01

In forensic casework, Y chromosome short tandem repeat markers (Y-STRs) are often used to identify a male donor DNA profile in the presence of excess quantities of female DNA, such as is found in many sexual assault investigations. Commercially available Y-STR multiplexes incorporating 12–17 loci are currently used in forensic casework (Promega's PowerPlex® Y and Applied Biosystems' AmpFlSTR® Yfiler®). Despite the robustness of these commercial multiplex Y-STR systems and the ability to discriminate two male individuals in most cases, the coincidence match probabilities between unrelated males are modest compared with the standard set of autosomal STR markers. Hence there is still a need to develop new multiplex systems to supplement these for those cases where additional discriminatory power is desired or where there is a coincidental Y-STR match between potential male participants. Over 400 Y-STR loci have been identified on the Y chromosome. While these have the potential to increase the discrimination potential afforded by the commercially available kits, many have not been well characterized. In the present work, 91 loci were tested for their relative ability to increase the discrimination potential of the commonly used ‘core’ Y-STR loci. The result of this extensive evaluation was the development of an ultra high discrimination (UHD) multiplex DNA typing system that allows for the robust co-amplification of 14 non-core Y-STR loci. Population studies with a mixed African American and American Caucasian sample set (n = 572) indicated that the overall discriminatory potential of the UHD multiplex was superior to all commercial kits tested. The combined use of the UHD multiplex and the Applied Biosystems' AmpFlSTR® Yfiler® kit resulted in 100% discrimination of all individuals within the sample set, which presages its potential to maximally augment currently available forensic casework markers. It could also find applications in human evolutionary genetics and genetic genealogy. PMID:17668066

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

PubMed

Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

2016-08-01

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Sensitive and reliable detection of Kit point mutation Asp 816 to Val in pathological material

PubMed Central

Kähler, Christian; Didlaukat, Sabine; Feller, Alfred C; Merz, Hartmut

2007-01-01

Background Human mastocytosis is a heterogenous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication. Methods The Kit point mutation Asp 816 to Val is heterozygous which means a problem in detection by PCR because the wild-type allele is also amplified and the number of cells which bear the point mutation is in most of the cases low. Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result. This is why point-mutated forward primers were designed and tested for efficiency in amplification of the mutated allele. Results One primer combination (A) fits the most for the introduced PCR assay. It was able just to amplify the mutated allele with high specificity from different patient's materials (FFPE or blood) of varying quality and quantity. Moreover, the sensitivity for this assay was convincing because 10 ng of DNA which bears the point mutation could be detected in a total volume of 200 ng of DNA. Conclusion The PCR assay is able to deal with different materials (blood and FFPE) this means quality and quantity of DNA and can be used for high-througput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory. PMID:17900365

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

PubMed

Casarin, Elisabetta; Lucchese, Laura; Grazioli, Santina; Facchin, Sonia; Realdon, Nicola; Brocchi, Emiliana; Morpurgo, Margherita; Nardelli, Stefano

2016-01-01

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

Forensic genetic study of 29 Y-STRs in Korean population.

PubMed

Jung, Ju Yeon; Park, Ji-Hye; Oh, Yu-Li; Kwon, Han-Sol; Park, Hyun-Chul; Park, Kyung-Hwa; Kim, Eun Hye; Lee, Dong-Sub; Lim, Si-Keun

2016-11-01

In this study, we compared two recently released commercial Y-chromosomal short tandem repeat (Y-STR) kits: the PowerPlex Y23 System (PPY23) and Yfiler® Plus PCR amplification kit (YPlus). We performed validation studies, including sensitivity, tolerance to PCR inhibitors, and mixture analysis, and a population genetics study using 306 unrelated South Korean males. PPY23 and YPlus showed similar sensitivity, but PPY23 showed higher tolerance to humic acid than YPlus. Furthermore, the detection rate of unique minor alleles called from male/male mixtures was higher for PPY23 than for YPlus. Comparing the newly added loci, the mean values of gene diversity for PPY23 and YPlus were 0.6715 and 0.8158, respectively. The discrimination capacity in the 306 unrelated South Korean males for PPY23 was 0.9837, and that for YPlus was 0.9935. These results will inform the selection of suitable Y-STR kits based on the purpose of forensic DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria.

PubMed

Chauhan, Arjun; Sharma, J N; Modgil, Manju; Siddappa, Sundaresha

2018-05-29

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus. Copyright © 2018 Elsevier B.V. All rights reserved.

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Point-of-care testing system enabling 30 min detection of influenza genes.

PubMed

Abe, Tomoteru; Segawa, Yuji; Watanabe, Hidetoshi; Yotoriyama, Tasuku; Kai, Shinichi; Yasuda, Akio; Shimizu, Norio; Tojo, Naoko

2011-03-21

We developed a portable and easy-to-use nucleic acid amplification test (NAT) system for use in point-of-care testing (POCT). The system shows sensitivity that is sufficiently higher than that of the currently available rapid diagnostic kit and is comparable to that of real-time reverse transcription polymerase chain reaction (RT-PCR) for influenza testing. This journal is © The Royal Society of Chemistry 2011

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.

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

PubMed Central

Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

2014-01-01

Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

PubMed

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-08-17

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

PubMed Central

Gouveia, Gisele Rodrigues; Ferreira, Suzete Cleusa; Ferreira, Jerenice Esdras; Siqueira, Sheila Aparecida Coelho; Pereira, Juliana

2014-01-01

The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens. PMID:25105117

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.

Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

PubMed Central

Garrido, Joseba M.; Molina, Elena; Geijo, María V.; Elguezabal, Natalia; Vázquez, Patricia; Juste, Ramón A.

2014-01-01

The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs. PMID:24727272

Technical note: developmental validation of a novel 6-dye typing system with 36 Y-STR loci.

PubMed

Du, Weian; Feng, Peipei; Huang, Hongyan; Wu, Weibin; Zhang, Lei; Guo, Yulin; Liu, Changhui; Liu, Hong; Liu, Chao; Chen, Ling

2018-05-30

Y-chromosomal short tandem repeats (Y-STRs) have proven to be very useful in investigating sexual assault cases and in paternity lineage differentiation. However, currently available commercial Y-STR multiplex amplification systems bear the limitations in the identification of related males from the same paternal lineage due to there being an insufficient number of loci in any single amplification kit. The aim of this study was to establish and validate a novel 6-dye, 36-plex Y-STR multiplex amplification system that incorporated all of the loci present in the Yfiler™ Plus kit (DYS19, DYS385a/b, DYF387S1, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, Y_GATA_H4) as well as a further nine highly polymorphic Y-STR loci (DYS388, DYS444, DYS447, DYS522, DYS527a/b, DYS549, DYS596, DYS643). The novel system was optimized and validated by a series of studies that tested the effect of different PCR-based conditions as well as the species specificity, sensitivity, stability, stutter precision, suitability for use on DNA mixtures, reproducibility, and parallel testing of the system, as well as its performance on casework samples and population analysis, according to the SWGDAM developmental validation guidelines. A total of 246 haplotypes were found for the 36 Y-STRs among 247 Guangdong Han unrelated males. Collectively, the results demonstrate that the developed 36-plex Y-STR system is sensitive, robust, reliable, and highly informative for use in forensic genetics.

NASBA: A detection and amplification system uniquely suited for RNA

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

Sooknanan, R.; Malek, L.T.

1995-06-01

The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal:more » sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.« less

[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.

Development of positive control materials for DNA-based detection of cystic fibrosis: Cloning and sequencing of 31 mutations

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

Iovannisci, D.; Brown, C.; Winn-Deen, E.

1994-09-01

The cloning and sequencing of the gene associated with cystic fibrosis (CF) now provides the opportunity for earlier detection and carrier screening through DNA-based detection schemes. To date, over 300 mutations have been reported to the CF Consortium; however, only 30 mutations have been observed frequently enough world-wide to warrant routine screening. Many of these mutations are not available as cloned material or as established tissue culture cell lines to aid in the development of DNA-based detection assays. We have therefore cloned the 30 most frequently reported mutations, plus the mutation R347H due to its association with male infertility (31more » mutations, total). Two approaches were employed: direct PCR amplification, where mutations were available from patient sources, and site-directed PCR mutagenesis of normal genomic DNA to generate the remaining mutations. After amplification, products were cloned into a sequencing vector, bacterial transformants were screened by a novel method (PCR/oligonucleotide litigation assay/sequence-coded separation), and plamid DNA sequences determined by automated fluorescent methods on the Applied Biosystems 373A. Mixing of the clones allows the construction of artificial genotypes useful as positive control material for assay validation. A second round of mutagenesis, resulting in the construction of plasmids bearing multiple mutations, will be evaluated for their utility as reagent control materials in kit development.« less

The development of the residential Fire H.E.L.P. tool kit: a resource to protect homebound older adults.

PubMed

Diekman, Shane; Huitric, Michele; Netterville, Linda

2010-01-01

This article describes the development of the Fire H.E.L.P. tool kit for training selected Meals On Wheels (MOW) staff in Texas to implement a fire safety program for homebound older adults. We used a formative evaluation approach during the tool kit's development, testing, and initial implementation stages. The tool kit includes instructional curricula on how to implement Fire H.E.L.P., a home assessment tool to determine a residence's smoke alarm needs, and fire safety educational materials. During the tool kit's pilot test, MOW participants showed enhanced fire safety knowledge and high levels of confidence about applying their newfound training skills. After the pilot test, MOW staff used the tool kit to conduct local training sessions, provide fire safety education, and install smoke alarms in the homes of older adults. We believe the approach used to develop this tool kit can be applied to education efforts for other, related healthy home topics.

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.

Automated forensic DNA purification optimized for FTA card punches and identifiler STR-based PCR analysis.

PubMed

Tack, Lois C; Thomas, Michelle; Reich, Karl

2007-03-01

Forensic labs globally face the same problem-a growing need to process a greater number and wider variety of samples for DNA analysis. The same forensic lab can be tasked all at once with processing mixed casework samples from crime scenes, convicted offender samples for database entry, and tissue from tsunami victims for identification. Besides flexibility in the robotic system chosen for forensic automation, there is a need, for each sample type, to develop new methodology that is not only faster but also more reliable than past procedures. FTA is a chemical treatment of paper, unique to Whatman Bioscience, and is used for the stabilization and storage of biological samples. Here, the authors describe optimization of the Whatman FTA Purification Kit protocol for use with the AmpFlSTR Identifiler PCR Amplification Kit.

Automated quantification of Epstein-Barr Virus in whole blood of hematopoietic stem cell transplant patients using the Abbott m2000 system.

PubMed

Salmona, Maud; Fourati, Slim; Feghoul, Linda; Scieux, Catherine; Thiriez, Aline; Simon, François; Resche-Rigon, Matthieu; LeGoff, Jérôme

2016-08-01

Accurate quantification of Epstein-Barr virus (EBV) load in blood is essential for the management of post-transplant lymphoproliferative disorders. The automation of DNA extraction and amplification may improve accuracy and reproducibility. We evaluated the EBV PCR Kit V1 with fully automated DNA extraction and amplification on the m2000 system (Abbott assay). Conversion factor between copies and international units (IU), lower limit of quantification, imprecision and linearity were determined in a whole blood (WB) matrix. Results from 339 clinical WB specimens were compared with a home-brew real-time PCR assay used in our laboratory (in-house assay). The conversion factor between copies and IU was 3.22 copies/IU. The lower limit of quantification (LLQ) was 1000 copies/mL. Intra- and inter-assay coefficients of variation were 3.1% and 7.9% respectively for samples with EBV load higher than the LLQ. The comparison between Abbott assay and in-house assay showed a good concordance (kappa = 0.77). Loads were higher with the Abbott assay (mean difference = 0.62 log10 copies/mL). The EBV PCR Kit V1 assay on the m2000 system provides a reliable and easy-to-use method for quantification of EBV DNA in WB. Copyright © 2016 Elsevier Inc. All rights reserved.

Forensic and population genetic analysis of Xinjiang Uyghur population on 21 short tandem repeat loci of 6-dye GlobalFiler™ PCR Amplification kit.

PubMed

Zhang, Honghua; Xia, Mingying; Qi, Lijie; Dong, Lei; Song, Shuang; Ma, Teng; Yang, Shuping; Jin, Li; Li, Liming; Li, Shilin

2016-05-01

Estimating the allele frequencies and forensic statistical parameters of commonly used short tandem repeat (STR) loci of the Uyghur population, which is the fifth largest group in China, provides a more precise reference database for forensic investigation. The 6-dye GlobalFiler™ Express PCR Amplification kit incorporates 21 autosomal STRs, which have been proven that could provide reliable DNA typing results and enhance the power of discrimination. Here we analyzed the GlobalFiler STR loci on 1962 unrelated individuals from Chinese Uyghur population of Xinjiang, China. No significant deviations from Hardy-Weinberg equilibrium and linkage disequilibrium were detected within and between the GlobalFiler STR loci. SE33 showed the greatest power of discrimination in Uyghur population, whereas TPOX showed the lowest. The combined power of discrimination was 99.999999999999999999999998746%. No significant difference was observed between Uyghur and the other two Uyghur populations at all tested STRs, as well as Dai and Mongolian. Significant differences were only observed between Uyghur and other Chinese populations at TH01, as well as Central-South Asian at D13S317, East Asian at TH01 and VWA. The phylogenetic analysis showed that Uyghur is genetically close to Chinese populations, as well as East Asian and Central-South Asian. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DNA recovery from microhymenoptera using six non-destructive methodologies with considerations for subsequent preparation of museum slides.

PubMed

Guzmán-Larralde, Adriana J; Suaste-Dzul, Alba P; Gallou, Adrien; Peña-Carrillo, Kenzy I

2017-01-01

Because of the tiny size of microhymenoptera, successful morphological identification typically requires specific mounting protocols that require time, skills, and experience. Molecular taxonomic identification is an alternative, but many DNA extraction protocols call for maceration of the whole specimen, which is not compatible with preserving museum vouchers. Thus, non-destructive DNA isolation methods are attractive alternatives for obtaining DNA without damaging sample individuals. However, their performance needs to be assessed in microhymenopterans. We evaluated six non-destructive methods: (A) DNeasy® Blood & Tissue Kit; (B) DNeasy® Blood & Tissue Kit, modified; (C) Protocol with CaCl 2 buffer; (D) Protocol with CaCl 2 buffer, modified; (E) HotSHOT; and (F) Direct PCR. The performance of each DNA extraction method was tested across several microhymenopteran species by attempting to amplify the mitochondrial gene COI from insect specimens of varying ages: 1 day, 4 months, 3 years, 12 years, and 23 years. Methods B and D allowed COI amplification in all insects, while methods A, C, and E were successful in DNA amplification from insects up to 12 years old. Method F, the fastest, was useful in insects up to 4 months old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.

Extending the phenotype of monosomy 1p36 syndrome and mapping of a critical region for obesity and hyperphagia.

PubMed

D'Angelo, Carla S; Kohl, Ilana; Varela, Monica Castro; de Castro, Cláudia I E; Kim, Chong A; Bertola, Débora R; Lourenço, Charles M; Koiffmann, Célia P

2010-01-01

Rearrangements of 1p36 are the most frequently detected abnormalities in diagnostic testing for chromosomal cryptic imbalances and include variably sized simple terminal deletions, derivative chromosomes, interstitial deletions, and complex rearrangements. These rearrangements result in the specific pattern of malformation and neurodevelopmental disabilities that characterizes monosomy 1p36 syndrome. Thus far, no individual gene within this region has been conclusively determined to be causative of any component of the phenotype. Nor is it known if the rearrangements convey phenotypes via a haploinsufficiency mechanism or through a position effect. We have used multiplex ligation-dependent probe amplification to screen for deletions of 1p36 in a group of 154 hyperphagic and overweight/obese, PWS negative individuals, and in a separate group of 83 patients initially sent to investigate a variety of other conditions. The strategy allowed the identification and delineation of rearrangements in nine subjects with a wide spectrum of clinical presentations. Our work reinforces the association of monosomy 1p36 and obesity and hyperphagia, and further suggests that these features may be associated with non-classical manifestations of this disorder in addition to a submicroscopic deletion of approximately 2-3 Mb in size. Multiplex ligation probe amplification using the monosomy 1p36 syndrome-specific kit coupled to the subtelomeric kit is an effective approach to identify and delineate rearrangements at 1p36.

Evaluation of whole genome amplified DNA to decrease material expenditure and increase quality.

PubMed

Bækvad-Hansen, Marie; Bybjerg-Grauholm, Jonas; Poulsen, Jesper B; Hansen, Christine S; Hougaard, David M; Hollegaard, Mads V

2017-06-01

The overall aim of this study is to evaluate whole genome amplification of DNA extracted from dried blood spot samples. We wish to explore ways of optimizing the amplification process, while decreasing the amount of input material and inherently the cost. Our primary focus of optimization is on the amount of input material, the amplification reaction volume, the number of replicates and amplification time and temperature. Increasing the quality of the amplified DNA and the subsequent results of array genotyping is a secondary aim of this project. This study is based on DNA extracted from dried blood spot samples. The extracted DNA was subsequently whole genome amplified using the REPLIg kit and genotyped on the PsychArray BeadChip (assessing > 570,000 SNPs genome wide). We used Genome Studio to evaluate the quality of the genotype data by call rates and log R ratios. The whole genome amplification process is robust and does not vary between replicates. Altering amplification time, temperature or number of replicates did not affect our results. We found that spot size i.e. amount of input material could be reduced without compromising the quality of the array genotyping data. We also showed that whole genome amplification reaction volumes can be reduced by a factor of 4, without compromising the DNA quality. Whole genome amplified DNA samples from dried blood spots is well suited for array genotyping and produces robust and reliable genotype data. However, the amplification process introduces additional noise to the data, making detection of structural variants such as copy number variants difficult. With this study, we explore ways of optimizing the amplification protocol in order to reduce noise and increase data quality. We found, that the amplification process was very robust, and that changes in amplification time or temperature did not alter the genotyping calls or quality of the array data. Adding additional replicates of each sample also lead to insignificant changes in the array data. Thus, the amount of noise introduced by the amplification process was consistent regardless of changes made to the amplification protocol. We also explored ways of decreasing material expenditure by reducing the spot size or the amplification reaction volume. The reduction did not affect the quality of the genotyping data.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for equipment-free detection of Cryptosporidium spp. oocysts in dairy cattle feces.

PubMed

Wu, Yao-Dong; Zhou, Dong-Hui; Zhang, Long-Xian; Zheng, Wen-Bin; Ma, Jian-Gang; Wang, Meng; Zhu, Xing-Quan; Xu, Min-Jun

2016-09-01

Cryptosporidium is a widespread protozoan parasite that infects a large number of vertebrate animals, resulting in varying degrees of diarrhea or even death. As dairy cattle feces is an important source of Cryptosporidium spp. infection, development of a handy and accurate detection method via its oocysts in dairy cattle feces would be interesting and necessary. We herein developed a quick detecting method using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip to detect DNA of Cryptosporidium oocysts in dairy cattle feces. The DNA was released by boiled water with 0.1 % N-lauroylsarcosine sodium salt (LSS). The established method was proven to be of higher sensitivity than normal polymerase chain reaction (PCR) amplification with the lowest detection of 0.5 oocyst per reaction, and specificity with no cross reactivity to other common protozoan species in the intestine of dairy cattle. The diagnostic method established herein is simple, rapid, and cost-effective, and has potential for further development as a diagnostic kit for the diagnosis of cryptosporidiosis of dairy cattle.

Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

PubMed

Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

2018-01-01

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

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.

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

PubMed

Olova, Nelly; Krueger, Felix; Andrews, Simon; Oxley, David; Berrens, Rebecca V; Branco, Miguel R; Reik, Wolf

2018-03-15

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

40 CFR 1045.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 33 2011-07-01 2011-07-01 false What special provisions apply for installing and removing altitude kits? 1045.655 Section 1045.655 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM SPARK-IGNITION PROPULSION...

40 CFR 1045.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 34 2012-07-01 2012-07-01 false What special provisions apply for installing and removing altitude kits? 1045.655 Section 1045.655 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM SPARK-IGNITION PROPULSION...

40 CFR 1045.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 32 2010-07-01 2010-07-01 false What special provisions apply for installing and removing altitude kits? 1045.655 Section 1045.655 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM SPARK-IGNITION PROPULSION...

40 CFR 1045.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 34 2013-07-01 2013-07-01 false What special provisions apply for installing and removing altitude kits? 1045.655 Section 1045.655 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM SPARK-IGNITION PROPULSION...

40 CFR 1045.655 - What special provisions apply for installing and removing altitude kits?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 33 2014-07-01 2014-07-01 false What special provisions apply for installing and removing altitude kits? 1045.655 Section 1045.655 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM SPARK-IGNITION PROPULSION...

Taxol and LPS Modulation of c-kit and nm23 Expression in Macrophages and Normal vs. Malignant Breast Cancer Cell Lines.

DTIC Science & Technology

1999-07-01

medium only, LPS (100 ng/ml), or paclitaxel (35 ^iM), concentrations found to induce maximal levels of mRNA in murine macrophages. Total RNA was...not detected in RNA derived from the DA-3 cells over an 8 h timecourse, even after 40 cycles of PCR amplification, without or with treatment...indicated times after stimulation with LPS or paclitaxel. Isolation of total cellular RNA . For in vitro experiments, culture supematants were removed

Improvement and automation of a real-time PCR assay for vaginal fluids.

PubMed

De Vittori, E; Giampaoli, S; Barni, F; Baldi, M; Berti, A; Ripani, L; Romano Spica, V

2016-05-01

The identification of vaginal fluids is crucial in forensic science. Several molecular protocols based on PCR amplification of mfDNA (microflora DNA) specific for vaginal bacteria are now available. Unfortunately mfDNA extraction and PCR reactions require manual optimization of several steps. The aim of present study was the verification of a partial automatization of vaginal fluids identification through two instruments widely diffused in forensic laboratories: EZ1 Advanced robot and Rotor Gene Q 5Plex HRM. Moreover, taking advantage of 5-plex thermocycler technology, the ForFluid kit performances were improved by expanding the mfDNA characterization panel with a new bacterial target for vaginal fluids and with an internal positive control (IPC) to monitor PCR inhibition. Results underlined the feasibility of a semi-automated extraction of mfDNA using a BioRobot and demonstrated the analytical improvements of the kit. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex ® ESX 17 and ESI 17 Systems.

PubMed

Hill, Carolyn R; Duewer, David L; Kline, Margaret C; Sprecher, Cynthia J; McLaren, Robert S; Rabbach, Dawn R; Krenke, Benjamin E; Ensenberger, Martin G; Fulmer, Patricia M; Storts, Douglas R; Butler, John M

2011-08-01

The PowerPlex(®) ESX 17 and ESI 17 Systems for short tandem repeat (STR) amplification were developed by the Promega Corporation to meet the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group recommendations for increasing the number of STR loci included in the European Standard Set (ESS). The PowerPlex ESX 17 and ESI 17 Systems utilize different PCR primer combinations to co-amplify the following 17 loci: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA, SE33, and the sex-typing locus amelogenin. A total of 1443 U.S. population samples were evaluated with pre-commercialization versions of both kits. Stutter and heterozygote peak height ratios have been used to characterize kit performance. Typing results have been used to estimate the match probabilities provided by the chosen loci as well as in concordance studies. Full concordance between the typing results for the two kits was observed in 99.994% (49,055 out of 49,062) STR allele calls compared. All genotyping discrepancies were confirmed by DNA sequence analysis. As a result of these comparisons, a second forward primer for the D22S1045 locus has been added to the PowerPlex ESX 17 System to address a primer binding site mutation and the D1S1656 locus reverse primer in the PowerPlex ESI 17 System was modified to eliminate an amplification-efficiency reducing primer dimer. Copyright © 2010 Elsevier Ireland Ltd. 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.

Technology assessment and strategy for development of a Rapid Field Water Microbiology Test Kit. Technical report

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

Preston, D.R.; Schaub, S.A.

1991-09-01

A literature and market search of existing technology for the detection, identification, and quantification of microorganisms in water was conducted. Based upon the availability of technologies and their configurations, an assessment of the appropriate strategies to pursue for the near and long term development plans in development of the Rapid Field Bacteriology Test Kit was performed. Near term technologies to improve the Army's capability to detect microorganisms would appear to be essentially improvements in versatility and measurement of coliform indicator organisms. New chromogenic and fluorogenic indicator substances associated with new substrates appear to be best suited for test kit developmentmore » either for quantitative membrane filter tests or presence/absence and multiple fermentation tests. Test times, incubator requirements, and operator involvement appear to be similar to older technologies. Long term development would appear to favor such technologies as genetic probes with amplification of the hydridized nucleic acid materials of positive samples, and some immunological based systems such as enzyme linked, immuno-sorbent assays. In both cases, the major problems would appear to be sample preparation and development of signal strengths from the reactions which would allow the user to see results in 1 hour.« less

77 FR 28851 - Foreign-Trade Zone 126-Reno, NV; Notification of Proposed Production Activity; Brightpoint North...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-16

...; Notification of Proposed Production Activity; Brightpoint North America L.P. (Cell Phone Kitting and... for cell phone kitting, warehousing and distribution operations. Production under FTZ procedures could... procedures that apply to cell phone kits (duty free) for the foreign status inputs noted below. Customs...

Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus.

PubMed

Niedbalski, W

2011-01-01

The aim of this study was to estimate the diagnostic value of different commercially available ELISA kits for the detection of bluetongue virus (BTV) antibodies in infected and vaccinated animals. The relative specificity of ELISA kits was evaluated using a panel of sera originating from healthy cattle, never vaccinated nor exposed to BTV. All ELISA kits applied had a high relative specificity (99.3 - 100%). The relative sensitivity of ELISA kits assessed using a panel of sera collected from BTV infected cattle was also high and similar for all the kits (97.3 - 100%). However, the relative sensitivity evaluated on the basis of testing vaccinated animals was different: the highest sensitivity was found for Ingenasa, PrioCHECK and ID VET ELISAs (96.5 - 98.3%). Slightly lower sensitivity was calculated for Pourquier and LSI kits (82.8% and 85.4%, respectively) and much lower sensitivity was found for VMRD ELISA kit (69.5%). The repeatability of BTV ELISA kits was expressed as a coefficient of variation (CV) of results of sera tested 5 times in the same day and in different days by the period of 2 months, by the same person, in the same conditions, and by using the same equipment. The CVs of sera tested in all ELISA kits ranged from 6.1 to 9.8% and were below 10% threshold adopted as a maximum for the acceptable repeatability of the method. In conclusion, it can be stated that the applied ELISA kits can be a valuable diagnostic tool for the serological monitoring studies in the BTV contaminated premises. All the methods are very specific and sensitive when testing BTV infected animals. Nevertheless, the Ingenasa and PrioCHECK can be the most useful in sero-surveillance of livestock following vaccination.

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.

Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia.

PubMed

Piera, Kim A; Aziz, Ammar; William, Timothy; Bell, David; González, Iveth J; Barber, Bridget E; Anstey, Nicholas M; Grigg, Matthew J

2017-01-13

Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection. Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs. The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9-100), P. falciparum (95% CI 83.2-100), and P. vivax (95% CI 29.2-100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL. The Eiken Loopamp™ MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.

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.

Detection of mycobacterium tuberculosis in paraffin-embedded pleural biopsy specimens by commercial ribosomal RNA and DNA amplification kits.

PubMed

Ruiz-Manzano, J; Manterola, J M; Gamboa, F; Calatrava, A; Monsó, E; Martínez, C; Ausina, V

2000-09-01

To evaluate the utility of two gene amplification systems in historical paraffin-embedded pleural biopsy (PEB) tissues from patients with pleural tuberculosis, and to compare the results to those obtained with conventional histologic and microbiological methods. A retrospective study. Seventy-four formalin-fixed PEB tissues collected and stored over 12 years (1984 through 1995) were retrieved. Gene amplifications were performed in 57 tissues from patients with diagnoses of pleural tuberculosis and in 17 from patients with carcinoma as controls, using the first version of the Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe; San Diego, CA) and the LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Abbott Park, IL). The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (p = not statistically significant). The specificity of both tests was 100%. Twenty tissue samples (35.1%) were positive by both systems, and 10 tissues (17.5%) were positive only by the AMTDT, while 16 tissues (28.1%) were positive only by the LCxMTB. Both tests gave negative results for 11 specimens (19.3%). When both tests were used, a positive diagnosis was achieved in 80.7% of the samples. Diagnosis of 73.7% of patient conditions had previously been made by smear examination of pleural biopsy and sputum, pleural liquid, or biopsy culture. The overall diagnostic yield with both culture and amplification techniques was 96.5% (55 of 57 patients) for pleural tuberculosis, with amplification techniques adding 22.8% of the diagnoses. Amplification techniques are useful in archival PEB tissues, providing additional diagnoses beyond culturing, although the sensitivity should be improved, possibly by standardizing protocols.

Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication.

PubMed

Aida, A A; Che Man, Y B; Wong, C M V L; Raha, A R; Son, R

2005-01-01

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy(®) Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.

Hotspot mutations in cancer genes may be missed in routine diagnostics due to neighbouring sequence variants.

PubMed

Bartels, Stephan; Schipper, Elisa; Hasemeier, Britta; Kreipe, Hans; Lehmann, Ulrich

2018-05-27

The detection of hotspot mutations in key cancer genes is now an essential part of the diagnostic work-up in molecular pathology. Nearly all assays for mutation detection involve an amplification step. A second single nucleotide variant (SNV) on the same allele adjacent to a mutational hotspot can interfere with primer binding, leading to unnoticed allele-specific amplification of the wild type allele and thereby false-negative mutation testing. We present two diagnostic cases with false negative sequence results for JAK2 and SRSF2. In both cases mutations would have escaped detection if only one strand of DNA had been analysed. Because many commercially available diagnostic kits rely on the analysis of only one DNA strand they are prone to fail in cases like these. Detailed protocols and quality control measures to prevent corresponding pitfalls are presented. Copyright © 2017. Published by Elsevier Inc.

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

PubMed

Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

2014-11-01

Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

Population genetic data of the AmpFℓSTR® Identifiler® Plus and PowerPlex® 16 HS STR loci in four Canadian populations.

PubMed

Laurin, Nancy; Milot, Emmanuel

2014-03-01

Allele frequencies and forensically relevant population statistics were estimated for the short tandem repeat (STR) loci of the AmpFℓSTR® Identifiler® Plus and PowerPlex® 16 HS amplification kits, including D2S1338, D19S433, Penta D, and Penta E, for three First Nations Aboriginal populations and for Caucasians in Canada. The cumulative power of discrimination was ≥ 0.999999999999984 and the cumulative power of exclusion was ≥ 0.999929363 for both amplification systems in all populations. No significant departure from Hardy-Weinberg equilibrium was detected for D2S1338, D19S433, Penta D, and Penta E or the 13 Combined DNA Index System core STR loci after correction for multiple testing. Significant genetic diversity was observed between these four populations. Comparison with published frequency data for other populations is also presented.

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

Paternity tests in Mexico: Results obtained in 3005 cases.

PubMed

García-Aceves, M E; Romero Rentería, O; Díaz-Navarro, X X; Rangel-Villalobos, H

2018-04-01

National and international reports regarding the paternity testing activity scarcely include information from Mexico and other Latin American countries. Therefore, we report different results from the analysis of 3005 paternity cases analyzed during a period of five years in a Mexican paternity testing laboratory. Motherless tests were the most frequent (77.27%), followed by trio cases (20.70%); the remaining 2.04% included different cases of kinship reconstruction. The paternity exclusion rate was 29.58%, higher but into the range reported by the American Association of Blood Banks (average 24.12%). We detected 65 mutations, most of them involving one-step (93.8% and the remaining were two-step mutations (6.2%) thus, we were able to estimate the paternal mutation rate for 17 different STR loci: 0.0018 (95% CI 0.0005-0.0047). Five triallelic patterns and 12 suspected null alleles were detected during this period; however, re-amplification of these samples with a different Human Identification (HID) kit confirmed the homozygous genotypes, which suggests that most of these exclusions actually are one-step mutations. HID kits with ≥20 STRs detected more exclusions, diminishing the rate of inconclusive results with isolated exclusions (<3 loci), and leading to higher paternity indexes (PI). However, the Powerplex 21 kit (20 STRs) and Powerplex Fusion kit (22 STRs) offered similar PI (p = 0.379) and average number of exclusions (PE) (p = 0.339) when a daughter was involved in motherless tests. In brief, besides to report forensic parameters from paternity tests in Mexico, results describe improvements to solve motherless paternity tests using HID kits with ≥20 STRs instead of one including 15 STRs. Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

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.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification.

PubMed

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 °C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 10(3) cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

Enzyme-free detection of sequence-specific microRNAs based on nanoparticle-assisted signal amplification strategy.

PubMed

Li, Ru-Dong; Wang, Qian; Yin, Bin-Cheng; Ye, Bang-Ce

2016-03-15

Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA-Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe-miRNA-CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu(2+)). Cu(2+) could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA-Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu(2+), which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5 pM to 1000 pM with a detection limit of ~0.73 pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit. Copyright © 2015 Elsevier B.V. All rights reserved.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

PubMed Central

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness. PMID:26208001

Identification of Skeletal Remains of Communist Armed Forces Victims During and After World War II: Combined Y-chromosome Short Tandem Repeat (STR) and MiniSTR Approach

PubMed Central

Marjanović, Damir; Durmić-Pašić, Adaleta; Kovačević, Lejla; Avdić, Jasna; Džehverović, Mirela; Haverić, Sanin; Ramić, Jasmin; Kalamujić, Belma; Bilela, Lada Lukić; Škaro, Vedrana; Projić, Petar; Bajrović, Kasim; Drobnič, Katja; Davoren, Jon; Primorac, Dragan

2009-01-01

Aim To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. Methods Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Škofja Loka area (Lovrenska Grapa and Žolšče) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpFℓSTR® MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex® Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. Results Following the Y-STR analysis, 1 of the “weak matches” previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Žološče, we were able to obtain 6 useful DNA profiles. Conclusion The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and miniSTR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II. PMID:19480024

Stochastic sampling effects in STR typing: Implications for analysis and interpretation.

PubMed

Timken, Mark D; Klein, Sonja B; Buoncristiani, Martin R

2014-07-01

The analysis and interpretation of forensic STR typing results can become more complicated when reduced template amounts are used for PCR amplification due to increased stochastic effects. These effects are typically observed as reduced heterozygous peak-height balance and increased frequency of undetected alleles (allelic "dropout"). To investigate the origins of these effects, a study was performed using the AmpFlSTR(®) Identifiler Plus(®) and MiniFiler(®) kits to amplify replicates from a dilution series of NIST Human DNA Quantitation Standard (SRM(®) 2372A). The resulting amplicons were resolved and detected on two different genetic analyzer platforms, the Applied Biosystems 3130xL and 3500 analyzers. Results from our study show that the four different STR/genetic analyzer combinations exhibited very similar peak-height ratio statistics when normalized for the amount of template DNA in the PCR. Peak-height ratio statistics were successfully modeled using the Poisson distribution to simulate pre-PCR stochastic sampling of the alleles, confirming earlier explanations that sampling is the primary source for peak-height imbalance in reduced template dilutions. In addition, template-based pre-PCR sampling simulations also successfully predicted allelic dropout frequencies, as modeled by logistic regression methods, for the low-template DNA dilutions. We discuss the possibility that an accurately quantified DNA template might be used to characterize the linear signal response for data collected using different STR kits or genetic analyzer platforms, so as to provide a standardized approach for comparing results obtained from different STR/CE combinations and to aid in validation studies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Sex and PRNP genotype determination in preimplantation caprine embryos.

PubMed

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Isolation, amplification and characterization of foodborne pathogen disease bacteria gene for rapid kit test development

NASA Astrophysics Data System (ADS)

Nurjayadi, M.; Santoso, I.; Kartika, I. R.; Kurniadewi, F.; Saamia, V.; Sofihan, W.; Nurkhasanah, D.

2017-07-01

There is a lot of public concern over food safety. Food-safety cases recently, including many food poisoning cases in both the developed and developing countries, considered to be the national security threats which involved police investigation. Quick and accurate detection methods are needed to handle the food poisoning cases with a big number of sufferers at the same time. Therefore, the research is aimed to develop a specific, sensitive, and rapid result molecular detection tool for foodborne pathogen bacteria. We, thus, propose genomic level approach with Polymerase Chain Reaction. The research has successfully produced a specific primer to perform amplification to fim-C S. typhi, E. coli, and pef Salmonella typhimurium genes. The electrophoresis result shows that amplification products are 95 base pairs, 121 base pairs, and 139 base pairs; and all three genes are in accordance with the size of the in silico to third genes bacteria. In conclusion, the research has been successfully designed a specific detection tool to three foodborne pathogen bacteria genes. Further stages test and the uses of Real-time PCR in the detection are still in the trial process for better detection method.

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

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

Yuzawa,S.; Opatowsky, Y.; Zhang, Z.

2007-01-01

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less

Performance testing of a semi-automatic card punch system, using direct STR profiling of DNA from blood samples on FTA™ cards.

PubMed

Ogden, Samantha J; Horton, Jeffrey K; Stubbs, Simon L; Tatnell, Peter J

2015-01-01

The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA(™) sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch. © 2014 American Academy of Forensic Sciences.

[Application of isothermal amplification technology for pathogen detection in parasitic and other diseases].

PubMed

Ting, Li; Kun, Yang

2018-04-16

The in vitro nucleic acid amplification technique based on polymerase chain reaction (PCR) has been successfully applied to scientific researches. In recent years, the emergence of isothermal amplification technology is increasingly applied in the molecular diagnosis and disease detection because of its advantages of constant temperature, high efficiency, short time-consuming, and less reliance on equipment and instruments. The principle, characteristics and application of the partial isothermal amplification technique in the pathogen detection in parasitic and other diseases are reviewed in this paper, and the prospects of the wide development of the technique are also discussed.

Recovery of human DNA profiles from poached deer remains part 2: improved recovery protocol without the need for LCN analysis.

PubMed

Tobe, Shanan S; Bailey, Stuart; Govan, James; Welch, Lindsey A

2013-03-01

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis. Samples from 10 deer (40 samples total - one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5pg±43.2pg, 31× greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpFℓSTR® SGM Plus® kit at 30cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690×10(-05) to 2.2706×10(-14). The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents. Copyright © 2012 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

[Rapid Detection of Adenovirus in Fecal Samples by Capillary Electrophoresis-laser Induced Fluorescence and Microchip Capillary Electrophoresis-laser Induced Fluorescence].

PubMed

Ruan, Jia; Ren, Dong-xia; Yang, Dan-ni; Long, Pin-pin; Zhao, Hong-yue; Wang, Yi-qi; Li, Yong-xin

2015-07-01

To establish a rapid and sensitive method based on polymerase chain reaction (PCR) combined with capillary electrophoresis-laser induced fluorescence (CE-LIF) and microchip capillary electrophoresis-laser induced fluorescence (MCE-LIF) for detecting adenoviruses in fecal samples. The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction. After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively, PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector. PCR amplification products could be detected within 9 min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition. The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI. The intraday and inter-day relative standard deviation (RSD) of the size (bp) of the target DNA was in the range of 1.14%-1.34% and 1.27%- 2.76%, respectively, for CE-LIF, and 1.18%-1.48% and 2.85%-4.06%, respectively, for MCE-LIF. The detection limits was 2.33 x 10(2) copies/mL for CE-LIF and 2.33 x 10(3) copies/mL for MCE-LIF. The two proposed methods were applied to detect fecal samples, both showing high accuracy. The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly, sensitively and specifically.

21 CFR 872.3560 - OTC denture reliner.

Code of Federal Regulations, 2010 CFR

2010-04-01

... applied as a permanent coating or lining on the base or tissue-contacting surface of a denture. The device... Reliners, Repair Kits, and Partially Fabricated Denture Kits.” [52 FR 30097, Aug. 12, 1987, as amended at...

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

Current state and future perspectives of loop-mediated isothermal amplification (LAMP)-based diagnosis of filamentous fungi and yeasts.

PubMed

Niessen, Ludwig

2015-01-01

Loop-mediated isothermal amplification is a rather novel method of enzymatic deoxyribonucleic acid amplification which can be applied for the diagnosis of viruses, bacteria, and fungi. Although firmly established in viral and bacterial diagnosis, the technology has only recently been applied to a noteworthy number of species in the filamentous fungi and yeasts. The current review gives an overview of the literature so far published on the topic by discussing the different groups of fungal organisms to which the method has been applied. Moreover, the method is described in detail as well as the different possibilities available for signal detection and quantification and sample preparation. Future perspective of loop-mediated isothermal amplification-based assays is discussed in the light of applicability for fungal diagnostics.

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.

Investigation of major genetic alterations in neuroblastoma.

PubMed

Costa, Régis Afonso; Seuánez, Héctor N

2018-06-01

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. This malignancy shows a wide spectrum of clinical outcome and its prognosis is conditioned by manifold biological and genetic factors. We investigated the tumor genetic profile and clinical data of 29 patients with NB by multiplex ligation-dependent probe amplification (MLPA) to assess therapeutic risk. In 18 of these tumors, MYCN status was assessed by fluorescence in situ hybridization (FISH). Copy number variation was also determined for confirming MLPA findings in two 6p loci. We found 2p, 7q and 17q gains, and 1p and 11q losses as the most frequent chromosome alterations in this cohort. FISH confirmed all cases of MYCN amplification detected by MLPA. In view of unexpected 6p imbalance, copy number variation of two 6p loci was assessed for validating MLPA findings. Based on clinical data and genetic profiles, patients were stratified in pretreatment risk groups according to international consensus. MLPA proved to be effective for detecting multiple genetic alterations in all chromosome regions as requested by the International Neuroblastoma Risk Group (INRG) for therapeutic stratification. Moreover, this technique proved to be cost effective, reliable, only requiring standard PCR equipment, and attractive for routine analysis. However, the observed 6p imbalances made PKHD1 and DCDC2 inadequate for control loci. This must be considered when designing commercial MLPA kits for NB. Finally, four patients showed a normal MLPA profile, suggesting that NB might have a more complex genetic pattern than the one assessed by presently available MLPA kits.

Validation of a reaction volume reduction protocol for analysis of Y chromosome haplotypes targeting DNA databases.

PubMed

Souza, C A; Oliveira, T C; Crovella, S; Santos, S M; Rabêlo, K C N; Soriano, E P; Carvalho, M V D; Junior, A F Caldas; Porto, G G; Campello, R I C; Antunes, A A; Queiroz, R A; Souza, S M

2017-04-28

The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA ® cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex ® Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.

Allele-Specific Polymerase Chain Reaction for the Imatinib-Resistant KIT D816V and D816F Mutations in Mastocytosis and Acute Myelogenous Leukemia

PubMed Central

Corless, Christopher L.; Harrell, Patina; Lacouture, Mario; Bainbridge, Troy; Le, Claudia; Gatter, Ken; White, Clifton; Granter, Scott; Heinrich, Michael C.

2006-01-01

Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM. PMID:17065430

Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias.

PubMed

Aydin-Schmidt, Berit; Xu, Weiping; González, Iveth J; Polley, Spencer D; Bell, David; Shakely, Delér; Msellem, Mwinyi I; Björkman, Anders; Mårtensson, Andreas

2014-01-01

Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6-782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94-99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0-4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1-98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2-99.9) and 76.9% (95%CI 46.2-95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1-100) in both study groups. Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.

Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

PubMed Central

González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

2014-01-01

Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings. PMID:25105591

Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP)

PubMed Central

Mukhtar, Maowia; Ali, Sababil S.; Boshara, Salah A.; Albertini, Audrey; Monnerat, Séverine; Bessell, Paul; Mori, Yasuyoshi; Kubota, Yutaka; Ndung’u, Joseph M.

2018-01-01

Background Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results. Methodology/Principal findings The Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%. Conclusions/Significance Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration. PMID:29444079

Sports Nutrition.

ERIC Educational Resources Information Center

Houtkooper, Linda; And Others

This kit provides coaches, physical education teachers, and health professionals with current nutrition information and guidelines for applying that information in classes and athletic training programs. The kit contains four components. A "Key Terms" section provides an index to nutrition-fitness terminology and concepts. The instructional…

Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

PubMed Central

John, Neetha; Rajasekhar, Moka; Girisha, Katta Mohan; Sharma, Podila Satya Venkata Narasimha; Gopinath, Puthiya Mundyat

2013-01-01

BACKGROUND: Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability. MATERIALS AND METHODS: A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland. RESULTS: All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients. CONCLUSION: Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR. PMID:24019617

Reduction of Powerplex(®) Y23 reaction volume for genotyping buccal cell samples on FTA(TM) cards.

PubMed

Raziel, Aliza; Dell'Ariccia-Carmon, Aviva; Zamir, Ashira

2015-01-01

PowerPlex(®) Y23 is a novel kit for Y-STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y-STR analysis. This study examined PCR amplification from 1.2-mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High-quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories. © 2014 American Academy of Forensic Sciences.

Comparison of an assay using signal amplification of the heat-dissociated p24 antigen with the Roche Monitor human immunodeficiency virus RNA assay.

PubMed

Pascual, Alvaro; Cachafeiro, Ada; Funk, Michele L; Fiscus, Susan A

2002-07-01

We compared an assay using signal amplification of a heat-dissociated p24 antigen (HDAg) with the Roche Monitor human immunodeficiency virus (HIV) RNA assay. The two assays gave comparable results when 130 specimens from 130 patients were tested (r = 0.60, P < 0.0001). The HDAg assay was almost as sensitive (85%) as the Roche HIV RNA kit (95%), just as specific (25 negative results from 25 HIV seronegative volunteers [100%]), less variable (mean log standard deviation of 0.07 compared to 0.11 when eight specimens were tested three or four times), and less expensive (reagent and labor costs, $8 versus $75). The assay appeared to be useful for monitoring established patients (n = 17) and identifying seroconverters (n = 4). HIV subtypes A to F were all recognized. This assay should be useful for monitoring patients in resource-poor countries and for monitoring vaccine recipients.

Allele frequency data of 15 autosomal STR loci in four major population groups of South Africa.

PubMed

Lucassen, Anton; Ehlers, Karen; Grobler, Paul J; Shezi, Adeline L

2014-03-01

Allele frequency distributions for 15 tetrameric short tandem repeat (STR) loci were determined using the AmpFlSTR® Identifiler Plus™ PCR amplification kit. There was little evidence of departures from Hardy-Weinberg equilibrium or association of alleles of different loci in the population samples. The probability of identity values for the different populations range from 1/3.3 × 10(17) (White) to 1/1.88 × 10(18) (Coloured). The combined probability of paternal exclusion for the different population groups ranges from 0.9995858 (Coloured) to 0.9997874 (Indian).

Recombinase polymerase amplification combined with lateral flow dipstick for equipment-free detection of Salmonella in shellfish.

PubMed

Gao, Weifang; Huang, Hailong; Zhu, Peng; Yan, Xiaojun; Fan, Jianzhong; Jiang, Jinpo; Xu, Jilin

2018-05-01

Salmonella is a major pathogen that causes acute foodborne outbreaks worldwide. Seafood, particularly shellfish, is a proven source of Salmonella spp. infection because many people prefer to eat it raw or lightly cooked. However, traditional identification methods are too time-consuming and complex to detect contamination of bacteria in the food chain in a timely manner, and few studies have aimed to identify Salmonella in shellfish early in the supply chain. We herein developed a method for rapid detection of Salmonella in shellfish based on the method of recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD), which targets the invasion gene A (invA). The RPA-LFD was able to function at 30-45 °C, and at the temperature of 40 °C, it only took 8 min of amplification to reach the test threshold of amplicons. The established method had both a good specificity and a sensitivity of 100 fg DNA per reaction (20 µL). Regarding practical performance, RPA-LFD performed better than real-time PCR. Another advantage of RPA-LFD is that it was capable of being performed without expensive equipments. Thus, RPA-LFD has potential for further development as a detection kit for Salmonella in shellfish and other foods under field conditions.

Y-chromosomal diversity of the Valachs from the Czech Republic: model for isolated population in Central Europe

PubMed Central

Ehler, Edvard; Vaněk, Daniel; Stenzl, Vlastimil; Vančata, Václav

2011-01-01

Aim To evaluate Y-chromosomal diversity of the Moravian Valachs of the Czech Republic and compare them with a Czech population sample and other samples from Central and South-Eastern Europe, and to evaluate the effects of genetic isolation and sampling. Methods The first sample set of the Valachs consisted of 94 unrelated male donors from the Valach region in northeastern Czech Republic border-area. The second sample set of the Valachs consisted of 79 men who originated from 7 paternal lineages defined by surname. No close relatives were sampled. The third sample set consisted of 273 unrelated men from the whole of the Czech Republic and was used for comparison, as well as published data for other 27 populations. The total number of samples was 3244. Y-short tandem repeat (STR) markers were typed by standard methods using PowerPlex® Y System (Promega) and Yfiler® Amplification Kit (Applied Biosystems) kits. Y-chromosomal haplogroups were estimated from the haplotype information. Haplotype diversity and other intra- and inter-population statistics were computed. Results The Moravian Valachs showed a lower genetic variability of Y-STR markers than other Central European populations, resembling more to the isolated Balkan populations (Aromuns, Csango, Bulgarian, and Macedonian Roma) than the surrounding populations (Czechs, Slovaks, Poles, Saxons). We illustrated the effect of sampling on Valach paternal lineages, which includes reduction of discrimination capacity and variability inside Y-chromosomal haplogroups. Valach modal haplotype belongs to R1a haplogroup and it was not detected in the Czech population. Conclusion The Moravian Valachs display strong substructure and isolation in their Y chromosomal markers. They represent a unique Central European population model for population genetics. PMID:21674832

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA ▿

PubMed Central

Liang, Zhanbei; Keeley, Ann

2011-01-01

Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102 oocysts g−1 of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104 C. parvum oocysts g−1 soil for sandy, loamy, and clay samples, respectively. PMID:21803904

Data on quantification of signaling pathways activated by KIT and PDGFRA mutants.

PubMed

Bahlawane, Christelle; Schmitz, Martine; Letellier, Elisabeth; Arumugam, Karthik; Nicot, Nathalie; Nazarov, Petr V; Haan, Serge

2016-12-01

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

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.

Internal validation of two new retrotransposons-based kits (InnoQuant® HY and InnoTyper® 21) at a forensic lab.

PubMed

Martins, Cátia; Ferreira, Paulo Miguel; Carvalho, Raquel; Costa, Sandra Cristina; Farinha, Carlos; Azevedo, Luísa; Amorim, António; Oliveira, Manuela

2018-02-01

Obtaining a genetic profile from pieces of evidence collected at a crime scene is the primary objective of forensic laboratories. New procedures, methods, kits, software or equipment must be carefully evaluated and validated before its implementation. The constant development of new methodologies for DNA testing leads to a steady process of validation, which consists of demonstrating that the technology is robust, reproducible, and reliable throughout a defined range of conditions. The present work aims to internally validate two new retrotransposon-based kits (InnoQuant ® HY and InnoTyper ® 21), under the working conditions of the Laboratório de Polícia Científica da Polícia Judiciária (LPC-PJ). For the internal validation of InnoQuant ® HY and InnoTyper ® 21 sensitivity, repeatability, reproducibility, and mixture tests and a concordance study between these new kits and those currently in use at LPC-PJ (Quantifiler ® Duo and GlobalFiler™) were performed. The results obtained for sensitivity, repeatability, and reproducibility tests demonstrated that both InnoQuant ® HY and InnoTyper ® 21 are robust, reproducible, and reliable. The results of the concordance studies demonstrate that InnoQuant ® HY produced quantification results in nearly 29% more than Quantifiler ® Duo (indicating that this new kit is more effective in challenging samples), while the differences observed between InnoTyper ® 21 and GlobalFiler™ are not significant. Therefore, the utility of InnoTyper ® 21 has been proven, especially by the successful amplification of a greater number of complete genetic profiles (27 vs. 21). The results herein presented allowed the internal validation of both InnoQuant ® HY and InnoTyper ® 21, and their implementation in the LPC-PJ laboratory routine for the treatment of challenging samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

PubMed

Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan

2015-12-01

The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively. Copyright © 2015. Published by Elsevier B.V.

Genotyping of 25 leukemia-associated genes in a single work flow by next-generation sequencing technology with low amounts of input template DNA.

PubMed

Rinke, Jenny; Schäfer, Vivien; Schmidt, Mathias; Ziermann, Janine; Kohlmann, Alexander; Hochhaus, Andreas; Ernst, Thomas

2013-08-01

We sought to establish a convenient, sensitive next-generation sequencing (NGS) method for genotyping the 26 most commonly mutated leukemia-associated genes in a single work flow and to optimize this method for low amounts of input template DNA. We designed 184 PCR amplicons that cover all of the candidate genes. NGS was performed with genomic DNA (gDNA) from a cohort of 10 individuals with chronic myelomonocytic leukemia. The results were compared with NGS data obtained from sequencing of DNA generated by whole-genome amplification (WGA) of 20 ng template gDNA. Differences between gDNA and WGA samples in variant frequencies were determined for 2 different WGA kits. For gDNA samples, 25 of 26 genes were successfully sequenced with a sensitivity of 5%, which was achieved by a median coverage of 492 reads (range, 308-636 reads) per amplicon. We identified 24 distinct mutations in 11 genes. With WGA samples, we reliably detected all mutations above 5% sensitivity with a median coverage of 506 reads (range, 256-653 reads) per amplicon. With all variants included in the analysis, WGA amplification by the 2 kits tested yielded differences in variant frequencies that ranged from -28.19% to +9.94% [mean (SD) difference, -0.2% (4.08%)] and from -35.03% to +18.67% [mean difference, -0.75% (5.12%)]. Our method permits simultaneous analysis of a wide range of leukemia-associated target genes in a single sequencing run. NGS can be performed after WGA of template DNA for reliable detection of variants without introducing appreciable bias.

A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples.

PubMed

Dobhal, S; Zhang, G; Rohla, C; Smith, M W; Ma, L M

2014-10-01

PCR is widely used in the routine detection of foodborne human pathogens; however, challenges remain in overcoming PCR inhibitors present in some sample matrices. The objective of this study was to develop a simple, sensitive, cost-effective and rapid method for processing large numbers of environmental and pecan samples for Salmonella detection. This study was also aimed at validation of a new protocol for the detection of Salmonella from in-shell pecans. Different DNA template preparation methods, including direct boiling, prespin, multiple washing and commercial DNA extraction kits, were evaluated with pure cultures of Salmonella Typhimurium and with enriched soil, cattle feces and in-shell pecan each spiked individually with Salmonella Typhimurium. PCR detection of Salmonella was conducted using invA and 16S rRNA gene (internal amplification control) specific primers. The effect of amplification facilitators, including bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and gelatin on PCR sensitivity, was also evaluated. Conducting a prespin of sample matrices in combination with the addition of 0·4% (w/v) BSA and 1% (w/v) PVP in PCR mix was the simplest, most rapid, cost-effective and sensitive method for PCR detection of Salmonella, with up to 40 CFU Salmonella per reaction detectable in the presence of over 10(9 ) CFU ml(-1) of background micro-organisms from enriched feces soil or pecan samples. The developed method is rapid, cost-effective and sensitive for detection of Salmonella from different matrices. This study provides a method with broad applicability for PCR detection of Salmonella in complex sample matrices. This method has a potential for its application in different research arenas and diagnostic laboratories. © 2014 The Society for Applied Microbiology.

A multisite trial comparing two cytomegalovirus (CMV) pp65 antigenemia test kits, biotest CMV brite and Bartels/Argene CMV antigenemia.

PubMed

St George, K; Boyd, M J; Lipson, S M; Ferguson, D; Cartmell, G F; Falk, L H; Rinaldo, C R; Landry, M L

2000-04-01

A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia.

[Genetic analysis of human remains exhumed during archaeological excavations on former military training ground Brus in Lodz].

PubMed

Debska, Ewelina; Nowakowski, Piotr A; Jacewicz, Renata; Babol-Pokora, Katarzyna; Prośniak, Adam; Jedrzejczyk, Maciej; Berent, Jarosław

2013-01-01

The aim of this study was the genetic identification of Nazi repression victims. Human remains were found in 2011 in the area of former military training ground BRUS in Lodz. Genetic tests were performed upon the request of the Departmental Commission for the Prosecution of Crimes against the Polish Nation of the Institute of National Remembrance in Lodz. The research material was provided by the Institute of Archaeology (University of Lodz). It consisted of bones and teeth which were exhumed from mass Grave No 7. As a reference material we used a buccal swab collected from the putative son of one of the victims. Genomic DNA was extracted from the skeletal samples using the PrepFiler BTA Forensic DNA Extraction Kit. DNA was amplified using the AmpFlSTR Identifiler Plus PCR Amplification Kit and analyzed using an AB 3500 genetic analyzer. The obtained results showed 12 male genetic profiles. The analysis excluded paternity of 10 investigated victims. The genetic data of the remaining samples did not allow for paternity settlement.

Complete genome sequence of a novel Plum pox virus strain W isolate determined by 454 pyrosequencing.

PubMed

Sheveleva, Anna; Kudryavtseva, Anna; Speranskaya, Anna; Belenikin, Maxim; Melnikova, Natalia; Chirkov, Sergei

2013-10-01

The near-complete (99.7 %) genome sequence of a novel Russian Plum pox virus (PPV) isolate Pk, belonging to the strain Winona (W), has been determined by 454 pyrosequencing with the exception of the thirty-one 5'-terminal nucleotides. This region was amplified using 5'RACE kit and sequenced by the Sanger method. Genomic RNA released from immunocaptured PPV particles was employed for generation of cDNA library using TransPlex Whole transcriptome amplification kit (WTA2, Sigma-Aldrich). The entire Pk genome has identity level of 92.8-94.5 % when compared to the complete nucleotide sequences of other PPV-W isolates (W3174, LV-141pl, LV-145bt, and UKR 44189), confirming a high degree of variability within the PPV-W strain. The isolates Pk and LV-141pl are most closely related. The Pk has been found in a wild plum (Prunus domestica) in a new region of Russia indicating widespread dissemination of the PPV-W strain in the European part of the former USSR.

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

Microfluidic concentration of bacteria by on-chip electrophoresis

PubMed Central

Puchberger-Enengl, Dietmar; Podszun, Susann; Heinz, Helene; Hermann, Carsten; Vulto, Paul; Urban, Gerald A.

2011-01-01

In this contribution, we present a system for efficient preconcentration of pathogens without affecting their viability. Development of miniaturized molecular diagnostic kits requires concentration of the sample, molecule extraction, amplification, and detection. In consequence of low analyte concentrations in real-world samples, preconcentration is a critical step within this workflow. Bacteria and viruses exhibit a negative surface charge and thus can be electrophoretically captured from a continuous flow. The concept of phaseguides was applied to define gel membranes, which enable effective and reversible collection of the target species. E. coli of the strains XL1-blue and K12 were used to evaluate the performance of the device. By suppression of the electroosmotic flow both strains were captured with efficiencies of up to 99%. At a continuous flow of 15 μl/min concentration factors of 50.17 ± 2.23 and 47.36 ± 1.72 were achieved in less than 27 min for XL1-blue and K12, respectively. These results indicate that free flow electrophoresis enables efficient concentration of bacteria and the presented device can contribute to rapid analyses of swab-derived samples. PMID:22207893

Genome wide analysis in a discordant monozygotic twin with caudal appendage and multiple congenital anomalies.

PubMed

Cogulu, O; Pariltay, E; Koroglu, O A; Aykut, A; Ozyurek, R; Levent, E; Kultursay, N; Ozkinay, F

2013-01-01

Caudal appendage is a rare dysmorphic feature of which etiologic mechanisms are not well understood. Here we report monozygotic (MZ) twin brothers who are discordant for the caudal appendage and multiple congenital anomalies. Twins were the product of a 33 weeks of gestation, monochorionic-diamniotic pregnancy. On admission the proband had micrognathia, beaked nose, hypospadias, caudal appendage and juxtaductal aorta coarctation. At birth, he was small for gestational age and he had transient hypothyroidism which was detected in the newborn period. Karyotype analysis showed 46,XY. Monozygosity was shown by 15 microsatellite markers plus amelogenin (AmpFlSTR Identifiler PCR Amplification Kit, Applied Biosystems). Genome-wide copy number analysis of the twins by DNA-DNA hybridization of whole genomic DNA (NimbleGen Human CGH 385K WG-T v2.0 array) showed a significant difference at two neighboring probes with Log2 ratio: 0.72088 which are located on chromosome 3p12.3. Further analysis by high resolution of chromosome 3 array (Roche NimbleGen Human HG18 CHR3 FT Median Probe Spacing 475 bp) and quantitative PCR analysis did not confirm the deletion.

Peace and the News Media: SANE'S Action Kit.

ERIC Educational Resources Information Center

SANE, Washington, DC.

Designed to encourage public action in the media, this "action kit" consists of articles and reports dealing with the subjects of peace and the news media. Included are an article by Federal Communications Commissioner Nicholas Jackson which argues that the "law of effective reform" should be applied to the Federal Communications Commission (FCC)…

Successful isolation and PCR amplification of DNA from National Institute of Standards and Technology herbal dietary supplement standard reference material powders and extracts.

PubMed

Cimino, Matthew T

2010-03-01

Twenty-four herbal dietary supplement powder and extract reference standards provided by the National Institute of Standards and Technology (NIST) were investigated using three different commercially available DNA extraction kits to evaluate DNA availability for downstream nucleotide-based applications. The material included samples of Camellia, Citrus, Ephedra, Ginkgo, Hypericum, Serenoa, And Vaccinium. Protocols from Qiagen, MoBio, and Phytopure were used to isolate and purify DNA from the NIST standards. The resulting DNA concentration was quantified using SYBR Green fluorometry. Each of the 24 samples yielded DNA, though the concentration of DNA from each approach was notably different. The Phytopure method consistently yielded more DNA. The average yield ratio was 22 : 3 : 1 (ng/microL; Phytopure : Qiagen : MoBio). Amplification of the internal transcribed spacer II region using PCR was ultimately successful in 22 of the 24 samples. Direct sequencing chromatograms of the amplified material suggested that most of the samples were comprised of mixtures. However, the sequencing chromatograms of 12 of the 24 samples were sufficient to confirm the identity of the target material. The successful extraction, amplification, and sequencing of DNA from these herbal dietary supplement extracts and powders supports a continued effort to explore nucleotide sequence-based tools for the authentication and identification of plants in dietary supplements. (c) Georg Thieme Verlag KG Stuttgart . New York.

Genetic polymorphisms of 15 STR loci within Turkish student population living in Sarajevo, Bosnia and Herzegovina.

PubMed

Dogan, Serkan; Kovacević, Lejla; Marjanović, Damir

2013-12-01

Allele frequencies of 15 STRs included in the PowerPlex 16 System (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX and FGA) were calculated from the referent sample of 100 unrelated individuals of both sexes from Turkish student population living in Sarajevo, Bosnia and Herzegovina. Buccal swab, as a source of DNA, was collected from the volunteers from whom the informed consent form was obtained. DNA extraction was performed using QIAamp DNA Micro kit by Qiagen. DNA template ranging from 0.5 to 2 ng was used to amplify 15 STR loci by PCR multiplex amplification which was performed by using the PowerPlex 16 kit (Promega Corp., Madison, WI, USA) according to the manufacturer's protocol. The amplifications were carried out in a PE Gene Amp PCR System thermal cycler (Applied Biosystems) and capillary electrophoresis was carried out in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) in accordance with the manufacturer's recommendations. The frequency of each locus was calculated from the numbers of each observed genotype. Deviation from Hardy-Weinberg equilibrium and observed heterozygosity were calculated. Data were analyzed by using Microsoft Excel workbook template--Powerstats V12 and the power of discrimination (PD), power of exclusion (PE), as well as other population genetic indices for the 15 STR loci were calculated. Obtained results contribute to existing Turkish DNA database, as well as insight of differences and similarities in comparison to population of Bosnia and Herzegovina. In addition, 13 autosomal STR loci frequencies (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSFIPO, Penta D, VWA, D8S1 179, TPOX, and FGA) were studied in 15 different worldwide populations (Turkish, Bosnian, Croatian, Serbian, Montenegrin, Macedonian, Albanian, Kosovan, Greek, Russian, Japanese, Korean, Lithuanian, Iraqi, Belarusian). For the proof of corresponding data, two different Turkish population STR data obtained from previously published articles were compared with our data and this showed that our data correspond to these 2 previously published data. Further, STR allele frequency data for 13 loci for each population were obtained from previous scientific articles and the allele frequencies and genetic diversity among the 15 sample populations were compared. In addition, even though the populations are from different nationalities, the STR data are similar among the geographically close populations. The phylogenetic tree established among worldwide populations and genetic distance values show a great affinity among the 15populations. Our data is useful for anthropological and further comparative genetic studies of populations.

Two brothers' alleged paternity for a child: who is the father?

PubMed

Dogan, Muhammed; Kara, Umut; Emre, Ramazan; Fung, Wing Kam; Canturk, Kemal Murat

2015-06-01

In paternity cases where individuals are close relatives, it may be necessary to evaluate mother's DNA profile (trio test) and to increase the number of polymorphic STR loci that are analyzed. In our case, two alleged fathers who are brothers and the child (duo case) were analyzed based on 20 STR loci; however, no exclusions could be achieved. Then trio test (with mother) was performed using the Identifiler Plus kit (Applied Biosystems) and no exclusions could be achieved again. Analysis performed with the ESS Plex Plus kit (Qiagen), the paternity of one of the two alleged fathers was rejected only on 2 STR loci. We made the calculations of power of exclusion values to interpret our results more properly. The probability of exclusion (PE) is calculated as 0.9776546 in 15 loci of Identifiler Plus kit without mother. The PE is calculated as 0.9942803, if 5 additional loci from ESS Plex Plus kit are typed. The PE becomes 0.9961048 for the Identifiler Plus kit in trio analysis. If both Identifiler Plus and ESS Plex Plus kits are used for testing, the PE is calculated as 0.999431654, which indicates that the combined kits are highly discriminating.

Indoor Air Quality: Tools for Schools Action Kit.

ERIC Educational Resources Information Center

Environmental Protection Agency, Washington, DC. Indoor Air Div.

Good indoor air quality contributes to a favorable learning environment for students, productivity for teachers and staff, and a sense of comfort, health, and well-being for all school occupants. The goal of this kit is to provide clear and easily applied guidance that will help prevent Indoor Air Quality (IAQ) problems and resolve such problems…

Using Control Heuristics as a Means to Explore the Educational Potential of Robotics Kits

ERIC Educational Resources Information Center

Gaudiello, Ilaria; Zibetti, Elisabetta

2013-01-01

The educational potential of robotics kits as a form of control technology will remain undervalued until meaningful observation parameters are identified to enable a better understanding of children?s control strategies. For this reason, this paper aims primarily to identify and classify the heuristics spontaneously applied by 6-10 year old…

77 FR 14000 - Foreign-Trade Zone 126-Reno, NV; Application for Temporary/Interim Manufacturing Authority...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-08

..., NV; Application for Temporary/ Interim Manufacturing Authority, Brightpoint North America L.P. (Cell... to produce cell phone kits (HTSUS 8517.12, 8517.69, duty free). Foreign components that would be used... customs entry procedures that apply to cell phone kits (duty free) for the foreign inputs noted above. In...

78 FR 39707 - Foreign-Trade Zone (FTZ) 32-Miami, Florida; Notification of Proposed Production Activity...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-02

..., Florida; Notification of Proposed Production Activity; Brightstar Corporation; (Cell Phone Kitting); Miami... FTZ 32. The facility is used for the kitting of cell phones and cell phone accessories. Pursuant to 15..., Brightstar would be able to choose the duty rates during customs entry procedures that apply to cell phones...

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

PubMed

Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

2016-04-01

Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

PubMed Central

LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

2011-01-01

Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. PMID:21573065

Genotyping of Plant and Animal Samples without Prior DNA Purification

PubMed Central

Chum, Pak Y.; Haimes, Josh D.; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

2012-01-01

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

A Measurement of Vocational Education’s Student Satisfaction in Learning Electronic Appliance Repair and Maintenance Course with CCTV Trainer Kit

NASA Astrophysics Data System (ADS)

Rakhmawati, Lusia; Irwansyah Febriyanto, Fariz

2018-04-01

This study aimed to investigate satisfaction levels, among of the eleventh-grade students majoring in audio-video technique, towards the instructional media CCTV trainer kit: The CCTV Prototype and job sheet on to and analyzed perspectives of the practical approach to learning using CCTV prototype to improve installation skills in the electronic appliance repair and maintenance class. Survey questionnaires and instructional media were applied to data collection. The students’ satisfaction towards the instructional media in teaching and learning process was presented in positive responses, 91.94 % satisfied. The finding reveals that the instructional media using trainer could better to overcome the need a practical approach to learning. Furthermore, using trainer kit media creates an environment where students can support each other and receive feedback from their peers. They performed practice activities that help them apply the new information from the steps on the job sheet.

Connecting University and Student Teaching Experiences through the Jackdaw Kit Project

ERIC Educational Resources Information Center

Marshall, Jill

2010-01-01

The author shares the challenges faced in her social studies methods class due to the constraints of NCLB. By incorporating the jackdaw kit project within her social studies methods course, her candidates were able to connect what they were talking about in methods and apply it to their students teaching situations where there was little time for…

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

PubMed

Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

2009-02-02

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

Study of genetic markers of CODIS and ESS systems in a population of individuals from Cabo Verde living in Lisboa.

PubMed

Resende, Ana; Amorim, António; da Silva, Cláudia Vieira; Ribeiro, Teresa; Porto, Maria João; Costa Santos, Jorge; Afonso Costa, Heloísa

2017-01-01

Twenty-two autosomal short tandem repeats included in the PowerPlex® Fusion System Amplification kit (Promega Corporation) were genotyped in a population sample of 500 unrelated individuals from Cabo Verde living in Lisboa. Allelic frequency data and forensic and statistical parameters were calculated and evaluated in this work. The genetic relationship among immigrant population from Cabo Verde living in Lisboa and other populations, such as Brazilian and Angola immigrants living in Lisboa; Afro-Americans, Caucasians, Hispanics and Asians living in the USA and the population from Lisboa was assessed, and a multidimensional scaling plot was drown to show these results.

Parametric amplification in a resonant sensing array

NASA Astrophysics Data System (ADS)

Yie, Zi; Miller, Nicholas J.; Shaw, Steven W.; Turner, Kimberly L.

2012-03-01

We demonstrate parametric amplification of a multidegree of freedom resonant mass sensing array via an applied base motion containing the appropriate frequency content and phases. Applying parametric forcing in this manner is simple and aligns naturally with the vibrational properties of the sensing structure. Using this technique, we observe an increase in the quality factors of the coupled array resonances, which provides an effective means of improving device sensitivity.

[Clinical application of testing methods on acid-fast bacteria].

PubMed

Ichiyama, Satoshi; Suzuki, Katsuhiro

2005-02-01

Clinical bacteriology pertaining to acid-fast bacteria has made marked advances over the past decade, initiated by the development of a DNA probe kit for identification of acid-fast bacteria. Wide-spread use of nucleic acid amplification for rapid detection of tubercle bacillus contributed more greatly than any other factor to such advances in this field. At present, 90% of all kits used for nucleic acid amplification in the world are consumed in Japan. Unfortunately, not a few clinicians in Japan have a false idea that the smear method and nucleic acid amplification are necessary but culture is not. In any event nucleic acid amplification has exerted significant impacts on the routine works at bacteriology laboratories. Among others, collecting bacteria by pretreatment with NALC-NaOH has simplified the introduction of the collective mode smear method and liquid media. Furthermore, as clinicians have become increasingly more experienced with various methods of molecular biology, it now seems possible to apply these techniques for detection of genes encoding drug resistance and for utilization of molecular epidemiology in routine laboratory works. Meanwhile, attempts to diagnose acid-fast bacteriosis by checking blood for antibody have also been made, primarily in Japan. At present, two kits for detecting antibodies to glycolipids (LAM, TDM, etc.) are covered by national health insurance in Japan. We have an impression that in Japan clinicians do not have adequate knowledge and skill to make full use of these new testing methods clinically. We, as the chairmen of this symposium, hope that this symposium will help clinicians increase their skill related to new testing methods, eventually leading to stimulation of advances in clinical practices related to acid-fast bacteria in Japan. 1. Smear microscopy by concentration method and broth culture system: Kazunari TSUYUGUCHI (Clinical Research Center, National Hospital Organization Kinki-chuo Chest Medical Center) Smear microscopy and culture still remain the cornerstone to diagnose tuberculosis. However, the classical methods in Japan using direct microscopy and Ogawa solid media were not sufficient for clinical use. In recent years substantial advance has been made in these fields. Concentration of clinical samples by centrifugation improves the sensitivity of smear microscopy with excellent reproducibility. The Mycobacteria Growth Indicator Tube (MGIT) system using liquid media yields high sensitivity and rapidity. Using these methods, more and more tuberculosis cases would be correctly diagnosed and treated adequately based on drug susceptibility testing. 2. New technologies for anti-tuberculosis drug susceptibility testing: Satoshi MITARAI (Bacteriology Division, Reference Centre for Mycobacterium, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) Several new technologies have been developed to obtain anti-tuberculosis drug susceptibility testing (AST) results rapidly, utilising liquid culture and molecular technologies. Mycobacterium Growth Indicator Tube (MGIT), as a popular liquid culturing and AST system, was evaluated for its accuracy and usefulness. As for isoniazid, MGIT showed 12.6% of discordant result comparing with standard method. These MGIT resistant and Ogawa susceptible strains had relatively high MICs ranging 0.13 to 2.0 microg/ml. The molecular detection of resistant gene mutation is also a useful method to estimate drug resistance rapidly. The rpoB mutation detection is reliable with high sensitivity and specificity. 3. Nucleic acid amplification and novel diagnostic methods: Shunji TAKAKURA (Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine) Sensitivities of nucleic acid amplification tests (NAATs) for the diagnosis of tuberculosis meet clinical requirement that patients with high-risk of transmission should be identified within a day. Comparison of the performance of various NAATs is difficult because of the difference in sample processing and in samples tested among methods and reports. Considering the limitations of NAATs (low sensitivity compared with culture, inability to differentiate dead bacilli from the living), further advances would be expected when novel technologies could confer additional information, such as drug susceptibility, quantity, viability, and genotype. 4. Serodiagnosis of Mycobacterium avium complex lung disease: Seigo KITADA (Department of Internal Medicine, National Hospital Organization Toneyama National Hospital) Mycobacterium avium complex (MAC) organisms are ubiquitous in environment and a contamination in respiratory tracts is sometimes observed, and that complex the diagnosis. We developed a serodiagnostic method for MAC disease using an enzyme immunoassay with the MAC-specific glycopeptidolipid (GPL) core as antigen. A significant increase in GPL core antibodies was detected in sera of patients with MAC pulmonary diseases compared to patients who were colonized with MAC, patients with M. kansasii disease and tuberculosis and healthy subjects. The serodiagnosis is useful for diagnosis of MAC lung disease. 5. Molecular epidemiologic tools for tuberculosis: IS6110 RFLP, Spoligotyping, and VNTR: Tomoshige MATSUMOTO, Hiromi ANO, Tetsuya TAKASHIMA, Izuo TSUYUGUCHI (Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) We have performed molecular typing on about 1,300 culture positive clinical isolates that made up the majority of tuberculosis strains in part of southeast Osaka since 2001 until now. By spoligotyping, about 75% of entire strains belonged to the Beijing strain. Particular spoligotyping descriptions, which were not described in SpolDBIII, were found in the strains with lower than 6 copies of IS6110 RFLP. We described them as Osaka type. We could also show that direct typing from Tb PCR positive sputum of patients with tuberculosis was possible by VNTR and that VNTR with 16 loci was useful in tuberculosis typing in Osaka.

Regulation of a mammalian gene bearing a CpG island promoter and a distal enhancer.

PubMed

Berrozpe, Georgina; Bryant, Gene O; Warpinski, Katherine; Ptashne, Mark

2013-08-15

A quantitative nucleosome occupancy assay revealed rules for nucleosome disposition in yeast and showed how disposition affects regulation of the GAL genes. Here, we show how those findings apply to the control of Kit, a mammalian gene. The Kit promoter lies in a CpG island, and its enhancer (active in mast cells) lies some 150 kb upstream. Nucleosomes form with especially high avidities at the Kit promoter, a reaction that, we surmise, ensures extremely low basal expression. In mast cells, transcriptional activators displace nucleosomes that are less tightly formed at the Kit enhancer. In turn, the active enhancer replaces a single Kit promoter nucleosome with the transcriptional machinery, thereby inducing transcription over 1,000-fold. As at the yeast GAL genes, the inhibitory effects of nucleosomes facilitate high factors of induction by mammalian activators working in the absence of specific repressors. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Variant Alleles, Triallelic Patterns, and Point Mutations Observed in Nuclear Short Tandem Repeat Typing of Populations in Bosnia and Serbia

PubMed Central

Huel, René L. M.; Bašić, Lara; Madacki-Todorović, Kamelija; Smajlović, Lejla; Eminović, Izet; Berbić, Irfan; Miloš, Ana; Parsons, Thomas J.

2007-01-01

Aim To present a compendium of off-ladder alleles and other genotyping irregularities relating to rare/unexpected population genetic variation, observed in a large short tandem repeat (STR) database from Bosnia and Serbia. Methods DNA was extracted from blood stain cards relating to reference samples from a population of 32 800 individuals from Bosnia and Serbia, and typed using Promega’s PowerPlex®16 STR kit. Results There were 31 distinct off-ladder alleles were observed in 10 of the 15 STR loci amplified from the PowerPlex®16 STR kit. Of these 31 alleles, 3 have not been previously reported. Furthermore, 16 instances of triallelic patterns were observed in 9 of the 15 loci. Primer binding site mismatches that affected amplification were observed in two loci, D5S818 and D8S1179. Conclusion Instances of deviations from manufacturer’s allelic ladders should be expected and caution taken to properly designate the correct alleles in large DNA databases. Particular care should be taken in kinship matching or paternity cases as incorrect designation of any of these deviations from allelic ladders could lead to false exclusions. PMID:17696304

[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.

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.

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

PubMed

Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L

2018-05-02

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.

Evaluation of the Loop Mediated Isothermal DNA Amplification (LAMP) Kit for Malaria Diagnosis in P. vivax Endemic Settings of Colombia

PubMed Central

Vallejo, Andrés F.; Martínez, Nora L.; González, Iveth J.; Arévalo-Herrera, Myriam; Herrera, Sócrates

2015-01-01

Background Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance. Methodology/Principal Findings First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms. Conclusions/Significance mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies. PMID:25569550

Development of Local Amplification Factors in the NEAM Region for Production of Regional Tsunami Hazard Maps

NASA Astrophysics Data System (ADS)

Harbitz, C. B.; Glimsdal, S.; Løvholt, F.; Orefice, S.; Romano, F.; Brizuela, B.; Lorito, S.; Hoechner, A.; Babeyko, A. Y.

2016-12-01

The standard way of estimating tsunami inundation is by applying numerical depth-averaged shallow-water run-up models. However, for a regional Probabilistic Tsunami Hazard Assessment (PTHA), applying such inundation models may be too time-consuming. A faster, yet less accurate procedure, is to relate the near-shore surface elevations at offshore points to maximum shoreline water levels by using a set of amplification factors based on the characteristics of the incident wave and the bathymetric slope. The surface elevation at the shoreline then acts as a rough approximation for the maximum inundation height or run-up height along the shoreline. An amplification-factor procedure based on a limited set of idealized broken shoreline segments has previously been applied to estimate the maximum inundation heights globally. Here, we present a study where this technique is developed further, by taking into account the local bathymetric profiles. We extract a large number of local bathymetric transects over a significant part of the North East Atlantic, the Mediterranean and connected seas (NEAM) region. For each bathymetric transect, we compute the wave amplification from an offshore control point to points close to the shoreline using a linear shallow-water model for waves of different period and polarity with a sinusoidal pulse wave as input. The amplification factors are then tabulated. We present maximum water levels from the amplification factor method, and compare these with results from conventional inundation models. Finally, we demonstrate how the amplification factor method can be convolved with PTHA results to provide regional tsunami hazard maps. This work has been supported by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement 603839 (Project ASTARTE), and the TSUMAPS-NEAM Project (http://www.tsumapsneam.eu/), co-financed by the European Union Civil Protection Mechanism, Agreement Number: ECHO/SUB/2015/718568/PREV26.

Development of Local Amplification Factors in the NEAM Region for Production of Regional Tsunami Hazard Maps

NASA Astrophysics Data System (ADS)

Glimsdal, Sylfest; Løvholt, Finn; Bonnevie Harbitz, Carl; Orefice, Simone; Romano, Fabrizio; Brizuela, Beatriz; Lorito, Stefano; Hoechner, Andreas; Babeyko, Andrey

2017-04-01

The standard way of estimating tsunami inundation is by applying numerical depth-averaged shallow-water run-up models. However, for a regional Probabilistic Tsunami Hazard Assessment (PTHA), applying such inundation models may be too time-consuming. A faster, yet less accurate procedure, is to relate the near-shore surface elevations at offshore points to maximum shoreline water levels by using a set of amplification factors based on the characteristics of the incident wave and the bathymetric slope. The surface elevation at the shoreline then acts as a rough approximation for the maximum inundation height or run-up height along the shoreline. An amplification-factor procedure based on a limited set of idealized broken shoreline segments has previously been applied to estimate the maximum inundation heights globally. Here, we present a study where this technique is developed further, by taking into account the local bathymetric profiles. We extract a large number of local bathymetric transects over a significant part of the North East Atlantic, the Mediterranean and connected seas (NEAM region). For each bathymetric transect, we compute the wave amplification from an offshore control point to points close to the shoreline using a linear shallow-water model for waves of different period and polarity with a sinusoidal pulse wave as input. The amplification factors are then tabulated. We present maximum water levels from the amplification factor method, and compare these with results from conventional inundation models. Finally, we demonstrate how the amplification factor method can be convolved with PTHA results to provide regional tsunami hazard maps. This work has been supported by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement 603839 (Project ASTARTE), and the TSUMAPS-NEAM Project (http://www.tsumapsneam.eu/), co-financed by the European Union Civil Protection Mechanism, Agreement Number: ECHO/SUB/2015/718568/PREV26.

[4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03) inhibits SCF/c-kit signaling in 501mel human melanoma cells and abolishes melanin production in mice and brownish guinea pigs.

PubMed

Na, Yong Joo; Baek, Heung Su; Ahn, Soo Mi; Shin, Hyun Jung; Chang, Ih-Seop; Hwang, Jae Sung

2007-09-01

It is well known that c-kit is related to pigmentation as well as to the oncology target protein. The objective of this study was to discover a skin-whitening agent that regulates c-kit activity. We have developed a high-throughput screening system using recombinant human c-kit protein. Approximately 10,000 synthetic compounds were screened for their effect on c-kit activity. Phenyl-imidazole sulfonamide derivatives showed inhibitory activity on c-kit phosphorylation in vitro. The effects of one derivative, [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03), on stem-cell factor (SCF)/c-kit cellular signaling in 501mel human melanoma cells were examined further. Pretreatment of 501mel cells with ISCK03 inhibited SCF-induced c-kit phosphorylation dose dependently. ISCK03 also inhibited p44/42 ERK mitogen-activated protein kinase (MAPK) phosphorylation, which is known to be involved in SCF/c-kit downstream signaling. However ISCK03 did not inhibit hepatocyte growth factor (HGF)-induced phosphorylation of p44/42 ERK proteins. To determine the in vivo potency of ISCK03, it was orally administered to depilated C57BL/6 mice. Interestingly, oral administration of ISCK03 induced the dose-dependent depigmentation of newly regrown hair, and this was reversed with cessation of ISCK03 treatment. Finally, to investigate whether the inhibitory effect of ISCK03 on SCF/c-kit signaling abolished UV-induced pigmentation, ISCK03 was applied to UV-induced pigmented spots on brownish guinea pig skin. The topical application of ISCK03 promoted the depigmentation of UV-induced hyperpigmented spots. Fontana-Masson staining analysis showed epidermal melanin was diminished in spots treated with ISCK03. These results indicate that phenyl-imidazole sulfonamide derivatives are potent c-kit inhibitors and might be used as skin-whitening agents.

Signal transduction and amplification through enzyme-triggered ligand release and accelerated catalysis.

PubMed

Goggins, Sean; Marsh, Barrie J; Lubben, Anneke T; Frost, Christopher G

2015-08-01

Signal transduction and signal amplification are both important mechanisms used within biological signalling pathways. Inspired by this process, we have developed a signal amplification methodology that utilises the selectivity and high activity of enzymes in combination with the robustness and generality of an organometallic catalyst, achieving a hybrid biological and synthetic catalyst cascade. A proligand enzyme substrate was designed to selectively self-immolate in the presence of the enzyme to release a ligand that can bind to a metal pre-catalyst and accelerate the rate of a transfer hydrogenation reaction. Enzyme-triggered catalytic signal amplification was then applied to a range of catalyst substrates demonstrating that signal amplification and signal transduction can both be achieved through this methodology.

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma.

PubMed

Takata, Minoru; Murata, Hiroshi; Saida, Toshiaki

2010-02-01

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark's multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking 'field melanocytes', which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.

Occurrence and characterisation of pathogenic Escherichia coli isolated from beef meat imported from South Africa and marketed in united Arab Emirates

NASA Astrophysics Data System (ADS)

Dulaimi, Samar Abdulkareem Khalifa Al; Ibrahim, Nazlina

2018-04-01

This study is aimed at determining the presence of Escherichia coli and characterizing the availability of Shiga toxin gene in beef meat samples imported from South Africa. Meat samples (n=47) were randomly collected from meat sections at different supermarkets in the United Arab Emirates (UAE) from the beginning of August 2016 till the end of March 2017. Samples were diluted and inoculated on MacConkey agar and Eosin methylene blue agar (EMB), and the colony forming units (CFU) were recorded. API 20E identification kit was used for biochemical identification of E. coli. Detection of the 16S rRNA and shiga toxin genes by PCR amplification was done. Our results revealed that 14 (29.7%) out of the total 47 samples were positive for E. coli. The bacterial burden of E. coli as determined by colony growth showed variable level of contamination. From the PCR amplification, the shiga toxin gene carried by the E. coli is the Stx2 gene. This study revealed moderately high contamination levels of E. coli in beef samples imported from South Africa and marketed in UAE which mostly carries the shiga toxin gene Stx2.

Optimising the diagnostic strategy for onychomycosis from sample collection to FUNGAL identification evaluation of a diagnostic kit for real-time PCR.

PubMed

Petinataud, Dimitri; Berger, Sibel; Ferdynus, Cyril; Debourgogne, Anne; Contet-Audonneau, Nelly; Machouart, Marie

2016-05-01

Onychomycosis is a common nail disorder mainly due to dermatophytes for which the conventional diagnosis requires direct microscopic observation and culture of a biological sample. Nevertheless, antifungal treatments are commonly prescribed without a mycological examination having been performed, partly because of the slow growth of dermatophytes. Therefore, molecular biology has been applied to this pathology, to support a quick and accurate distinction between onychomycosis and other nail damage. Commercial kits are now available from several companies for improving traditional microbiological diagnosis. In this paper, we present the first evaluation of the real-time PCR kit marketed by Bio Evolution for the diagnosis of dermatophytosis. Secondly, we compare the efficacy of the kit on optimal and non-optimal samples. This study was conducted on 180 nails samples, processed by conventional methods and retrospectively analysed using this kit. According to our results, this molecular kit has shown high specificity and sensitivity in detecting dermatophytes, regardless of sample quality. On the other hand, and as expected, optimal samples allowed the identification of a higher number of dermatophytes by conventional mycological diagnosis, compared to non-optimal samples. Finally, we have suggested several strategies for the practical use of such a kit in a medical laboratory for quick pathogen detection. © 2016 Blackwell Verlag GmbH.

Reversible Gating of Plasmonic Coupling for Optical Signal Amplification.

PubMed

Khoury, Christopher G; Fales, Andrew M; Vo-Dinh, Tuan

2016-07-20

Amplification of optical signals is useful for a wide variety of applications, ranging from data signal transmission to chemical sensing and biomedical diagnostics. One such application in chemical sensing is surface-enhanced Raman scattering (SERS), an important technique for increasing the Raman signal using the plasmonic effect of enhanced electromagnetic fields associated with metallic nanostructures. One of the most important limitations of SERS-based amplification is the difficulty to reproducibly control the SERS signal. Here, we describe the design and implementation of a unique hybrid system capable of producing reversible gating of plasmonic coupling for Raman signal amplification. The hybrid system is composed of two subsystems: (1) colloidal magneto-plasmonic nanoparticles for SERS enhancement and (2) a micromagnet substrate with an externally applied magnetic field to modulate the colloidal nanoparticles. For this proof of concept demonstration, the nanoparticles were labeled with a Raman-active dye, and it was shown that the detected SERS signal could be reproducibly modulated by controlling the externally applied magnetic field. The developed system provides a simple, robust, inexpensive, and reusable device for SERS signal modulation. These properties will open up new possibilities for optical signal amplification and gating as well for high-throughput, reproducible SERS detection.

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.

Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors.

PubMed

Yan, Weixin; Zhang, Aiguo; Powell, Michael J

2016-07-21

Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived "driver" and "drug-resistant" alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called "liquid biopsy" allows for the dynamic monitoring of the patients' tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

Detection of Hepatitis B Virus DNA among Chronic and potential Occult HBV patients in resource-limited settings by Loop-Mediated Isothermal Amplification assay.

PubMed

Akram, Arifa; Islam, S M Rashedul; Munshi, Saif Ullah; Tabassum, Shahina

2018-05-16

Transmission of Hepatitis B Virus (HBV) usually occurs due to the transfusion of blood or blood products from chronic HBV (CHB) or occult HBV infected (OBI) patients. Besides serological tests e.g. HBsAg and anti-HBc (total), detection of HBV-DNA is necessary for the diagnosis of OBI patients. Different nucleic acid tests (NATs) including real-time-Polymerase Chain Reaction (qPCR) are used for the detect HBV-DNA. The NATs are expensive and require technical expertise which are barriers to introducing them in resource-limited settings. This study was undertaken to evaluate the use of Loop-Mediated Isothermal Amplification (LAMP) assay as an alternative to qPCR for the detection of HBV-DNA in CHB and potential OBI patients in resource-limited settings. Following the published protocols with some modifications, a LAMP assay was developed for detection of HBV-DNA by either using a heat block followed by detection in an agarose gel or using a qPCR thermocycler. The LAMP assay was applied to supernatant prepared from heat treated serum collected from CHB and potential OBI patients. HBV viral load in serum was measured by qPCR using a single step HBV-DNA quantification kit. Among 200 samples tested, qPCR was capable to detect HBV-DNA in 25.5% of cases, whereas LAMP assay detected HBV-DNA in 43.5% cases. The qPCR was able to detect 11 (9.16%) potential OBI cases, whereas LAMP assay identified HBV-DNA in 43 (35.83%) cases. In addition to tests for HBsAg and/or anti-HBc (total), detection of HBV-DNA by LAMP assay may aid in preventing post-transfusion HBV infection in resource-limited settings. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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.

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

PubMed Central

Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

2012-01-01

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.) In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders. We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity. PMID:22806215

Habitat evaluation using GIS a case study applied to the San Joaquin Kit Fox

USGS Publications Warehouse

Gerrard, R.; Stine, P.; Church, R.; Gilpin, M.

2001-01-01

Concern over the fate of plant and animal species throughout the world has accelerated over recent decades. Habitat loss is considered the main culprit in reducing many species' abundance and range, leading to numerous efforts to plan and manage habitat preservation. Our work uses Geographic Information Systems (GIS) data and modeling to define a spatially explicit analysis of habitat value, using the San Joaquin Kit Fox (Vulpes macrotis mutica) of California (USA) as an example. Over the last 30 years, many field studies and surveys have enhanced our knowledge of the life history, behavior, and needs of the kit fox, which has been proposed as an umbrella or indicator species for grassland habitat in the San Joaquin Valley of California. There has yet been no attempt to convert much of this field knowledge into a model of spatial habitat value useful for planning purposes. This is a significant omission given the importance and visibility of the imperiled kit fox and increasing trends toward spatially explicit modeling and planning. In this paper we apply data from northern California to derive a small-cell GIS raster of habitat value for the kit fox that incorporates both intrinsic habitat quality and neighborhood context, as well the effects of barriers such as roads. Such a product is a useful basis for assessing the presence and amounts of good (and poor) quality habitat and for eventually constructing GIS representations of viable animal territories that could be included in future reserves. ?? 2001 Elsevier Science B.V.

Amplification in Technical Manuals: Theory and Practice.

ERIC Educational Resources Information Center

Killingsworth, M. Jimmie; And Others

1989-01-01

Examines how amplification (rhetorical techniques by which discourse is extended to enhance its appeal and information value) tends to increase and improve the coverage, rationale, warnings, behavioral alternatives, examples, previews, and general emphasis of technical manuals. Shows how classical and modern rhetorical theories can be applied to…

APPLIED AUDIOLOGY FOR CHILDREN. SECOND EDITION.

ERIC Educational Resources Information Center

DALE, D.M.C.

THE PURPOSE OF THE BOOK IS TO HELP TEACHERS, PARENTS, DOCTORS, AND WORKERS IN AUDIOLOGY CLINICS MAKE THE MOST OF SOUND IN THE EDUCATIONAL AND SOCIAL TREATMENT OF DEAFNESS. ASPECTS OF SOUND AMPLIFICATION CONSIDERED ARE THE NATURE OF SOUND, ELECTRICAL AMPLIFICATION, AND VARIOUS TYPES OF HEARING AIDS (INDIVIDUAL, GROUP, INDUCTION LOOP, SPEECH…

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy.

PubMed

Wang, Xiaoying; Shu, Guofang; Gao, Chanchan; Yang, Yu; Xu, Qian; Tang, Meng

2014-12-01

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs-PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs-PA6-PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs-ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

PubMed

Shanks, Orin C; Kelty, Catherine A; Oshiro, Robin; Haugland, Richard A; Madi, Tania; Brooks, Lauren; Field, Katharine G; Sivaganesan, Mano

2016-05-01

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures.

PubMed

Weiler, Natalie E C; Matai, Anuska S; Sijen, Titia

2012-01-01

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpFℓSTR(®) Identifiler(®) amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpFℓSTR(®) Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Female users of internet-based screening for rectal STIs: descriptive statistics and correlates of positivity.

PubMed

Ladd, Jessica; Hsieh, Yu-Hsiang; Barnes, Mathilda; Quinn, Nicole; Jett-Goheen, Mary; Gaydos, Charlotte A

2014-09-01

Internet-based screening for vaginal sexually transmitted infections (STI) has been shown to reach high-risk populations. Published studies of internet-based screening for rectal STIs in women are needed. Our objectives were to describe the female users of a rectal internet-based screening intervention and assess what factors correlated with rectal positivity for STIs. The website http://www.iwantthekit.org offers free STI testing via home self-sampling kits. Women could order vaginal and rectal kits, both containing questionnaires. Rectal and vaginal swabs were tested for Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis using nucleic acid amplification tests. Data were analysed from 205 rectal kits from January 2009 through February 2011. Self-reported characteristics of participants were examined, and correlates of rectal STI positivity were analysed. Of the 205 rectal samples returned and eligible for testing, 38 (18.5%) were positive for at least one STI. The women were young (mean age 25.8 years), mostly African-American (50.0%), and only 14.0% always used condoms. After adjusting for age and race, Black race (AOR=3.06) and vaginal STI positivity (AOR=40.6) were significantly correlated with rectal STI positivity. Of women testing positive for rectal STIs who also submitted vaginal swabs, 29.4% were negative in the vaginal sample. Internet-based rectal screening can reach populations that appear to be at high risk for rectal STIs (18.5% prevalence) and led to the diagnosis of STIs in women who would not have been diagnosed vaginally. Black race and vaginal STI positivity were highly correlated with rectal STI positivity. 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.

SAS molecular tests Escherichia coli O157 detection kit. Performance tested method 031203.

PubMed

Bapanpally, Chandra; Montier, Laura; Khan, Shah; Kasra, Akif; Brunelle, Sharon L

2014-01-01

The SAS Molecular tests Escherichia coli O157 Detection method, a loop-mediated isothermal amplification method, performed as well as or better than the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, bagged mixed lettuce, and fresh spinach. Ground beef (30% fat, 25 g test portion) was validated for 7-8 h enrichment, leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was also shown to be acceptable under conditions of co-enrichment with Salmonella. Thus, after a short co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. The SAS Molecular tests Salmonella Detection Kit was validated using the same test portions as for the SAS Molecular tests E. coli O157 Detection Kit and those results are presented in a separate report. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 E. coli 0157 strains, including H7 and non-motile strains, and 30 non-E. coli O157 strains examined. Finally, the method was shown to be robust when variations to DNA extract hold time and DNA volume were varied. The method comparison and robustness data suggest a full 7 h enrichment time should be used for 25 g ground beef test portions.

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay.

PubMed

Varlet-Marie, Emmanuelle; Sterkers, Yvon; Perrotte, Marina; Bastien, Patrick

2018-05-01

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10 5  tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at -20 °C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer's recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1 T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples. Copyright © 2018. Published by Elsevier Ltd.

DNA typing for personal identification of urine after long-term preservation for testing in doping control.

PubMed

Aoki, Kimiko; Tanaka, Hiroyuki; Ueki, Makoto

2017-08-01

When the tampering of a urine sample is suspected in doping control, personal identification of the sample needs to be determined by short tandem repeat (STR) analysis using DNA. We established a method for extracting DNA from urine samples stored at -20 °C without using any additives or procedures, which is consistent with how samples are required to be managed for doping control. The method, using the Puregene® Blood Core kit followed by NucleoSpin® gDNA Clean-up or NucleoSpin® gDNA Clean-up XS kit, does not need any special instrument and can provide a purified extract with high-quality DNA from up to 40 mL of urine suitable for STR analysis using an AmpFlSTR® Identifiler® PCR amplification kit. Storing urine at -20 °C is detrimental to the stability of DNA. The DNA concentration of preserved urine could not be predicted by specific gravity or creatinine level at the time of urine collection. The DNA concentration of a purified extract (10 μL) was required to be >0.06 ng/μL to ensure a successful STR analysis. Thus, the required extraction volumes of urine preserved for 3-7 years at -20 °C were estimated to be 30 mL and 20 mL to succeed in at least 86% of men and 91% of women, respectively. Considering the long half-life of DNA during long-term preservation, our extraction method is applicable to urine samples stored even for 10 years, which is currently the storage duration allowed (increased from 8 years) before re-examination in doping control. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

First Evaluation of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Vivax Malaria Endemic Regions in the Republic of Korea

PubMed Central

Goo, Youn-Kyoung; Ji, So-Young; Shin, Hyun-Il; Moon, Jun-Hye; Cho, Shin-Hyung; Lee, Won-Ja; Kim, Jung-Yeon

2014-01-01

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK). Methods Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK. Results Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants. Conclusions No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine. PMID:24853873

First evaluation of glucose-6-phosphate dehydrogenase (G6PD) deficiency in vivax malaria endemic regions in the Republic of Korea.

PubMed

Goo, Youn-Kyoung; Ji, So-Young; Shin, Hyun-Il; Moon, Jun-Hye; Cho, Shin-Hyung; Lee, Won-Ja; Kim, Jung-Yeon

2014-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK). Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK. Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants. No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.

Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection

PubMed Central

Konrad, Anna; Ashok, Nikhil; Pontén, Fredrik; Hober, Sophia; Asplund, Anna

2013-01-01

Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues. PMID:23920108

Applying the Miniature Dyna-Metric Model for Segmenting War Readiness Spares Kits: A User’s Guide

DTIC Science & Technology

1988-09-01

Reserve Materiel (WRM) concept. VRX is the extra, or additive, materiel required to augment peacetime assets to completely support the forces. missions...determines requirements for VRX through a number, of different forecasting systems, however the overall source of VRM authorizations, also called VRN...fluctuate as overall WRX spares requirements and funding change (32). Follow-on Spares Kits (FOSK) are in some ways a hybrid of both VRX and peacetime

[Principle of LAMP method--a simple and rapid gene amplification method].

PubMed

Ushikubo, Hiroshi

2004-06-01

So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

Genome amplification of single sperm using multiple displacement amplification.

PubMed

Jiang, Zhengwen; Zhang, Xingqi; Deka, Ranjan; Jin, Li

2005-06-07

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions.

PubMed

Hagiwara, Ken'ichi; Iwamaru, Yoshifumi; Tabeta, Naoko; Yokoyama, Takashi; Tobiume, Minoru

2017-03-04

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

PubMed Central

Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

PubMed

Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Optically Controlled Signal Amplification for DNA Computation.

PubMed

Prokup, Alexander; Hemphill, James; Liu, Qingyang; Deiters, Alexander

2015-10-16

The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF → ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF → ON or ON → OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification.

Magnetic micro/nanoparticle flocculation-based signal amplification for biosensing

PubMed Central

Mzava, Omary; Taş, Zehra; İçöz, Kutay

2016-01-01

We report a time and cost efficient signal amplification method for biosensors employing magnetic particles. In this method, magnetic particles in an applied external magnetic field form magnetic dipoles, interact with each other, and accumulate along the magnetic field lines. This magnetic interaction does not need any biomolecular coating for binding and can be controlled with the strength of the applied magnetic field. The accumulation can be used to amplify the corresponding pixel area that is obtained from an image of a single magnetic particle. An application of the method to the Escherichia coli 0157:H7 bacteria samples is demonstrated in order to show the potential of the approach. A minimum of threefold to a maximum of 60-fold amplification is reached from a single bacteria cell under a magnetic field of 20 mT. PMID:27354793

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

Population genetic study for 24 STR loci and Y indel (GlobalFiler™ PCR Amplification kit and PowerPlex® Fusion system) in 1000 Korean individuals.

PubMed

Park, Hyun-Chul; Kim, Kicheol; Nam, Younhyoung; Park, Jihye; Lee, Jinmyung; Lee, Hyehyeon; Kwon, Hansol; Jin, Hanjun; Kim, Wook; Kim, Won; Lim, Sikeun

2016-07-01

Allele frequencies for 23 autosomal short tandem repeat loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, TH01, FGA, D5S818, D13S317, D7S820, D2S441, D19S433, D22S1045, D10S1248, D1S1656, D12S391, D2S1338, SE33, Penta D, Penta E), 1 Y-chromosome short tandem repeat locus (DYS391) and Y indel were obtained from 1000 unrelated individuals of the Korean population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

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.

Short tandem repeat analysis in Japanese population.

PubMed

Hashiyada, M

2000-01-01

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.

[ISSR analysis for genetic polymorphism of Aconitum leucostomum from different habitats].

PubMed

Gao, Fu-chun; Sun, Yun; Zhang, Jing; Zhang, Fan

2014-01-01

To investigate the genetic diversities and variations of Aconitum leucostomum,and to supply essential characteristics for identifying Aconitum crude drugs. Plant genome extraction kit was applied to extract DNA,and ultraviolet spectrophotometer was used to detect the concentrations and purity of DNA. 60 ISSR primers were screened to analyze the DNA of Aconitum leucostomum from 10 habitats. Biosoftwares including POPGEN32 and NTSYS-PC were used to analyze the polymorphic bands obtained, and hence to yield the genetic similarity coefficient of the 10 habitats and map the related graphics, and cluster analysis were performed by UPGMA method. 11 primers selected from 60 ISSR primers were used for amplification and a total of 101 DNA bands were obtained, including 89 polymorphic bands,the average percentage of polymorphic bands (PPB) was 88.1%. Shannon information index (I) was 0.5298, the genetic similarity coefficient (H) was 0.3648, observed number of alleles was 1.8911, and effective number of alleles was 1.6555. The genetic identity was from 0.4950 to 0.6931, and the genetic distances were from 0.3666 to 0.7031. According to cluster analysis result of ISSR, the 10 habitats of Aconitum leucostomum were classified into five groups. Germplasm resources of Aconitum leucostomum show abundant polymorphism and higher genetic variation, which might supply molecular level basis, and provide basis for building DNA fingerprint.

Multifunctional sample preparation kit and on-chip quantitative nucleic acid sequence-based amplification tests for microbial detection.

PubMed

Zhao, Xinyan; Dong, Tao

2012-10-16

This study reports a quantitative nucleic acid sequence-based amplification (Q-NASBA) microfluidic platform composed of a membrane-based sampling module, a sample preparation cassette, and a 24-channel Q-NASBA chip for environmental investigations on aquatic microorganisms. This low-cost and highly efficient sampling module, having seamless connection with the subsequent steps of sample preparation and quantitative detection, is designed for the collection of microbial communities from aquatic environments. Eight kinds of commercial membrane filters are relevantly analyzed using Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus aureus as model microorganisms. After the microorganisms are concentrated on the membrane filters, the retentate can be easily conserved in a transport medium (TM) buffer and sent to a remote laboratory. A Q-NASBA-oriented sample preparation cassette is originally designed to extract DNA/RNA molecules directly from the captured cells on the membranes. Sequentially, the extract is analyzed within Q-NASBA chips that are compatible with common microplate readers in laboratories. Particularly, a novel analytical algorithmic method is developed for simple but robust on-chip Q-NASBA assays. The reported multifunctional microfluidic system could detect a few microorganisms quantitatively and simultaneously. Further research should be conducted to simplify and standardize ecological investigations on aquatic environments.

Use of Sequenom Sample ID Plus® SNP Genotyping in Identification of FFPE Tumor Samples

PubMed Central

Miller, Jessica K.; Buchner, Nicholas; Timms, Lee; Tam, Shirley; Luo, Xuemei; Brown, Andrew M. K.; Pasternack, Danielle; Bristow, Robert G.; Fraser, Michael; Boutros, Paul C.; McPherson, John D.

2014-01-01

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76–139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework. PMID:24551080

Use of Sequenom sample ID Plus® SNP genotyping in identification of FFPE tumor samples.

PubMed

Miller, Jessica K; Buchner, Nicholas; Timms, Lee; Tam, Shirley; Luo, Xuemei; Brown, Andrew M K; Pasternack, Danielle; Bristow, Robert G; Fraser, Michael; Boutros, Paul C; McPherson, John D

2014-01-01

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.

Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome.

PubMed

Jaroenram, Wansadaj; Owens, Leigh

2014-11-01

Penaeus stylirostris densovirus (PstDV) is an important shrimp pathogen that causes mortality in P. stylirostris and runt deformity syndrome (RDS) in Penaeus vannamei and Penaeus monodon. Recently, PstDV-related sequences were found in the genome of P. monodon and P. vannamei. This led to false positive results by PCR-based detection system. Here, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed for detecting PstDV. Under the optimal conditions, 30 min at 37°C for RPA followed by 5 min at room temperature for LFD, the protocol was 10 times more sensitive than the Saksmerphrome et al's interim 3-tube nested PCR and showed no cross-reaction with other shrimp viruses. It also reduced false positive results arising from viral inserts to ∼5% compared to 76-78% by the IQ2000™ nested PCR kit and the 309F/R PCR protocol currently recommended by World Organization for Animal Health (OIE) for PstDV detection. Together with simplicity and portability, the protocol serves as an alternative tool to PCR for primarily screening PstDV, which is suitable for both laboratory and field application. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.

PubMed

Faye, Oumar; Faye, Ousmane; Soropogui, Barré; Patel, Pranav; El Wahed, Ahmed Abd; Loucoubar, Cheikh; Fall, Gamou; Kiory, Davy; Magassouba, N'Faly; Keita, Sakoba; Kondé, Mandy Kader; Diallo, Alpha Amadou; Koivogui, Lamine; Karlberg, Helen; Mirazimi, Ali; Nentwich, Oliver; Piepenburg, Olaf; Niedrig, Matthias; Weidmann, Manfred; Sall, Amadou Alpha

2015-01-01

In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of the combined SE and EBOV-RT-RPA were tested in a mobile laboratory consisting of a mobile glovebox and a Diagnostics-in-a-Suitcase powered by a battery and solar panel, deployed to Matoto Conakry, Guinea as part of the reinforced surveillance strategy in April 2015 to reach the goal of zero cases. The EBOV-RT-RPA was evaluated in comparison to two real-time PCR assays. Of 928 post-mortem swabs, 120 tested positive, and the combined SE and EBOV-RT-RPA yielded a sensitivity and specificity of 100% in reference to one real-time RT-PCR assay. Another widely used real-time RT-PCR was much less sensitive than expected. Results were provided very fast within 30 to 60 min, and the field deployment of the mobile laboratory helped improve burial management and community engagement.

Development and Use of an Internal Positive Control for Detection of Bordetella pertussis by PCR

PubMed Central

Herwegh, Stéphanie; Carnoy, Christophe; Wallet, Frédéric; Loïez, Caroline; Courcol, René J.

2005-01-01

An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and coamplified with the target gene. This detection was successfully applied to the diagnosis of whooping cough by amplification of a fragment of Bordetella pertussis IS481. PMID:15872283

A pure DNA hydrogel with stable catalytic ability produced by one-step rolling circle amplification.

PubMed

Huang, Yishun; Xu, Wanlin; Liu, Guoyuan; Tian, Leilei

2017-03-09

A rolling-circle-amplification method was developed to produce DNA hydrogels with horseradish-peroxidase-like catalytic capability. The catalytic hydrogel exhibits highly improved stability at elevated temperatures or during a long-term storage. Integrated with glucose oxidase, the complex hydrogel can be applied to the sensitive and reliable detection of glucose.

Validation of the Applied Biosystems RapidFinder Shiga Toxin-Producing E. coli (STEC) Detection Workflow.

PubMed

Cloke, Jonathan; Matheny, Sharon; Swimley, Michelle; Tebbs, Robert; Burrell, Angelia; Flannery, Jonathan; Bastin, Benjamin; Bird, Patrick; Benzinger, M Joseph; Crowley, Erin; Agin, James; Goins, David; Salfinger, Yvonne; Brodsky, Michael; Fernandez, Maria Cristina

2016-11-01

The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant differences were observed between the reference method and the individual or combined kits forming the candidate assay using either of the DNA preparation kits (manual or automated extraction). For the inclusivity and exclusivity evaluation, the RapidFinder STEC Detection Workflow, comprising both RapidFinder STEC screening and confirmation kits, correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains for both of the assays evaluated. The results of these studies demonstrate the sensitivity and selectivity of the RapidFinder STEC Detection Workflow for the detection of E. coli O157:H7 and the "Big 6" STEC serotypes in both raw ground beef and beef trim. The robustness testing demonstrated that minor variations in the method parameters did not impact the accuracy of the assay and highlighted the importance of following the correct incubation temperatures.

Applied Computational Chemistry for the Blind and Visually Impaired

ERIC Educational Resources Information Center

Wedler, Henry B.; Cohen, Sarah R.; Davis, Rebecca L.; Harrison, Jason G.; Siebert, Matthew R.; Willenbring, Dan; Hamann, Christian S.; Shaw, Jared T.; Tantillo, Dean J.

2012-01-01

We describe accommodations that we have made to our applied computational-theoretical chemistry laboratory to provide access for blind and visually impaired students interested in independent investigation of structure-function relationships. Our approach utilizes tactile drawings, molecular model kits, existing software, Bash and Perl scripts…

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method

PubMed Central

2012-01-01

Background The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification. Methods A total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in. Results Performing DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification. Conclusions The loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited. PMID:22748106

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR Yfiler PCR amplification kit.

PubMed

Goedbloed, Miriam; Vermeulen, Mark; Fang, Rixun N; Lembring, Maria; Wollstein, Andreas; Ballantyne, Kaye; Lao, Oscar; Brauer, Silke; Krüger, Carmen; Roewer, Lutz; Lessig, Rüdiger; Ploski, Rafal; Dobosz, Tadeusz; Henke, Lotte; Henke, Jürgen; Furtado, Manohar R; Kayser, Manfred

2009-11-01

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR Yfiler polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730-1,764 DNA-confirmed father-son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son's birth) of fathers with mutations was with 34.40 (+/-11.63) years higher than that of fathers without ones at 30.32 (+/-10.22) years, a difference that is highly statistically significant (p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father's age on a statistically significant level (alpha = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father-son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses.

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.

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

PubMed

Besuschio, Susana A; Llano Murcia, Mónica; Benatar, Alejandro F; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, María de Los Ángeles; Kubota, Yutaka; Wehrendt, Diana P; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M; Schijman, Alejandro G

2017-07-01

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples

PubMed Central

Besuschio, Susana A.; Llano Murcia, Mónica; Benatar, Alejandro F.; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, María de los Ángeles; Kubota, Yutaka; Wehrendt, Diana P.; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M.

2017-01-01

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after “Boil & Spin” rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response. PMID:28727723

Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.

PubMed

Barreda-García, Susana; González-Álvarez, María José; de-Los-Santos-Álvarez, Noemí; Palacios-Gutiérrez, Juan José; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

2015-06-15

A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiple Cross Displacement Amplification Combined with Gold Nanoparticle-Based Lateral Flow Biosensor for Detection of Vibrio parahaemolyticus

PubMed Central

Wang, Yi; Li, Hui; Li, Dongxun; Li, Kewei; Wang, Yan; Xu, Jianguo; Ye, Changyun

2016-01-01

Vibrio parahaemolyticus (V. parahaemolyticus) is a marine seafood-borne pathogen causing severe illnesses in humans and aquatic animals. In the present study, multiple cross displacement amplification was combined with a lateral flow biosensor (MCDA-LFB) to detect the toxR gene of V. parahaemolyticus in DNA extracts from pure cultures and spiked oyster homogenates. Amplification was carried out at a constant temperature (62°C) for only 30 min, and amplification products were directly applied to the biosensor. The entire process, including oyster homogenate processing (30 min), isothermal amplification (30 min) and results indicating (∼2 min), could be completed within 65 min. Amplification product was detectable from as little as 10 fg of pure V. parahaemolyticus DNA and from approximately 4.2 × 102 CFU in 1 mL of oyster homogenate. No cross-reaction with other Vibrio species and with non-Vibrio species was observed. Therefore, the MCDA-LFB method established in the current report is suitable for the rapid screening of V. parahaemolyticus in clinical, food, and environmental samples. PMID:28066368

Gloss and surface roughness produced by polishing kits on resin composites.

PubMed

Sadidzadeh, Ramtin; Cakir, Deniz; Ramp, Lance C; Burgess, John O

2010-08-01

To compare in vitro the surface roughness (Ra) and gloss (G) produced by three conventional and one experimental polishing kits on four resin composites. 24 discs were prepared (d = 12 mm, t = 4 mm) for each resin composite: Filtek Supreme Plus Body/A2 (FSB), Yellow Translucent (FST), Heliomolar/A2 (HM), and EsthetX/A2 (EX) following the manufacturers' instructions. They were finished with 320 grit silicon carbide paper for 80 seconds each. Polishing systems: Sof-Lex, Enhance-Pogo, Astropol and Experimental Discs/EXL-695, were applied following manufacturers' instructions. Each specimen was ultrasonically cleaned with distilled water and dried. Gloss and Ra were measured with a small area glossmeter (Novo-curve) and non-contact profilometer (Proscan 2000) following ISO 4288, respectively. The results were evaluated by two-way ANOVA followed by separate one-way ANOVA and Tukey/Kramer test (P = 0.05). There was a significant interaction of surface roughness and gloss between the composites and polishing systems (P < 0.05). The lowest surface roughness was recorded for FST polished with the Experimental kit. The highest gloss was obtained for FSB composite polished with the Experimental kit. The experimental polishing system produced smoothest surfaces (P < 0.05). The Enhance-Pogo and the experimental polishing kit produced highest gloss (P < 0.05).

Rapid diagnosis of tuberculosis in dromedary camel (Camelus dromedarius) using lateral flow assay-based kit.

PubMed

Ranjan, Rakesh; Narnaware, Shirish D; Nath, Kashi; Sawal, R K; Patil, N V

2018-04-01

Accurate early antemortem diagnosis of tuberculosis in dromedary camels is difficult due to the lack of reliable diagnostic test. The present study aimed to evaluate a lateral flow assay-based kit (rapid assay kit) in tuberculosis diagnosis that employs immuno-chromatographic detection of antibodies in serum, plasma, or whole blood. In a dromedary camel herd comprising 337 animals located at Bikaner, Rajasthan, India, 50 adult weak camels (11 males and 39 females) were tested by applying a single intradermal tuberculin test (SIDT) and rapid assay kit. A total of 14 animals (2 males, 12 females) were found positive in rapid assay. In SIDT, four animals revealed a positive reaction in the neck region and seven animals in the tail base. Another male animal was found SIDT positive but negative in rapid assay; it died after 12 months. Nine rapid assay positive animals died asymptomatically in 1- to 11-month period revealing postmortem tuberculosis lesions that were confirmed by Ziehl-Neelsen staining and histopathology. No tuberculous lesion was evident in the animal found positive in SIDT alone. Results of the present study indicated that serological tests like rapid assay kit can serve as a reliable test for antemortem diagnosis of tuberculosis in dromedary camel.

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

NASA Astrophysics Data System (ADS)

Guan, Fengyi; Lu, Jiaju; Wang, Xiumei

2017-03-01

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH2) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.

A framework for high-school teacher support in Geosciences

NASA Astrophysics Data System (ADS)

Bookhagen, B.; Mair, A.; Schaller, G.; Koeberl, C.

2012-04-01

To attract future geoscientists in the classroom and share the passion for science, successful geoscience education needs to combine modern educational tools with applied science. Previous outreach efforts suggest that classroom-geoscience teaching tremendously benefits from structured, prepared lesson plans in combination with hands-on material. Building on our past experience, we have developed a classroom-teaching kit that implements interdisciplinary exercises and modern geoscientific application to attract high-school students. This "Mobile Phone Teaching Kit" analyzes the components of mobile phones, emphasizing the mineral compositions and geologic background of raw materials. Also, as geoscience is not an obligatory classroom topic in Austria, and university training for upcoming science teachers barely covers geoscience, teacher training is necessary to enhance understanding of the interdisciplinary geosciences in the classroom. During the past year, we have held teacher workshops to help implementing the topic in the classroom, and to provide professional training for non-geoscientists and demonstrate proper usage of the teaching kit. The material kit is designed for classroom teaching and comes with a lesson plan that covers background knowledge and provides worksheets and can easily be adapted to school curricula. The project was funded by kulturkontakt Austria; expenses covered 540 material kits, and we reached out to approximately 90 schools throughout Austria and held a workshop in each of the nine federal states in Austria. Teachers received the training, a set of the material kit, and the lesson plan free of charge. Feedback from teachers was highly appreciative. The request for further material kits is high and we plan to expand the project. Ultimately, we hope to enlighten teachers and students for the highly interdisciplinary variety of geosciences and a link to everyday life.

19 CFR 122.131 - Application.

Code of Federal Regulations, 2010 CFR

2010-04-01

... AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.131 Application. (a) Liquor and tobacco. Subpart M...) Aircraft. Subpart M applies to all commercial aircraft on domestic or foreign flights operating into, from...

[Effect of different oxygen concentrations on biological properties of bone marrow hematopoietic stem cells of mice].

PubMed

Ma, Yi-Ran; Ren, Si-Hua; He, Yu-Xin; Wang, Lin-Lin; Jin, Li; Hao, Yi-Wen

2012-10-01

This study purposed to investigate the effects of different oxygen concentrations and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and their possible mechanisms through simulating oxygen environment to which the peripheral blood HSC are subjected in peripheral blood HSCT. The proliferation ability, cell cycle, directed differentiation ability, ROS level and hematopoietic reconstitution ability of Lin(-)c-kit(+)Sca-1(+) BMHSC were detected by using in vitro amplification test, directional differentiation test, cell cycle analysis, ROS assay and transplantation of Lin(-)c-kit(+)Sca-1(+) HSC from sublethally irradiated mice respectively. The results showed that oxygen concentrations lower than normal oxygen concentration, especially in hypoxic oxygen environment, could reduce ROS generation and amplify more primitive CD34(+)AC133(+) HSC and active CD34(+) HSC, and maintain more stem cells in the G(0)/G(1) phase, which is more helpful to the growth of CFU-S and viability of mice. At the same time, BMHSC exposed to normal oxygen level or inconstant and greatly changed oxygen concentrations could produce a high level of ROS, and the above-mentioned features and functional indicators are relatively low. It is concluded that ROS levels of HSC in BMHSCT are closely related with the oxygen concentration surrounding the cells and its stability. Low oxygen concentration and antioxidant intervention are helpful to transplantation of BMHSC.

Efficient isolation method for high-quality genomic DNA from cicada exuviae.

PubMed

Nguyen, Hoa Quynh; Kim, Ye Inn; Borzée, Amaël; Jang, Yikweon

2017-10-01

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.

Mitochondrial and Y-chromosomal profile of the Kazakh population from East Kazakhstan

PubMed Central

Tarlykov, Pavel V.; Zholdybayeva, Elena V.; Akilzhanova, Ainur R.; Nurkina, Zhannur M.; Sabitov, Zhaxylyk M.; Rakhypbekov, Tolebay K.; Ramanculov, Erlan M.

2013-01-01

Aim To study the genetic relationship of Kazakhs from East Kazakhstan to other Eurasian populations by examining paternal and maternal DNA lineages. Methods Whole blood samples were collected in 2010 from 160 unrelated healthy Kazakhs residing in East Kazakhstan. Genomic DNA was extracted with Wizard® genomic DNA Purification Kit. Nucleotide sequence of hypervariable segment I of mitochondrial DNA (mtDNA) was determined and analyzed. Seventeen Y-short tandem repeat (STR) loci were studied in 67 samples with the AmpFiSTR Y-filer PCR Amplification Kit. In addition, mtDNA data for 2701 individuals and Y-STR data for 677 individuals were retrieved from the literature for comparison. Results There was a high degree of genetic differentiation on the level of mitochondrial DNA. The majority of maternal lineages belonged to haplogroups common in Central Asia. In contrast, Y-STR data showed very low genetic diversity, with the relative frequency of the predominant haplotype of 0.612. Conclusion The results revealed different migration patterns in the population sample, showing there had been more migration among women. mtDNA genetic diversity in this population was equivalent to that in other Central Asian populations. Genetic evidence suggests the existence of a single paternal founder lineage in the population of East Kazakhstan, which is consistent with verbal genealogical data of the local tribes. PMID:23444242

Responses to crizotinib in patients with ALK-positive lung adenocarcinoma who tested immunohistochemistry (IHC)-positive and fluorescence in situ hybridization (FISH)-negative.

PubMed

Ma, Di; Wang, Zheng; Yang, Lin; Mu, Xinlin; Wang, Yan; Zhao, Xinming; Li, Junling; Lin, Dongmei

2016-09-27

Although the Ventana immunohistochemistry (IHC) platform for detecting anaplastic lymphoma kinase gene (ALK) (D5F3) expression was recently approved by the US Food and Drugs Administration (FDA), fluorescence in situ hybridization (FISH) is still the "gold-standard" method recommended by the US National Comprehensive Cancer Network (NCCN) guideline for NSCLC. We evaluated 6 ALK-positive lung adenocarcinoma patients who tested Ventana IHC-positive and FISH-negative and assessed their clinical responses to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Histologic and cytologic specimens from the 6 patients were stained with Ventana anti-ALK(D5F3) rabbit monoclonal primary antibody using the OptiView™ DAB IHC detection kit and OptiView™ amplification kit on a Ventana BenchMark XT processor. In addition, they were also tested by FISH, qRT-PCR, next-generation sequencing (NGS), and RNAscope ISH analysis. All patients received crizotinib treatment and their follow-up clinical data were recorded. The objective response rate achieved with crizotinib therapy was 66.7% (4/6 partial responses and 2/6 stable disease). One patient in whom a new fusion type (EML4->EXOC6B->ALK fusion) was identified obtained a partial response. These findings indicate that patients with ALK-positive lung adenocarcinoma who test Ventana IHC-positive and FISH-negative may still respond to crizotinib therapy.

Genetic examination of the putative skull of Jan Kochanowski reveals its female sex

PubMed Central

Kupiec, Tomasz; Branicki, Wojciech

2011-01-01

We report the results of genetic examination of the putative skull of Jan Kochanowski (1530-1584), a great Polish renaissance poet. The skull was retrieved in 1791 by historian Tadeusz Czacki from the Kochanowski family tomb and became the property of the Czartoryskis Museum in Krakow. An anthropological study in 1926 questioned its male origin, which raised doubts about its authenticity. Our report presents genetic evidence that resolves this dispute. From the sole tooth we obtained a sufficient amount of DNA to perform the analysis of nuclear markers. The analysis of the sex-informative part of intron 1 in amelogenin, genotyped using AmpFiSTR® NGM PCR Amplification Kit and Powerplex® ESI17 Kit human identification systems, revealed the female origin of the tooth. The female origin was further confirmed by the analysis of a portion of amelogenin intron 2, a microsatellite marker located on the X chromosome, as well as by a lack of signal from Y chromosomal microsatellite markers and the sex-determining region Y marker. Data obtained for two hypervariable regions, HVI and HVII, in mitochondrial DNA showed that mtDNA haplotype was relatively frequent among contemporary Europeans. The analysis of a set of single nucleotide polymorphisms relevant for prediction of the iris color indicated an 87% probability that the woman had hazel or brown eye color. PMID:21674838

Genetic examination of the putative skull of Jan Kochanowski reveals its female sex.

PubMed

Kupiec, Tomasz; Branicki, Wojciech

2011-06-01

We report the results of genetic examination of the putative skull of Jan Kochanowski (1530-1584), a great Polish renaissance poet. The skull was retrieved in 1791 by historian Tadeusz Czacki from the Kochanowski family tomb and became the property of the Czartoryskis Museum in Krakow. An anthropological study in 1926 questioned its male origin, which raised doubts about its authenticity. Our report presents genetic evidence that resolves this dispute. From the sole tooth we obtained a sufficient amount of DNA to perform the analysis of nuclear markers. The analysis of the sex-informative part of intron 1 in amelogenin, genotyped using AmpFiSTR® NGM PCR Amplification Kit and Powerplex® ESI17 Kit human identification systems, revealed the female origin of the tooth. The female origin was further confirmed by the analysis of a portion of amelogenin intron 2, a microsatellite marker located on the X chromosome, as well as by a lack of signal from Y chromosomal microsatellite markers and the sex-determining region Y marker. Data obtained for two hypervariable regions, HVI and HVII, in mitochondrial DNA showed that mtDNA haplotype was relatively frequent among contemporary Europeans. The analysis of a set of single nucleotide polymorphisms relevant for prediction of the iris color indicated an 87% probability that the woman had hazel or brown eye color.

Improved Method for Direct Detection of Environmental Microorganisms Using an Amplification of 16S rDNA Region

NASA Astrophysics Data System (ADS)

Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.

2004-12-01

The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection of microorganisms with important genes from more other environments.

2D dynamic studies combined with the surface curvature analysis to predict Arias Intensity amplification

NASA Astrophysics Data System (ADS)

Torgoev, Almaz; Havenith, Hans-Balder

2016-07-01

A 2D elasto-dynamic modelling of the pure topographic seismic response is performed for six models with a total length of around 23.0 km. These models are reconstructed from the real topographic settings of the landslide-prone slopes situated in the Mailuu-Suu River Valley, Southern Kyrgyzstan. The main studied parameter is the Arias Intensity (Ia, m/sec), which is applied in the GIS-based Newmark method to regionally map the seismically-induced landslide susceptibility. This method maps the Ia values via empirical attenuation laws and our studies investigate a potential to include topographic input into them. Numerical studies analyse several signals with varying shape and changing central frequency values. All tests demonstrate that the spectral amplification patterns directly affect the amplification of the Ia values. These results let to link the 2D distribution of the topographically amplified Ia values with the parameter called as smoothed curvature. The amplification values for the low-frequency signals are better correlated with the curvature smoothed over larger spatial extent, while those values for the high-frequency signals are more linked to the curvature with smaller smoothing extent. The best predictions are provided by the curvature smoothed over the extent calculated according to Geli's law. The sample equations predicting the Ia amplification based on the smoothed curvature are presented for the sinusoid-shape input signals. These laws cannot be directly implemented in the regional Newmark method, as 3D amplification of the Ia values addresses more problem complexities which are not studied here. Nevertheless, our 2D results prepare the theoretical framework which can potentially be applied to the 3D domain and, therefore, represent a robust basis for these future research targets.

Dental Sealants Prevent Cavities

MedlinePlus

... Digital Press Kit Read the MMWR Science Clips Dental Sealants Prevent Cavities Effective protection for children Language: ... more use of sealants and reimbursement of services. Dental care providers can Apply sealants to children at ...

Amplification without instability: applying fluid dynamical insights in chemistry and biology

NASA Astrophysics Data System (ADS)

McCoy, Jonathan H.

2013-11-01

While amplification of small perturbations often arises from instability, transient amplification is possible locally even in asymptotically stable systems. That is, knowledge of a system's stability properties can mislead one's intuition for its transient behaviors. This insight, which has an interesting history in fluid dynamics, has more recently been rediscovered in ecology. Surprisingly, many nonlinear fluid dynamical and ecological systems share linear features associated with transient amplification of noise. This paper aims to establish that these features are widespread in many other disciplines concerned with noisy systems, especially chemistry, cell biology and molecular biology. Here, using classic nonlinear systems and the graphical language of network science, we explore how the noise amplification problem can be reframed in terms of activatory and inhibitory interactions between dynamical variables. The interaction patterns considered here are found in a great variety of systems, ranging from autocatalytic reactions and activator-inhibitor systems to influential models of nerve conduction, glycolysis, cell signaling and circadian rhythms.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

Describing Site Amplification for Surface Waves in Realistic Basins

NASA Astrophysics Data System (ADS)

Bowden, D. C.; Tsai, V. C.

2017-12-01

Standard characterizations of site-specific site response assume a vertically-incident shear wave; given a 1D velocity profile, amplification and resonances can be calculated based on conservation of energy. A similar approach can be applied to surface waves, resulting in an estimate of amplification relative to a hard rock site that is different in terms of both amount of amplification and frequency. This prediction of surface-wave site amplification has been well validated through simple simulations, and in this presentation we explore the extent to which a 1D profile can explain observed amplifications in more realistic scenarios. Comparisons of various simple 2D and 3D simulations, for example, allow us to explore the effect of different basin shapes and the relative importance of effects such as focusing, conversion of wave-types and lateral surface wave resonances. Additionally, the 1D estimates for vertically-incident shear waves and for surface waves are compared to spectral ratios of historic events in deep sedimentary basins to demonstrate the appropriateness of the two different predictions. This difference in amplification responses between the wave types implies that a single measurement of site response, whether analytically calculated from 1D models or empirically observed, is insufficient for regions where surface waves play a strong role.

Amplification of a seed pumped by a chirped laser in the strong coupling Brillouin regime

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

Schluck, F.; Lehmann, G.; Spatschek, K. H.

Seed amplification via Brillouin backscattering of a long pump laser-pulse is considered. The interaction takes place in the so called strong coupling regime. Pump chirping is applied to mitigate spontaneous Raman backscattering of the pump before interacting with the seed. The strong coupling regime facilitates stronger exponential growth and narrower seeds compared to the so called weak coupling regime, although in the latter the scaling with pump amplitude is stronger. Strong coupling is achieved when the pump laser amplitude exceeds a certain threshold. It is shown how the chirp influences both the linear as well as the nonlinear amplification process.more » First, linear amplification as well as the seed profiles are determined in dependence of the chirping rate. In contrast to the weak coupling situation, the evolution is not symmetric with respect to the sign of the chirping rate. In the nonlinear stage of the amplification, we find an intrinsic chirp of the seed pulse even for an un-chirped pump. We show that chirping the pump may have a strong influence on the shape of the seed in the nonlinear amplification phase. Also, the influence of pump chirp on the efficiency of Brillouin seed amplification is discussed.« less

Site Amplification Characteristics of the Several Seismic Stations at Jeju Island, in Korea, using S-wave Energy, Background Noise, and Coda waves from the East Japan earthquake (Mar. 11th, 2011) Series.

NASA Astrophysics Data System (ADS)

Seong-hwa, Y.; Wee, S.; Kim, J.

2016-12-01

Observed ground motions are composed of 3 main factors such as seismic source, seismic wave attenuation and site amplification. Among them, site amplification is also important factor and should be considered to estimate soil-structure dynamic interaction with more reliability. Though various estimation methods are suggested, this study used the method by Castro et. al.(1997) for estimating site amplification. This method has been extended to background noise, coda waves and S waves recently for estimating site amplification. This study applied the Castro et. al.(1997)'s method to 3 different seismic waves, that is, S-wave Energy, Background Noise, and Coda waves. This study analysed much more than about 200 ground motions (acceleration type) from the East Japan earthquake (March 11th, 2011) Series of seismic stations at Jeju Island (JJU, SGP, HALB, SSP and GOS; Fig. 1), in Korea. The results showed that most of the seismic stations gave similar results among three types of seismic energies. Each station showed its own characteristics of site amplification property in low, high and specific resonance frequency ranges. Comparison of this study to other studies can give us much information about dynamic amplification of domestic sites characteristics and site classification.

Bleeding

MedlinePlus

... Vaginal bleeding (heavier than usual or after menopause) First Aid First aid is appropriate for external bleeding. If bleeding is ... pant leg or sleeve. If you have a first-aid kit that comes with a tourniquet, apply it ...

[Recombinase Polymerase Amplification and its Applications in Parasite Detection].

PubMed

ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

2015-10-01

Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

HER2, MET and FGFR2 oncogenic driver alterations define distinct molecular segments for targeted therapies in gastric carcinoma.

PubMed

Liu, Y J; Shen, D; Yin, X; Gavine, P; Zhang, T; Su, X; Zhan, P; Xu, Y; Lv, J; Qian, J; Liu, C; Sun, Y; Qian, Z; Zhang, J; Gu, Y; Ni, X

2014-03-04

Gastric cancer (GC) is a leading cause of cancer deaths worldwide. Since the approval of trastuzumab, targeted therapies are emerging as promising treatment options for the disease. This study aimed to explore the molecular segmentation of several known therapeutics targets, human epidermal growth factor receptor 2 (HER2), MET and fibroblast growth factor receptor 2 (FGFR2), within GC using clinically approved or investigational kits and scoring criteria. Knowledge of how these markers are segmented in the same cohort of GC patients could improve future clinical trial designs. Using immunohistochemistry (IHC) and FISH methods, overexpression and amplification of HER2, FGFR2 and MET were profiled in a cohort of Chinese GC samples. The correlations between anti-tumour sensitivity and the molecular segments of HER2, MET and FGFR2 alterations were further tested in a panel of GC cell lines and the patient-derived GC xenograft (PDGCX) model using the targeted inhibitors. Of 172 GC patients, positivity for HER2, MET and FGFR2 alternations was found in 23 (13.4%), 21 (12.2%) and 9 (5.2%) patients, respectively. Positivity for MET was found in 3 of 23 HER2-positive GC patients. Co-positivity for FGFR2 and MET was found in 1 GC patient, and amplification of the two genes was found in different tumour cells. Our study in a panel of GC cell lines showed that in most cell lines, amplification or high expression of a particular molecular marker was mutually exclusive and in vitro sensitivity to the targeted agents lapatinib, PD173074 and crizotinib was only observed in cell lines with the corresponding high expression of the drugs' target protein. SGC031, an MET-positive PDGCX mouse model, responded to crizotinib but not to lapatinib or PD173074. Human epidermal growth factor receptor 2, MET and FGFR2 oncogenic driver alterations (gene amplification and overexpression) occur in three largely distinct molecular segments in GC. A significant proportion of HER2-negative patients may potentially benefit from MET- or FGFR2-targeted therapies.

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

Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency,more » the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.« less

Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification.

PubMed

Tang, Juan; Huang, Yapei; Liu, Huiqiong; Zhang, Cengceng; Tang, Dianping

2016-12-01

A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) in foodstuff. The system consisted of anti-AFB 1 antibody labeled DNA 1 (Ab-DNA 1 ), AFB 1 -bovine serum albumin (BSA)-conjugated DNA 2 (AFB 1 -DNA 2 ), and methylene blue functionalized hairpin DNA. Owing to a specific antigen-antibody reaction between anti-AFB 1 and AFB 1 -BSA, the immunocomplex formed assisted the proximity hybridization of DNA 1 with DNA 2 , thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB 1 , the analyte competed with AFB 1 -DNA 2 for the conjugated anti-AFB 1 on the Ab-DNA 1 , subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB 1 in a dynamic working range of 0.01-30 ng mL -1 with a detection limit of 4.8 pg mL -1 . In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB 1 -spiked peanut samples, giving results that matched well with those obtained with a commercial AFB 1 ELISA kit.

An economic analysis of adherence engineering to improve use of best practices during central line maintenance procedures.

PubMed

Nelson, Richard E; Angelovic, Aaron W; Nelson, Scott D; Gleed, Jeremy R; Drews, Frank A

2015-05-01

Adherence engineering applies human factors principles to examine non-adherence within a specific task and to guide the development of materials or equipment to increase protocol adherence and reduce human error. Central line maintenance (CLM) for intensive care unit (ICU) patients is a task through which error or non-adherence to protocols can cause central line-associated bloodstream infections (CLABSIs). We conducted an economic analysis of an adherence engineering CLM kit designed to improve the CLM task and reduce the risk of CLABSI. We constructed a Markov model to compare the cost-effectiveness of the CLM kit, which contains each of the 27 items necessary for performing the CLM procedure, compared with the standard care procedure for CLM, in which each item for dressing maintenance is gathered separately. We estimated the model using the cost of CLABSI overall ($45,685) as well as the excess LOS (6.9 excess ICU days, 3.5 excess general ward days). Assuming the CLM kit reduces the risk of CLABSI by 100% and 50%, this strategy was less costly (cost savings between $306 and $860) and more effective (between 0.05 and 0.13 more quality-adjusted life-years) compared with not using the pre-packaged kit. We identified threshold values for the effectiveness of the kit in reducing CLABSI for which the kit strategy was no longer less costly. An adherence engineering-based intervention to streamline the CLM process can improve patient outcomes and lower costs. Patient safety can be improved by adopting new approaches that are based on human factors principles.

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

PubMed

Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

2017-04-26

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

Adiabatic Amplification of Plasmons and Demons in 2D Systems

NASA Astrophysics Data System (ADS)

Sun, Zhiyuan; Basov, D. N.; Fogler, M. M.

2016-08-01

We theoretically investigate charged collective modes in a two-dimensional conductor with hot electrons where the instantaneous mode frequencies gradually increase or decrease with time. We show that the loss compensation or even amplification of the modes may occur. We apply our theory to two types of collective modes in graphene, the plasmons and the energy waves, which can be probed in optical pump-probe experiments.

Adiabatic Amplification of Plasmons and Demons in 2D Systems.

PubMed

Sun, Zhiyuan; Basov, D N; Fogler, M M

2016-08-12

We theoretically investigate charged collective modes in a two-dimensional conductor with hot electrons where the instantaneous mode frequencies gradually increase or decrease with time. We show that the loss compensation or even amplification of the modes may occur. We apply our theory to two types of collective modes in graphene, the plasmons and the energy waves, which can be probed in optical pump-probe experiments.

[Possibility of the species identification using blood stains located on the material evidences and bone fragments with the method of solid phase enzyme immunoassay with "IgG general-EIA-BEST" kit and human immunoglobulin G].

PubMed

Sidorov, V L; Shvetsova, I V; Isakova, I V

2007-01-01

The authors give the comparative analysis of Russian and foreign forensic medical methods of species character identification of the blood from the stains on the material evidences and bone fragments. It is shown that for this purpose it is feasible to apply human immunoglobulin G (IgG) and solid phase enzyme immunoassay (EIA) with the kit "IgG general-EIA-BEST". In comparison with the methods used in Russia this method is more sensitive, convenient for objective registration and computer processing. The results of experiments shown that it is possible to use the kit "IgG general-EIA-BEST" in forensic medicine for the species character identification of the blood from the stains on the material evidences and bone fragments.

Optimization of PCR for quantification of simian immunodeficiency virus (SIV) genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA

PubMed Central

Monjure, C. J.; Tatum, C. D.; Panganiban, A. T.; Arainga, M.; Traina-Dorge, V.; Marx, P. A.; Didier, E. S.

2014-01-01

Introduction Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. Methods The PVL quantification procedure was optimized by inclusion of an exogenous control Hepatitis C Virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660 and the LTR region of SIVagmSAB were also optimized. Results Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV-RNA in the same samples using the “industry standard” method of branched-DNA (bDNA) signal amplification. Conclusions Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs. PMID:24266615

Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA.

PubMed

Monjure, C J; Tatum, C D; Panganiban, A T; Arainga, M; Traina-Dorge, V; Marx, P A; Didier, E S

2014-02-01

Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. The PVL quantification procedure was optimized by inclusion of an exogenous control hepatitis C virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660, and the LTR region of SIVagmSAB were also optimized. Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV RNA in the same samples using the 'industry standard' method of branched-DNA (bDNA) signal amplification. Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Isothermal recombinase polymerase amplification assay applied to the detection of group B streptococci in vaginal/anal samples.

PubMed

Daher, Rana K; Stewart, Gale; Boissinot, Maurice; Bergeron, Michel G

2014-04-01

Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid-based diagnostics at the point of care.

Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome

PubMed Central

Favero, F.; McGranahan, N.; Salm, M.; Birkbak, N. J.; Sanborn, J. Z.; Benz, S. C.; Becq, J.; Peden, J. F.; Kingsbury, Z.; Grocok, R. J.; Humphray, S.; Bentley, D.; Spencer-Dene, B.; Gutteridge, A.; Brada, M.; Roger, S.; Dietrich, P.-Y.; Forshew, T.; Gerlinger, M.; Rowan, A.; Stamp, G.; Eklund, A. C.; Szallasi, Z.; Swanton, C.

2015-01-01

Background Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. Methods We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. Results We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. Conclusion This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success. PMID:25732040

Diagnostic value of a novel fully automated immunochemistry assay for detection of ALK rearrangement in primary lung adenocarcinoma.

PubMed

Ying, J; Guo, L; Qiu, T; Shan, L; Ling, Y; Liu, X; Lu, N

2013-10-01

To evaluate the diagnostic value of a novel fully automated immunohistochemistry (IHC) assay for detection of anaplastic lymphoma kinase (ALK) fusion in a large number of ALK-positive lung adenocarcinoma (ADC) patients. We tested 196 lung ADCs for ALK rearrangement by two IHC assays (Ventana pre-diluted ALK D5F3 antibody with the Optiview DAB IHC detection kit and Optiview Amplification kit, D5F3 by Cell Signaling Technology (CST) with Ultraview DAB detection kit by Ventana), fluorescence in situ hybridization (FISH) and real-time reverse transcription-PCR (RT-PCR). CST ALK IHC was scored using the scoring scheme of 0, no staining; 1+, faint; 2+, moderate; and 3+, strong cytoplasmic reactivity in ≥ 10% of tumor cells. As for Ventana IHC, a binary scoring system (positive or negative for ALK status) was adopted for evaluating the staining results. Among 196 cases tested, 63 (32%), 65 (33%), 70 (36%), and 69 (35%) cases were ALK positive by FISH, Ventana IHC, CST IHC, and RT-PCR, respectively. The sensitivity and specificity of Ventana IHC were 100% and 98%, respectively. Two Ventana IHC-positive cases, which were also CST IHC score of 3+, showed FISH negative, but their ALK rearrangement was confirmed by RT-PCR and direct sequencing. The sensitivity and specificity of CST IHC with staining intensity score of 1+ or more were 100% and 95%, respectively. Five (25%, of 20) patients with CST IHC score of 1+ were both FISH and RT-PCR negative. The sensitivity and specificity of RT-PCR for detection of ALK fusion were 98% and 95%, respectively. The total accordance rate between ALK RT-PCR and Ventana IHC was 97%. The novel fully automated IHC assay is a reliable screening tool in routine pathologic laboratories for identification of patients with ALK rearrangement for targeted therapy in lung ADC.

Development, validation and testing costs of an in-house real-time PCR assay for the detection of Chlamydia trachomatis.

PubMed

Santos, Camila Gurgel Dos; Sabidó, Meritxell; Leturiondo, André Luiz; Ferreira, Cynthia de Oliveira; da Cruz, Thielle Pereira; Benzaken, Adele Schwartz

2017-03-01

To improve the screening of Chlamydia trachomatis(C. trachomatis) in Brazil, an accurate and affordable method is needed. The objective of this study was to develop and assess the performance and costs of a new in-house real-time PCR (qPCR) assay for the diagnosis of C. trachomatis infection. Asymptomatic women aged 14-25 years who attended primary health services in Manaus, Brazil, were screened for C. trachomatis using the Digene Hybrid Capture II CT-ID (HCII CT-ID) DNA test. A subset of cervical specimens were tested using an in-house qPCR and a commercial qPCR, ArtusC. trachomatis Plus RG PCR 96 CE (Artus qPCR) kit, as a reference test. A primer/probe based on the sequence of cryptic plasmid (CP) was designed. An economic evaluation was conducted from the provider's perspective. The primers were considered specific for C. trachomatis because they did not amplify any product from non-sexually transmitted bacterial species tested. Overall, 292 specimens were tested by both the commercial kit (Artus qPCR) and the in-house qPCR. Of those, one resulted in no amplification and was excluded from the analysis. The sensitivity, specificity, and positive and negative predictive values of the in-house qPCR were 99.5 % [95 % confidence interval (CI): 97.1-100], 95.1 % (95 % CI: 89-98.4), 97.4 % (95 % CI: 94-99.1) and 99.0 % (95 % CI: 94.5-100), respectively. The cost per case of C. trachomatis was £0.44 ($0.55) for HCII CT-ID, £1.16 ($1.45) for Artus qPCR and £1.06 ($1.33) for in-house qPCR. We have standardized an in-house qPCR to detect cervical C. trachomatis targeting CP. The in-house qPCR showed excellent accuracy and was more affordable than the commercial qPCR kit.

49 CFR 565.21 - Applicability.

Code of Federal Regulations, 2013 CFR

2013-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS Alternative VIN... applicability of this subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, and motorcycles. Vehicles imported into...

49 CFR 565.21 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS Alternative VIN... applicability of this subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, and motorcycles. Vehicles imported into...

49 CFR 565.21 - Applicability.

Code of Federal Regulations, 2012 CFR

2012-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS Alternative VIN... applicability of this subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, and motorcycles. Vehicles imported into...

49 CFR 565.11 - Applicability.

Code of Federal Regulations, 2013 CFR

2013-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS VIN Requirements... subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, low speed vehicles, and motorcycles manufactured on or after...

49 CFR 565.11 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS VIN Requirements... subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, low speed vehicles, and motorcycles manufactured on or after...

49 CFR 565.11 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS VIN Requirements... subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, low speed vehicles, and motorcycles manufactured on or after...

49 CFR 565.11 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS VIN Requirements... subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, low speed vehicles, and motorcycles manufactured on or after...

49 CFR 565.21 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS Alternative VIN... applicability of this subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, and motorcycles. Vehicles imported into...

49 CFR 565.21 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS Alternative VIN... applicability of this subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, and motorcycles. Vehicles imported into...

49 CFR 565.11 - Applicability.

Code of Federal Regulations, 2012 CFR

2012-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IDENTIFICATION NUMBER (VIN) REQUIREMENTS VIN Requirements... subpart. This part applies to passenger cars, multipurpose passenger vehicles, trucks, buses, trailers (including trailer kits), incomplete vehicles, low speed vehicles, and motorcycles manufactured on or after...

21 CFR 872.3570 - OTC denture repair kit.

Code of Federal Regulations, 2013 CFR

2013-04-01

... powder and liquid glues, that is intended to be applied permanently to a denture to mend cracks or breaks. The device may be available for purchase over-the counter. (b) Classification. Class II. The special...

21 CFR 872.3570 - OTC denture repair kit.

Code of Federal Regulations, 2010 CFR

2010-04-01

... powder and liquid glues, that is intended to be applied permanently to a denture to mend cracks or breaks. The device may be available for purchase over-the counter. (b) Classification. Class II. The special...

21 CFR 872.3570 - OTC denture repair kit.

Code of Federal Regulations, 2012 CFR

2012-04-01

... powder and liquid glues, that is intended to be applied permanently to a denture to mend cracks or breaks. The device may be available for purchase over-the counter. (b) Classification. Class II. The special...

21 CFR 872.3570 - OTC denture repair kit.

Code of Federal Regulations, 2011 CFR

2011-04-01

... powder and liquid glues, that is intended to be applied permanently to a denture to mend cracks or breaks. The device may be available for purchase over-the counter. (b) Classification. Class II. The special...

21 CFR 872.3570 - OTC denture repair kit.

Code of Federal Regulations, 2014 CFR

2014-04-01

... powder and liquid glues, that is intended to be applied permanently to a denture to mend cracks or breaks. The device may be available for purchase over-the counter. (b) Classification. Class II. The special...

The DBBC environment for millimeter radioastronomy

NASA Astrophysics Data System (ADS)

Tuccari, Gino; Comoretto, Giovanni; Melis, Andrea; Buttaccio, Salvo

2012-09-01

The Digital Base Band Converter project developed in the last decade produced a general architecture and a class of boards, firmware and software, giving the possibility to build a general purpose back-end system for VLBI or single-dish observational activities. Such approach suggests the realization of a digital radio system, i.e. a receiver with conversion not realized with analogue techniques, maintaining only amplification stages in the analogue domain. This solution can be applied until a maximum around 16 GHz, the present limit for the instantaneous input band in the latest version of the DBBC project, while in the millimeter frequency range this maximum limit of 0.5-2 GHz of the previous versions allows the intermediate frequency to be processed in the digital domain. A description of the elements developed in the DBBC project is presented, with their use in different environments. The architecture is composed of a PC controlled mainframe, and of different modules that can be combined in a very flexible way in order to realize different instruments. The instrument can be expanded or retrofitted to meet increasing observational demands. Available modules include ADC converters, processing boards, physical interfaces (VSI and 10G Ethernet). Several applications have already been implemented and used in radioastronomic observations: a DDC (Direct Digital Conversion) for VLBI observations, a Polyphase Digital Filter Bank, and a Multiband Scansion Spectrometer. Other applications are currently studied for additional functionalities like a spectropolarimeter, a linear-to-circular polarization converter, a RFI-mitigation tool, and a phase-reference holographic tool-kit.

[The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

PubMed

Sil'veĭstrova, O Iu; Domonova, É A; Shipulina, O Iu

2014-04-01

The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

Mathematical simulation of the amplification of 1790-nm laser radiation in a nuclear-excited He - Ar plasma containing nanoclusters of uranium compounds

NASA Astrophysics Data System (ADS)

Kosarev, V. A.; Kuznetsova, E. E.

2014-02-01

The possibility of applying dusty active media in nuclearpumped lasers has been considered. The amplification of 1790-nm radiation in a nuclear-excited dusty He - Ar plasma is studied by mathematical simulation. The influence of nanoclusters on the component composition of the medium and the kinetics of the processes occurring in it is analysed using a specially developed kinetic model, including 72 components and more than 400 reactions. An analysis of the results indicates that amplification can in principle be implemented in an active laser He - Ar medium containing 10-nm nanoclusters of metallic uranium and uranium dioxide.

Four photon parametric amplification. [in unbiased Josephson junction

NASA Technical Reports Server (NTRS)

Parrish, P. T.; Feldman, M. J.; Ohta, H.; Chiao, R. Y.

1974-01-01

An analysis is presented describing four-photon parametric amplification in an unbiased Josephson junction. Central to the theory is the model of the Josephson effect as a nonlinear inductance. Linear, small signal analysis is applied to the two-fluid model of the Josephson junction. The gain, gain-bandwidth product, high frequency limit, and effective noise temperature are calculated for a cavity reflection amplifier. The analysis is extended to multiple (series-connected) junctions and subharmonic pumping.

Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

PubMed Central

Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

2013-01-01

Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PubMed

Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

2014-09-01

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

Inventions leading to the development of the diagnostic test kit industry--from the modern pregnancy test to the sandwich assays.

PubMed

Wide, Leif

2005-01-01

The universities are encouraged by the government nowadays to stimulate innovations and also to provide the proper machinery for assisting the protection and commercialisation of innovations. A better understanding of the innovation process may help to create an atmosphere suitable for inventions at the university. Examples can be taken from successful innovations previously made at the university. During the 1960's I made a series of inventions, which ultimately led to the development of the diagnostic test kit industry. The first, which I made as an undergraduate, was a simple and reliable test kit for diagnosis of pregnancy. This was followed by the solid phase radioimmunoassay and a solid phase assay for vitamin B12; next, the dual specific non-competitive sandwich assay and the in-vitro test for diagnosis of allergy, called RAST (Radioallergosorbent test). Organon in Holland with the pregnancy test kit, and Pharmacia in Sweden with test kits for radioimmunoassay, became pioneers among the diagnostic test kit industries. Pharmacia Diagnostics later became one of the leading diagnostic test kit companies in the world and has continued to be so in the field of allergy diagnosis. Each one of these inventions started with a few unique observations leading to a technical development. The pregnancy test as well as the allergy test emerged from the development of assay methods with unique qualities with the subsequent search for appropriate applications. The foreseeing of a commercial value on a future market was a very important step. This was followed by the search for a suitable industry interested to exploit the invention with its new business opportunity i.e. apply for a patent, produce and market the products, which in my case consisted of the necessary reagents and equipments for particular diagnostic tests. Finally, an agreement had to be settled between the entrepreneur and the inventors. This report describes these inventions and particularly discusses some crucial steps of the innovation processes.

Mutant KIT as imatinib-sensitive target in metastatic sinonasal carcinoma.

PubMed

Dieter, S M; Heining, C; Agaimy, A; Huebschmann, D; Bonekamp, D; Hutter, B; Ehrenberg, K R; Fröhlich, M; Schlesner, M; Scholl, C; Schlemmer, H-P; Wolf, S; Mavratzas, A; Jung, C S; Gröschel, S; von Kalle, C; Eils, R; Brors, B; Penzel, R; Kriegsmann, M; Reuss, D E; Schirmacher, P; Stenzinger, A; Federspil, P A; Weichert, W; Glimm, H; Fröhling, S

2017-01-01

Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy. We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors. Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459_2462delATTCinsG, p.D820_S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST. These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

77 FR 64311 - Foreign-Trade Zone 176-Rockford, IL, Notification of Proposed Production Activity, AndersonBrecon...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-19

... procedures that applies to SURGIFLO[supreg] Hemostatic Matrix Kits and SURGIFLO[supreg] Hemostatic Matrix... from abroad include: Sterile water in vials/non-needled syringes, hemostatic gelatin matrix pouches...

DNA database of populations from different parts in the Kingdom of Thailand.

PubMed

Shotivaranon, Jittima; Chirachariyavej, Thamrong; Leetrakool, Nipapan; Rerkamnuaychoke, Budsaba

2009-12-01

The polymorphism of 15 short tandem repeat (STR) loci-D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA from AmpFlSTR Identifiler PCR amplification kit were analysed in 929 unrelated individuals living in the north, northeast, central and south of Thailand. The comparison between these four subpopulations demonstrated that subpopulations in the north and northeast were different in two loci from all paired groups while those in the north, central and south were closely related. The inter-population comparisons between combined Thai population and other ethnic groups including Eastern Chinese, Japanese, Iraq and Egyptian revealed that Eastern Chinese and Thai were closely related.

Amplification of the flgE gene provides evidence for the existence of a Brazilian borreliosis.

PubMed

Mantovani, Elenice; Marangoni, Roberta G; Gauditano, Giancarla; Bonoldi, Virgínia L N; Yoshinari, Natalino H

2012-01-01

The symptoms of Brazilian borreliosis resemble the clinical manifestations of Lyme disease (LD). However, there are differences between the two in terms of epidemiological and laboratory findings. Primers usually employed to diagnose LD have failed to detect Borrelia strains in Brazil. We aimed to identify the Brazilian Borrelia using a conserved gene that synthesizes the flagellar hook (flgE) of Borrelia burgdorferi sensu lato. Three patients presenting with erythema migrans and positive epidemiological histories were recruited for the study. Blood samples were collected, and the DNA was extracted by commercial kits. The gene flgE was amplified from DNA of all selected patients. Upon sequencing, these positive samples revealed 99% homology to B. burgdorferi flgE. These results support the existence of borreliosis in Brazil. However, it is unclear whether this borreliosis is caused by a genetically modified B. burgdorferi sensu stricto or by a new species of Borrelia spp.

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 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.

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection.

PubMed

Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

2018-01-01

Highly sensitive testing of nucleic acids is essential to improve the detection of pathogens, which pose a major threat for public health worldwide. Currently available molecular assays, mainly based on PCR, have a limited utility in point-of-need control or resource-limited settings. Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detection of these harmful microorganisms. Since its description in 2004, isothermal helicase-dependent amplification (HDA) has been successfully applied in the development of novel molecular-based technologies for rapid, sensitive, and selective detection of viruses and bacteria. In this review, we highlight relevant analytical systems using this simple nucleic acid amplification methodology that takes place at a constant temperature and that is readily compatible with microfluidic technologies. Different strategies for monitoring HDA amplification products are described. In addition, we present technological advances for integrating sample preparation, HDA amplification, and detection. Future perspectives and challenges toward point-of-need use not only for clinical diagnosis but also in food safety testing and environmental monitoring are also discussed. Graphical Abstract Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA).

Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

PubMed Central

Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

2011-01-01

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

Diagnosis of Clostridium difficile-associated disease: examination of multiple algorithms using toxin EIA, glutamate dehydrogenase EIA and loop-mediated isothermal amplification.

PubMed

Bamber, A I; Fitzsimmons, K; Cunniffe, J G; Beasor, C C; Mackintosh, C A; Hobbs, G

2012-01-01

The laboratory diagnosis of Clostridium difficile infection (CDI) needs to be accurate and timely to ensure optimal patient management, infection control and reliable surveillance. Three methods are evaluated using 810 consecutive stool samples against toxigenic culture: CDT TOX A/B Premier enzyme immunoassay (EIA) kit (Meridian Bioscience, Europe), Premier EIA for C. difficile glutamate dehydrogenase (GDH) (Meridian Bioscience, Europe) and the Illumigene kit (Meridian Bioscience, Europe), both individually and within combined testing algorithms. The study revealed that the CDT TOX A/B Premier EIA gave rise to false-positive and false-negative results and demonstrated poor sensitivity (56.47%), compared to Premier EIA for C. difficile GDH (97.65%), suggesting this GDH EIA can be a useful negative screening method. Results for the Illumigene assay alone showed sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of 91.57%, 98.07%, 99.03% and 84.44%, respectively. A two-stage algorithm using Premier EIA for C. difficile GDH/Illumigene assay yielded superior results compared with other testing algorithms (91.57%, 98.07%, 99.03% and 84.44%, respectively), mirroring the Illumigene performance. However, Illumigene is approximately half the cost of current polymerase chain reaction (PCR) methods, has a rapid turnaround time and requires no specialised skill base, making it an attractive alternative to assays such as the Xpert C. difficile assay (Cepheid, Sunnyvale, CA). A three-stage algorithm offered no improvement and would hamper workflow.

Rare genomic rearrangement in a boy with Williams-Beuren syndrome associated to XYY syndrome and intriguing behavior.

PubMed

Dutra, Roberta L; Piazzon, Flavia B; Zanardo, Évelin A; Costa, Thais Virginia Moura Machado; Montenegro, Marília M; Novo-Filho, Gil M; Dias, Alexandre T; Nascimento, Amom M; Kim, Chong Ae; Kulikowski, Leslie D

2015-12-01

Williams-Beuren syndrome (WBS) is caused by a hemizygous contiguous gene microdeletion of 1.55-1.84 Mb at 7q11.23 region. Approximately, 28 genes have been shown to contribute to classical phenotype of SWB with presence of dysmorphic facial features, supravalvular aortic stenosis (SVAS), intellectual disability, and overfriendliness. With the use of Microarray-based comparative genomic hybridization and other molecular cytogenetic techniques, is possible define with more accuracy partial or atypical deletion and refine the genotype-phenotype correlation. Here, we report on a rare genomic structural rearrangement in a boy with atypical deletion in 7q11.23 and XYY syndrome with characteristic clinical signs, but not sufficient for the diagnosis of WBS. Cytogenetic analysis of G-banding showed a karyotype 47,XYY. Analysis of DNA with the technique of MLPA (Multiplex Ligation-dependent Probe Amplification) using kits a combination of kits (P064, P036, P070, and P029) identified an atypical deletion on 7q11.23. In addition, high resolution SNP Oligonucleotide Microarray Analysis (SNP-array) confirmed the alterations found by MLPA and revealed others pathogenic CNVs, in the chromosomes 7 and X. The present report demonstrates an association not yet described in literature, between Williams-Beuren syndrome and 47,XYY. The identification of atypical deletion in 7q11.23 concomitant to additional pathogenic CNVs in others genomic regions allows a better comprehension of clinical consequences of atypical genomic rearrangements. © 2015 Wiley Periodicals, Inc.

Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

PubMed Central

Conde, Esther; Suárez-Gauthier, Ana; Benito, Amparo; Garrido, Pilar; García-Campelo, Rosario; Biscuola, Michele; Paz-Ares, Luis; Hardisson, David; de Castro, Javier; Camacho, M. Carmen; Rodriguez-Abreu, Delvys; Abdulkader, Ihab; Ramirez, Josep; Reguart, Noemí; Salido, Marta; Pijuán, Lara; Arriola, Edurne; Sanz, Julián; Folgueras, Victoria; Villanueva, Noemí; Gómez-Román, Javier; Hidalgo, Manuel; López-Ríos, Fernando

2014-01-01

Background Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. Results All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations. PMID:25248157

[Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

PubMed

Cao, Y; Zou, K N; Huang, J P; Ma, K; Ping, Y

2017-08-01

To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine

Highly Effective DNA Extraction Method for Nuclear Short Tandem Repeat Testing of Skeletal Remains from Mass Graves

PubMed Central

Davoren, Jon; Vanek, Daniel; Konjhodzić, Rijad; Crews, John; Huffine, Edwin; Parsons, Thomas J.

2007-01-01

Aim To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves. Methods DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit. Results DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles. Conclusions The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method. PMID:17696302

17 to 23: A novel complementary mini Y-STR panel to extend the Y-STR databases from 17 to 23 markers for forensic purposes.

PubMed

Núñez, Carolina; Baeta, Miriam; Ibarbia, Nerea; Ortueta, Urko; Jiménez-Moreno, Susana; Blazquez-Caeiro, José Luis; Builes, Juan José; Herrera, Rene J; Martínez-Jarreta, Begoña; de Pancorbo, Marian M

2017-04-01

A Y-STR multiplex system has been developed with the purpose of complementing the widely used 17 Y-STR haplotyping (AmpFlSTR Y Filer® PCR Amplification kit) routinely employed in forensic and population genetic studies. This new multiplex system includes six additional STR loci (DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643) to reach the 23 Y-STR of the PowerPlex® Y23 System. In addition, this kit includes the DYS456 and DYS385 loci for traceability purposes. Male samples from 625 individuals from ten worldwide populations were genotyped, including three sample sets from populations previously published with the 17 Y-STR system to expand their current data. Validation studies demonstrated good performance of the panel set in terms of concordance, sensitivity, and stability in the presence of inhibitors and artificially degraded DNA. The results obtained for haplotype diversity and discrimination capacity with this multiplex system were considerably high, providing further evidences of the suitability of this novel Y-STR system for forensic purposes. Thus, the use of this multiplex for samples previously genotyped with 17 Y-STRs will be an efficient and low-cost alternative to complete the set of 23 Y-STRs and improve allele databases for population and forensic purposes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

DNA and bone structure preservation in medieval human skeletons.

PubMed

Coulson-Thomas, Yvette M; Norton, Andrew L; Coulson-Thomas, Vivien J; Florencio-Silva, Rinaldo; Ali, Nadir; Elmrghni, Samir; Gil, Cristiane D; Sasso, Gisela R S; Dixon, Ronald A; Nader, Helena B

2015-06-01

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Performance scaling via passive pulse shaping in cavity-enhanced optical parametric chirped-pulse amplification.

PubMed

Siddiqui, Aleem M; Moses, Jeffrey; Hong, Kyung-Han; Lai, Chien-Jen; Kärtner, Franz X

2010-06-15

We show that an enhancement cavity seeded at the full repetition rate of the pump laser can automatically reshape small-signal gain across the interacting pulses in an optical parametric chirped-pulse amplifier for close-to-optimal operation, significantly increasing both the gain bandwidth and the conversion efficiency, in addition to boosting gain for high-repetition-rate amplification. Applied to a degenerate amplifier, the technique can provide an octave-spanning gain bandwidth.

Study Unveils New Method for Universal Extraction and PCR Amplification of Fungal DNA

DTIC Science & Technology

2014-06-12

Wickes noted that there are methods to extract fungi from soil , for example, and "once you get down to pure DNA, everything else is the same," he said...rare or hard to identify fungal infections. The new extraction and amplification method can be universally applied to fungi , according to the...best treatments. In addition, rare fungi , or species with phenotypic doppelgangers, can stump medical mycologists, so molecular methods are critical

75 FR 81563 - Notice of Petitions by Firms for Determination of Eligibility To Apply for Trade Adjustment...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-28

..., CO 80027. mountings, fittings, and other machined metal components for aerospace applications. Foam.... custom packaging kits, gaskets, seals, sheets, blocks, etc., of all types of foam materials. Gulf Fish...

Real-time correction of tsunami site effect by frequency-dependent tsunami-amplification factor

NASA Astrophysics Data System (ADS)

Tsushima, H.

2017-12-01

For tsunami early warning, I developed frequency-dependent tsunami-amplification factor and used it to design a recursive digital filter that can be applicable for real-time correction of tsunami site response. In this study, I assumed that a tsunami waveform at an observing point could be modeled by convolution of source, path and site effects in time domain. Under this assumption, spectral ratio between offshore and the nearby coast can be regarded as site response (i.e. frequency-dependent amplification factor). If the amplification factor can be prepared before tsunamigenic earthquakes, its temporal convolution to offshore tsunami waveform provides tsunami prediction at coast in real time. In this study, tsunami waveforms calculated by tsunami numerical simulations were used to develop frequency-dependent tsunami-amplification factor. Firstly, I performed numerical tsunami simulations based on nonlinear shallow-water theory from many tsuanmigenic earthquake scenarios by varying the seismic magnitudes and locations. The resultant tsunami waveforms at offshore and the nearby coastal observing points were then used in spectral-ratio analysis. An average of the resulted spectral ratios from the tsunamigenic-earthquake scenarios is regarded as frequency-dependent amplification factor. Finally, the estimated amplification factor is used in design of a recursive digital filter that can be applicable in time domain. The above procedure is applied to Miyako bay at the Pacific coast of northeastern Japan. The averaged tsunami-height spectral ratio (i.e. amplification factor) between the location at the center of the bay and the outside show a peak at wave-period of 20 min. A recursive digital filter based on the estimated amplification factor shows good performance in real-time correction of tsunami-height amplification due to the site effect. This study is supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant 15K16309.

Three-input majority function as the unique optimal function for the bias amplification using nonlocal boxes

NASA Astrophysics Data System (ADS)

Mori, Ryuhei

2016-11-01

Brassard et al. [Phys. Rev. Lett. 96, 250401 (2006), 10.1103/PhysRevLett.96.250401] showed that shared nonlocal boxes with a CHSH (Clauser, Horne, Shimony, and Holt) probability greater than 3/+√{6 } 6 yield trivial communication complexity. There still exists a gap with the maximum CHSH probability 2/+√{2 } 4 achievable by quantum mechanics. It is an interesting open question to determine the exact threshold for the trivial communication complexity. Brassard et al.'s idea is based on recursive bias amplification by the three-input majority function. It was not obvious if another choice of function exhibits stronger bias amplification. We show that the three-input majority function is the unique optimal function, so that one cannot improve the threshold 3/+√{6 } 6 by Brassard et al.'s bias amplification. In this work, protocols for computing the function used for the bias amplification are restricted to be nonadaptive protocols or a particular adaptive protocol inspired by Pawłowski et al.'s protocol for information causality [Nature (London) 461, 1101 (2009), 10.1038/nature08400]. We first show an adaptive protocol inspired by Pawłowski et al.'s protocol, and then show that the adaptive protocol improves upon nonadaptive protocols. Finally, we show that the three-input majority function is the unique optimal function for the bias amplification if we apply the adaptive protocol to each step of the bias amplification.

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

Relativistic Eulerian Vlasov simulations of the amplification of seed pulses by Brillouin backscattering in plasmas

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

Shoucri, M., E-mail: Shoucri.Magdi@ireq.ca; Matte, J.-P.; Vidal, F.

We apply an Eulerian Vlasov code to study the amplification by Brillouin scattering of a short seed laser pulse by a long pump laser pulse in an underdense plasma. The stimulated Brillouin backscattering interaction is the coupling of the pump and seed electromagnetic waves propagating in opposite directions, and the ion plasma wave. The code solves the one-dimensional relativistic Vlasov-Maxwell set of equations. Large amplitude ion waves are generated. In the simulations we present, the density plateau of the plasma is n{sub e}=0.3 n{sub c} (n{sub c} is the critical density), which excludes spurious stimulated Raman scattering amplification (which can occurmore » only if n{sub e}« less

Methods for applying accurate digital PCR analysis on low copy DNA samples.

PubMed

Whale, Alexandra S; Cowen, Simon; Foy, Carole A; Huggett, Jim F

2013-01-01

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

PubMed Central

Whale, Alexandra S.; Cowen, Simon; Foy, Carole A.; Huggett, Jim F.

2013-01-01

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA. PMID:23472156

Genetic progression of malignant melanoma.

PubMed

Tímár, J; Vizkeleti, L; Doma, V; Barbai, T; Rásó, E

2016-03-01

Malignant melanoma of the skin is the most aggressive human cancer given that a primary tumor a few millimeters in diameter frequently has full metastatic competence. In view of that, revealing the genetic background of this potential may also help to better understand tumor dissemination in general. Genomic analyses have established the molecular classification of melanoma based on the most frequent driver oncogenic mutations (BRAF, NRAS, KIT) and have also revealed a long list of rare events, including mutations and amplifications as well as genetic microheterogeneity. At the moment, it is unclear whether any of these rare events have role in the metastasis initiation process since the major drivers do not have such a role. During lymphatic and hematogenous dissemination, the clonal selection process is evidently reflected by differences in oncogenic drivers in the metastases versus the primary tumor. Clonal selection is also evident during lymphatic progression, though the genetic background of this immunoselection is less clear. Genomic analyses of metastases identified further genetic alterations, some of which may correspond to metastasis maintenance genes. The natural genetic progression of melanoma can be modified by targeted (BRAF or MEK inhibitor) or immunotherapies. Some of the rare events in primary tumors may result in primary resistance, while further new genetic lesions develop during the acquired resistance to both targeted and immunotherapies. Only a few genetic lesions of the primary tumor are constant during natural or therapy-modulated progression. EGFR4 and NMDAR2 mutations, MITF and MET amplifications and PTEN loss can be considered as metastasis drivers. Furthermore, BRAF and MITF amplifications as well as PTEN loss are also responsible for resistance to targeted therapies, whereas NRAS mutation is the only founder genetic lesion showing any association with sensitivity to immunotherapies. Unfortunately, there are hardly any data on the possible organ-specific metastatic drivers in melanoma. These observations suggest that clinical management of melanoma patients must rely on the genetic analysis of the metastatic lesions to be able to monitor progression-associated changes and to personalize therapies.

Laser-ablated poly(methyl methacrylate) microdevices for sub-microliter DNA amplification suitable for micro-total analysis systems

NASA Astrophysics Data System (ADS)

Lounsbury, Jenny A.; Poe, Brian L.; Do, Michael; Landers, James P.

2012-08-01

Biochemical techniques, such as the polymerase chain reaction (PCR), can take up to 3.5 h for completion using a commercial bench-top instrument, creating a bottleneck in sample preparation processes. PCR has been successfully adapted to microfluidic devices, reducing the time needed to as little as 7-10 min. Recently, a trend in the field is to use alternative substrates, such as poly(methyl methacyrlate) (PMMA), for the fabrication of microfluidic devices. PMMA has several advantages over more expensive substrates including rigidity without fragility, disposability, and it is easy to fabricate, using techniques such as hot embossing or CO2 laser ablation. Here, we report the fabrication of PMMA microdevices to explore their effectiveness for PCR amplification. Several types of PMMA microdevices were fabricated using a CO2 laser ablation system, with two or three PMMA layers of different thicknesses. Bonding of the microdevices was significantly improved through the use of a grid system etched into the device, allowing for trapped air to escape, eliminating leakage. Using infrared thermal cycling, the ramping rates were determined to be dependent on the thickness of the PMMA used in fabrication, allowing for customization of cycling conditions. Further reduction of the thermal mass by isolation of the chambers provided a significant increase in the heating and cooling rates (up to 6.19 (±0.32 °C s-1) and -7.99 (±0.06 °C s-1), respectively). Bubble formation, a chronic problem in microfluidic systems in general and problematic during the heating phase of PCR, was minimized through the use of a biocompatible adhesive/manifold combination to seal the reservoirs. Finally, successful PCR amplification was demonstrated with both a fragment from the β-globin gene and 15 tetra-nucleotide repeat regions with a sex-typing marker in a conventional STR kit with the latter facilitated by the dynamic coating of the fluidic architecture with poly(ethylene glycol) (PEG).

Compression stockings for treating venous leg ulcers: measurement of interface pressure under a new ulcer kit.

PubMed

Partsch, B; Partsch, H

2008-01-01

The aim of this study was to measure the interface pressure of a newly designed two-layer compression stocking (Mediven ulcer kit Medi QMBH, Bayreuth, Germany) in different body positions and to compare the values with those obtained with another two-layer product. Interface pressure was measured on the distal medial leg in 16 legs of volunteers, with the basic layer alone and with the whole stocking kit in the supine, sitting and standing position for both stocking systems. The literature concerning ulcer-healing rates is reviewed. Mediven ulcerkit produced statistically significant higher pressure values than the ulcer stocking with a median resting value of 35.5 mmHg in the supine and 42.5 mmHg in the standing position. The pressure while standing comes close to values exerted by bandages. The basic layer alone applies a pressure of 20.5 mmHg. Especially designed compression stockings exerting sufficient interface pressure may be indicated in patients with small ulcers of short duration.

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

Nested-PCR and a new ELISA-based NovaLisa test kit for malaria diagnosis in an endemic area of Thailand.

PubMed

Thongdee, Pimwan; Chaijaroenkul, Wanna; Kuesap, Jiraporn; Na-Bangchang, Kesara

2014-08-01

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.

A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

NASA Astrophysics Data System (ADS)

Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

2015-06-01

Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification.

PubMed

Cheng, Wei; Zhang, Wei; Yan, Yurong; Shen, Bo; Zhu, Dan; Lei, Pinhua; Ding, Shijia

2014-12-15

A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin-strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1 fM to 100 pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Signal amplification of FISH for automated detection using image cytometry.

PubMed

Truong, K; Boenders, J; Maciorowski, Z; Vielh, P; Dutrillaux, B; Malfoy, B; Bourgeois, C A

1997-05-01

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy.

PubMed

Li, Ying; Yu, Chuanfeng; Han, Huixia; Zhao, Caisheng; Zhang, Xiaoru

2016-07-15

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitivity analysis of reactive ecological dynamics.

PubMed

Verdy, Ariane; Caswell, Hal

2008-08-01

Ecological systems with asymptotically stable equilibria may exhibit significant transient dynamics following perturbations. In some cases, these transient dynamics include the possibility of excursions away from the equilibrium before the eventual return; systems that exhibit such amplification of perturbations are called reactive. Reactivity is a common property of ecological systems, and the amplification can be large and long-lasting. The transient response of a reactive ecosystem depends on the parameters of the underlying model. To investigate this dependence, we develop sensitivity analyses for indices of transient dynamics (reactivity, the amplification envelope, and the optimal perturbation) in both continuous- and discrete-time models written in matrix form. The sensitivity calculations require expressions, some of them new, for the derivatives of equilibria, eigenvalues, singular values, and singular vectors, obtained using matrix calculus. Sensitivity analysis provides a quantitative framework for investigating the mechanisms leading to transient growth. We apply the methodology to a predator-prey model and a size-structured food web model. The results suggest predator-driven and prey-driven mechanisms for transient amplification resulting from multispecies interactions.

49 CFR 173.161 - Chemical kits and first aid kits.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Chemical kits and First aid kits must conform to... 10 kg. (b) Chemical kits and First aid kits are excepted from the specification packaging...

Loop-Mediated Isothermal Amplification on Crude DNA as a Point-of-Care Test for the Diagnosis of Mycoplasma-Related Vaginitis During Early Pregnancy.

PubMed

Wang, Yichao; Zhang, Bumei; Sun, Yan; Liu, Yunde; Gu, Yajun

2017-12-20

Mycoplasma-related vaginitis gradually has been growing as a threat in adults-genitourinary infection contributes to funisitis, spontaneous abortion, and low birth weight. Until now, use of loop-mediated isothermal amplification (LAMP) to detect Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), or Mycoplasma genitalium (MG) has been reported by some researchers. However, previous studies focused on purified DNA as the template for LAMP assay, which is usually extracted via commercial kit. We developed a LAMP assay for rapid detection of UU, MH, and MG genital mycoplasmas using a simple boiling method for DNA extraction, in a cohort of pregnant women with mycoplasma-related vaginitis. We monitored amplicons with the naked eye using SYBR Green I. The cohort in our study showed a prevalence of 22.6% in pregnant women, as detected by UU-LAMP assay. Compared to the polymerase chain reaction (PCR) test with purified DNA, the sensitivity of the UU-LAMP in clinical specimens with crude DNA was 87.5% (95% confidence interval [CI], 64.6%->99.9). For crude DNA specimens, UU-LAMP was more sensitive and reliable than PCR, with a higher agreement rate (96.8%) and Youden index value (0.88). As a point-of-care test, LAMP is a useful, specific, and efficient way to detect genital mycoplasmas in resource-limited settings, especially for crude DNA. © American Society for Clinical Pathology 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan.

PubMed

Chishti, Hafsah Muhammad; Ansar, Muhammad; Ajmal, Muhammad; Hameed, Abdul

2014-09-15

Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies. Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers. All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥0.9 (except TPOX locus). Variation from Hardy-Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients. The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic analysis results of mature cystic teratomas of the ovary in Taiwan disagree with the previous origin theory of this tumor.

PubMed

Wang, Wen-Chung; Lai, Yen-Chein

2016-06-01

The most accepted theory regarding mature cystic teratomas of the ovary is that they are of parthenogenetic origin from oocyte after the completion of first division. Our previous study demonstrated that the origin of mature cystic teratoma of the uterus is not related to the parthenogenetic process, but is most likely pluripotential stem cell or primordial germ cell before meiosis I. Further studies are needed to clarify the origin of benign mature cystic teratomas of the ovary in Taiwan. In the present study, we investigated the DNA profiles of 9 mature cystic teratomas of the ovary using short tandem repeat analysis with AmpFLSTR SGM Plus, Profiler PCR amplification kits. The methylation statuses of the HhaI sites in the SNRPN, H19DMR, and KvDMR regions were determined on methylation-sensitive multiplex ligation-dependent probe amplification analysis. DNA profiling data from the 9 mature cystic teratomas of the ovary excluded parthenogenetic origin, as most of the 15 short tandem repeat loci were heterozygous on genotyping. There were varying degrees of hypermethylation of SNRPN gene and KvDMR locus in the presence of maternal uniparental disomy in all 9 mature cystic teratomas of the ovary. In light of these results, we further postulated that the origin of these mature cystic teratomas of the ovary is oogonia or primary oocyte before germinal vesicle stage failure of meiosis I. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic analysis of haplotype data for 23 Y-chromosome short tandem repeat loci in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina.

PubMed

Dogan, Serkan; Primorac, Dragan; Marjanović, Damir

2014-10-01

To explore the distribution and polymorphisms of 23 short tandem repeat (STR) loci on the Y chromosome in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina and to investigate its genetic relationships with the homeland Turkish population and neighboring populations. This study included 100 healthy unrelated male individuals from the Turkish population living in Sarajevo. Buccal swab samples were collected as a DNA source. Genomic DNA was extracted using the salting out method and amplification was performed using PowerPlex Y 23 amplification kit. The studied population was compared to other populations using pairwise genetic distances, which were represented with a multi-dimensional scaling plot. Haplotype and allele frequencies of the sample population were calculated and the results showed that all 100 samples had unique haplotypes. The most polymorphic locus was DYS458, and the least polymorphic DYS391. The observed haplotype diversity was 1.0000 ± 0.0014, with a discrimination capacity of 1.00 and the match probability of 0.01. Rst values showed that our sample population was closely related in both dimensions to the Lebanese and Iraqi populations, while it was more distant from Bosnian, Croatian, and Macedonian populations. Turkish population residing in Sarajevo could be observed as a representative Turkish population, since our results were consistent with those previously published for the homeland Turkish population. Also, this study once again proved that geographically close populations were genetically more related to each other.

A factorial design experiment as a pilot study for noninvasive genetic sampling.

PubMed

Renan, Sharon; Speyer, Edith; Shahar, Naama; Gueta, Tomer; Templeton, Alan R; Bar-David, Shirli

2012-11-01

Noninvasive genetic sampling has increasingly been used in ecological and conservation studies during the last decade. A major part of the noninvasive genetic literature is dedicated to the search for optimal protocols, by comparing different methods of collection, preservation and extraction of DNA from noninvasive materials. However, the lack of quantitative comparisons among these studies and the possibility that different methods are optimal for different systems make it difficult to decide which protocol to use. Moreover, most studies that have compared different methods focused on a single factor - collection, preservation or extraction - while there could be interactions between these factors. We designed a factorial experiment, as a pilot study, aimed at exploring the effect of several collection, preservation and extraction methods, and the interactions between them, on the quality and amplification success of DNA obtained from Asiatic wild ass (Equus hemionus) faeces in Israel. The amplification success rates of one mitochondrial DNA and four microsatellite markers differed substantially as a function of collection, preservation and extraction methods and their interactions. The most efficient combination for our system integrated the use of swabs as a collection method with preservation at -20 °C and with the Qiagen DNA Stool Kit with modifications as the DNA extraction method. The significant interaction found between the collection, preservation methods and the extraction methods reinforces the importance of conducting a factorial design experiment, rather than examining each factor separately, as a pilot study before initiating a full-scale noninvasive research project. © 2012 Blackwell Publishing Ltd.

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

Apparatus and method for enabling quantum-defect-limited conversion efficiency in cladding-pumped Raman fiber lasers

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

Heebner, John E.; Sridharan, Arun K.; Dawson, Jay Walter

Cladding-pumped Raman fiber lasers and amplifiers provide high-efficiency conversion efficiency at high brightness enhancement. Differential loss is applied to both single-pass configurations appropriate for pulsed amplification and laser oscillator configurations applied to high average power cw source generation.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

PubMed

Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

2018-05-15

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

49 CFR 173.161 - Chemical kits and first aid kits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 2 2012-10-01 2012-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Applicability. Chemical kits and first aid kits... assigned to the chemical kit and first aid kit as a whole must be the most stringent packing group assigned...

DAKOTA JAGUAR 3.0 user's manual.

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

Adams, Brian M.; Bauman, Lara E; Chan, Ethan

2013-05-01

JAGUAR (JAva GUi for Applied Research) is a Java software tool providing an advanced text editor and graphical user interface (GUI) to manipulate DAKOTA (Design Analysis Kit for Optimization and Terascale Applications) input specifications. This document focuses on the features necessary to use JAGUAR.

JAGUAR developer's manual.

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

Chan, Ethan

2011-06-01

JAGUAR (JAva GUi for Applied Research) is a Java software tool providing an advanced text editor and graphical user interface (GUI) to manipulate DAKOTA (Design Analysis Kit for Optimization and Terascale Applications) input specifications. This document focuses on the technical background necessary for a developer to understand JAGUAR.

Diagnostic performance of a novel loop-mediated isothermal amplification (LAMP) assay targeting the apicoplast genome for malaria diagnosis in a field setting in sub-Saharan Africa.

PubMed

Oriero, Eniyou C; Okebe, Joseph; Jacobs, Jan; Van Geertruyden, Jean-Pierre; Nwakanma, Davis; D'Alessandro, Umberto

2015-10-09

New diagnostic tools to detect reliably and rapidly asymptomatic and low-density malaria infections are needed as their treatment could interrupt transmission. Isothermal amplification techniques are being explored for field diagnosis of malaria. In this study, a novel molecular tool (loop-mediated isothermal amplification-LAMP) targeting the apicoplast genome of Plasmodium falciparum was evaluated for the detection of asymptomatic malaria-infected individuals in a rural setting in The Gambia. A blood was collected from 341 subjects (median age 9 years, range 1-68 years) screened for malaria. On site, a rapid diagnostic test (RDT, SD Bioline Malaria Antigen P.f) was performed, thick blood films (TBF) slides for microscopy were prepared and dry blood spots (DBS) were collected on Whatman(®) 903 Specimen collection paper. The TBF and DBS were transported to the field laboratory where microscopy and LAMP testing were performed. The latter was done on DNA extracted from the DBS using a crude (methanol/heating) extraction method. A laboratory-based PCR amplification was done on all the samples using DNA extracted with the Qiagen kit and its results were taken as reference for all the other tests. Plasmodium falciparum malaria prevalence was 37 % (127/341) as detected by LAMP, 30 % (104/341) by microscopy and 37 % (126/341) by RDT. Compared to the reference PCR method, sensitivity was 92 % for LAMP, 78 % for microscopy, and 76 % for RDT; specificity was 97 % for LAMP, 99 % for microscopy, and 88 % for RDT. Area under the receiver operating characteristic (ROC) curve in comparison with the reference standard was 0.94 for LAMP, 0.88 for microscopy and 0.81 for RDT. Turn-around time for the entire LAMP assay was approximately 3 h and 30 min for an average of 27 ± 9.5 samples collected per day, compared to a minimum of 10 samples an hour per operator by RDT and over 8 h by microscopy. The LAMP assay could produce reliable results the same day of the screening. It could detect a higher proportion of low density malaria infections than the other methods tested and may be used for large campaigns of systematic screening and treatment.

DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.

PubMed

Gaget, V; Keulen, A; Lau, M; Monis, P; Brookes, J D

2017-01-01

Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing. © 2016 The Society for Applied Microbiology.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

PubMed

Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

2014-11-01

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Inefficiency of Signal Amplification by Post-selection

NASA Astrophysics Data System (ADS)

Tanaka, Saki; Yamamoto, Naoki

Basing the two-state vector formalism, Aharonov, Albert and Vaidman found a measurement way such that spin 1/2 particle can turn out 100 [1]. The measurement result is called weak value and this value depends on pre-and post- selected states. The weak value becomes infinitely large when the post- selected state is orthogonal to pre-selected state. By using this feature, the weak measurement has been applied to amplification technique. However, the success of the post-selection depends on luck and this technique does not always work. We take into account of loss by post-selection, and evaluate this amplification by quantum estimation theory. As a result, we get an inequality which means that post-selection does not improve estate accuracy when the number of states is limited.

Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: an immunoelectron microscopic study

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

Stierhof, Y.D.; Humbel, B.M.; Schwarz, H.

1991-03-01

In order to exploit the recently introduced 1 nm gold colloids in routine electron microscopic labeling experiments, an efficient enhancement step for a better visualization of this small marker is a prerequisite. Efficiency and reproducibility of enhancement as well as growth homogeneity of gold particles were evaluated for three different silver intensifying solutions: silver lactate/hydroquinone/gum arabic, and the commercially available IntenSE M silver enhancement kit. The best results were obtained by using the silver lactate/hydroquinone/gum arabic mixture. The quality of enhancement of the IntenSE M kit was considerably increased by the addition of the protective colloid gum arabic.

Multiscale image contrast amplification (MUSICA)

NASA Astrophysics Data System (ADS)

Vuylsteke, Pieter; Schoeters, Emile P.

1994-05-01

This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

Field-effect amperometric immuno-detection of protein biomarker.

PubMed

Wang, Jiapeng; Yau, Siu-Tung

2011-11-15

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work. Copyright © 2011 Elsevier B.V. All rights reserved.

CucCAP - Developing genomic resources for the cucurbit community

USDA-ARS?s Scientific Manuscript database

The U.S. cucurbit community has initiated a USDA-SCRI funded cucurbit genomics project, CucCAP: Leveraging applied genomics to increase disease resistance in cucurbit crops. Our primary objectives are: develop genomic and bioinformatic breeding tool kits for accelerated crop improvement across the...

Theory of multiwave mixing within the superconducting kinetic-inductance traveling-wave amplifier

NASA Astrophysics Data System (ADS)

Erickson, R. P.; Pappas, D. P.

2017-03-01

We present a theory of parametric mixing within the coplanar waveguide (CPW) of a superconducting nonlinear kinetic-inductance traveling-wave (KIT) amplifier engineered with periodic dispersion loadings. This is done by first developing a metamaterial band theory of the dispersion-engineered KIT using a Floquet-Bloch construction and then applying it to the description of mixing of the nonlinear RF traveling waves. Our theory allows us to calculate signal gain versus signal frequency in the presence of a frequency stop gap, based solely on loading design. We present results for both three-wave mixing (3WM), with applied dc bias, and four-wave mixing (4WM), without dc. Our theory predicts an intrinsic and deterministic origin to undulations of 4WM signal gain with signal frequency, apart from extrinsic sources, such as impedance mismatch, and shows that such undulations are absent from 3WM signal gain achievable with dc. Our theory is extensible to amplifiers based on Josephson junctions in a lumped LC-ladder transmission line (TWPA).

A microsatellite study for determination of allelic variation of Kurdish population-Kurdistan region-Iraq

NASA Astrophysics Data System (ADS)

Murad, Media J.; Amin, Bushra K.

2017-09-01

The purpose of this study was detecting genetic variations for the Kurdish population in Kurdistan region-Iraq, using fifteen autosomal STR loci. Buccal swabs were collected and depositing on Nucleic Card (Copan, Italia Spa) from 302 healthy unrelated Iraqi Kurds in five provinces of Kurdistan region-Iraq. Fifteen autosomal STR loci are D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA and Amelogenin included in the AmpFlSTR Identifiler® Direct PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). No significant departure from Hardy Weinberg Equilibrium (HWE) expectations were observed in 10 from 15 STR loci analyzed (a 5% significance level was taken). The exceptions were the CSF1PO, D3S1358, D13S317, D16S539 and D2S1338 loci. Statistical parameters of forensic efficiencies were estimated for the loci, based on allelic frequencies. The mean of observed heterozygosity, expected heterozygosity and PIC values across the 15 loci were 0.762, 0.797 and 0.768 respectively, indicating high gene diversity. The combined probability of exclusion, power of discrimination, probability of matching value for all the 15 STR loci were 0.9999968; 0.9999999 and 4.966×10-17, respectively. These parameters indicated the importance of the loci for forensic genetic purposes and paternity testing.

The protective rate of the feline immunodeficiency virus vaccine: An Australian field study.

PubMed

Westman, M E; Malik, R; Hall, E; Harris, M; Norris, J M

2016-09-07

A case-control field study was undertaken to determine the level of protection conferred to client-owned cats in Australia against feline immunodeficiency virus (FIV) using a commercial vaccine. 440 cats with outdoor access from five Australian states/territories underwent testing, comprising 139 potential cases (complete course of primary FIV vaccinations and annual boosters for three or more years), and 301 potential controls (age, sex and postcode matched FIV-unvaccinated cats). FIV status was determined using a combination of antibody testing (using point-of-care test kits) and nucleic acid amplification, as well as virus isolation in cases where results were discordant and in all suspected FIV-vaccinated/FIV-infected cats ('vaccine breakthroughs'). Stringent inclusion criteria were applied to both 'cases' and 'controls'; 89 FIV-vaccinated cats and 212 FIV-unvaccinated cats ultimately satisfied the inclusion criteria. Five vaccine breakthroughs (5/89; 6%), and 25 FIV-infected controls (25/212; 12%) were identified, giving a vaccine protective rate of 56% (95% CI -20 to 84). The difference in FIV prevalence rates between the two groups was not significant (P=0.14). Findings from this study raise doubt concerning the efficacy of Fel-O-Vax FIV® under field conditions. Screening for FIV infection may be prudent before annual FIV re-vaccination and for sick FIV-vaccinated cats. Owners should not rely on vaccination alone to protect cats against the risk of acquiring FIV infection; other measures such as cat curfews, the use of 'modular pet parks' or keeping cats exclusively indoors, are recommended. Copyright © 2016. Published by Elsevier Ltd.

Renal trematode infection due to Paratanaisia bragai in zoo housed Columbiformes and a red bird-of-paradise (Paradisaea rubra).

PubMed

Unwin, Steve; Chantrey, Julian; Chatterton, James; Aldhoun, Jitka A; Littlewood, D Timothy J

2013-12-01

Trematode infections affect a diverse range of avian species and the organs that are parasitised are also very varied. The family Eucotylidae contains seven genera of renal flukes that parasitise various birds. In birds, mild to severe lesions have been reported for species of the genus Paratanaisia, which was originally described from columbiform and galliform specimens collected in South America and has been identified in a number of wild avian species. This paper investigates eight cases of renal trematode infection at Chester Zoo in the UK due to Paratanaisia bragai in five previously unreported species: red bird-of-paradise, Socorro dove, Mindanao bleeding heart dove, laughing dove and emerald dove. Pathological changes, which varied between species, are discussed. A known intermediate snail host Allopeas clavulinum was present in the enclosures but there was no direct evidence of trematode infection. The size of the snails, possible low prevalence and the difficulty of visualising sporocysts contributed to this. Thus the development and application of further molecular diagnostic markers that can be applied to snail tissues is warranted. Parasite identification was confirmed utilizing DNA amplification from formalin-fixed paraffin-embedded tissues using PCR and trematode specific primers. Sequencing full ssrDNA and D1-D3 lsrDNA confirmed the identity in all cases as P. bragai. However, the short 310 bp fragment used provides insufficient variation or sequence length for wider application. The epidemiology, pathology and consequences for the management of these endangered species are discussed. Preliminary work on developing an effective ante mortem diagnostic PCR test kit is also highlighted.

Homogeneous electrochemical detection of ochratoxin A in foodstuff using aptamer-graphene oxide nanosheets and DNase I-based target recycling reaction.

PubMed

Sun, Ai-Li; Zhang, Yan-Fang; Sun, Guo-Peng; Wang, Xuan-Nian; Tang, Dianping

2017-03-15

A simple and feasible homogeneous electrochemical sensing protocol was developed for the detection of ochratoxin A (OTA) in foodstuff on the immobilization-free aptamer-graphene oxide nanosheets coupling with DNase I-based cycling signal amplification. Thionine-labeled OTA aptamers were attached to the surface of nanosheets because of the strong noncovalent binding of graphene oxide nanosheets with nucleobases and aromatic compounds. The electronic signal was acquired via negatively charged screen-printed carbon electrode (SPCE) toward free thionine molecules. Initially, the formed thionine-aptamer/graphene nanocomposites were suspended in the detection solution and far away from the electrode, thereby resulting in a weak electronic signal. Upon addition of target OTA, the analyte reacted with the aptamer and caused the dissociation of thionine-aptamer from the graphene oxide nanosheets. The newly formed thionine-aptamer/OTA could be readily cleaved by DNase I and released target OTA, which could retrigger thionine-aptamer/graphene nanocomposites with target recycling to generate numerous free thionine molecules. Free thionine molecules were captured by negatively charged SPCE, each of which could produce an electrochemical signal within the applied potentials. Under optimal conditions, graphene-based aptasensing platform could exhibit good electrochemical responses for the detection of OTA at a concentration as low as 5.6pg/mL. The reproducibility, precision and selectivity of the system were acceptable. Importantly, the method accuracy was comparable with commercialized OTA ELISA kit when using for quantitative monitoring of contaminated wheat samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of four commercial biuret reagent kits of serum total protein by the American Association for Clinical Chemistry reference measurement procedure.

PubMed

He, Meilin; Zhang, Jie

2011-06-01

In China, the traceability of clinical chemistry methods is still immature. Therefore, it is necessary to establish a reference measurement procedure and evaluate commercial reagent kits using such established procedures. We reproduced the reference measurement procedure for serum total protein, as recommended by the American Association for Clinical Chemistry (AACC). We evaluated the performance by Clinical Laboratory Standard Institute (CLSI) guidelines EP15-A and EP6-A. Subsequently, four commercial reagent kits were evaluated by the reproduced reference procedure following CLSI guideline EP9-A2. The performance of the reproduced reference procedure was as follows: CVs ranged from 0.47% to 0.85% at medical decision levels (X(c)) of 45 g/L, 60 g/L and 80 g/L. Linearity was Y=1.0022X-0.2121 (r=0.9999), and recovery ranged from 100.2% to 102.4%. The External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine (RELA) was applied, and the result was within the limit of equivalence. The linear relationships of four commercial reagent kits, Merit Choice, KHB, Leadman, and Olympus, were, respectively: Y=0.9922X+0.5776 (r=0.9961); Y=0.9936X+0.4316 (r=0.9992); Y=0.9949X+0.9129 (r=0.9987) and Y=0.9923X+0.8876 (r=0.9989). KHB showed slight negative bias, and the mean±SD was -0.03±0.60 g/L. Merit Choice, Leadman, and Olympus all showed positive bias, and the mean±SDs were 0.02±0.63 g/L, 0.55±0.77 g/L and 0.34±0.71 g/L, respectively. The correlation and bias of four commercial reagent kits for serum total protein were found to be acceptable. Thus, these reagent kits can be used reliably in China.

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.

A large ungated TPC with GEM amplification

NASA Astrophysics Data System (ADS)

Berger, M.; Ball, M.; Fabbietti, L.; Ketzer, B.; Arora, R.; Beck, R.; Böhmer, F. V.; Chen, J.-C.; Cusanno, F.; Dørheim, S.; García, F.; Hehner, J.; Herrmann, N.; Höppner, C.; Kaiser, D.; Kis̆, M.; Kleipa, V.; Konorov, I.; Kunkel, J.; Kurz, N.; Leifels, Y.; Müllner, P.; Münzer, R.; Neubert, S.; Rauch, J.; Schmidt, C. J.; Schmitz, R.; Soyk, D.; Vandenbroucke, M.; Voss, B.; Walther, D.; Zmeskal, J.

2017-10-01

A Time Projection Chamber (TPC) is an ideal device for the detection of charged particle tracks in a large volume covering a solid angle of almost 4 π. The high density of hits on a given particle track facilitates the task of pattern recognition in a high-occupancy environment and in addition provides particle identification by measuring the specific energy loss for each track. For these reasons, TPCs with Multiwire Proportional Chamber (MWPC) amplification have been and are widely used in experiments recording heavy-ion collisions. A significant drawback, however, is the large dead time of the order of 1 ms per event generated by the use of a gating grid, which is mandatory to prevent ions created in the amplification region from drifting back into the drift volume, where they would severely distort the drift path of subsequent tracks. For experiments with higher event rates this concept of a conventional TPC operating with a triggered gating grid can therefore not be applied without a significant loss of data. A continuous readout of the signals is the more appropriate way of operation. This, however, constitutes a change of paradigm with considerable challenges to be met concerning the amplification region, the design and bandwidth of the readout electronics, and the data handling. A mandatory prerequisite for such an operation is a sufficiently good suppression of the ion backflow from the avalanche region, which otherwise limits the tracking and particle identification capabilities of such a detector. Gas Electron Multipliers (GEM) are a promising candidate to combine excellent spatial resolution with an intrinsic suppression of ions. In this paper we describe the design, construction and the commissioning of a large TPC with GEM amplification and without gating grid (GEM-TPC). The design requirements have driven innovations in the construction of a light-weight field-cage, a supporting media flange, the GEM amplification and the readout system, which are presented in this paper. We further describe the support infrastructure such as gas, cooling and slow control. Finally, we report on the operation of the GEM-TPC in the FOPI experiment, and describe the calibration procedures which are applied to achieve the design performance of the device.

Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria

PubMed Central

Perera, Rushini S.; Ding, Xavier C.; Tully, Frank; Oliver, James; Bright, Nigel; Bell, David; Chiodini, Peter L.; Gonzalez, Iveth J.; Polley, Spencer D.

2017-01-01

Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. Results The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7–100), and 99.7% (95% CI, 99.2–100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1–100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1–100) and specificity was 99.7% (95% CI, 99.2–100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3–99.5) and 100% respectively. Conclusions The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns. PMID:28166235

Bismuth-based quadruple Helicobacter pylori eradication regimen alters the composition of gut microbiota.

PubMed

Yildiz, Serap Süzük; Yalinay, Meltem; Karakan, Tarkan

2018-06-01

Microbiota is a dynamic system showing individual differences in both the number and species of microorganisms. Dietary habits, lifestyle, age, genetic predisposition of the host and use of antibiotics are effective on microbiota. The aim of our research was to carry out a quantitative comparison of Bifidobacterium spp, Bacteroides fragilis, Lactobacillus spp, Akkermansia mucinophilia and Faecalibacterium prausnitzii, important bacterial microbiota species, before and after antibiotic therapy treated with tetracycline and metronidazole in patients who are diagnosed as positive for Helicobacter pylori (HP), and to determine the effects of antibiotic use on the microbiota. Eighteen HP-positive patients were enrolled in this study. A special extraction kit (QIAmp DNA Stool Mini Kit, QIAgen, Germany) was used for the DNA isolation procedure. Primers specific to the 16S rRNA region of the bacteria included in the study were used for the amplification of the target region. All the bacteria were subjected to real-time quantification procedure with PCR method on RotorGene® 20 device (Qiagen, Germany). According to quantification before and after antibiotic use in patients receiving HP treatment, statistically significant decreases were observed in Bifidobacterium spp (p=0.001), B. fragilis (p=0.001), Lactobacillus spp (p=0.001), A. mucinophilia (p=0.001) and F. prausnitzii (p=0.001). We were unable to identify B. fragilis in the microbiota of five patients after treatment. Based on the data obtained, it can be concluded that antibiotics used to treat HP can prepare the ground that could result in dysbiosis in microbiota.

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

PubMed

Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

2012-11-20

The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

PubMed Central

2012-01-01

The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

PubMed Central

Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

2014-01-01

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

Optical parametric amplification of arbitrarily polarized light in periodically poled LiNbO3.

PubMed

Shao, Guang-hao; Song, Xiao-shi; Xu, Fei; Lu, Yan-qing

2012-08-13

Optical parametric amplification (OPA) of arbitrarily polarized light is proposed in a multi-section periodically poled Lithium Niobate (PPLN). External electric field is applied on selected sections to induce the polarization rotation of involved lights, thus the quasi-phase matched optical parametric processes exhibit polarization insensitivity under suitable voltage. In addition to the amplified signal wave, an idler wave with the same polarization is generated simultaneously. As an example, a ~10 times OPA showing polarization independency is simulated. Applications of this technology are also discussed.

Mid Course Space Experiment (MSX) Environmental Assessment

DTIC Science & Technology

1992-03-06

8217____ a ~~ACTrv=T _ _ _ _ _ _I wTLL USU/SDL =H/APL OSFC VAPE ITP Fabrication -SBV -SPIRIT ft1 -Satellites -Reference -UVISI ___________ Objects...Vandenberg Air Force Base ( VAPE ) Jobit Hopkins University/Applied Physics Laboratoty MM/UAPL) Kausi Tea Facility (KIT 3 Satellite trussn assucttue, OSDP

Women in Sports, Spring 1977. Sports Kit.

ERIC Educational Resources Information Center

Women's Equity Action League, Washington, DC.

Information concerning the Washington-based Women's Equity Action League (WEAL), its legislative recommendations, and internship program is presented. Excerpts from Title IX sections applying to sport and a memorandum from the Department of Health, Education, and Welfare (HEW) regarding the legal obligations of school officials are included, as…

Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome.

PubMed

Favero, F; McGranahan, N; Salm, M; Birkbak, N J; Sanborn, J Z; Benz, S C; Becq, J; Peden, J F; Kingsbury, Z; Grocok, R J; Humphray, S; Bentley, D; Spencer-Dene, B; Gutteridge, A; Brada, M; Roger, S; Dietrich, P-Y; Forshew, T; Gerlinger, M; Rowan, A; Stamp, G; Eklund, A C; Szallasi, Z; Swanton, C

2015-05-01

Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries.

PubMed

Jehee, Fernanda Sarquis; Takamori, Jean Tetsuo; Medeiros, Paula F Vasconcelos; Pordeus, Ana Carolina B; Latini, Flavia Roche M; Bertola, Débora Romeo; Kim, Chong Ae; Passos-Bueno, Maria Rita

2011-01-01

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cut-off for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

PubMed Central

Bhadra, Sanchita; Ellington, Andrew D.

2014-01-01

Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer ‘Spinach’ that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits. PMID:24493736

Amplification and Attenuation Across USArray Using Ambient Noise Wavefront Tracking

NASA Astrophysics Data System (ADS)

Bowden, Daniel C.; Tsai, Victor C.; Lin, Fan-Chi

2017-12-01

As seismic traveltime tomography continues to be refined using data from the vast USArray data set, it is advantageous to also exploit the amplitude information carried by seismic waves. We use ambient noise cross correlation to make observations of surface wave amplification and attenuation at shorter periods (8-32 s) than can be observed with only traditional teleseismic earthquake sources. We show that the wavefront tracking approach can be successfully applied to ambient noise correlations, yielding results quite similar to those from earthquake observations at periods of overlap. This consistency indicates that the wavefront tracking approach is viable for use with ambient noise correlations, despite concerns of the inhomogeneous and unknown distribution of noise sources. The resulting amplification and attenuation maps correlate well with known tectonic and crustal structure; at the shortest periods, our amplification and attenuation maps correlate well with surface geology and known sedimentary basins, while our longest period amplitudes are controlled by crustal thickness and begin to probe upper mantle materials. These amplification and attenuation observations are sensitive to crustal materials in different ways than traveltime observations and may be used to better constrain temperature or density variations. We also value them as an independent means of describing the lateral variability of observed Rayleigh wave amplitudes without the need for 3-D tomographic inversions.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

PubMed

Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

2018-02-15

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

PubMed

Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

2017-05-01

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10 -15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Optimization of whole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysis.

PubMed

Wu, Jieying; Gao, Weimin; Zhang, Weiwen; Meldrum, Deirdre R

2011-01-01

Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities. Copyright © 2010 Elsevier B.V. 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.

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.

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

A micromechanical analogue mixer with dynamic displacement amplification

NASA Astrophysics Data System (ADS)

Erismis, M. A.

2018-06-01

A new micromechanical device is proposed which is capable of modulation, demodulation and filtering operations. The device uses a patented 3-mass coupled micromechanical resonator which dynamically amplifies the displacement within a frequency range of interest. Modulation can be obtained by exciting different masses of the resonator with the data and the carrier signals. Demodulation can be obtained similarly by exciting the actuator with the input and carrier signals at the same time. With the help of dynamic motion amplification, filtering and signal amplification can be achieved simultaneously. A generic design approach is introduced which can be applied from kHz to MHz regime frequencies of interest. A sample mixer design for an silicon on insulator-based process is provided. A SPICE (Simulation Program with Integrated Circuit Emphasis)-based electro-mechanical co-simulation platform is also developed and the proposed mixer is simulated.

Final Technical Report "Study of Efficiency of Raman Backscattering Amplification in Plasma"

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

Suckewer, Szymon

2014-03-31

General : Our major scientific achievements in Raman Backscattering (RBS) amplification and compression of short laser pulses in plasma. The laser system based on RBS steps in where the current technology of chirped pulse amplification (CPA) (extremely successful in developing ultra-short and ultra-intense laser pulses in last 2 decades) becomes difficult and very expensive to apply. Good base for such RBS laser was created by our recent experiments, which were supported by GPS grants. The main objective of the present grant was: improvement efficiency of energy transfer from pump to seed. The results surpassed our expectations; we improved the efficiencymore » of energy transfer from pump to seed by a factor of 6 compared to the best of our previous results and amplified seed pulse compressed down to about 50 fsec.« less

Techni-kits and Techni-kit Building Systems

NASA Technical Reports Server (NTRS)

Callender, E. D.; Hartsough, C.; Morris, R. V.; Yamamoto, Y.

1985-01-01

Techni-kits consists of theories, methods, standards and computer based tools that assist in design of information-intensive systems. Techni-kit "building system" is techni-kit that builds techni-kits.

Optimized thermal amplification in a radiative transistor

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

Prod'homme, Hugo; Ordonez-Miranda, Jose; Ezzahri, Younes, E-mail: younes.ezzahri@univ-poitiers.fr

The thermal performance of a far-field radiative transistor made up of a VO{sub 2} base in between a blackbody collector and a blackbody emitter is theoretically studied and optimized. This is done by using the grey approximation on the emissivity of VO{sub 2} and deriving analytical expressions for the involved heat fluxes and transistor amplification factor. It is shown that this amplification factor can be maximized by tuning the base temperature close to its critical one, which is determined by the temperature derivative of the VO{sub 2} emissivity and the equilibrium temperatures of the collector and emitter. This maximization ismore » the result of the presence of two bi-stable temperatures appearing during the heating and cooling processes of the VO{sub 2} base and enables a thermal switching (temperature jump) characterized by a sizeable variation of the collector-to-base and base-to-emitter heat fluxes associated with a slight change of the applied power to the base. This switching effect leads to the optimization of the amplification factor and therefore it could be used for thermal modulation purposes.« less

Improving Mobile Phone Speech Recognition by Personalized Amplification: Application in People with Normal Hearing and Mild-to-Moderate Hearing Loss.

PubMed

Kam, Anna Chi Shan; Sung, John Ka Keung; Lee, Tan; Wong, Terence Ka Cheong; van Hasselt, Andrew

In this study, the authors evaluated the effect of personalized amplification on mobile phone speech recognition in people with and without hearing loss. This prospective study used double-blind, within-subjects, repeated measures, controlled trials to evaluate the effectiveness of applying personalized amplification based on the hearing level captured on the mobile device. The personalized amplification settings were created using modified one-third gain targets. The participants in this study included 100 adults of age between 20 and 78 years (60 with age-adjusted normal hearing and 40 with hearing loss). The performance of the participants with personalized amplification and standard settings was compared using both subjective and speech-perception measures. Speech recognition was measured in quiet and in noise using Cantonese disyllabic words. Subjective ratings on the quality, clarity, and comfortableness of the mobile signals were measured with an 11-point visual analog scale. Subjective preferences of the settings were also obtained by a paired-comparison procedure. The personalized amplification application provided better speech recognition via the mobile phone both in quiet and in noise for people with hearing impairment (improved 8 to 10%) and people with normal hearing (improved 1 to 4%). The improvement in speech recognition was significantly better for people with hearing impairment. When the average device output level was matched, more participants preferred to have the individualized gain than not to have it. The personalized amplification application has the potential to improve speech recognition for people with mild-to-moderate hearing loss, as well as people with normal hearing, in particular when listening in noisy environments.

Innovation Online Teaching Module Plus Digital Engineering Kit with Proteus Software through Hybrid Learning Method to Improve Student Skills

NASA Astrophysics Data System (ADS)

Kholis, Nur; Syariffuddien Zuhrie, Muhamad; Rahmadian, Reza

2018-04-01

Demands the competence (competence) needs of the industry today is a competent workforce to the field of work. However, during this lecture material Digital Engineering (Especially Digital Electronics Basics and Digital Circuit Basics) is limited to the delivery of verbal form of lectures (classical method) is dominated by the Lecturer (Teacher Centered). Though the subject of Digital Engineering requires learning tools and is required understanding of electronic circuits, digital electronics and high logic circuits so that learners can apply in the world of work. One effort to make it happen is by creating an online teaching module and educational aids (Kit) with the help of Proteus software that can improve the skills of learners. This study aims to innovate online teaching modules plus kits in Proteus-assisted digital engineering courses through hybrid learning approaches to improve the skills of learners. The process of innovation is done by considering the skills and mastery of the technology of students (students) Department of Electrical Engineering - Faculty of Engineering – Universitas Negeri Surabaya to produce quality graduates Use of online module plus Proteus software assisted kit through hybrid learning approach. In general, aims to obtain adequate results with affordable cost of investment, user friendly, attractive and interactive (easily adapted to the development of Information and Communication Technology). With the right design, implementation and operation, both in the form of software both in the form of Online Teaching Module, offline teaching module, Kit (Educational Viewer), and e-learning learning content (both online and off line), the use of the three tools of the expenditure will be able to adjust the standard needs of Information and Communication Technology world, both nationally and internationally.

Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4.

PubMed

Nguyen, Lam Thanh; Nakaishi, Kazunari; Motojima, Keiko; Ohkawara, Ayako; Minato, Erina; Maruyama, Junki; Hiono, Takahiro; Matsuno, Keita; Okamatsu, Masatoshi; Kimura, Takashi; Takada, Ayato; Kida, Hiroshi; Sakoda, Yoshihiro

2017-01-01

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

PubMed

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

40 CFR 1054.115 - What other requirements apply?

Code of Federal Regulations, 2010 CFR

2010-07-01

... POLLUTION CONTROLS CONTROL OF EMISSIONS FROM NEW, SMALL NONROAD SPARK-IGNITION ENGINES AND EQUIPMENT... § 1054.145(c), engines must meet applicable emission standards at all specified atmospheric pressures, except that for atmospheric pressures below 94.0 kPa you may rely on an altitude kit for all testing if...

Applied Anatomy and Physiology. Student's Manual [and] Instructor's Guide.

ERIC Educational Resources Information Center

Williams, Catherine

The student manual in this two-part instructional kit contains basic concepts and specific information needed for understanding anatomy and physiology, with emphasis on those areas of particular interest to health occupations students. The student manual is organized in 10 lessons, each containing objectives, new terms and definitions, technical…

A Large Population Genetic Study of 15 Autosomal Short Tandem Repeat Loci for Establishment of Korean DNA Profile Database

PubMed Central

Yoo, Seong Yeon; Cho, Nam Soo; Park, Myung Jin; Seong, Ki Min; Hwang, Jung Ho; Song, Seok Bean; Han, Myun Soo; Lee, Won Tae; Chung, Ki Wha

2011-01-01

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10-17. This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications. PMID:21597912

A large population genetic study of 15 autosomal short tandem repeat loci for establishment of Korean DNA profile database.

PubMed

Yoo, Seong Yeon; Cho, Nam Soo; Park, Myung Jin; Seong, Ki Min; Hwang, Jung Ho; Song, Seok Bean; Han, Myun Soo; Lee, Won Tae; Chung, Ki Wha

2011-07-01

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10(-17). This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications.

Molecular characterization and RAPD analysis of Juniperus species from Iran.

PubMed

Kasaian, J; Behravan, J; Hassany, M; Emami, S A; Shahriari, F; Khayyat, M H

2011-06-07

The genus Juniperus L. (Cupressaceae), an aromatic evergreen plant, consists of up to 68 species around the world. We classified five species of Juniperus found in Iran using molecular markers to provide a means for molecular identification of Iranian species. Plants were collected (three samples of each species) from two different provinces of Iran (Golestan and East Azarbayejan). The DNA was extracted from the leaves using a Qiagen Dneasy Plant Mini Kit. Amplification was performed using 18 ten-mer RAPD primers. Genetic distances were estimated based on 187 RAPD bands to construct a dendrogram by means of unweighted pair group method of arithmetic means. It was found that J. communis and J. oblonga were differentiated from the other species. Genetic distance values ranged from 0.19 (J. communis and J. oblonga) to 0.68 (J. communis and J. excelsa). Juniperus foetidissima was found to be most similar to J. sabina. Juniperus excelsa subspecies excelsa and J. excelsa subspecies polycarpos formed a distinct group.

Analysis of an "off-ladder" allele at the Penta D short tandem repeat locus.

PubMed

Yang, Y L; Wang, J G; Wang, D X; Zhang, W Y; Liu, X J; Cao, J; Yang, S L

2015-11-25

Kinship testing of a father and his son from Guangxi, China, the location of the Zhuang minority people, was performed using the PowerPlex® 18D System with a short tandem repeat typing kit. The results indicated that both the father and his son had an off-ladder allele at the Penta D locus, with a genetic size larger than that of the maximal standard allelic ladder. To further identify this locus, monogenic amplification, gene cloning, and genetic sequencing were performed. Sequencing analysis demonstrated that the fragment size of the Penta D-OL locus was 469 bp and the core sequence was [AAAGA]21, also called Penta D-21. The rare Penta D-21 allele was found to be distributed among the Zhuang population from the Guangxi Zhuang Autonomous Region of China; therefore, this study improved the range of DNA data available for this locus and enhanced our ability for individual identification of gene loci.

Microfluidic system for the identification of bacterial pathogens causing urinary tract infections

NASA Astrophysics Data System (ADS)

Becker, Holger; Hlawatsch, Nadine; Haraldsson, Tommy; van der Wijngaart, Wouter; Lind, Anders; Malhotra-Kumar, Surbi; Turlej-Rogacka, Agata; Goossens, Herman

2015-03-01

Urinary tract infections (UTIs) are among the most common bacterial infections and pose a significant healthcare burden. The growing trend in antibiotic resistance makes it mandatory to develop diagnostic kits which allow not only the determination of a pathogen but also the antibiotic resistances. We have developed a microfluidic cartridge which takes a direct urine sample, extracts the DNA, performs an amplification using batch-PCR and flows the sample over a microarray which is printed into a microchannel for fluorescence detection. The cartridge is injection-molded out of COP and contains a set of two-component injection-molded rotary valves to switch between input and to isolate the PCR chamber during thermocycling. The hybridization probes were spotted directly onto a functionalized section of the outlet microchannel. We have been able to successfully perform PCR of E.coli in urine in this chip and perform a fluorescence detection of PCR products. An upgraded design of the cartridge contains the buffers and reagents in blisters stored on the chip.

[Usefulness of conventional polymerase chain reaction for the detection of Mycoplasma hominis, Ureaplasma spp. and Trichomonas vaginalis in female outpatient's genital samples].

PubMed

Alarcón, Gonzalo; Barraza, Gabriela; Vera, Andrea; Wozniak, Aniela; García, Patricia

2016-02-01

Trichomonas vaginalis, Mycoplasma hominis and Ureaplasma spp. are microorganisms responsible for genitourinary and pregnancy pathologies. Nucleic acid amplification methods have shown several advantages, but have not been widely studied for the detection of these microorganisms. To implement a conventional polymerase chain reaction (PCR) for the detection of the microorganisms and to compare its results versus the methods currently used at our laboratory. 91 available samples were processed by PCR, culture (M. hominis y Ureaplasma spp.) and wet mount (T vaginalis). Results were compared and statistically analyzed by kappa agreement test. 85, 80 and 87 samples resulted in agreement for the detection of M. hominis, Ureaplasma spp. y T. vaginalis, respectively. For M. hominis and Ureaplasma spp., agreement was substantial, whereas for T. vaginalis it was moderate, however, for the latter, PCR detected more cases than wet mount. We recommend the implementation of PCR for detection of T. vaginalis whereas culture kit is still a useful method for the other microorganisms.

Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus.

PubMed

Su, Zi Dan; Shi, Cheng Yin; Huang, Jie; Shen, Gui Ming; Li, Jin; Wang, Sheng Qiang; Fan, Chao

2015-09-26

Red-spotted grouper nervous necrosis virus (RGNNV) is an important pathogen that causes diseases in many species of fish in marine aquaculture. The larvae and juveniles are more easily infected by RGNNV and the cumulative mortality is as high as 100 % after being infected with RGNNV. This virus imposes a serious threat to aquaculture of grouper fry. This study aimed to establish a simple, accurate and highly sensitive method for rapid detection of RGNNV on the spot. In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification (CPA) system was established. The product amplified by CPA was detected through visualization with lateral flow dipstick (LFD). Three important parameters, including the amplification temperature, the concentration of dNTPs and the concentration of Mg(2+) for the CPA system, were optimized. The sensitivity and specificity of this method for RGNNV were tested and compared with those of the conventional RT-PCR and real-time quantitative RT-PCR (qRT-PCR). The optimized conditions for the CPA amplification system were determined as follows: the optimal amplification temperature, the optimized concentration of dNTPs and the concentration for Mg(2+) were 69 °C, 1.2 mmol/L and 5 mmol/L, respectively. The lowest limit of detection (LLOD) of this method for RGNNV was 10(1) copies/μL of RNA sample, which was 10 times lower than that of conventional RT-PCR and comparable to that of RT-qPCR. This method was specific for RGNNV in combination with SJNNV and had no cross-reactions with 8 types of virus and bacterial strains tested. This method was successfully applied to detect RGNNV in fish samples. This study established a CPA-LFD method for detection of RGNNV. This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of this virus on the spot.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony.

PubMed

Yan, Muxia; Li, Weidong; Zhou, Zhenwen; Peng, Hongxia; Luo, Ziyan; Xu, Ling

2017-01-01

In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 45 min with 25 μl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 10 2 and 10 4 or 10 5  CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Amplification of the basic reproduction number in cattle farm networks

PubMed Central

2018-01-01

The popularly known 20–80 rule or Pareto rule states that 20% of efforts leads to 80% of results. This rule has been applied to the study of infection transmission in contact networks, and specifically, contact networks between cattle farms. Woolhouse and collaborators showed that targeting interventions for disease control and prevention to the 20% of the farms that contribute the most to the basic reproduction number (Ro), could reduce it by 80%. The rule results from the number of incoming and outgoing contacts per farm being highly heterogeneous. Besides, Woolhouse and collaborators showed that this high contact heterogeneity, together with a high positive correlation between the number of incoming and outgoing animal movements per farm leads to an amplification in the Ro. Two previous studies carried out with Scottish cattle transport data found either no correlation or only a weak correlation (rho up to 0.33) when using weighted data. Using data from the contacts between Swiss cattle farms in 2015, we found that the 20–80 rule applies with respect to Ro, although the proportion of highly active farms is smaller (11%). Besides, a positive strong correlation (rho = 0.64, weighted data) between the incoming and outgoing contacts of farms exists. This means that the amplification of Ro (due to the contact heterogeneities and the existing correlation) in cattle contact networks can be much higher than known until now. Our results highlight the importance of an effective active surveillance, more so than in other countries were these amplification mechanisms are absent. PMID:29672512

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors

PubMed Central

Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

2017-01-01

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs. PMID:28192400

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.

Cardiac c-Kit Biology Revealed by Inducible Transgenesis.

PubMed

Gude, Natalie A; Firouzi, Fareheh; Broughton, Kathleen M; Ilves, Kelli; Nguyen, Kristine P; Payne, Christina R; Sacchi, Veronica; Monsanto, Megan M; Casillas, Alexandria R; Khalafalla, Farid G; Wang, Bingyan J; Ebeid, David E; Alvarez, Roberto; Dembitsky, Walter P; Bailey, Barbara A; van Berlo, Jop; Sussman, Mark A

2018-06-22

Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress. c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings. © 2018 American Heart Association, Inc.

Genetic analysis of haplotype data for 23 Y-chromosome short tandem repeat loci in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina

PubMed Central

Dogan, Serkan; Primorac, Dragan; Marjanović, Damir

2014-01-01

Aim To explore the distribution and polymorphisms of 23 short tandem repeat (STR) loci on the Y chromosome in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina and to investigate its genetic relationships with the homeland Turkish population and neighboring populations. Methods This study included 100 healthy unrelated male individuals from the Turkish population living in Sarajevo. Buccal swab samples were collected as a DNA source. Genomic DNA was extracted using the salting out method and amplification was performed using PowerPlex Y 23 amplification kit. The studied population was compared to other populations using pairwise genetic distances, which were represented with a multi-dimensional scaling plot. Results Haplotype and allele frequencies of the sample population were calculated and the results showed that all 100 samples had unique haplotypes. The most polymorphic locus was DYS458, and the least polymorphic DYS391. The observed haplotype diversity was 1.0000 ± 0.0014, with a discrimination capacity of 1.00 and the match probability of 0.01. Rst values showed that our sample population was closely related in both dimensions to the Lebanese and Iraqi populations, while it was more distant from Bosnian, Croatian, and Macedonian populations. Conclusion Turkish population residing in Sarajevo could be observed as a representative Turkish population, since our results were consistent with those previously published for the homeland Turkish population. Also, this study once again proved that geographically close populations were genetically more related to each other. PMID:25358886

Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

PubMed

Yang, Qi; Franco, Christopher M M; Zhang, Wei

2015-10-01

Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory▿

PubMed Central

Linton, Christopher J.; Borman, Andrew M.; Cheung, Grace; Holmes, Ann D.; Szekely, Adrien; Palmer, Michael D.; Bridge, Paul D.; Campbell, Colin K.; Johnson, Elizabeth M.

2007-01-01

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species. PMID:17251397

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.

Evaluation of the kinase domain of c-KIT in canine cutaneous mast cell tumors

PubMed Central

Webster, Joshua D; Kiupel, Matti; Yuzbasiyan-Gurkan, Vilma

2006-01-01

Background Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD) mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors. Methods In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16–20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations. Results No mutations or polymorphisms were identified in exons 16–20 of any of the MCTs examined. Conclusion In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs. PMID:16579858

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

PubMed

Chen, Zhongming; Zhu, Wuqiang; Bender, Ingrid; Gong, Wuming; Kwak, Il-Youp; Yellamilli, Amritha; Hodges, Thomas J; Nemoto, Natsumi; Zhang, Jianyi; Garry, Daniel J; van Berlo, Jop H

2017-12-12

Although cardiac c-kit + cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit + cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit + cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit + cells. We used single-cell sequencing and genetic lineage tracing of c-kit + cells to determine whether various pathological stimuli would result in different fates of c-kit + cells. Single-cell sequencing of cardiac CD45 - c-kit + cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit + cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit + cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit + cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit + cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit + cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit + cells. © 2017 American Heart Association, Inc.

Single Cell Total RNA Sequencing through Isothermal Amplification in Picoliter-Droplet Emulsion.

PubMed

Fu, Yusi; Chen, He; Liu, Lu; Huang, Yanyi

2016-11-15

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, "easier-seq", to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 × 10 5 aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing.

Single laser pulse compression via strongly coupled stimulated Brillouin scattering in plasma

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

Peng, H.; Wu, Z. H.; Zhang, Z. M.

2016-07-15

Laser amplification in plasma, including stimulated Raman scattering amplification and strongly coupled stimulated Brillouin scattering (sc-SBS) amplification, is very promising to generate ultrahigh-power and ultrashort laser pulses. But both are quite complex in experiments: at least three different laser pulses must be prepared; temporal delay and spatial overlap of these three pulses are difficult. We propose a single pulse compression scheme based on sc-SBS in plasma. Only one moderately long laser is applied, the front part of which ionizes the gas to produced plasma, and gets reflected by a plasma mirror at the end of the gas channel. The reflectedmore » front quickly depletes the remaining part of the laser by sc-SBS in the self-similar regime. The output laser is much stronger and shorter. This scheme is at first considered theoretically, then validated by using 1D PIC simulations.« less

Ultrasensitive Detection of Low-Abundance Surface-Marker Protein using Isothermal Rolling Circle Amplification in Microfluidic Nano-Liter Platform

PubMed Central

Konry, Tania; Yarmush, Joel M.; Irimia, Daniel

2011-01-01

With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer. PMID:21294269

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

PubMed

Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

2011-08-15

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

Broad-Spectrum Molecular Detection of Fungal Nucleic Acids by PCR-Based Amplification Techniques.

PubMed

Czurda, Stefan; Lion, Thomas

2017-01-01

Over the past decade, the incidence of life-threatening invasive fungal infections has dramatically increased. Infections caused by hitherto rare and emerging fungal pathogens are associated with significant morbidity and mortality among immunocompromised patients. These observations render the coverage of a broad range of clinically relevant fungal pathogens highly important. The so-called panfungal or, perhaps more correctly, broad-range nucleic acid amplification techniques do not only facilitate sensitive detection of all clinically relevant fungal species but are also rapid and can be applied to analyses of any patient specimens. They have therefore become valuable diagnostic tools for sensitive screening of patients at risk of invasive fungal infections. This chapter summarizes the currently available molecular technologies employed in testing of a wide range of fungal pathogens, and provides a detailed workflow for patient screening by broad-spectrum nucleic acid amplification techniques.

Application of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cow Components Adulterated in Buffalo Milk/Meat.

PubMed

Deb, Rajib; Sengar, Gyanendra Singh; Singh, Umesh; Kumar, Sushil; Alyethodi, R R; Alex, Rani; Raja, T V; Das, A K; Prakash, B

2016-12-01

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1 h 40 min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis. The test can detect up to 5 % level of cow milk/meat mixed in buffalo counterparts. Due to the simplicity and specificity, the developed LAMP test can be easily adapted in any laboratory for rapid detection of cow species identification in livestock by products.

Three-wave interaction solitons in optical parametric amplification.

PubMed

Ibragimov, E; Struthers, A A; Kaup, D J; Khaydarov, J D; Singer, K D

1999-05-01

This paper applies three-wave interaction (TWI)-soliton theory to optical parametric amplification when the signal, idler, and pump wave can all contain TWI solitons. We use an analogy between two different velocity regimes to compare the theory with output from an experimental synchronously pumped optical parametric amplifier. The theory explains the observed inability to compress the intermediate group-velocity wave and 20-fold pulse compression in this experiment. The theory and supporting numerics show that one can effectively control the shape and energy of the optical pulses by shifting the TWI solitons in the pulses.

Bacteriophage Amplification-Coupled Detection and Identification of Bacterial Pathogens

NASA Astrophysics Data System (ADS)

Cox, Christopher R.; Voorhees, Kent J.

Current methods of species-specific bacterial detection and identification are complex, time-consuming, and often require expensive specialized equipment and highly trained personnel. Numerous biochemical and genotypic identification methods have been applied to bacterial characterization, but all rely on tedious microbiological culturing practices and/or costly sequencing protocols which render them impractical for deployment as rapid, cost-effective point-of-care or field detection and identification methods. With a view towards addressing these shortcomings, we have exploited the evolutionarily conserved interactions between a bacteriophage (phage) and its bacterial host to develop species-specific detection methods. Phage amplification-coupled matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was utilized to rapidly detect phage propagation resulting from species-specific in vitro bacterial infection. This novel signal amplification method allowed for bacterial detection and identification in as little as 2 h, and when combined with disulfide bond reduction methods developed in our laboratory to enhance MALDI-TOF-MS resolution, was observed to lower the limit of detection by several orders of magnitude over conventional spectroscopy and phage typing methods. Phage amplification has been combined with lateral flow immunochromatography (LFI) to develop rapid, easy-to-operate, portable, species-specific point-of-care (POC) detection devices. Prototype LFI detectors have been developed and characterized for Yersinia pestis and Bacillus anthracis, the etiologic agents of plague and anthrax, respectively. Comparable sensitivity and rapidity was observed when phage amplification was adapted to a species-specific handheld LFI detector, thus allowing for rapid, simple, POC bacterial detection and identification while eliminating the need for bacterial culturing or DNA isolation and amplification techniques.

Association between somatic amplification, anxiety, depression, stress and migraine

PubMed Central

2013-01-01

Background The aim of this study is to investigate the associations between migraine related disability and somatosensory amplification, depression, anxiety, and stress. Method Fifty-five migraine patients who applied to the outpatient unit of the Neurology Department of Acibadem University School of Medicine, Maslak Hospital in Istanbul, Turkey, and twenty-eight subjects without migraine were recruited for the study. The participants were asked to complete a sociodemographic form, Migraine Disability Assessment Scale (MIDAS), Depression Anxiety Stress Scale, Somatosensory Amplification Scale (SSAS). Results Somatosensory amplification scores were significantly higher in the migraineurs than in the control group (29.85+/−6.63 vs 26.07+/−7.1; p=0.027). Somatosensory amplification scores and depression scores were significantly higher in migraineurs with moderate and severe disability than in patients with minimal and mild disability (31.7+/−6.4 vs 27.71+/−5.49; p=0.01, 11.27+/−8.7 vs 7.38+/−8.11; p=0.04, respectively). A significant positive correlation was found between the frequency of migraine attacks for at least three consecutive months (MIDAS A scores) and the SSAS scores (r=0.363, p=0.007) in migraineurs. The MIDAS total scores were also significantly correlated with the DASS depression subcale scores (r=0.267, p=0.04), and the DASS stress subscale scores (r=0.268, p=0.05). Conclusion Psychological factors, and vulnerability to bodily sensations may incease the burden of migraine. We point out that the timely assessing of somatic amplification and the evaluation of mental status would help improve the quality of life of in migraineurs. PMID:23799958

Numerical simulation of stress amplification induced by crack interaction in human femur bone

NASA Astrophysics Data System (ADS)

Alia, Noor; Daud, Ruslizam; Ramli, Mohammad Fadzli; Azman, Wan Zuki; Faizal, Ahmad; Aisyah, Siti

2015-05-01

This research is about numerical simulation using a computational method which study on stress amplification induced by crack interaction in human femur bone. Cracks in human femur bone usually occur because of large load or stress applied on it. Usually, the fracture takes longer time to heal itself. At present, the crack interaction is still not well understood due to bone complexity. Thus, brittle fracture behavior of bone may be underestimated and inaccurate. This study aims to investigate the geometrical effect of double co-planar edge cracks on stress intensity factor (K) in femur bone. This research focuses to analyze the amplification effect on the fracture behavior of double co-planar edge cracks, where numerical model is developed using computational method. The concept of fracture mechanics and finite element method (FEM) are used to solve the interacting cracks problems using linear elastic fracture mechanics (LEFM) theory. As a result, this study has shown the identification of the crack interaction limit (CIL) and crack unification limit (CUL) exist in the human femur bone model developed. In future research, several improvements will be made such as varying the load, applying thickness on the model and also use different theory or method in calculating the stress intensity factor (K).

Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells

PubMed Central

Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

2016-01-01

c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

PubMed

Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

2014-03-01

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

PubMed

Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, M Jesús

2016-12-01

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

Direct detection of microRNAs using isothermal amplification and molecular beacon with excellent sensitivity and specificity

NASA Astrophysics Data System (ADS)

Zhang, Wancun; Zhang, Qi; Qian, Zhiyu; Gu, Yueqing

2017-02-01

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets. In this article, we develop a highly sensitive, and specific miRNA assay based on the two-stage isothermal amplification reactions and molecular beacon. The two-stage isothermal amplification reactions involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide(Y). Importantly, different miRNAs can be converted to the same Y seperately, which can hybridize with the same set of molecular beacon to generate fluorescent signals. This assay is highly sensitive and specific with a detection limit of 1 fM and can even discriminate single-nucleotide differences. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same molecular beacon. This method has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.

Allele frequency distribution for 21 autosomal STR loci in Bhutan.

PubMed

Kraaijenbrink, Thirsa; van Driem, George L; Tshering of Gaselô, Karma; de Knijff, Peter

2007-07-20

We studied the allele frequency distribution of 21 autosomal STR loci contained in the AmpFlSTR Identifiler (Applied Biosystems), the Powerplex 16 (Promega) and the FFFL (Promega) multiplex PCR kits among 936 individuals from the Royal Kingdom of Bhutan. As such these are the first published autosomal DNA results from this country.

NREL Announces Third Round of Start-Ups to Participate in the Wells Fargo

Science.gov Websites

innovative commercial building technologies Photo of NREL researchers talking. George Lee and Steven Low that provide scalable solutions to reduce the energy impact of commercial buildings. Including Round 3 kit for commercial buildings. Referred to apply to program by University of Colorado Boulder Software

Fiscal Year 1999 At-a-Glance. Office of Justice Programs.

ERIC Educational Resources Information Center

Department of Justice, Washington, DC. Office of Justice Programs.

This publication provides brief descriptions of all the funding opportunities of the bureaus and offices of the Office of Justice Programs. Each description lists the amount of funding available, tells who can apply, and gives the status of program regulations, guidelines, reports, and application kits. The citations also contain the name of the…

Industrial Microbiology.

ERIC Educational Resources Information Center

Demain, Arnold L.; Solomon, Nadine A.

1981-01-01

Presents an overview of the field of industrial microbiology, providing historical backgrounds of scientific discoveries in the field and descriptions of industrially important microorganisms. Applied research in industry is also detailed, with mention of gene amplification, DNA recombination, pharmaceutical approaches, and detoxification and…

A 1J LD pumped Nd:YAG pulsed laser system

NASA Astrophysics Data System (ADS)

Yi, Xue-bin; Wang, Bin; Yang, Feng; Li, Jing; Liu, Ya-Ping; Li, Hui-Jun; Wang, Yu; Chen, Ren

2017-11-01

A 1J LD pumped Nd;YAG pulsed laser was designed. The laser uses an oscillation and two-staged amplification structure, and applies diode bar integrated array as side-pump. The TEC temperature control device combing liquid cooling system is organized to control the temperature of the laser system. This study also analyzed the theoretical threshold of working material, the effect of thermal lens and the basic principle of laser amplification. The results showed that the laser system can achieve 1J, 25Hz pulse laser output, and the laser pulse can be output at two width: 6-7ns and 10ns, respectively, and the original beam angle is 1.2mrad. The laser system is characterized by small size, light weight, as well as good stability, which make it being applied in varied fields such as photovoltaic radar platform and etc

Controlled Microwave Heating Accelerates Rolling Circle Amplification.

PubMed

Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

2015-01-01

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781

Amphibian molecular ecology and how it has informed conservation.

PubMed

McCartney-Melstad, Evan; Shaffer, H Bradley

2015-10-01

Molecular ecology has become one of the key tools in the modern conservationist's kit. Here we review three areas where molecular ecology has been applied to amphibian conservation: genes on landscapes, within-population processes, and genes that matter. We summarize relevant analytical methods, recent important studies from the amphibian literature, and conservation implications for each section. Finally, we include five in-depth examples of how molecular ecology has been successfully applied to specific amphibian systems. © 2015 John Wiley & Sons Ltd.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

PubMed Central

Furitsu, T; Tsujimura, T; Tono, T; Ikeda, H; Kitayama, H; Koshimizu, U; Sugahara, H; Butterfield, J H; Ashman, L K; Kanayama, Y

1993-01-01

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells. Images PMID:7691885

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

PubMed Central

Shi, Xiarong; Sousa, Leiliane P.; Mandel-Bausch, Elizabeth M.; Tome, Francisco; Reshetnyak, Andrey V.; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

2016-01-01

Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

Megakaryocytes compensate for Kit insufficiency in murine arthritis.

PubMed

Cunin, Pierre; Penke, Loka R; Thon, Jonathan N; Monach, Paul A; Jones, Tatiana; Chang, Margaret H; Chen, Mary M; Melki, Imene; Lacroix, Steve; Iwakura, Yoichiro; Ware, Jerry; Gurish, Michael F; Italiano, Joseph E; Boilard, Eric; Nigrovic, Peter A

2017-05-01

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

Trichomonas vaginalis infection in women who submit self-obtained vaginal samples after internet recruitment.

PubMed

Gaydos, Charlotte A; Hsieh, Yu-Hsiang; Barnes, Mathilda; Quinn, Nicole; Agreda, Patricia; Jett-Goheen, Mary; Whittle, Pamela; Hogan, Terry

2011-09-01

Submission of self-obtained vaginal samples (SOVs) collected at home could remove barriers that women face in getting tested for sexually transmitted infections (STIs). Internet recruitment of SOVs is highly acceptable. Sexually active women ≥14 years were recruited by an educational Internet program, available at: www.iwantthekit.org (IWTK), which offered free testing for trichomonas as part of a panel, which also offered testing for chlamydia and gonorrhea. Kits were ordered online, SOVs were sent through US mail to the laboratory, and tested by nucleic acid amplification tests. Demographics and sexual risk factors were accessed by questionnaires. Women called or were contacted to receive their results. Of women requesting kits, 1525 (43%) returned swabs by mail. In all, 61% were <25 years, 52% were black, and 80% were single. Vaginal discharge was reported by 44%, prevalence for trichomonas was 10% (10% for chlamydia, 1% for gonorrhea), and 18% had at least one prevalent STI. Multivariate logistic regression demonstrated several significantly associated risks factors as follows: adjusted odds ratio for black race was 2.69; for residence of Illinois, 3.85; for not having health insurance, 1.57; for lack of a bachelor's degree, 5.53; for having 2 to 15 partners, 1.60; for having ≥16 partners in previous year, 3.51; for being bisexual, 2.0; for not always using condoms, 3.04; and for having a partner who had a previous STI, 1.71. Age was not associated with trichomonas infection. All infected women were treated. A high prevalence of trichomonas and high sexual risk factors were demonstrated. Internet recruitment was a useful method of screening women for trichomonas infection.

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.

PubMed

Wu, Gary D; Lewis, James D; Hoffmann, Christian; Chen, Ying-Yu; Knight, Rob; Bittinger, Kyle; Hwang, Jennifer; Chen, Jun; Berkowsky, Ronald; Nessel, Lisa; Li, Hongzhe; Bushman, Frederic D

2010-07-30

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.

Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin.

PubMed

Knet, Malgorzata; Wartalski, Kamil; Hoja-Lukowicz, Dorota; Tabarowski, Zbigniew; Slomczynska, Maria; Duda, Malgorzata

2015-10-01

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed. Copyright © 2015 Elsevier Inc. All rights reserved.

Forensic validation of the PowerPlex® ESI 16 STR Multiplex and comparison of performance with AmpFlSTR® SGM Plus®.

PubMed

Tucker, Valerie C; Kirkham, Amanda J; Hopwood, Andrew J

2012-05-01

We describe the forensic validation of Promega's PowerPlex® European Standard Investigator 16 (ESI 16) multiplex kit and compare results generated with the AmpFlSTR® SGM Plus® (SGM+) multiplex. ESI 16 combines the loci contained within the SGM+ multiplex with five additional loci: D2S441, D10S1248, D22S1045, D1S1656, and D12S391. A relative reduction in amplicon size of the SGM+ loci facilitates an increased robustness and amplification success of these amplicons with degraded DNA samples. Tests performed herein supplement ESI 16 data published previously with sensitivity, profile quality, mock casework, inhibitor and mixture study data collected in our laboratories in alignment with our internal technical and quality guidelines and those issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), the DNA Advisory Board (DAB) and the DNA working group (DNAWG) of the European Network of Forensic Science Institutes (ENFSI). Full profiles were routinely generated from a fully heterozygous single source DNA template using 62.5 pg for ESI 16 and 500 pg for SGM+. This increase in sensitivity has a consequent effect on mixture analyses and the detection of minor mixture components. The improved PCR chemistry confers enhanced tolerance to high levels of laboratory prepared inhibitors compared with SGM+ results. In summary, our results demonstrate that the ESI 16 multiplex kit is more robust and sensitive compared with SGM+ and will be a suitable replacement system for the analysis of forensic DNA samples providing compliance with the European standard set of STR loci.

A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.

PubMed

Karataylı, Ersin; Altunoğlu, Yasemin Çelik; Karataylı, Senem Ceren; Alagöz, S Gökçe K; Cınar, Kubilay; Yalçın, Kendal; Idilman, Ramazan; Yurdaydın, Cihan; Bozdayı, A Mithat

2014-05-01

Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

PubMed

Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

PubMed Central

Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics. PMID:21655320

EGFR amplification and expression in oral squamous cell carcinoma in young adults.

PubMed

Costa, V; Kowalski, L P; Coutinho-Camillo, C M; Begnami, M D; Calsavara, V F; Neves, J I; Kaminagakura, E

2018-07-01

The aim of this study was to investigate epidermal growth factor receptor (EGFR) gene alterations in two groups of patients with oral squamous cell carcinoma (OSCC) (a test group of subjects aged ≤40 years and a control group of subjects aged ≥50 years) and to associate the results with EGFR immunostaining, clinicopathological features, and the prognosis. Sixty cases of OSCC were selected (test group, n=21; control group, n=39). The tissue microarray technique was applied to ensure the uniformity of results. Gene amplification was analyzed by fluorescence in situ hybridization (FISH), and immunohistochemical staining for EGFR was analyzed using an automated imaging system. EGFR amplification was higher in the test group than in the control group (P=0.018) and was associated with advanced clinical stage (P=0.013), regardless of age. Patients with EGFR overexpression had worse survival rates, as did patients who had T3-T4 tumours and positive margins. EGFR overexpression has a negative impact on disease progression. Despite the higher amplification of EGFR in young adults, it does not significantly impact the survival rates of affected patients. Copyright © 2018 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

PubMed Central

Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.

2015-01-01

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. PMID:25856093

Novel electrochemiluminescence of perylene derivative and its application to mercury ion detection based on a dual amplification strategy.

PubMed

Zhao, Jing; Lei, Yan-Mei; Chai, Ya-Qin; Yuan, Ruo; Zhuo, Ying

2016-12-15

In this paper, a novel covalently crosslinked perylene derivative (PTC-PEI) composed of polyethylenimine (PEI) and perylenetetracarboxylic acid (PTCA) has been first investigated for cathodic electrochemiluminescence (ECL) in an aqueous system with dissolved O2 as coreactant. The promising novel ECL materials of PTC-PEI exhibited admirable physical and chemical stability and high ECL intensity, which held an alternative way to construct ECL sensor with improved sensitivity. Thus, it was applied to construct a dual amplified "signal-on" mercury ion (Hg(2+)) sensor by the employment of nicking endonuclease (NEase)-assisted target recycling and rolling circle amplification (RCA) for signal amplification. Herein, a long G-rich sequence was generated by RCA process to capture abundant hemin on the electrode surface, and then a significantly amplified ECL signal of PTC-PEI was obtained. Based on dual signal amplification strategy, the devised sensor showed a linear range from 0.1pM to 0.1μΜ with a detection limit down to 33fM (S/N=3), and was successfully used in the direct detection of real water sample with high sensitivity and selectivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Helicase dependent OnChip-amplification and its use in multiplex pathogen detection.

PubMed

Andresen, Dennie; von Nickisch-Rosenegk, Markus; Bier, Frank F

2009-05-01

The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent OnChip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the OnChip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. We have successfully shown the OnChip-HDA and applied it for single- and duplex-detection of the pathogens N. gonorrhoeae and S. aureus. We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.

Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

PubMed

Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

2014-02-06

Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. Copyright © 2013 Elsevier B.V. All rights reserved.

A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target recycling.

PubMed

Yan, Zhongdan; Gan, Ning; Li, Tianhua; Cao, Yuting; Chen, Yinji

2016-04-15

A multiplex electrochemical aptasensor was developed for simultaneous detection of two antibiotics such as chloramphenicol (CAP) and oxytetracycline (OTC), and high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted target recycling was used to improve sensitivity. The cascade amplification process consists of the exonuclease-assisted target recycling amplification and metal ions encoded magnetic hollow porous nanoparticles (MHPs) to produce voltammetry signals. Upon the specific recognition of aptamers to targets (CAP and OTC), exonuclease I (Exo I) selectively digested the aptamers which were bound with CAP and OTC, then the released CAP and OTC participated new cycling to produce more single DNA, which can act as trigger strands to hybrid with nanotracers to generate further signal amplification. MHPs were used as carriers to load more amounts of metal ions and coupling with Exo I assisted cascade target recycling can amplify the signal for about 12 folds compared with silica based nanotracers. Owing to the dual signal amplification, the linear range between signals and the concentrations of CAP and OTC were obtained in the range of 0.0005-50 ng mL(-1). The detection limits of CAP and OTC were 0.15 and 0.10 ng mL(-1) (S/N=3) which is more than 2 orders lower than commercial enzyme-linked immunosorbent immunoassay (ELISA) method, respectively. The proposed method was successfully applied to simultaneously detection of CAP and OTC in milk samples. Besides, this aptasensor can be applied to other antibiotics detection by changing the corresponding aptamer. The whole scheme is facile, selective and sensitive enough for antibiotics screening in food safety. Copyright © 2015 Elsevier B.V. All rights reserved.

Renal trematode infection due to Paratanaisia bragai in zoo housed Columbiformes and a red bird-of-paradise (Paradisaea rubra)☆

PubMed Central

Unwin, Steve; Chantrey, Julian; Chatterton, James; Aldhoun, Jitka A.; Littlewood, D. Timothy J.

2012-01-01

Trematode infections affect a diverse range of avian species and the organs that are parasitised are also very varied. The family Eucotylidae contains seven genera of renal flukes that parasitise various birds. In birds, mild to severe lesions have been reported for species of the genus Paratanaisia, which was originally described from columbiform and galliform specimens collected in South America and has been identified in a number of wild avian species. This paper investigates eight cases of renal trematode infection at Chester Zoo in the UK due to Paratanaisia bragai in five previously unreported species: red bird-of-paradise, Socorro dove, Mindanao bleeding heart dove, laughing dove and emerald dove. Pathological changes, which varied between species, are discussed. A known intermediate snail host Allopeas clavulinum was present in the enclosures but there was no direct evidence of trematode infection. The size of the snails, possible low prevalence and the difficulty of visualising sporocysts contributed to this. Thus the development and application of further molecular diagnostic markers that can be applied to snail tissues is warranted. Parasite identification was confirmed utilizing DNA amplification from formalin-fixed paraffin-embedded tissues using PCR and trematode specific primers. Sequencing full ssrDNA and D1-D3 lsrDNA confirmed the identity in all cases as P. bragai. However, the short 310 bp fragment used provides insufficient variation or sequence length for wider application. The epidemiology, pathology and consequences for the management of these endangered species are discussed. Preliminary work on developing an effective ante mortem diagnostic PCR test kit is also highlighted. PMID:24533313

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

Germline mutations of KIT in gastrointestinal stromal tumor (GIST) and mastocytosis.

PubMed

Ke, Hengning; Kazi, Julhash U; Zhao, Hui; Sun, Jianmin

2016-01-01

Somatic mutations of KIT are frequently found in mastocytosis and gastrointestinal stromal tumor (GIST), while germline mutations of KIT are rare, and only found in few cases of familial GIST and mastocytosis. Although ligand-independent activation is the common feature of KIT mutations, the phenotypes mediated by various germline KIT mutations are different. Germline KIT mutations affect different tissues such as interstitial cells of Cajal (ICC), mast cells or melanocytes, and thereby lead to GIST, mastocytosis, or abnormal pigmentation. In this review, we summarize germline KIT mutations in familial mastocytosis and GIST and discuss the possible cellular context dependent transforming activity of KIT mutations.

The challenges of lean manufacturing implementation in kitting assembly

NASA Astrophysics Data System (ADS)

Fansuri, A. F. H.; Rose, A. N. M.; Nik Mohamed, N. M. Z.; Ahmad, H.

2017-10-01

Literature studies shows that lean manufacturing goes way back with the original founder Eli Whitney in year 1799. The main purpose of lean manufacturing is to identify and eliminate waste in production. The application of lean manufacturing can be carried out in any industrial processes with regards to the understanding of lean principles, theories and practices. Kitting is one of the important aspects in a successful production. The continuous supply of materials from store to production has to be systematic and able to achieve lean standard for it to be successful. The objective of this paper is to review the implementation of lean manufacturing in kitting assembly. Previous papers show that, the implementation of lean manufacturing in kitting assembly may be beneficial to the organization such as reduce in space occupancy, part shortages, lead time and manpower. Based on previous research, some industries may tend to change between kitting and line stocking which are due to lack of understanding when implementing kitting and causes longer lead time and materials overflow in store. With a proper understanding on what to kit, where to kit, how to kit, why to kit and who kits the material with a standardised process flow may ensure the success of kitting.

No GIST-type c-kit gain of function mutations in neuroblastic tumours

PubMed Central

Korja, M; Finne, J; Salmi, T T; Haapasalo, H; Tanner, M; Isola, J

2005-01-01

Aims: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. Methods: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). Results: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. Conclusions: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated. PMID:15976348

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.

Low Cost DIY Lenses kit For High School Teaching

NASA Astrophysics Data System (ADS)

Thepnurat, Meechai; Saphet, Parinya; Tong-on, Anusorn

2017-09-01

A set of lenses was fabricated from a low cost materials in a DIY (Do it yourself) process. The purpose was to demonstrate to teachers and students in high schools how to construct lenses by themselves with the local available materials. The lenses could be applied in teaching Physics, about the nature of a lens such as focal length and light rays passing through lenses in either direction, employing a set of simple laser pointers. This instrumental kit was made from a transparent 2 mm thick of acrylic Perspex. It was cut into rectangular pieces with dimensions of 2x15 cm2 and bent into curved shape by a hot air blower on a cylindrical wooden rod with curvature radii of about 3-4.5 cm. Then a pair of these Perspex were formed into a hollow thick lenses with a base supporting platform, so that any appropriate liquids could be filled in. The focal length of the lens was measured from laser beam drawing on a paper. The refractive index, n (n) of a filling liquid could be calculated from the measured focal length (f). The kit was low cost and DIY but was greatly applicable for optics teaching in high school laboratory.

Nanostructured catalysts applied to degrade atrazine in aqueous phase by heterogeneous photo-Fenton process.

PubMed

Benzaquén, Tamara B; Barrera, Deicy A; Carraro, Paola M; Sapag, Karim; Alfano, Orlando M; Eimer, Griselda A

2018-06-02

SBA-15 and KIT-6 materials have been synthesized and modified with iron salts by the wet impregnation method with different metal loadings. The different mesostructures obtained were characterized by N 2 adsorption-desorption at 77 K, X-ray diffraction, temperature-programmed reduction, and ultraviolet-visible spectroscopy. These iron-containing mesostructured materials have been successfully tested for the heterogeneous photo-Fenton degradation of aqueous solutions of dangerous herbicides, such as atrazine, using UV-visible light irradiation, at room temperature and close to neutral pH. The results showed that the Fe/SBA-15 (10%) and Fe/KIT-6 (5%) catalysts exhibited the highest activities. However, the Fe/KIT-6 (5%) catalyst with minor Fe loading than Fe/SBA-15 (10%) presented a higher degradation of atrazine (above 98% in a reaction time of 240 min). Therefore, the interconnectivity of the cage-like mesopores had an important influence on the catalytic activity, favoring probably mass-transfer effects. Thus, the high performance of these materials indicates that the heterogeneous via of photo-Fenton process can also be efficiently employed to treat wastewaters containing pollutants such as herbicides, in order to reduce them to simplest and less toxic molecules.

Pim1 kinase regulates c-Kit gene translation.

PubMed

An, Ningfei; Cen, Bo; Cai, Houjian; Song, Jin H; Kraft, Andrew; Kang, Yubin

2016-01-01

Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1 -/- mice and Pim1 -/- 2 -/- 3 -/- triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 -/- and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35 S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Experimental evaluation of a new system for laser tissue welding applied on damaged lungs.

PubMed

Schiavon, Marco; Marulli, Giuseppe; Zuin, Andrea; Lunardi, Francesca; Villoresi, Paolo; Bonora, Stefano; Calabrese, Fiorella; Rea, Federico

2013-05-01

Alveolar air leaks represent a challenging problem in thoracic surgery, leading to increased patient morbidity and prolonged hospitalization. Several methods have been used, but no ideal technique exists yet. We investigated the lung-sealing capacity of an experimental kit for laser tissue welding. The kit is composed of a semiconductor laser system applied on a protein substrate associated with a chromophore that increases absorption. In vitro tests on porcine lung tissue were done to define ideal laser parameters (power 100 Å, frequency 50 Hz, pulse duration 400 µs) and protein substrate dilution (50%). For in vivo tests, through a left thoracotomy, 14 pigs received two different lung damages: a linear incision and a circular incision. Protein substrate applied on damaged areas was treated with laser to obtain a layer that reconstituted the integrity of the visceral pleura. Air leaks were intraoperatively evaluated by water submersion test with an airway pressure of 20 cmH2O. Animals were sacrificed at postoperative days 0 and 7 to study early and late pathological features. After applying laser treatment, no air leaks were seen in all proofs except in 2 cases in which a second application was required. At time 0, pathological damage mostly consisted of superficial alveolar necrotic tissue covered by protein membrane. At time 7, a complete recovery of lung lesions by fibrous scar with slight inflammatory reaction of adjacent lung tissue was seen. This experimental study demonstrated the effectiveness of laser tissue welding applied to seal air leaks after lung surgery. Further studies are needed to verify acceptability for human application.

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.

Early kit mortality and growth in farmed mink are affected by litter size rather than nest climate.

PubMed

Schou, T M; Malmkvist, J

2017-09-01

We investigated the effects of nest box climate on early mink kit mortality and growth. We hypothesised that litters in warm nest boxes experience less hypothermia-induced mortality and higher growth rates during the 1st week of life. This study included data from 749, 1-year-old breeding dams with access to nesting materials. Kits were weighed on days 1 and 7, dead kits were collected daily from birth until day 7 after birth, and nest climate was measured continuously from days 1 to 6. We tested the influences of the following daily temperature (T) and humidity (H) parameters on the number of live-born kit deaths and kit growth: T mean, T min, T max, T var (fluctuation) and H mean. The nest microclimate experienced by the kits was buffered against the ambient climate, with higher temperatures and reduced climate fluctuation. Most (77.0%) live-born kit deaths in the 1st week occurred on days 0 and 1. Seven of 15 climate parameters on days 1 to 3 had significant effects on live-born kit mortality. However, conflicting effects among days, marginal effects and late effects indicated that climate was not the primary cause of kit mortality. Five of 30 climate parameters had significant effects on kit growth. Few and conflicting effects indicated that the climate effect on growth was negligible. One exception was that large nest temperature fluctuations on day 1 were associated with reduced deaths of live-born kit (P<0.001) and increased kit growth (P=0.003). Litter size affected kit vitality; larger total litter size at birth was associated with greater risks of kit death (P<0.001) and reduced growth (P<0.001). The number of living kits in litters had the opposite effect, as kits in large liveborn litters had a reduced risk of death (P<0.001) and those with large mean litter size on days 1 to 7 had increased growth (P=0.026). Nest box temperature had little effect on early kit survival and growth, which could be due to dams' additional maternal behaviour. Therefore, we cannot confirm that temperature is the primary reason for kit mortality, under the conditions of plenty straw access for maternal nest building. Instead, prenatal and/or parturient litter size is the primary factor influencing early kit vitality. The results indicate that the focus should be on litter size and dam welfare around the times of gestation and birth to increase early kit survival in farmed mink.

The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.

PubMed

Ma, Xuhui; Wu, Yunteng; Zhang, Tian; Song, Hao; Jv, Houyu; Guo, Wei; Ren, Guoxin

2017-10-10

c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.

Intratumoral CD3+ T-Lymphocytes Immunoexpression and Its Association with c-Kit, Angiogenesis, and Overall Survival in Malignant Canine Mammary Tumors

PubMed Central

Carvalho, Maria Isabel; Pires, Isabel; Dias, Marlene; Prada, Justina; Gregório, Hugo; Lobo, Luis; Queiroga, Felisbina

2015-01-01

In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor. PMID:26346272

Comparison of sensor structures for the signal amplification of surface plasmon resonance immunoassay using enzyme precipitation

NASA Astrophysics Data System (ADS)

Yang, Chih-Tsung; Thierry, Benjamin

2015-12-01

Surface plasmon resonance (SPR) biosensing has been successfully applied for the label-free detection of a broad range of bioanalytes ranging from bacteria, cell, exosome, protein and nucleic acids. When it comes to the detection of small molecules or analytes found at low concentration, amplification schemes are desirable to enhance binding signals and in turn increase sensitivity. A number of SPR signal amplification schemes have been developed and validated; however, little effort has been devoted to understanding the effect of the SPR sensor structures on the amplification of binding signals and therefore on the overall sensing performance. The physical phenomenon of long-range SPR (LRSPR) relies on the propagation of coupled surface plasmonic waves on the opposite sides of a nanoscale-thick noble metal film suspended between two dielectrics with similar refractive indices. Importantly, as compared with commonly used conventional SPR (cSPR), LRSPR is not only characterized by a longer penetration depth of the plasmonic waves in the analyzed medium but also by a greater sensitivity to bulk refractive index changes. In this work, an immunoassay signal amplification platform based on horseradish peroxidase (HRP) catalyzed precipitation was utilized to investigate the sensing performance with regards to cSPR and LRSPR. The enzymatic precipitation of 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 was used to amplify SPR signals. The structure-function relationship of cSPR and LRSPR sensors is presented for both standard refractometric measurements and the enzymatic precipitation scheme. Experimental data shows that despite its inherent higher sensitivity to bulk refractive index changes and higher figure of merit, LRSPR was characterized by a lower angular sensitivity in the model enzymatic amplification scheme used here.

External and semi-internal controls for PCR amplification of homologous sequences in mixed templates.

PubMed

Kalle, Elena; Gulevich, Alexander; Rensing, Christopher

2013-11-01

In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. © 2013 Elsevier B.V. All rights reserved.

Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP).

PubMed

Chen, Changguo; Zhao, Qiangyuan; Guo, Jianwei; Li, Yanjun; Chen, Qiuyuan

2017-08-01

The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg 2+ concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg 2+ concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.

Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones

PubMed Central

2016-01-01

Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

PubMed

Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

2015-06-02

The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. Copyright © 2015 Elsevier B.V. All rights reserved.

Amplification of the 20q Chromosomal Arm Occurs Early in Tumorigenic Transformation and May Initiate Cancer

PubMed Central

Buganim, Yosef; Solomon, Hilla; Goldfinger, Naomi; Hovland, Randi; Ke, Xi-Song; Oyan, Anne M.; Kalland, Karl-H.; Rotter, Varda; Domany, Eytan

2011-01-01

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification. PMID:21297939

Fundamentals of quality and safety in diagnostic radiology.

PubMed

Bruno, Michael A; Nagy, Paul

2014-12-01

The most fundamental aspects of quality and safety in radiology are reviewed, including a brief history of the quality and safety movement as applied to radiology, the overarching considerations of organizational culture, team building, choosing appropriate goals and metrics, and the radiologist's quality "tool kit." Copyright © 2014 American College of Radiology. Published by Elsevier Inc. All rights reserved.

The Council of Europe's Citizenship Conception in "Education for Democratic Citizenship": A Critical Discourse Analysis of Two Textbooks

ERIC Educational Resources Information Center

Ververi, Olga

2017-01-01

This paper presents a neocommunitarian conception of citizenship identified in two textbooks of the programme "Education for Democratic Citizenship," organised by the Council of Europe. Critical discourse analysis is applied to the key themes of the textbooks "T-Kit 7: Under construction: Citizenship Youth and Europe" and…

Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change

DOEpatents

Miles, Robin R [Danville, CA; Belgrader, Phillip [Severna Park, MD; Fuller, Christopher D [Oakland, CA

2007-01-02

Impedance measurements are used to detect the end-point for PCR DNA amplification. A pair of spaced electrodes are located on a surface of a microfluidic channel and an AC or DC voltage is applied across the electrodes to produce an electric field. An ionically labeled probe will attach to a complementary DNA segment, and a polymerase enzyme will release the ionic label. This causes the conductivity of the solution in the area of the electrode to change. This change in conductivity is measured as a change in the impedance been the two electrodes.

A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

PubMed

Nkouawa, Agathe; Sako, Yasuhito; Li, Tiaoying; Chen, Xingwang; Nakao, Minoru; Yanagida, Tetsuya; Okamoto, Munehiro; Giraudoux, Patrick; Raoul, Francis; Nakaya, Kazuhiro; Xiao, Ning; Qiu, Jiamin; Qiu, Dongchuan; Craig, Philip S; Ito, Akira

2012-12-01

In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours.

PubMed

Costa Casagrande, T A; de Oliveira Barros, L M; Fukumasu, H; Cogliati, B; Chaible, L M; Dagli, M L Z; Matera, J M

2015-03-01

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) and the rate of tumour recurrence and tumour-related deaths in dogs affected with mast cell tumour (MCT). Kaplan-Meier curves were constructed to compare tumour recurrence and tumour-related death between patients. The log-rank test was used to check for significant differences between curves. KIT-I, KIT-II and KIT-III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour-related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT-PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis. © 2013 Blackwell Publishing Ltd.