Simultaneous detection of multiple biomarkers by means of SERS on polymer nanopillar gold arrays

NASA Astrophysics Data System (ADS)

Morasso, Carlo; Picciolini, Silvia; Mehn, Dora; Pellacani, Paola; Frangolho, Ana; Marchesini, Gerardo; Vanna, Renzo; Gualerzi, Alice; Bedoni, Marzia; Marabelli, Franco; Gramatica, Furio

2016-03-01

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.

A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies.

PubMed

Guldager, Daniel Kring Rasmussen; von Stemann, Jakob Hjorth; Larsen, Rune; Bay, Jakob Thaning; Galle, Pia Søndergaard; Svenson, Morten; Ullum, Henrik; Hansen, Morten Bagge

2015-10-01

To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-10 (IL-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon α (IFNα). Because the c-aAbs can be transferred to patients through blood transfusion, the assay was used to assess c-aAb levels in a cohort of patients who were receiving blood transfusions and subsequently presented with or without febrile reactions. The microsphere-based Luminex platform was used. Recombinant forms of human IL-1α, IL-6, IL-10, GM-CSF, and IFNα were gently coupled to MAG-PLEX beads. Plasma IgG binding was measured with phycoerythrin (PE)-labeled secondary antibodies. Previously confirmed c-aAb positive and negative donor plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay. The assay detected specific and saturable immunoglobulin G (IgG) binding to each of the tested cytokines in previously confirmed c-aAb positive plasmas and in preparations of pooled normal immunoglobulin. Confirmed c-aAb negative plasmas gave no saturable binding. The detection limit of the cytokine autoantibodies was estimated to be between 1 pM and 10 pM. The recovery of confirmed cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients with or without febrile transfusion reactions. The c-aAb level before and after the blood transfusions varied only slightly and in an irregular manner. This assay simultaneously detected up to five different c-aAbs in pooled human IgG and in plasma from individual blood donors, and it was deemed suitable for larger screenings. Based on confirmed antibody binding characteristics and the resultant reactivity in this multiplex assay, a classification of the c-aAb levels was suggested. The screening results of the recipients who received blood transfusions indicate that more studies are needed to clarify the role of antibodies, if any, in transfusion medicine and in high-dose immunoglobulin treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

Validation of a method to detect cocaine and its metabolites in nails by gas chromatography-mass spectrometry.

PubMed

Valente-Campos, Simone; Yonamine, Mauricio; de Moraes Moreau, Regina Lucia; Silva, Ovandir Alves

2006-06-02

The objective of the present work was to compare previously published methods and provide validation data to detect simultaneously cocaine (COC), benzoylecgonine (BE) and norcocaine (NCOC) in nail. Finger and toenail samples (5mg) were cut in very small pieces and submitted to an initial procedure for external decontamination. Methanol (3 ml) was used to release analytes from the matrix. A cleanup step was performed simultaneously by solid-phase extraction (SPE) and the residue was derivatized with pentafluoropropionic anhydride/pentafluoropropanol (PFPA/PFP). Gas chromatography-mass spectrometry (GC-MS) was used to detect the analytes in selected ion monitoring mode (SIM). Confidence parameters of validation of the method were: recovery, intra- and inter-assay precision, as well as limit of detection (LOD) of the analytes. The limits of detection were: 3.5 ng/mg for NCOC and 3.0 ng/mg for COC and BE. Good intra-assay precision was observed for all detected substances (coefficient of variation (CV)<11%). The inter-assay precision for norcocaine and benzoylecgonine were <4%. For intra- and inter-assay precision deuterated internal standards were used. Toenail and fingernail samples from eight declared cocaine users were submitted to the validated method.

A dye-assisted paper-based point-of-care assay for fast and reliable blood grouping.

PubMed

Zhang, Hong; Qiu, Xiaopei; Zou, Yurui; Ye, Yanyao; Qi, Chao; Zou, Lingyun; Yang, Xiang; Yang, Ke; Zhu, Yuanfeng; Yang, Yongjun; Zhou, Yang; Luo, Yang

2017-03-15

Fast and simultaneous forward and reverse blood grouping has long remained elusive. Forward blood grouping detects antigens on red blood cells, whereas reverse grouping identifies specific antibodies present in plasma. We developed a paper-based assay using immobilized antibodies and bromocresol green dye for rapid and reliable blood grouping, where dye-assisted color changes corresponding to distinct blood components provide a visual readout. ABO antigens and five major Rhesus antigens could be detected within 30 s, and simultaneous forward and reverse ABO blood grouping using small volumes (100 μl) of whole blood was achieved within 2 min through on-chip plasma separation without centrifugation. A machine-learning method was developed to classify the spectral plots corresponding to dye-based color changes, which enabled reproducible automatic grouping. Using optimized operating parameters, the dye-assisted paper assay exhibited comparable accuracy and reproducibility to the classical gel-card assays in grouping 3550 human blood samples. When translated to the assembly line and low-cost manufacturing, the proposed approach may be developed into a cost-effective and robust universal blood-grouping platform. Copyright © 2017, American Association for the Advancement of Science.

Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

NASA Astrophysics Data System (ADS)

Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

2012-10-01

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

Demonstration of four immunoassay formats using the array biosensor

NASA Technical Reports Server (NTRS)

Sapsford, Kim E.; Charles, Paul T.; Patterson, Charles H Jr; Ligler, Frances S.

2002-01-01

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

PubMed

Li, D Q; Zhao, J; Li, S P

2014-06-06

Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O₂(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplex hydrolysis probe real-time PCR for simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

2014-05-30

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV.

Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

PubMed

Nadal, Anna; Esteve, Teresa; Pla, Maria

2009-01-01

A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

PubMed

Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

2006-09-01

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti.

PubMed

Chan, Kamfai; Marras, Salvatore A E; Parveen, Nikhat

2013-12-20

The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients' blood samples. Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

PubMed Central

2013-01-01

Background The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. Results In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients’ blood samples. Conclusion Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner. PMID:24359556

Optical heterogeneous bioassay for the detection of the inflammatory biomarker suPAR

NASA Astrophysics Data System (ADS)

Tombelli, S.; Trono, C.; Adinolfi, B.; Chiavaioli, F.; Giannetti, A.; Eugen-Olsen, J.; Bernini, R.; Grimaldi, I. A.; Persichetti, G.; Testa, G.; Baldini, F.

2015-03-01

Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory protein present in blood and a marker of disease presence, severity and prognosis. A heterogeneous sandwich assay is proposed for quantifying suPAR by employing a capture antibody from rat and a biotinylated detection antibody from mouse. Optical detection was achieved by a successive exposure of the biotinylated sandwich to streptavidin labelled with ATTO647N. The heterogeneous assay was implemented on a multichannel polymethylmetacrylate (PMMA) optical biochip, potentially capable of the simultaneous detection of more than one analyte. Capture antibody was immobilized on the PMMA surface of the microfluidic channel and the assay was performed with the following protocol: i) surface blocking with BSA, ii) incubation with suPAR or PBS, iii) incubation with biotinylated suPAR detection Ab and iv) incubation with streptavidin-ATTO647N. Promising preliminary results were obtained with this protocol. Moreover, an improved optical setup is proposed which avoids the mechanical scanning of the chip and consequently the in-series fluorescence excitation and read out, allowing the simultaneous measurement of the fluorescence on all the channels of the microfluidic chip.

Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues

PubMed Central

Anderson, Courtney M.; Zhang, Bingqing; Miller, Melanie; Butko, Emerald; Wu, Xingyong; Laver, Thomas; Kernag, Casey; Kim, Jeffrey; Luo, Yuling; Lamparski, Henry; Park, Emily; Su, Nan

2016-01-01

ABSTRACT Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:27191821

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

Simultaneous determination of 13 fluoroquinolone and 22 sulfonamide residues in milk by a dual-colorimetric enzyme-linked immunosorbent assay

USDA-ARS?s Scientific Manuscript database

Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor the two classes of antimicrobial residues in different food matrices. In th...

A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

PubMed Central

Gelaye, Esayas; Mach, Lukas; Kolodziejek, Jolanta; Grabherr, Reingard; Loitsch, Angelika; Achenbach, Jenna E.; Nowotny, Norbert; Diallo, Adama; Lamien, Charles Euloge

2017-01-01

Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus]. The assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans. PMID:28216667

Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus

PubMed Central

Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

2014-01-01

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV. PMID:24886818

Simultaneous inhibition assay for human and microbial kinases via MALDI-MS/MS.

PubMed

Smith, Anne Marie E; Brennan, John D

2014-03-03

Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

PubMed Central

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathy L.; Marks, James D.; Varnum, Susan M.

2012-01-01

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples. PMID:22935296

Development and evaluation of a new lateral flow assay for simultaneous detection of antibodies against African Horse Sickness and Equine Infectious Anemia viruses.

PubMed

Costa, Sofia; Sastre, Patricia; Pérez, Teresa; Tapia, Istar; Barrandeguy, María; Sánchez-Vizcaíno, José M; Sánchez-Matamoros, Almudena; Wigdorovitz, Andrés; Sanz, Antonio; Rueda, Paloma

2016-11-01

African horse sickness (AHS) and equine infectious anemia (EIA) are both notifiable equid specific diseases that may present similar clinical signs. Considering the increased global movement of horses and equine products over the past decades, together with the socio-economic impact of previous AHS and EIA outbreaks, there is a clear demand for an early discrimination and a strict control of their transmission between enzootic and AHS/EIA-free regions. Currently, the individual control and prevention of AHS or EIA relies on a series of measures, including the restriction of animal movements, vector control, and the use of several laboratory techniques for viral identification, amongst others. Despite being widely employed in surveillance programmes and in the control of animal movements, the available serological assays can only detect AHS- or EIA-specific antibodies individually. In this work, a duplex lateral flow assay (LFA) for simultaneous detection and differentiation of specific antibodies against AHS virus (AHSV) and EIA virus (EIAV) was developed and evaluated with experimental and field serum samples. The duplex LFA was based on the AHSV-VP7 outer core protein and the EIAV-P26 major core protein. The results indicated that the duplex LFA presented a good analytical performance, detecting simultaneously and specifically antibodies against AHSV and EIAV. The initial diagnostic evaluation revealed a good agreement with results from the AHS and EIA tests prescribed by the OIE, and it highlighted the usefulness of the new AHSV/EIAV duplex LFA for an on-field and point-of-care first diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

A new PCR-CGE (size and color) method for simultaneous detection of genetically modified maize events.

PubMed

Nadal, Anna; Coll, Anna; La Paz, Jose-Luis; Esteve, Teresa; Pla, Maria

2006-10-01

We present a novel multiplex PCR assay for simultaneous detection of multiple transgenic events in maize. Initially, five PCR primers pairs specific to events Bt11, GA21, MON810, and NK603, and Zea mays L. (alcohol dehydrogenase) were included. The event specificity was based on amplification of transgene/plant genome flanking regions, i.e., the same targets as for validated real-time PCR assays. These short and similarly sized amplicons were selected to achieve high and similar amplification efficiency for all targets; however, its unambiguous identification was a technical challenge. We achieved a clear distinction by a novel CGE approach that combined the identification by size and color (CGE-SC). In one single step, all five targets were amplified and specifically labeled with three different fluorescent dyes. The assay was specific and displayed an LOD of 0.1% of each genetically modified organism (GMO). Therefore, it was adequate to fulfill legal thresholds established, e.g., in the European Union. Our CGE-SC based strategy in combination with an adequate labeling design has the potential to simultaneously detect higher numbers of targets. As an example, we present the detection of up to eight targets in a single run. Multiplex PCR-CGE-SC only requires a conventional sequencer device and enables automation and high throughput. In addition, it proved to be transferable to a different laboratory. The number of authorized GMO events is rapidly growing; and the acreage of genetically modified (GM) varieties cultivated and commercialized worldwide is rapidly increasing. In this context, our multiplex PCR-CGE-SC can be suitable for screening GM contents in food.

Using the Larval Zebrafish Locomotor Assay in Functional Neurotoxicity Screening: Light Intensity and the Order of Stimulus Presentation Affect the Outcome

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals using 6 day old zebrafish (Danio rerio) as an alternative test model for detecting neurotoxic chemicals. We use a behavioral testing paradigm that simultaneously tes...

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

PubMed

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

2014-06-15

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

Simultaneous determination of fluoxetine and norfluoxetine in dried blood spots using high-performance liquid chromatography-tandem mass spectrometry.

PubMed

da Silva, Anne Caroline Cezimbra; Raasch, Juliana Raquel; Vargas, Tainara Gomes; Peteffi, Giovana Piva; Hahn, Roberta Zilles; Antunes, Marina Venzon; Perassolo, Magda Susana; Linden, Rafael

2018-02-01

Therapeutic drug monitoring (TDM) of the widely prescribed antidepressant fluoxetine (FLU) is recommended in certain situations, such as occurrence of toxicity, inadequate response or suspect of poor adherence. Dried blood spot (DBS) sampling is an increasingly studied alternative for TDM, particularly for outpatients, due to its ease of collection and inherent stability. The aim of this study was to develop and validate an LC-MS/MS assay for the simultaneous quantification of FLU and norfluoxetine (NFLU) in DBS. The assay is based on a liquid extraction of single DBS with 8mm of diameter, using FLU-D6 as the internal standard, followed by reversed phase separation in an Accucore® C18 column (100×2.1mm, 2.6μm). Mobile phase was composed of water and acetonitrile (gradient from 80:20 to 50:50, v/v), both containing formic acid 0.1%. The assay was validated and applied to 30 patients under FLU pharmacotherapy. The assay was linear in the range 10-750ngmL -1 . Precision assays presented CV% of 3.13-9.61 and 3.54-7.99 for FLU and NFLU, respectively, and accuracy in the range of 97.98-110.44% and 100.25-105.8%. FLU and NFLU were stable at 25 and 45°C for 7days. The assay was evaluated in 30 patients under FLU treatment. Concentrations of both compounds were higher in DBS than in plasma, and the use of the multiplying factors 0.71 and 0.68 for FLU and NFLU, respectively, allowed acceptable estimation of plasma concentrations, with median prediction bias of -0.55 to 0.55% and mean differences of 0.4 to 2.2ngmL -1 . The presented data support the clinical use of DBS for therapeutic drug monitoring of FLU. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

PubMed

Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

2012-11-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses.

PubMed

Santiago, Gilberto A; Vázquez, Jesús; Courtney, Sean; Matías, Katia Y; Andersen, Lauren E; Colón, Candimar; Butler, Angela E; Roulo, Rebecca; Bowzard, John; Villanueva, Julie M; Muñoz-Jordan, Jorge L

2018-04-11

The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 10 3 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.

Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses.

PubMed

Kodani, Maja; Mixson-Hayden, Tonya; Drobeniuc, Jan; Kamili, Saleem

2014-10-01

Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No false-positives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). TAC assay was concordant with corresponding individual NATs for hepatitis A-E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly. Published by Elsevier B.V.

The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide

PubMed Central

Hu, Xiaofang; Zheng, Wei; Wang, Lu; Wan, Nan; Wang, Bing; Li, Weiwei; Hua, Hui; Hu, Xu; Shan, Fengping

2012-01-01

The aim of present research is to analyze the detailed changes of dendritic cells (DCs) induced by pidotimod(PTD). These impacts on DCs of both bone marrow derived DCs and established DC2.4 cell line were assessed with use of conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We demonstrated the ability of PTD to induce DC phynotypic and functional maturation as evidenced by higher expression of key surface molecules such as MHC II, CD80 and CD86. The functional tests proved the downregulation of ACP inside the DCs, occurred when phagocytosis of DCs decreased, with simultaneously antigen presentation increased toward maturation. Finally, PTD also stimulated production of more cytokine IL-12 and less TNF-α. Therefore it is concluded that PTD can markedly exert positive induction to murine DCs. PMID:22863756

Optimization and qualification of an Fc Array assay for assessments of antibodies against HIV-1/SIV.

PubMed

Brown, Eric P; Weiner, Joshua A; Lin, Shu; Natarajan, Harini; Normandin, Erica; Barouch, Dan H; Alter, Galit; Sarzotti-Kelsoe, Marcella; Ackerman, Margaret E

2018-04-01

The Fc Array is a multiplexed assay that assesses the Fc domain characteristics of antigen-specific antibodies with the potential to evaluate up to 500 antigen specificities simultaneously. Antigen-specific antibodies are captured on antigen-conjugated beads and their functional capacity is probed via an array of Fc-binding proteins including antibody subclassing reagents, Fcγ receptors, complement proteins, and lectins. Here we present the results of the optimization and formal qualification of the Fc Array, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. Assay conditions were optimized for performance and reproducibility, and the final version of the assay was then evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Barcoded microchips for biomolecular assays.

PubMed

Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

2015-01-20

Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

Net analyte signal standard addition method (NASSAM) as a novel spectrofluorimetric and spectrophotometric technique for simultaneous determination, application to assay of melatonin and pyridoxine

NASA Astrophysics Data System (ADS)

Asadpour-Zeynali, Karim; Bastami, Mohammad

2010-02-01

In this work a new modification of the standard addition method called "net analyte signal standard addition method (NASSAM)" is presented for the simultaneous spectrofluorimetric and spectrophotometric analysis. The proposed method combines the advantages of standard addition method with those of net analyte signal concept. The method can be applied for the determination of analyte in the presence of known interferents. The accuracy of the predictions against H-point standard addition method is not dependent on the shape of the analyte and interferent spectra. The method was successfully applied to simultaneous spectrofluorimetric and spectrophotometric determination of pyridoxine (PY) and melatonin (MT) in synthetic mixtures and in a pharmaceutical formulation.

An Automatic Lab-on-Disc System for Blood Typing.

PubMed

Chang, Yaw-Jen; Fan, Yi-Hua; Chen, Shia-Chung; Lee, Kuan-Hua; Lou, Liao-Yong

2018-04-01

A blood-typing assay is a critical test to ensure the serological compatibility of a donor and an intended recipient prior to a blood transfusion. This article presents a lab-on-disc blood-typing system to conduct a total of eight assays for a patient, including forward-typing tests, reverse-typing tests, and irregular-antibody tests. These assays are carried out in a microfluidic disc simultaneously. A blood-typing apparatus was designed to automatically manipulate the disc. The blood type can be determined by integrating the results of red blood cell (RBC) agglutination in the microchannels. The experimental results of our current 40 blood samples show that the results agree with those examined in the hospital. The accuracy reaches 97.5%.

Development and in-use evaluation of a novel Luminex MicroPlex microsphere-based (TRIOL) assay for simultaneous identification of Mycobacterium tuberculosis and detection of first-line and second-line anti-tuberculous drug resistance in China.

PubMed

Yin, Feifei; Chan, Jasper Fuk-Woo; Zhu, Qixuan; Fu, Ruijia; Chen, Jonathan Hon-Kwan; Choi, Garnet Kwan-Yue; Tee, Kah-Meng; Li, Lihua; Qian, Shiuyun; Yam, Wing-Cheong; Lu, Gang; Yuen, Kwok-Yung

2017-04-01

Rapid and accurate diagnostic assays with simultaneous microbial identification and drug resistance detection are essential for optimising treatment and control of tuberculosis. We developed a novel multiplex (TRIOL, Tuberculosis-Rifampicin-Isoniazid-Ofloxacin-Luminex) assay using the Luminex xMAP system that simultaneously identifies Mycobacterium tuberculosis and detects resistance to first-line and second-line anti-tuberculous drugs, and compared its performance with that by PCR sequencing, using phenotypic drug susceptibility testing as the gold standard. Identification of M. tuberculosis by the TRIOL assay was highly sensitive (100%) and specific (100%). The overall drug-specific specificities were excellent (100%). The overall sensitivity of the TRIOL assay was lower than that of the PCR-sequencing assays (72.4% vs 82.8%) because of a lower sensitivity of detecting rifampicin resistance (71.4% vs 92.9%). The sensitivity of detecting isoniazid and ofloxacin resistance was as good as the PCR-sequencing assays. Importantly, the TRIOL assay did not miss any mutations that were included in the assay. All of the resistant isolates that were missed had uncommon mutations or unknown resistance mechanisms that were not included in the assay. The TRIOL assay has higher throughput, lower cost and is less labour intensive than the PCR-sequencing assays. The TRIOL assay is advantageous in having the capability to detect resistance to multiple drugs and an open-architecture system that allows addition of more specific primers to detect uncommon mutations. Inclusion of additional primers for the identification of non-tuberculous mycobacteria, spoligotyping and improvement of rifampicin resistance detection would enhance the use of the TRIOL assay in future clinical and epidemiological studies. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Simultaneous detection and serotyping of dengue infection using single tube multiplex CDC Dengue Real-Time RT-PCR from India.

PubMed

Sharma, Shashi; Tandel, Kundan; Danwe, Surabhi; Bhatt, Puneet; Dash, P K; Ranjan, Praveer; Rathi, K R; Gupta, Rajiv Mohan; Parida, M M

2018-03-01

Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well serotyping assay in near future for rapid identification of dengue suspected sample screening.

Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.

PubMed

Barbau-Piednoir, Elodie; Botteldoorn, Nadine; Yde, Marc; Mahillon, Jacques; Roosens, Nancy H

2013-05-01

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

Simultaneous amplification of two bacterial genes: more reliable method of Helicobacter pylori detection in microbial rich dental plaque samples.

PubMed

Chaudhry, Saima; Idrees, Muhammad; Izhar, Mateen; Butt, Arshad Kamal; Khan, Ayyaz Ali

2011-01-01

Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Miniature silicon electronic biological assay chip and applications for rapid battlefield diagnostics

NASA Astrophysics Data System (ADS)

Cunningham, Brian T.; Regan, Robert A.; Clapp, Christopher; Hildebrant, Eric; Weinberg, Marc S.; Williams, John

1999-07-01

Assessing the medical condition of battlefield personnel requires the development of rapid, portable biological diagnostic assays for a wide variety of antigens and enzymes. Ideally, such an assay would be inexpensive, small, and require no added reagents while maintaining the sensitivity and accuracy of laboratory-based assays. In this work, a microelectromechanical (MEMS) based biological assay sensor is presented which is expected to meet the above requirements. The sensor is a thin silicon membrane resonator (SMR) which registers a decrease in resonant frequency when mass is adsorbed onto its surface. By coating the sensor surface with a monolayer of antibody, for example, we have detected the corresponding antigen with a detection resolution of 0.25 ng/ml in phosphate buffer solution. Micromachining techniques are being used to integrate many (64 elements on the first test chip) identical SMR sensors into a single silicon chip which would be capable of simultaneously performing a wide variety of biomedical assays. The sensors require only a small printed circuit board and 8V power supply to operate and provide a readout. The presentation will describe the operation of the SMR sensor, the fabrication of the sensor array, and initial test results using commercially available animal immunoglobulins in laboratory-prepared test solutions.

Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

EPA Science Inventory

High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

A laboratory-scale pretreatment and hydrolysis assay for determination of reactivity in cellulosic biomass feedstocks.

PubMed

Wolfrum, Edward J; Ness, Ryan M; Nagle, Nicholas J; Peterson, Darren J; Scarlata, Christopher J

2013-11-14

The rapid determination of the release of structural sugars from biomass feedstocks is an important enabling technology for the development of cellulosic biofuels. An assay that is used to determine sugar release for large numbers of samples must be robust, rapid, and easy to perform, and must use modest amounts of the samples to be tested.In this work we present a laboratory-scale combined pretreatment and saccharification assay that can be used as a biomass feedstock screening tool. The assay uses a commercially available automated solvent extraction system for pretreatment followed by a small-scale enzymatic hydrolysis step. The assay allows multiple samples to be screened simultaneously, and uses only ~3 g of biomass per sample. If the composition of the biomass sample is known, the results of the assay can be expressed as reactivity (fraction of structural carbohydrate present in the biomass sample released as monomeric sugars). We first present pretreatment and enzymatic hydrolysis experiments on a set of representative biomass feedstock samples (corn stover, poplar, sorghum, switchgrass) in order to put the assay in context, and then show the results of the assay applied to approximately 150 different feedstock samples covering 5 different materials. From the compositional analysis data we identify a positive correlation between lignin and structural carbohydrates, and from the reactivity data we identify a negative correlation between both carbohydrate and lignin content and total reactivity. The negative correlation between lignin content and total reactivity suggests that lignin may interfere with sugar release, or that more mature samples (with higher structural sugars) may have more recalcitrant lignin. The assay presented in this work provides a robust and straightforward method to measure the sugar release after pretreatment and saccharification that can be used as a biomass feedstock screening tool. We demonstrated the utility of the assay by identifying correlations between feedstock composition and reactivity in a population of 150 samples.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

PubMed

Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

2014-07-05

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Simultaneous determination of CRP and D-dimer in human blood plasma samples with White Light Reflectance Spectroscopy.

PubMed

Koukouvinos, Georgios; Petrou, Panagiota; Misiakos, Konstantinos; Drygiannakis, Dimitris; Raptis, Ioannis; Stefanitsis, Gerasimos; Martini, Spyridoula; Nikita, Dimitra; Goustouridis, Dimitrios; Moser, Isabella; Jobst, Gerhard; Kakabakos, Sotirios

2016-10-15

A dual-analyte assay for the simultaneous determination of C-reactive protein (CRP) and D-dimer in human blood plasma based on a white light interference spectroscopy sensing platform is presented. Measurement is accomplished in real-time by scanning the sensing surface, on which distinct antibody areas have been created, with a reflection probe used both for illumination of the surface and collection of the reflected interference spectrum. The composition of the transducer, the sensing surface chemical activation and biofunctionalization procedures were optimized with respect to signal magnitude and repeatability. The assay format involved direct detection of CRP whereas for D-dimer a two-site immunoassay employing a biotinylated reporter antibody and reaction with streptavidin was selected. The assays were sensitive with detection limits of 25ng/mL for both analytes, precise with intra- and inter-assay CV values ranging from 3.6% to 7.7%, and from 4.8% to 9.5%, respectively, for both assays, and accurate with recovery values ranging from 88.5% to 108% for both analytes. Moreover, the values determined for the two analytes in 35 human plasma samples were in excellent agreement with those received for the same samples by standard diagnostic laboratory instrumentation employing commercial kits. The excellent agreement of the results supported the validity of the proposed system for clinical application for the detection of multiple analytes since it was demonstrated that up to seven antibody areas can be created on the sensing surface and successfully interrogated with the developed optical set-up. Copyright © 2015. Published by Elsevier B.V.

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

PubMed

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food.

PubMed

Lee, Nari; Kwon, Kyung Yoon; Oh, Su Kyung; Chang, Hyun-Joo; Chun, Hyang Sook; Choi, Sung-Wook

2014-07-01

A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

Case report of a false positive result of the Xpert® MTB/RIF assay for rifampicin resistance in Mycobacterium tuberculosis complex.

PubMed

Claessens, Jolien; Mathys, Vanessa; Derdelinckx, Inge; Saegeman, Veroniek

2017-06-01

In the present case, we report a false positive result for the detection of rifampicin (RIF) resistance by the Xpert ® MTB/RIF assay, version G4.Miliary Mycobacterium tuberculosis infection (miliary TB) was suspected in a 50-year old Angolan woman. Imaging of the thorax and abdomen displayed diffuse lesions. The Xpert ® MTB/RIF assay conducted on the broncho-alveolar lavage (BAL) fluid was positive for TB and positive for RIF resistance. Confirmatory molecular tests and the phenotypic drug susceptibility determination supported the diagnosis of TB but not RIF resistance. The patient was treated successfully with a conventional therapeutic scheme. Because, the Xpert ® MTB/RIF assay allows the simultaneous detection of TB and RIF resistance, the World Health Organisation (WHO) recommends its use as initial diagnostic test, over microscopy, culture and phenotypic drug susceptibility testing. Even though specificity of the Xpert ® MTB/RIF assay version G4 is high, false positive test results remain possible and have to be considered for the interpretation of the RIF resistance detection by Xpert ® MTB/RIF assay.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

PubMed

Wang, Jianhao; Qin, Yuqin; Qin, Haifang; Liu, Li; Ding, Shumin; Teng, Yiwan; Ji, Junling; Qiu, Lin; Jiang, Pengju

2016-08-01

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new automated multiple allergen simultaneous test-chemiluminescent assay (MAST-CLA) using an AP720S analyzer.

PubMed

Lee, Sungsil; Lim, Hwan Sub; Park, Jungyong; Kim, Hyon Suk

2009-04-01

In the diagnosis of atopic diseases, allergen detection is a crucial step. Multiple allergen simultaneous test-chemiluminescent assay (MAST-CLA) is a simple and noninvasive method for in vitro screening of allergen-specific IgE antibodies. The Korean Inhalant Panel test on 20 patients and Food Panel test on 19 patients were performed using the conventional manual MAST-CLA kit and the new automated MAST-CLA method (automated AP720S system for the Optigen Assay; Hitachi Chemical Diagnostics, Inc., USA) simultaneously. The results were evaluated for positive reactivity and concordance. The results of inhalant panel gave a relatively higher class level result than the food panel. The 8 patients out of 20 (40%) of the inhalation panel, and 9 patients out of 18 (47.4%) of the food panel showed 100% concordance between the 2 systems. Eighteen patients (90%) of the Inhalation Panel and sixteen patients (84.2%) of the Food Panel showed more than 91% concordance. These results suggest that the MAST-CLA assay using the new, automated AP720S analyzer performs well, showing a high concordance rate with conventional MAST-CLA. Compared to manual MAST-CLA, the automated AP720S system has a shorter assay time and uses a smaller serum volume (500 microl) along with other conveniences.

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

USDA-ARS?s Scientific Manuscript database

Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

PubMed Central

Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

2012-01-01

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests. PMID:22539973

Utility of high-resolution accurate MS to eliminate interferences in the bioanalysis of ribavirin and its phosphate metabolites.

PubMed

Wei, Cong; Grace, James E; Zvyaga, Tatyana A; Drexler, Dieter M

2012-08-01

The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5´-triphosphate (RTP), and also possibly through ribavirin 5´-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5´-diphosphate and RMP). In a recombinant human UGT1A1 assay, the assay buffer components uridine and its phosphorylated derivatives are isobaric with RBV and its phosphorylated metabolites, leading to significant interference when analyzed by LC-MS with the nominal mass resolution mode. Presented here is a LC-MS method employing LC coupled with full-scan high-resolution accurate MS analysis for the simultaneous quantitative determination of RBV, RMP, ribavirin 5´-diphosphate and RTP by differentiating RBV and its phosphorylated metabolites from uridine and its phosphorylated derivatives by accurate mass, thus avoiding interference. The developed LC-high-resolution accurate MS method allows for quantitation of RBV and its phosphorylated metabolites, eliminating the interferences from uridine and its phosphorylated derivatives in recombinant human UGT1A1 assays.

Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing

EPA Science Inventory

In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. ...

Development of a multiplex real-time PCR for the simultaneous detection of herpes simplex and varicella zoster viruses in cerebrospinal fluid and lesion swab specimens.

PubMed

Wong, Anita A; Pabbaraju, Kanti; Wong, Sallene; Tellier, Raymond

2016-03-01

Herpes simplex viruses (HSV) and varicella zoster virus (VZV) can have very similar and wide-ranging clinical presentations. Rapid identification is necessary for timely antiviral therapy, especially with infections involving the central nervous system, neonates, and immunocompromised individuals. Detection of HSV-1, HSV-2 and VZV was combined into one real-time PCR reaction utilizing hydrolysis probes. The assay was validated on the LightCycler(®) (Roche) and Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific Inc.) to detect alphaherpesviruses in cerebral spinal fluid (CSF) and lesion swab specimens, respectively. Validation data on blood and tissue samples are also presented. The multiplex assay showed excellent sensitivity, specificity and reproducibility when compared to two singleplex real-time PCR assays for CSF samples and direct fluorescent antigen/culture for lesion swab samples. Implementation of the multiplex assay has facilitated improved sensitivity and accuracy as well as reduced turn-around-times and costs. The results from a large data set of 16,622 prospective samples tested between August 16, 2012 to February 1, 2014 at the Provincial Laboratory for Public Health (Alberta, Canada) are presented here. Copyright © 2015 Elsevier B.V. All rights reserved.

Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.

PubMed

Kilic, Abdullah; Basustaoglu, A Celal

2011-12-01

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min. Copyright © 2011 Institut Pasteur. All rights reserved.

Analytical and clinical performance characteristics of the Abbott RealTime MTB RIF/INH Resistance, an assay for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis in pulmonary specimens.

PubMed

Kostera, Joshua; Leckie, Gregor; Tang, Ning; Lampinen, John; Szostak, Magdalena; Abravaya, Klara; Wang, Hong

2016-12-01

Clinical management of drug-resistant tuberculosis patients continues to present significant challenges to global health. To tackle these challenges, the Abbott RealTime MTB RIF/INH Resistance assay was developed to accelerate the diagnosis of rifampicin and/or isoniazid resistant tuberculosis to within a day. This article summarizes the performance of the Abbott RealTime MTB RIF/INH Resistance assay; including reliability, analytical sensitivity, and clinical sensitivity/specificity as compared to Cepheid GeneXpert MTB/RIF version 1.0 and Hain MTBDRplus version 2.0. The limit of detection (LOD) of the Abbott RealTime MTB RIF/INH Resistance assay was determined to be 32 colony forming units/milliliter (cfu/mL) using the Mycobacterium tuberculosis (MTB) strain H37Rv cell line. For rifampicin resistance detection, the Abbott RealTime MTB RIF/INH Resistance assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Cepheid GeneXpert MTB/RIF. For isoniazid resistance detection, the assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Hain MTBDRplus. The performance data presented herein demonstrate that the Abbott RealTime MTB RIF/INH Resistance assay is a sensitive, robust, and reliable test for realtime simultaneous detection of first line anti-tuberculosis antibiotics rifampicin and isoniazid in patient specimens. Copyright © 2016 The Author. Published by Elsevier Ltd.. All rights reserved.

Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product

PubMed Central

Hassan, Mostafa A.; Zaghary, Wafaa A.

2018-01-01

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm–260.4 nm, amplitudes at 260.4 nm–274.0 nm, and mean-centered values at 287.6 nm–257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5–50 μg/mL and 2.5–25 μg/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD. PMID:29629213

Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product.

PubMed

Mowaka, Shereen; Ayoub, Bassam M; Hassan, Mostafa A; Zaghary, Wafaa A

2018-01-01

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm-260.4 nm, amplitudes at 260.4 nm-274.0 nm, and mean-centered values at 287.6 nm-257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5-50  μ g/mL and 2.5-25  μ g/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD.

Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes.

PubMed

Naserpour Farivar, Taghi; Najafipour, Reza; Johari, Pouran; Aslanimehr, Masoumeh; Peymani, Amir; Jahani Hashemi, Hoasan; Mirzaui, Baman

2014-10-01

We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods. Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Simultaneous point-of-care detection of anemia and sickle cell disease in Tanzania: the RAPID study.

PubMed

Smart, Luke R; Ambrose, Emmanuela E; Raphael, Kevin C; Hokororo, Adolfine; Kamugisha, Erasmus; Tyburski, Erika A; Lam, Wilbur A; Ware, Russell E; McGann, Patrick T

2018-02-01

Both anemia and sickle cell disease (SCD) are highly prevalent across sub-Saharan Africa, and limited resources exist to diagnose these conditions quickly and accurately. The development of simple, inexpensive, and accurate point-of-care (POC) assays represents an important advance for global hematology, one that could facilitate timely and life-saving medical interventions. In this prospective study, Robust Assays for Point-of-care Identification of Disease (RAPID), we simultaneously evaluated a POC immunoassay (Sickle SCAN™) to diagnose SCD and a first-generation POC color-based assay to detect anemia. Performed at Bugando Medical Center in Mwanza, Tanzania, RAPID tested 752 participants (age 1 day to 20 years) in four busy clinical locations. With minimally trained medical staff, the SCD POC assay diagnosed SCD with 98.1% sensitivity and 91.1% specificity. The hemoglobin POC assay had 83.2% sensitivity and 74.5% specificity for detection of severe anemia (Hb ≤ 7 g/dL). Interobserver agreement was excellent for both POC assays (r = 0.95-0.96). Results for the hemoglobin POC assay have informed the second-generation assay design to be more suitable for low-resource settings. RAPID provides practical feasibility data regarding two novel POC assays for the diagnosis of anemia and SCD in real-world field evaluations and documents the utility and potential impact of these POC assays for sub-Saharan Africa.

Diagnostic tools based on minor groove binder probe technology for rapid identification of vaccinal and field strains of canine parvovirus type 2b.

PubMed

Decaro, Nicola; Martella, Vito; Elia, Gabriella; Desario, Costantina; Campolo, Marco; Buonavoglia, Domenico; Bellacicco, Anna Lucia; Tempesta, Maria; Buonavoglia, Canio

2006-12-01

TaqMan-based diagnostic tests have been developed for the identification of canine parvovirus type 2 (CPV-2) strains in the faeces of dogs with diarrhoea, including a minor groove binder (MGB) probe assay for identification of type 2-based vaccines and field strains (types 2a, 2b and 2c). Since type 2b vaccines have been licensed recently in Europe, two novel MGB assays were developed for discrimination between type 2b vaccines and field strains of CPV. Such assays have been found to be highly sensitive, specific and reproducible, allowing for simultaneous detection of type 2b vaccinal and field strains present in the same specimens. These new assays will help resolution of the diagnostic problems related to the detection of a type 2b strain in the faeces of dogs shortly after the administration of a type 2b vaccine.

Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN-γ.

PubMed

Duran, Maria Carolina; Willenbrock, Saskia; Müller, Jessika-M V; Nolte, Ingo; Feige, Karsten; Murua Escobar, Hugo

2013-04-01

Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ). Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples. Cytokine detection ranged from 31.5-5,000 pg/ml and 15-10,000 pg/ml for eIL-12 and eIFN-γ, respectively. eIL-12 was detected in supernatants of stimulated peripheral blood mononuclear cells (PBMCs) and supernatants/cell lysates of eIL-12 expression plasmid-transfected cells. Low or undetectable cytokine concentrations were measured in negative controls. In equine serum samples, the mean measured eIL-12 concentration was 1,374 ± 8 pg/ml. The bead-based assay and ELISA for eIFN-γ used to measure eIFN-γ concentrations, showed similar concentrations. Results demonstrate, to our knowledge for the first time, that cross-reactive antibody pairs to eIL-12 and eIFN-γ and Luminex bead-based technology allow for accurate, simultaneous and multiplexed quantification of these key cytokines in biological samples.

A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.

PubMed

Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S

2013-10-01

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

PubMed Central

Kumar, Sunil; Alibhai, Dominic; Margineanu, Anca; Laine, Romain; Kennedy, Gordon; McGinty, James; Warren, Sean; Kelly, Douglas; Alexandrov, Yuriy; Munro, Ian; Talbot, Clifford; Stuckey, Daniel W; Kimberly, Christopher; Viellerobe, Bertrand; Lacombe, Francois; Lam, Eric W-F; Taylor, Harriet; Dallman, Margaret J; Stamp, Gordon; Murray, Edward J; Stuhmeier, Frank; Sardini, Alessandro; Katan, Matilda; Elson, Daniel S; Neil, Mark A A; Dunsby, Chris; French, Paul M W

2011-01-01

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated. PMID:21337485

Detection of Orthopoxvirus DNA by Real-Time PCR and Identification of Variola Virus DNA by Melting Analysis

PubMed Central

Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

2004-01-01

Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification. PMID:15004077

Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

PubMed

Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

2014-03-26

A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new facility for non-destructive assay using a 252Cf source.

PubMed

Stevanato, L; Caldogno, M; Dima, R; Fabris, D; Hao, Xin; Lunardon, M; Moretto, S; Nebbia, G; Pesente, S; Pino, F; Sajo-Bohus, L; Viesti, G

2013-03-01

A new laboratory facility for non-destructive analysis (NDA) using a time-tagged (252)Cf source is presented. The system is designed to analyze samples having maximum size of about 20 × 25 cm(2), the material recognition being obtained by measuring simultaneously total and energy dependent transmission of neutrons and gamma rays. The equipment technical characteristics and performances of the NDA system are presented, exploring also limits due to the sample thickness. Some recent applications in the field of cultural heritage are presented. Copyright © 2012 Elsevier Ltd. All rights reserved.

Epidemiology of Rifampicin Resistant Tuberculosis and Common Mutations in rpoB Gene of Mycobacterium tuberculosis: A Retrospective Study from Six Districts of Punjab (India) Using Xpert MTB/RIF Assay.

PubMed

Kaur, Ramandeep; Jindal, Neerja; Arora, Shilpa; Kataria, Shajla

2016-01-01

Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in <2 h. The RIF resistance pattern in Malwa region of Punjab, India, is not documented. Here, we report the epidemiology of RIF-resistant TB and mutations in rpoB gene of Mycobacterium tuberculosis (MTB). A total of 1612 specimens received between October 2013 and February 2015 were tested by Xpert MTB/RIF assay following manufacturer's instructions. The results thus obtained were analyzed using SPSS version 20.0.0 (SPSS Inc., Chicago, IL, USA) statistical software. RIF resistance was statistically higher in previously treated patients in comparison to the new patients (P = 0.006) and in patients with acid fast-Bacilli (AFB) positive smears to AFB-negative smears (P = 0.048). RIF resistance mutations in 130 specimens revealed frequency of E 73/130 (56%), B 28/130 (21.5%), D 18/130 (13.8%), A 11/130 (8.4%), and C 1/130 (0.7%) while in one specimen, mutation combination, i.e., mutations associated with more than one probe (A and B both) was present. Xpert MTB/RIF assay is a user-friendly screening tool for detection of MTB and RIF resistance from suspected TB/MDR cases in a shorter period of time. It could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of RIF resistance in MTB isolates.

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

PubMed

Paoletta, Martina Soledad; López Arias, Ludmila; de la Fournière, Sofía; Guillemi, Eliana Carolina; Luciani, Carlos; Sarmiento, Néstor Fabián; Mosqueda, Juan; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-02-01

Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

Continuously tunable nucleic acid hybridization probes.

PubMed

Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

2015-12-01

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Development and Validation of a Simultaneous RP-HPLCUV/DAD Method for Determination of Polyphenols in Gels Containing S. terebinthifolius Raddi (Anacardiaceae)

PubMed Central

Carvalho, Melina G.; Aragão, Cícero F. S; Raffin, Fernanda N.; de L. Moura, Túlio F. A.

2017-01-01

Topical gels containing extracts of Schinus terebinthifolius have been used to treat bacterial vaginosis. It has been reported that this species has antimicrobial, anti-inflammatory and anti-ulcerogenic properties, which can be attributed to the presence of phenolic compounds. In this work, a sensitive and selective reversed-phase HPLC-UV/DAD method for the simultaneous assay of six polyphenols that could be present in S. terebinthifolius was developed. The method was shown to be accurate and precise. Peak purity and similarity index both exceeded 0.99. Calibration curves were linear over the concentration range studied, with correlation coefficients between 0.9931 and 0.9974. This method was used to determine the polyphenol content of a hydroalcoholic extract and pharmacy-compounded vaginal gel. Although the method is useful to assess the 6 phenolic compounds, some compounds could not be detected in the products. SUMMARY A sensitive, selective, accurate and precise reversed-phase HPLC-UV/DAD method for the simultaneous assay of six polyphenols in S. terebinthifolius Raddi Abbreviations used: RP-HPLC-UV/DAD: Reverse Phase High Performance Liquid Chromatograph with Ultraviolet and Diode Array Detector, HPLC: High Performance Liquid Chromatograph, HPLC-UV: High Performance Liquid Chromatograph with Ultraviolet Detector, ANVISA: Brazilian National Health Surveillance Agency, LOD: Limit of detection, LOQ: Limit of quantitation PMID:28539726

The Current Incidence of Viral Disease in Korean Sweet Potatoes and Development of Multiplex RT-PCR Assays for Simultaneous Detection of Eight Sweet Potato Viruses

PubMed Central

Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

2014-01-01

Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded. PMID:25506306

Simultaneous real-time PCR detection of Fusarium asiaticum, F. ussurianum and F. vorosii, representing the Asian clade of the F. graminearum species complex.

PubMed

Fernández-Ortuño, Dolores; Waalwijk, Cees; Van der Lee, Theo; Fan, Jieru; Atkins, Sarah; West, Jonathan S; Fraaije, Bart A

2013-08-16

Due to the repeated discovery of new members of the Fusarium graminearum species complex (FGSC), some of the F. graminearum sensu stricto (s.s.)-specific qPCR assays developed to date have since been shown to be non-specific. In this study, a probe-based qPCR method was developed, targeting a sterol 14-alpha demethylase (CYP51) paralogue, CYP51C unique to the genus Fusarium, for the simultaneous detection of F. asiaticum, F. ussurianum and F. vorosii. Specificity of the assay was demonstrated for a wide range of Fusarium species, including all tested FGSC members (n=6), originating from different hosts and geographic regions. Alongside a previously published assay for detection of F. graminearum, we were able to show that members of the Asian clade of the FGSC (i.e. F. asiaticum, F. ussurianum and F. vorosii) were the primary etiological agent in wheat seeds samples originating from Central-East China. The grain samples from the UK tested negative for the presence of the FGSC's Asian clade and positive for presence of F. graminearum. It is likely that only F. graminearum s.s. is present in the UK, but the presence of other FGSC members cannot be ruled out and need further investigation. Copyright © 2013 Elsevier B.V. All rights reserved.

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

PubMed

Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

PubMed

Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

2016-05-01

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

PubMed Central

Aouri, Manel; Moradpour, Darius; Cavassini, Matthias; Mercier, Thomas; Buclin, Thierry; Csajka, Chantal; Telenti, Amalio; Rauch, Andri

2013-01-01

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy. PMID:23629707

Rapid LC-MRM-MS assay for simultaneous quantification of choline, betaine, trimethylamine, trimethylamine N-oxide, and creatinine in human plasma and urine.

PubMed

Zhao, Xueqing; Zeisel, Steven H; Zhang, Shucha

2015-06-17

There is a growing interest in analyzing choline, betaine, and their gut microbial metabolites including trimethylamine (TMA) and trimethylamine N-oxide (TMAO) in body fluids due to the high relevance of these compounds for human health and diseases. A stable isotope dilution (SID)-LC-MRM-MS assay was developed for the simultaneous determination of choline, betaine, TMA, TMAO, and creatinine in human plasma and urine. The assay was validated using quality control (QC) plasma samples, spiked at low, medium, and high levels. Freeze-thaw stability was also evaluated. The utility of this assay for urine was demonstrated using a nutritional clinical study on the effect of various egg doses on TMAO production in humans. This assay has a wide dynamic range (R 2 > 0.994) for all the analytes (choline: 0.122-250 μM; betaine: 0.488-1000 μM; TMA: 0.244-250 μM; TMAO: 0.061-62.5 μM; and creatinine: 0.977-2000 μM). High intra- and inter-day precision (CV < 6%) and high accuracy (< 15% error) were observed from the QC plasma samples. The assay is reliable for samples undergoing multiple freeze-thaw cycles (tested up to eight cycles). The assay also works for urine samples as demonstrated by a clinical study in which we observed a significant, positive linear response to various egg doses for urinary concentrations of all the analytes except creatinine. A rapid SID-LC-MRM-MS assay for simultaneous quantification of choline, betaine, TMA, TMAO, and creatinine has been developed and validated, and is expected to find wide application in nutrition and cardiovascular studies as well as diagnosis and management of trimethylaminuria. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses

PubMed Central

Wan, Zhenzhou; Zhang, Ya’nan; He, Zhixiang; Liu, Jia; Lan, Ke; Hu, Yihong; Zhang, Chiyu

2016-01-01

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 102 copies for HCoV-OC43, and 3 × 101 copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples. PMID:27886052

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses.

PubMed

Wan, Zhenzhou; Zhang, Ya'nan; He, Zhixiang; Liu, Jia; Lan, Ke; Hu, Yihong; Zhang, Chiyu

2016-11-23

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10² copies for HCoV-OC43, and 3 × 10¹ copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen

PubMed Central

van der Wal, Fimme J.; Achterberg, René P.; van Solt-Smits, Conny; Bergervoet, Jan H. W.; de Weerdt, Marjanne; Wisselink, Henk J.

2017-01-01

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population. PMID:28980519

Simultaneous assay of multiple antibiotics in human plasma by LC-MS/MS: importance of optimizing formic acid concentration.

PubMed

Chen, Feng; Hu, Zhe-Yi; Laizure, S Casey; Hudson, Joanna Q

2017-03-01

Optimal dosing of antibiotics in critically ill patients is complicated by the development of resistant organisms requiring treatment with multiple antibiotics and alterations in systemic exposure due to diseases and extracorporeal drug removal. Developing guidelines for optimal antibiotic dosing is an important therapeutic goal requiring robust analytical methods to simultaneously measure multiple antibiotics. An LC-MS/MS assay using protein precipitation for cleanup followed by a 6-min gradient separation was developed to simultaneously determine five antibiotics in human plasma. The precision and accuracy were within the 15% acceptance range. The formic acid concentration was an important determinant of signal intensity, peak shape and matrix effects. The method was designed to be simple and successfully applied to a clinical pharmacokinetic study.

Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use.

PubMed

Kim, Mi-Ju; Kim, Hae-Yeong

2018-04-25

A multiple loop-mediated isothermal amplification (LAMP) method was developed to detect cow and goat milk in the field using a portable fluorescence device. For rapid on-site detection, this duplex LAMP assay was used in combination with direct amplification, without DNA extraction. The cow- and goat-specific LAMP primer sets were designed based on the mitochondrial cytochrome b gene, and showed specificity against 13 other animal species in the reactions. The sensitivity of the duplex LAMP assay for cow and goat was 0.1 and 1 pg, respectively. The detection limit for both target species in milk mixtures was 2%. This assay successfully amplified and identified the two target species in 24 samples of commercial milk and yogurt products, with 30 min sampling-to-result analysis time. Therefore, this direct duplex real-time LAMP assay is useful for on-site simultaneous detection of cow and goat milk in commercial products, a capability needed to confirm accurate labeling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens.

PubMed

Cheng, Fang; Wu, Jiajie; Zhang, Jin; Pan, Aihu; Quan, Sheng; Zhang, Dabing; Kim, HaeYeong; Li, Xiang; Zhou, Shan; Yang, Litao

2016-05-15

Food allergies cause health risks to susceptible consumers and regulations on labeling of food allergen contents have been implemented in many countries and regions. To achieve timely and accurate food allergen labeling, the development of fast and effective allergen detection methods is very important. Herein, a decaplex polymerase chain reaction (PCR) assay combined with capillary electrophoresis was developed to detect simultaneously 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan. The absolute limit of detection (LODa) of this system is between 2 and 20 copies of haploid genome, and the relative LOD (LODr) is as low as 0.005% (w/w) in simulated food mixtures. The developed assay was subsequently applied to 20 commercial food products and verified the allergen ingredients stated on the labels. Furthermore, results using this decaplex PCR assay was successfully replicated in three other laboratories, demonstrating the repeatability and applicability of this assay in routine analysis of the 10 food allergens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays

PubMed Central

Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

2014-01-01

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins. PMID:24633798

Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

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

Cary,; Bruce, R; Stubben, Christopher J

The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Klein, H. P.; Jahnke, L.

1979-01-01

Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.

PubMed

Wang, Yi; Li, Dongxun; Wang, Yan; Li, Kewei; Ye, Changyun

2016-01-19

Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.

Multiple reaction monitoring assay based on conventional liquid chromatography and electrospray ionization for simultaneous monitoring of multiple cerebrospinal fluid biomarker candidates for Alzheimer's disease

PubMed Central

Choi1, Yong Seok; Lee, Kelvin H.

2016-01-01

Alzheimer's disease (AD) is the most common type of dementia, but early and accurate diagnosis remains challenging. Previously, a panel of cerebrospinal fluid (CSF) biomarker candidates distinguishing AD and non-AD CSF accurately (> 90%) was reported. Furthermore, a multiple reaction monitoring (MRM) assay based on nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) was developed to help validate putative AD CSF biomarker candidates including proteins from the panel. Despite the good performance of the MRM assay, wide acceptance may be challenging because of limited availability of nLC-MS/MS systems laboratories. Thus, here, a new MRM assay based on conventional LC-MS/MS is presented. This method monitors 16 peptides representing 16 (of 23) biomarker candidates that belonged to the previous AD CSF panel. A 30-times more concentrated sample than the sample used for the previous study was loaded onto a high capacity trap column, and all 16 MRM transitions showed good linearity (average R2 = 0.966), intra-day reproducibility (average coefficient of variance (CV) = 4.78%), and inter-day reproducibility (average CV = 9.85%). The present method has several advantages such as a shorter analysis time, no possibility of target variability, and no need for an internal standard. PMID:26404792

Multiple reaction monitoring assay based on conventional liquid chromatography and electrospray ionization for simultaneous monitoring of multiple cerebrospinal fluid biomarker candidates for Alzheimer's disease.

PubMed

Choi, Yong Seok; Lee, Kelvin H

2016-03-01

Alzheimer's disease (AD) is the most common type of dementia, but early and accurate diagnosis remains challenging. Previously, a panel of cerebrospinal fluid (CSF) biomarker candidates distinguishing AD and non-AD CSF accurately (>90 %) was reported. Furthermore, a multiple reaction monitoring (MRM) assay based on nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) was developed to help validate putative AD CSF biomarker candidates including proteins from the panel. Despite the good performance of the MRM assay, wide acceptance may be challenging because of limited availability of nLC-MS/MS systems in laboratories. Thus, here, a new MRM assay based on conventional LC-MS/MS is presented. This method monitors 16 peptides representing 16 (of 23) biomarker candidates that belonged to the previous AD CSF panel. A 30-times more concentrated sample than the sample used for the previous study was loaded onto a high capacity trap column, and all 16 MRM transitions showed good linearity (average R(2) = 0.966), intra-day reproducibility (average coefficient of variance (CV) = 4.78 %), and inter-day reproducibility (average CV = 9.85 %). The present method has several advantages such as a shorter analysis time, no possibility of target variability, and no need for an internal standard.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis.

PubMed

Sulyok, Kinga M; Bekő, Katinka; Kreizinger, Zsuzsa; Wehmann, Enikő; Jerzsele, Ákos; Rónai, Zsuzsanna; Turcsányi, Ibolya; Makrai, László; Szeredi, Levente; Jánosi, Szilárd; Nagy, Sára Ágnes; Gyuranecz, Miklós

2018-01-01

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis. The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 10 2 -10 5 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3-4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping. In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

PubMed Central

Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

2015-01-01

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

PubMed

Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

2015-01-01

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

Simultaneous determination of five systemic azoles in plasma by high-performance liquid chromatography with ultraviolet detection.

PubMed

Gordien, Jean-Baptiste; Pigneux, Arnaud; Vigouroux, Stephane; Tabrizi, Reza; Accoceberry, Isabelle; Bernadou, Jean-Marc; Rouault, Audrey; Saux, Marie-Claude; Breilh, Dominique

2009-12-05

A simple, specific and automatable HPLC assay was developed for a simultaneous determination of systemic azoles (fluconazole, posaconazole, voriconazole, itraconazole and its metabolite hydroxyl-itraconazole, and ketoconazole) in plasma. The major advantage of this assay was sample preparation by a fully automatable solid phase extraction with Varian Plexa cartridges. C6-phenyl column was used for chromatographic separation, and UV detection was set at a wavelength of 260 nm. Linezolid was used as an internal standard. The assay was specific and linear over the concentration range of 0.05 to 40 microg/ml excepted for fluconazole which was between 0.05 and 100 microg/ml, and itraconazole between 0.1 and 40 microg/ml. Validation data for accuracy and precision for intra- and inter-day were good and satisfied FDA's guidance: CV between 0.24% and 11.66% and accuracy between 93.8% and 108.7% for all molecules. This assay was applied to therapeutic drug monitoring on patients hospitalized in intensive care and onco-hematologic units.

Simultaneous assessment of iodine, iron, vitamin A, malarial antigenemia, and inflammation status biomarkers via a multiplex immunoassay method on a population of pregnant women from Niger.

PubMed

Brindle, Eleanor; Lillis, Lorraine; Barney, Rebecca; Hess, Sonja Y; Wessells, K Ryan; Ouédraogo, Césaire T; Stinca, Sara; Kalnoky, Michael; Peck, Roger; Tyler, Abby; Lyman, Christopher; Boyle, David S

2017-01-01

Deficiencies of vitamin A, iron, and iodine are major public health concerns in many low- and middle-income countries, but information on their status in populations is often lacking due to high costs and logistical challenges associated with assessing micronutrient status. Accurate, user-friendly, and low-cost analytical tools are needed to allow large-scale population surveys on micronutrient status. We present the expansion of a 7-plex protein microarray tool for the simultaneous measurement of up to seven biomarkers with relevance to the assessment of the key micronutrients iron, iodine, and vitamin A, and inflammation and malaria biomarkers: α-1-acid glycoprotein, C-reactive protein, ferritin, retinol binding protein 4, soluble transferrin receptor, thyroglobulin, and histidine-rich protein II. Assay performance was assessed using international reference standards and then verified by comparing the multiplexed and conventional immunoassay results on a training panel of plasma samples collected from US adults. These data were used to assign nominal concentrations to the calibrators of the assay to further improve performance which was then assessed by interrogating plasma samples from a cohort of pregnant women from Niger. The correlation between assays for each biomarker measured from this cohort was typically good, with the exception of thyroglobulin, and the sensitivity ranged from 74% to 93%, and specificity from 81% to 98%. The 7-Plex micronutrient assay has the potential for use as an affordable tool for population surveillance of vitamin A, iron, and iodine deficiencies as well as falciparum malarial parasitemia infectivity and inflammation. The assay is easy-to-use, requires minimal sample volume, and is scalable, rapid, and accurate-needing only a low-cost reader and basic equipment present in most reference laboratory settings and so may be employed by low and middle income countries for micronutrient surveillance to inform on status in key populations. Micronutrient deficiencies including iron, iodine, and vitamin A affect a significant portion of the world's population. Efforts to assess the prevalence of these deficiencies in vulnerable populations are challenging, partly due to measurement tools that are inadequate for assessing multiple micronutrients in large-scale population surveys. We have developed a 7-plex immunoassay for the simultaneous measurement of seven biomarkers relevant to assessing iodine, iron, and vitamin A status, inflammation and Plasmodium falciparum parasitemia by measuring levels of thyroglobulin, ferritin, soluble transferrin receptor, retinol binding protein 4, α-1-acid glycoprotein, C-reactive protein, and histidine-rich protein II. This 7-plex immunoassay technique has potential as a rapid and effective tool for use in large-scale surveys and assessments of nutrition intervention programs in low- and middle-income countries.

Application of Bioassays for the Ecotoxicity Assessment of Contaminated Soils

NASA Astrophysics Data System (ADS)

Fernández, María D.; Babín, Mar; Tarazona, José V.

The use of bioassays for soil characterization is receiving significant attention as a complementary tool to chemical analysis. Bioassays consist of direct toxicity assays of environmental samples that are transferred to the laboratory and analyzed for toxicity against selected organisms. Such soil samples contain the combination of the different pollutants present in situ and enable factors such as the bioavailability of contaminants or the interactions (synergic and antagonic) between them to be simultaneously studied.

Sandwich ELISA Microarrays: Generating Reliable and Reproducible Assays for High-Throughput Screens

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

Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

The sandwich ELISA microarray is a powerful screening tool in biomarker discovery and validation due to its ability to simultaneously probe for multiple proteins in a miniaturized assay. The technical challenges of generating and processing the arrays are numerous. However, careful attention to possible pitfalls in the development of your antibody microarray assay can overcome these challenges. In this chapter, we describe in detail the steps that are involved in generating a reliable and reproducible sandwich ELISA microarray assay.

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

PubMed

Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

2017-02-14

In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

Performance of transport and selective media for swine Bordetella bronchiseptica recovery and it comparison to polymerase chain reaction detection

PubMed Central

Coutinho, Tania Alen; Bernardi, Mari Lourdes; de Itapema Cardoso, Marisa Ribeiro; Borowski, Sandra Maria; Moreno, Andrea Micke; de Barcellos, David Emilio Santos Neves

2009-01-01

Three comparative assays were performed seeking to improve the sensitivity of the diagnosis of Bordetella bronchiseptica infection analyzing swine nasal swabs. An initial assay compared the recovery of B. bronchiseptica from swabs simultaneously inoculated with B. bronchiseptica and some interfering bacteria, immersed into three transport formulations (Amies with charcoal, trypticase soy broth and phosphate buffer according to Soerensen supplemented with 5% of bovine fetal serum) and submitted to different temperatures (10°C and 27°C) and periods of incubation (24, 72 and 120 hours). A subsequent assay compared three selective media (MacConkey agar, modified selective medium G20G and a ceftiofur medium) for their recovery capabilities from clinical specimens. One last assay compared the polymerase chain reaction to the three selective media. In the first assay, the recovery of B. bronchiseptica from transport systems was better at 27°C and the three formulations had good performances at this temperature, but the collection of qualitative and quantitative analysis indicated the advantage of Amies medium for nasal swabs transportation. The second assay indicated that MacConkey agar and modified G20G had similar results and were superior to the ceftiofur medium. In the final assay, polymerase chain reaction presented superior capability of B. bronchiseptica detection to culture procedures. PMID:24031390

Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.

PubMed

Jakob, Thilo; Forstenlechner, Peter; Matricardi, Paolo; Kleine-Tebbe, Jörg

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient's medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization. Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described. The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

Detection and measurement of surface contamination by multiple antineoplastic drugs using multiplex bead assay

PubMed Central

Smith, Jerome P; Sammons, Deborah L; Robertson, Shirley A; Pretty, Jack; Debord, D Gayle; Connor, Thomas H; Snawder, John

2015-01-01

Objectives Contamination of workplace surfaces by antineoplastic drugs presents an exposure risk for healthcare workers. Traditional instrumental methods to detect contamination such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) are sensitive and accurate but expensive. Since immunochemical methods may be cheaper and faster than instrumental methods, we wanted to explore their use for routine drug residue detection for preventing worker exposure. Methods In this study we examined the feasibility of using fluorescence covalent microbead immunosorbent assay (FCMIA) for simultaneous detection and semi-quantitative measurement of three antineoplastic drugs (5-fluorouracil, paclitaxel, and doxorubicin). The concentration ranges for the assay were 0–1000 ng/ml for 5-fluorouracil, 0–100 ng/ml for paclitaxel, and 0–2 ng/ml for doxorubicin. The surface sampling technique involved wiping a loaded surface with a swab wetted with wash buffer, extracting the swab in storage/blocking buffer, and measuring drugs in the extract using FCMIA. Results There was no significant cross reactivity between these drugs at the ranges studied indicated by a lack of response in the assay to cross analytes. The limit of detection (LOD) for 5-fluorouracil on the surface studied was 0.93 ng/cm2 with a limit of quantitation (LOQ) of 2.8 ng/cm2, the LOD for paclitaxel was 0.57 ng/cm2 with an LOQ of 2.06 ng/cm2, and the LOD for doxorubicin was 0.0036 ng/cm2 with an LOQ of 0.013 ng/cm2. Conclusion The use of FCMIA with a simple sampling technique has potential for low cost simultaneous detection and semi-quantitative measurement of surface contamination from multiple antineoplastic drugs. PMID:25293722

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

PubMed Central

Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

2013-01-01

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula. PMID:23825624

Rapid detection and simultaneous genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula using real-time PCR and high resolution melting (HRM) analysis.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

2013-01-01

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10(2) CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.

Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers.

PubMed

Okolie, Charles E; Wooldridge, Karl G; Turner, David P; Cockayne, Alan; James, Richard

2015-06-01

Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n = 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n = 43) containing S. aureus and CoNS in mono-microbial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 × 10(1) to 3.0 × 10(1) colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

PubMed

Gomgnimbou, Michel Kiréopori; Abadia, Edgar; Zhang, Jian; Refrégier, Guislaine; Panaiotov, Stefan; Bachiyska, Elizabeta; Sola, Christophe

2012-10-01

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Independent assessment of candidate HIV incidence assays on specimens in the CEPHIA repository

PubMed Central

Kassanjee, Reshma; Pilcher, Christopher D.; Keating, Sheila M.; Facente, Shelley N.; McKinney, Elaine; Price, Matthew A.; Martin, Jeffrey N.; Little, Susan; Hecht, Frederick M.; Kallas, Esper G.; Welte, Alex; Busch, Michael P.; Murphy, Gary

2014-01-01

Objective: Cross-sectional HIV incidence surveillance, using assays that distinguish ‘recent’ from ‘nonrecent’ infections, has been hampered by inadequate performance and characterization of incidence assays. In this study, the Consortium for the Evaluation and Performance of HIV Incidence Assays presents results of the first independent evaluation of five incidence assays (BED, Limiting Antigen Avidity, Less-sensitive Vitros, Vitros Avidity and BioRad Avidity). Design: A large repository of diverse specimens from HIV-positive patients was established, multiple assays were run on 2500 selected specimens, and data were analyzed to estimate assay characteristics relevant for incidence surveillance. Methods: The mean duration of recent infection (MDRI, average time ‘recent’ while infected for less than some time cut-off T) was estimated from longitudinal data on seroconverters by regression. The false-recent rate (FRR, probability of testing ‘recent’ when infected for longer than T) was explored by measuring the proportions of ‘recent’ results in various subsets of patients. Results: Assays continue to fail to attain the simultaneously large MDRI and small FRR demanded by existing performance guidelines. All assays produce high FRRs amongst virally suppressed patients (>40%), including elite controllers and treated patients. Conclusions: Results from this first independent evaluation provide valuable information about the current performance of assays, and suggest the need for further optimization. Variation of ‘recent’/‘nonrecent’ thresholds and the use of multiple antibody-maturation assays, as well as other biomarkers, can now be explored, using the rich data generated by the Consortium for the Evaluation and Performance of HIV Incidence Assays. Consistently high FRRs amongst those virally suppressed suggest that viral load will be a particularly valuable supplementary marker. Video abstract: PMID:25144218

An automated sequential injection analysis system for the determination of trace endotoxin levels in water.

PubMed

Samanta, Gautam; Zhang, Shuming; Dasgupta, Purnendu

2003-01-01

A new automated instrument based on the Limulus amebocyte lysate (LAL)-chromogenic substrate kinetic assay for the determination of bacterial endotoxins is reported. A computer controlled syringe pump-multiport valve combination was used to aspirate the sample and other reagents into a holding coil. The syringe was always filled with air; liquid did not enter the syringe. The valve could address up to four individual radial paths, fully referenced optical fiber LED-based absorbance detectors that were housed collectively in a single, small (20 x 20 x 30 mm) metal block with a heater and temperature sensor, and maintained at 37 degrees C. Assay mixtures containing sample or standards were delivered to the individual detector cells for the simultaneous collection of the time based absorbance data. The automated system determined endotoxins with good accuracy and reproducibility in the range of 0.005-0.5 endotoxin units (EU)/mL (r2 > or = 0.99). Based on three times the standard deviation of the blank and the slope of the calibration curve, the lower limit of detection was < or = 0.003 EU/mL. The variability of the assay method is less than 5% (n = 10). Analysis time required for a 0.005 EU/mL standard was <100 min. Appropriately diluted tap water samples were simultaneously analyzed by the present system and a manually loaded commercial microplate reader based instrument; the data were statistically indistinguishable at the 95% confidence level.

Polymer-Based Dense Fluidic Networks for High Throughput Screening with Ultrasensitive Fluorescence Detection

PubMed Central

Okagbare, Paul I.; Soper, Steven A.

2011-01-01

Microfluidics represents a viable platform for performing High Throughput Screening (HTS) due to its ability to automate fluid handling and generate fluidic networks with high number densities over small footprints appropriate for the simultaneous optical interrogation of many screening assays. While most HTS campaigns depend on fluorescence, readers typically use point detection and serially address the assay results significantly lowering throughput or detection sensitivity due to a low duty cycle. To address this challenge, we present here the fabrication of a high density microfluidic network packed into the imaging area of a large field-of-view (FoV) ultrasensitive fluorescence detection system. The fluidic channels were 1, 5 or 10 μm (width), 1 μm (depth) with a pitch of 1–10 μm and each fluidic processor was individually addressable. The fluidic chip was produced from a molding tool using hot embossing and thermal fusion bonding to enclose the fluidic channels. A 40X microscope objective (numerical aperture = 0.75) created a FoV of 200 μm, providing the ability to interrogate ~25 channels using the current fluidic configuration. An ultrasensitive fluorescence detection system with a large FoV was used to transduce fluorescence signals simultaneously from each fluidic processor onto the active area of an electron multiplying charge-coupled device (EMCCD). The utility of these multichannel networks for HTS was demonstrated by carrying out the high throughput monitoring of the activity of an enzyme, APE1, used as a model screening assay. PMID:20872611

Clinical evaluation of a helicase-dependant amplification (HDA)-based commercial assay for the simultaneous detection of HSV-1 and HSV-2.

PubMed

Teo, Jeanette W P; Chiang, Donald; Jureen, Roland; Lin, Raymond T P

2015-11-01

In this study, we evaluate the performance of a commercial assay, the AmpliVue HSV 1+2 Assay (Quidel), which employs HDA for the detection of both HSV-1 and HSV-2. The assay was tested on 307 clinical specimens (genital, oral, and dermal). When compared to shell vial virus culture and immunofluorescence typing of HSV, the positive percent agreement and negative percent agreement values were 98.2% and 90.9%, respectively. Excellent assay performance was demonstrated. Copyright © 2015 Elsevier Inc. All rights reserved.

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

Simultaneous Delivery of Highly Diverse Bioactive Compounds from Blend Electrospun Fibers for Skin Wound Healing.

PubMed

Peh, Priscilla; Lim, Natalie Sheng Jie; Blocki, Anna; Chee, Stella Min Ling; Park, Heyjin Chris; Liao, Susan; Chan, Casey; Raghunath, Michael

2015-07-15

Blend emulsion electrospinning is widely perceived to destroy the bioactivity of proteins, and a blend emulsion of water-soluble and nonsoluble molecules is believed to be thermodynamically unstable to electrospin smoothly. Here we demonstrate a method to retain the bioactivity of disparate fragile biomolecules when electrospun. Using bovine serum albumin as a carrier protein; water-soluble vitamin C, fat soluble vitamin D3, steroid hormone hydrocortisone, peptide hormone insulin, thyroid hormone triiodothyronine (T3), and peptide epidermal growth factor (EGF) were simultaneously blend-spun into PLGA-collagen nanofibers. Upon release, vitamin C maintained the ability to facilitate Type I collagen secretion by fibroblasts, EGF stimulated skin fibroblast proliferation, and insulin potentiated adipogenic differentiation. Transgenic cell reporter assays confirmed the bioactivity of vitamin D3, T3, and hydrocortisone. These factors concertedly increased keratinocyte and fibroblast proliferation while maintaining keratinocyte basal state. This method presents an elegant solution to simultaneously deliver disparate bioactive biomolecules for wound healing applications.

Automating quantum dot barcode assays using microfluidics and magnetism for the development of a point-of-care device.

PubMed

Gao, Yali; Lam, Albert W Y; Chan, Warren C W

2013-04-24

The impact of detecting multiple infectious diseases simultaneously at point-of-care with good sensitivity, specificity, and reproducibility would be enormous for containing the spread of diseases in both resource-limited and rich countries. Many barcoding technologies have been introduced for addressing this need as barcodes can be applied to detecting thousands of genetic and protein biomarkers simultaneously. However, the assay process is not automated and is tedious and requires skilled technicians. Barcoding technology is currently limited to use in resource-rich settings. Here we used magnetism and microfluidics technology to automate the multiple steps in a quantum dot barcode assay. The quantum dot-barcoded microbeads are sequentially (a) introduced into the chip, (b) magnetically moved to a stream containing target molecules, (c) moved back to the original stream containing secondary probes, (d) washed, and (e) finally aligned for detection. The assay requires 20 min, has a limit of detection of 1.2 nM, and can detect genetic targets for HIV, hepatitis B, and syphilis. This study provides a simple strategy to automate the entire barcode assay process and moves barcoding technologies one step closer to point-of-care applications.

xMAP Technology: Applications in Detection of Pathogens

PubMed Central

Reslova, Nikol; Michna, Veronika; Kasny, Martin; Mikel, Pavel; Kralik, Petr

2017-01-01

xMAP technology is applicable for high-throughput, multiplex and simultaneous detection of different analytes within a single complex sample. xMAP multiplex assays are currently available in various nucleic acid and immunoassay formats, enabling simultaneous detection and typing of pathogenic viruses, bacteria, parasites and fungi and also antigen or antibody interception. As an open architecture platform, the xMAP technology is beneficial to end users and therefore it is used in various pharmaceutical, clinical and research laboratories. The main aim of this review is to summarize the latest findings and applications in the field of pathogen detection using microsphere-based multiplex assays. PMID:28179899

Prospects and challenges of quantitative phase imaging in tumor cell biology

NASA Astrophysics Data System (ADS)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Portable Biomarker Detection with Magnetic Nanotags

PubMed Central

Hall, Drew A.; Wang, Shan X.; Murmann, Boris; Gaster, Richard S.

2012-01-01

This paper presents a hand-held, portable biosensor platform for quantitative biomarker measurement. By combining magnetic nanoparticle (MNP) tags with giant magnetoresistive (GMR) spin-valve sensors, the hand-held platform achieves highly sensitive (picomolar) and specific biomarker detection in less than 20 minutes. The rapid analysis and potential low cost make this technology ideal for point-of-care (POC) diagnostics. Furthermore, this platform is able to detect multiple biomarkers simultaneously in a single assay, creating a promising diagnostic tool for a vast number of applications. PMID:22495252

A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

PubMed Central

Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

2015-01-01

We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

Continuously Tunable Nucleic Acid Hybridization Probes

PubMed Central

Wu, Lucia R.; Wang, J. Sherry; Fang, John Z.; Reiser, Emily; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J.; Beechem, Joseph; Zhang, David Yu

2015-01-01

In silico designed nucleic acid probes and primers often fail to achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. Here, we present a novel, on-the-fly method of tuning probe affinity and selectivity via the stoichiometry of auxiliary species, allowing independent and decoupled adjustment of hybridization yield for different probes in multiplexed assays. Using this method, we achieve near-continuous tuning of probe effective free energy (0.03 kcal·mol−1 granularity). As applications, we enforced uniform capture efficiency of 31 DNA molecules (GC content 0% – 100%), maximized signal difference for 11 pairs of single nucleotide variants, and performed tunable hybrid-capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples (FFPE). PMID:26480474

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

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

Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang

2010-01-22

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potentialmore » entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.« less

Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

PubMed

Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

2013-12-01

A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

PubMed Central

Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

2016-01-01

We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

Estimating rates of denitrification enzyme activity in wetland soils and direct simultaneous quantification of nitrogen and nitrous oxide by membrane inlet mass spectrometry

EPA Science Inventory

Denitrification enzyme activity (DEA) was measured in short-term (4 h) anaerobic assays using Membrane Inlet Mass Spectrometry (MIMS) and electron capture gas chromatography (GC-ECD). Using MIMS, modifications of the instrument and sample handling allowed for the simultaneous me...

Development of a High Throughput Assay for Rapid and Accurate 10-Plex Detection of Citrus Pathogens

USDA-ARS?s Scientific Manuscript database

The need to reliably detect and identify multiple plant pathogens simultaneously, especially in woody perennial hosts, has led to development of new molecular diagnostic approaches. In this study, a Luminex-based system was developed that provided a robust and sensitive test for simultaneous detect...

Simultaneous detection of multiple chemical residues in milk using broad-specifity antibodies in a hybrid immunosorbent assay

USDA-ARS?s Scientific Manuscript database

The wide array of applications using quantum dots (QDs) for detection of multiple analytes reflects the versatility of the technology. In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different low-molecular...

Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

PubMed

Yang, Yu; Wang, Jing; Wen, Haiyan; Liu, Hengchuan

2012-01-01

We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

PubMed

Cross, Kristen E; Mercante, Jeffrey W; Benitez, Alvaro J; Brown, Ellen W; Diaz, Maureen H; Winchell, Jonas M

2016-07-01

Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease. Published by Elsevier Inc.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

PubMed

Moustacas, Valéria S; Silva, Teane M A; Costa, Luciana F; Xavier, Mariana N; Carvalho, Custódio A; Costa, Érica A; Paixão, Tatiane A; Santos, Renato L

2013-03-21

Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III.

PubMed

Furfaro, Lucy L; Chang, Barbara J; Payne, Matthew S

2017-09-01

Streptococcus agalactiae is the leading cause of early-onset neonatal sepsis. Culture-based screening methods lack the sensitivity of molecular assays and do not indicate serotype; a potentially important virulence marker. We aimed to develop a multiplex PCR to detect S. agalactiae while simultaneously identifying serotypes Ia, Ib, and III; commonly associated with infant disease. Primers were designed to target S. agalactiae serotype-specific cps genes and the dltS gene. The assay was validated with 512 vaginal specimens from pregnant women. 112 (21.9%) were dltS positive, with 14.3%, 0.9%, and 6.3% of these identified as cps Ia, Ib, and III, respectively. Our assay is a specific and sensitive method to simultaneously detect S. agalactiae and serotypes Ia, Ib, and III in a single step. It is of high significance for clinical diagnostic applications and also provides epidemiological data on serotype, information that may be important for vaccine development and other targeted non-antibiotic therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

PubMed Central

Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

2015-01-01

Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

Novel real-time polymerase chain reaction assay for simultaneous detection of recurrent fusion genes in acute myeloid leukemia.

PubMed

Dolz, Sandra; Barragán, Eva; Fuster, Óscar; Llop, Marta; Cervera, José; Such, Esperanza; De Juan, Inmaculada; Palanca, Sarai; Murria, Rosa; Bolufer, Pascual; Luna, Irene; Gómez, Inés; López, María; Ibáñez, Mariam; Sanz, Miguel A

2013-09-01

The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Detection of ricin in food using electrochemiluminescence-based technology.

PubMed

Garber, Eric A E; O'Brien, Thomas W

2008-01-01

Ricin is a toxic ribosome inactivating protein (RIP-II) present in beans of the castor plant, Ricinus communis. Its potential as a biodefense threat has made the rapid, sensitive detection of ricin in food important to the U.S. Food and Drug Administration. Samples of juice, dairy products, soda, vegetables, bakery products, chocolate, and condiments were spiked with varying concentrations of ricin and analyzed using a 96-well format, electrochemiluminescence (ECL) immunoassay. Assay configurations included the use of a monoclonal capture antibody coupled with either a polyclonal or monoclonal detector antibody. The samples and detector antibodies were either added sequentially or in combination during the capture step. Using the polyclonal antibody, 0.04 ng/mL ricin was detected in analytical samples prepared from several beverages. By simultaneously incubating the sample with detector antibody, it was possible to decrease the assay time to a single 20 min incubation step with a limit of detection <10 ng/mL. Assays run according to this single incubation step exhibited a hook effect (decrease in signal at high concentrations of ricin), but because of the large signal-to-noise ratio associated with the ECL assay, the response remained above background and detectable. Thus, the ECL assay was uniquely suited for the screening of samples for ricin.

A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

PubMed

Lehtoranta, Liisa; Villberg, Anja; Santanen, Riitta; Ziegler, Thedi

2009-08-01

A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.

Clinical accuracy of a PLEX-ID flu device for simultaneous detection and identification of influenza viruses A and B.

PubMed

Tang, Yi-Wei; Lowery, Kristin S; Valsamakis, Alexandra; Schaefer, Virginia C; Chappell, James D; White-Abell, Jill; Quinn, Criziel D; Li, Haijing; Washington, Cicely A; Cromwell, Jenna; Giamanco, Chantel M; Forman, Michael; Holden, Jeffery; Rothman, Richard E; Parker, Michelle L; Ortenberg, Elaine V; Zhang, Lei; Lin, Yea-Lin; Gaydos, Charlotte A

2013-01-01

Respiratory tract infections caused by influenza A and B viruses often present nonspecifically, and a rapid, high-throughput laboratory technique that can identify influenza viruses is clinically and epidemiologically desirable. The PLEX-ID Flu assay (Abbott Molecular Inc., Des Plaines, IL) incorporates multilocus PCR and electrospray ionization-mass spectrometry to detect and differentiate influenza A 2009 H1N1 (H1N1-p), seasonal H1N1 (H1N1-s), influenza A H3N2, and influenza B viruses in nasopharyngeal swab (NPS) specimens. The clinical performance characteristics of the PLEX-ID Flu assay in symptomatic patients were determined in this multicenter trial. A total of 2,617 prospectively and retrospectively collected NPS specimens from patients with influenza-like illness between February 2008 and 28 May 2010 were eligible for inclusion in the study. Each specimen was tested in parallel by the PLEX-ID Flu assay and by the Prodesse ProFLU+ assay (Prodesse Inc., Madison, WI), to detect influenza A and B viruses. Specimens testing positive for influenza A virus by ProFLU+ were subtyped as H1N1-p, H1N1-s, or H3N2 by using the ProFAST+ assay (Gen-Probe Prodesse Inc.). The reproducibility of the PLEX-ID Flu assay ranged from 98.3 to 100.0%, as determined by testing a nine-specimen panel at three clinical sites on each of 5 days. Positive percent agreements (PPAs) and negative percent agreements (NPAs) of the PLEX-ID Flu assay were 94.5% and 99.0% for influenza A virus and 96.0% and 99.9% for influenza B virus, respectively. For the influenza A virus subtyping characterization, the PLEX-ID Flu assay had PPAs and NPAs of 98.3% and 97.5% for H1N1-p, 88.6% and 100.0% for H1N1-s, and 98.0% and 99.9% for H3N2, respectively. The overall agreements between the PLEX-ID and Prodesse ProFLU+/ProFAST+ assays were 97.1 to 100.0%. Bidirectional Sanger sequencing analysis revealed that 87.5% of 96 discrepant results between the PLEX-ID Flu and ProFLU+/ProFAST+ assays were found upon influenza A virus detection and H1N1-p subtyping. The PLEX-ID Flu assay demonstrated a high level of accuracy for the simultaneous detection and identification of influenza A and B viruses in patient specimens, providing a new laboratory tool for the rapid diagnosis and management of influenza A and B virus infections.

A Simultaneously Antimicrobial, Protein-Repellent, and Cell-Compatible Polyzwitterion Network.

PubMed

Kurowska, Monika; Eickenscheidt, Alice; Guevara-Solarte, Diana-Lorena; Widyaya, Vania Tanda; Marx, Franziska; Al-Ahmad, Ali; Lienkamp, Karen

2017-04-10

A simultaneously antimicrobial, protein-repellent, and cell-compatible surface-attached polymer network is reported, which reduces the growth of bacterial biofilms on surfaces through its multifunctionality. The coating was made from a poly(oxonorbornene)-based zwitterion (PZI), which was surface-attached and cross-linked in one step by simultaneous UV-activated CH insertion and thiol-ene reaction. The process was applicable to both laboratory surfaces like silicon, glass, and gold and real-life surfaces like polyurethane foam wound dressings. The chemical structure and physical properties of the PZI surface and the two reference surfaces SMAMP ("synthetic mimic of an antimicrobial peptide"), an antimicrobial but protein-adhesive polymer coating, and PSB (poly(sulfobetaine)), a protein-repellent but not antimicrobial polyzwitterion coating were characterized by Fourier transform infrared spectroscopy, ellipsometry, contact angle measurements, photoelectron spectroscopy, swellability measurements (using surface plasmon resonance spectroscopy, SPR), zeta potential measurements, and atomic force microscopy. The time-dependent antimicrobial activity assay (time-kill assay) confirmed the high antimicrobial activity of the PZI; SPR was used to demonstrate that it was also highly protein-repellent. Biofilm formation studies showed that the material effectively reduced the growth of Escherichia coli and Staphylococcus aureus biofilms. Additionally, it was shown that the PZI was highly compatible with immortalized human mucosal gingiva keratinocytes and human red blood cells using the Alamar Blue assay, the live-dead stain, and the hemolysis assay. PZI thus may be an attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.

A high-throughput multiplex method adapted for GMO detection.

PubMed

Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

2008-12-24

A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

Single-tube, non-isotopic, multiplex PCR/OLA assay and sequence-coded separation for simultaneous screening of 31 cystic fibrosis mutations

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

Brinson, E.C.; Adriano, T.; Bloch, W.

1994-09-01

We have developed a rapid, single-tube, non-isotopic assay that screens a patient sample for the presence of 31 cystic fibrosis (CF) mutations. This assay can identify these mutations in a single reaction tube and a single electrophoresis run. Sample preparation is a simple, boil-and-go procedure, completed in less than an hour. The assay is composed of a 15-plex PCR, followed by a 61-plex oligonucleotide ligation assay (OLA), and incorporates a novel detection scheme, Sequence Coded Separation. Initially, the multiplex PCR amplifies 15 relevant segments of the CFTR gene, simultaneously. These PCR amplicons serve as templates for the multiplex OLA, whichmore » detects the normal or mutant allele at all loci, simultaneously. Each polymorphic site is interrogated by three oligonucleotide probes, a common probe and two allele-specific probes. Each common probe is tagged with a fluorescent dye, and the competing normal and mutant allelic probes incorporate different, non-nucleotide, mobility modifiers. These modifiers are composed of hexaethylene oxide (HEO) units, incorporated as HEO phosphoramidite monomers during automated DNA synthesis. The OLA is based on both probe hybridization and the ability of DNA ligase to discriminate single base mismatches at the junction between paired probes. Each single tube assay is electrophoresed in a single gel lane of a 4-color fluorescent DNA sequencer (Applied Biosystems, Model 373A). Each of the ligation products is identified by its unique combination of electrophoretic mobility and one of three colors. The fourth color is reserved for the in-lane size standard, used by GENESCAN{sup TM} software (Applied Biosystems) to size the OLA electrophoresis products. The Genotyper{sub TM} software (Applied Biosystems) decodes these Sequence-Coded-Separation data to create a patient summary report for all loci tested.« less

Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

NASA Technical Reports Server (NTRS)

Goodridge, Lawrence (Inventor)

2007-01-01

The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

PubMed Central

Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

2014-01-01

The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay

PubMed Central

Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

2016-01-01

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 104 CFU mL−1 or 105 CFU mL−1 for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R2) of 0.916–0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water. PMID:26884128

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

PubMed

Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

2016-02-17

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

Combined Effects of Nonylphenol and Bisphenol A on the Human Prostate Epithelial Cell Line RWPE-1

PubMed Central

Gan, Weidong; Zhou, Ming; Xiang, Zou; Han, Xiaodong; Li, Dongmei

2015-01-01

The xenoestrogens nonylphenol (NP) and bisphenol A (BPA) are regarded as endocrine disrupting chemicals (EDCs) which have widespread occurrence in our daily life. In the present study, the purpose was to analyze the combined effects of NP and BPA on the human prostate epithelial cell line RWPE-1 using two mathematical models based on the Loewe additivity (LA) theory and the Bliss independence (BI) theory. RWPE-1 cells were treated with NP (0.01–100 µM) and BPA (1–5000 µM) in either a single or a combined format. A cell viability assay and lactate dehydrogenase (LDH) leakage rate assay were employed as endpoints. As predicted by the two models and based on the cell viability assay, significant synergism between NP and BPA were observed. However, based on the LDH assay, the trends were reversed. Given that environmental contaminants are frequently encountered simultaneously, these data indicated that there were potential interactions between NP and BPA, and the combined effects of the chemical mixture might be stronger than the additive values of individual chemicals combined, which should be taken into consideration for the risk assessment of EDCs. PMID:25874684

A simple, one-tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria.

PubMed

Godwin, Rosamond M; Morgan, Jess A T

2014-02-01

Coccidiosis is a costly worldwide enteric disease of chickens caused by parasites of the genus Eimeria. At present, there are seven described species that occur globally and a further three undescribed, operational taxonomic units (OTUs X, Y, and Z) that are known to infect chickens from Australia. Species of Eimeria have both overlapping morphology and pathology and frequently occur as mixed-species infections. This makes definitive diagnosis with currently available tests difficult and, to date, there is no test for the detection of the three OTUs. This paper describes the development of a PCR-based assay that is capable of detecting all ten species of Eimeria, including OTUs X, Y, and Z in field samples. The assay is based on a single set of generic primers that amplifies a single diagnostic fragment from the mitochondrial genome of each species. This one-tube assay is simple, low-cost, and has the capacity to be high throughput. It will therefore be of great benefit to the poultry industry for Eimeria detection and control, and the confirmation of identity and purity of vaccine strains. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of mitogen-induced lymphocyte proliferation by bromodeoxyuridine (BrdU) incorporation in the chicken.

PubMed

Motobu, Maki; El-Abasy, Moshira; Na, Ki-Jeong; Hirota, Yoshikazu

2002-04-01

3H-thymidine (3H-TdR) incorporation assay has been generally used to measure lymphocyte proliferation in the chicken. Disadvantages of this assay are that radioisotope is biological hazard to the person and environment and that it can not measure which subset of lymphocytes proliferates. In this study, bromodeoxyuridine (BrdU) incorporation assay by flow cytometry was compared with 3H-TdR incorporation assay. As a result, BrdU incorporation assay showed a strong correlation with 3H-TdR incorporation assay, and it could be applied simultaneously to detect BrdU incorporation and expression of cell surface marker antigens. These results suggest that the BrdU incorporation assay by flow cytometry is useful to analyze lymphocyte proliferation in detail.

Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes.

PubMed

Okolie, Charles Emeka; Wooldridge, Karl G; Turner, David P J; Cockayne, Alan; James, Richard

2015-08-05

Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x10(3) CFU/mL for the 16S rRNA marker and 1.0x10(4) CFU/mL for six other markers and completes cycling in less than one hour. The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR.

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.

PubMed

Cremonesi, Paola; Vanoni, Laura; Morandi, Stefano; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

2011-03-30

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species. Copyright © 2011 Elsevier B.V. All rights reserved.

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

PubMed

Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

2012-09-01

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

NASA Astrophysics Data System (ADS)

St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

2016-02-01

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

Integrated Magneto-Electrochemical Sensor for Exosome Analysis.

PubMed

Jeong, Sangmoo; Park, Jongmin; Pathania, Divya; Castro, Cesar M; Weissleder, Ralph; Lee, Hakho

2016-02-23

Extracellular vesicles, including exosomes, are nanoscale membrane particles that carry molecular information on parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magneto-electrochemical assay: exosomes are immunomagnetically captured from patient samples and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables (i) highly sensitive, cell-specific exosome detection and (ii) sensor miniaturization and scale-up for high-throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the simultaneous profiling of multiple protein markers within an hour, outperforming conventional methods in assay sensitivity and speed.

Integrated Magneto-Electrochemical Sensor for Exosome Analysis

PubMed Central

Jeong, Sangmoo; Park, Jongmin; Pathania, Divya; Castro, Cesar M.; Weissleder, Ralph; Lee, Hakho

2016-01-01

Extracellular vesicles, including exosomes, are nanoscale vesicles that carry molecular information of parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magnetic-electrochemical assay: exosomes are immunomagnetically captured from patient samples, and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables i) highly sensitive, cell-specific exosome detection, and ii) sensor miniaturization and scale-up for high throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device, and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the profiling of multiple protein markers simultaneously within an hour, outperforming conventional methods in assay sensitivity and speed. PMID:26808216

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food.

PubMed

Nguyen, Thuy Trang; Van Giau, Vo; Vo, Tuong Kha

2016-12-01

The rapid detection of pathogens in food is becoming increasingly critical for ensuring the safety of consumers, since the majority of food-borne illnesses and deaths are caused by pathogenic bacteria. Hence, rapid, sensitive, inexpensive and convenient approaches to detect food-borne pathogenic bacteria is essential in controlling food safety. In this study, a multiplex PCR assay for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes was established. The invA, stx and hlyA genes specifically amplified DNA fragments of 284, 404 and 510 bp from Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity of the multiplex PCR were performed by testing different strains. The multiplex PCR assay was able to specifically simultaneously detect ten colony-forming unit/mL of each pathogen in artificially inoculated samples after enrichment for 12 h. The whole process took less than 24 h to complete, indicating that the assay is suitable for reliable and rapid identification of these three food-borne pathogens, which could be suitable in microbial epidemiology investigation.

Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

PubMed

Cortés-Gutiérrez, Elva I; Fernández, José Luis; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Gosálvez, Jaime

2017-01-01

A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

Development, validation and clinical application of a LC-MS/MS method for the simultaneous quantification of hydroxychloroquine and its active metabolites in human whole blood.

PubMed

Soichot, Marion; Mégarbane, Bruno; Houzé, Pascal; Chevillard, Lucie; Fonsart, Julien; Baud, Frédéric J; Laprévote, Olivier; Bourgogne, Emmanuel

2014-11-01

A rapid, sensitive and specific method using liquid chromatography coupled to tandem mass spectrometry was developed for the simultaneous quantification of hydroxychloroquine (HCQ) and its three major metabolites in human whole blood. The assay, using a sample volume of 100μL, was linear in a dynamic 25-2000ng/mL range (R(2)>0.99) for all four compounds and suitable for the determination of elevated HCQ concentrations up to 20,000ng/mL, after appropriate sample dilution. Inter- and intra-assay precisions were <18.2% and accuracies were between 84% and 113% for any analyte. No matrix effects were observed. The assay was successfully applied to a blood sample obtained from one poisoned patient following a massive HCQ self-ingestion resulting in an estimated concentration of 19,500ng/mL on hospital admission. In this patient, HCQ metabolites were identified and quantified at 1123, 465 and 91ng/mL for monodesethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, respectively. Further investigations are still required to assess the usefulness of the simultaneous measurement of blood concentrations of HCQ and its three active metabolites for monitoring HCQ treatment and managing HCQ poisoning. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

PubMed Central

Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

2011-01-01

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

Prevalence of respiratory viruses in Iranian patients with idiopathic pulmonary fibrosis.

PubMed

Keyvani, Hossein; Moghoofei, Mohsen; Bokharaei-Salim, Farah; Mostafaei, Shayan; Javad Mousavi, Seyed-Ali; Monavari, Seyed Hamidreza; Esghaei, Maryam

2017-11-01

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal lung disease. One of the risk factors involved in the acquisition of IPF is viral infections, especially respiratory viruses. In the present study, we investigated the detection of respiratory viruses and the possible relationship between these viruses and IPF. This cross-sectional study was supported by the Iran University of Medical Sciences (IUMS), Tehran, Iran. A total of 40 respiratory samples (five nasopharyngeal and 35 bronchoalveolar lavage specimens) were obtained from IPF patients referred to IUMS hospitals between April 2015 and December 2016. Assays were performed using the CLART Pneumovir DNA array assay, which made it possible to detect five genera of respiratory viruses simultaneously.Results/Key findings. Altogether, 22 of the 40 participants were male. Respiratory syncytial virus (RSV), parainfluenza, rhino, corona and influenza viruses were found in 2.5 % (1/40), 7.5 % (3/40), 10 % (4/40), 2.5 % (1/40) and 0% (0/40) of cases, respectively. Determining a correlation between the viruses and IPF is not an easy task, and therefore, this will require more research. In addition, the CLART Pneumovir DNA array can be considered as a useful method for simultaneous detection of several viral respiratory infections.

Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

PubMed Central

Chaisi, Mamohale E.; Janssens, Michiel E.; Vermeiren, Lieve; Oosthuizen, Marinda C.; Collins, Nicola E.; Geysen, Dirk

2013-01-01

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle. PMID:24146782

Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

PubMed

Chaisi, Mamohale E; Janssens, Michiel E; Vermeiren, Lieve; Oosthuizen, Marinda C; Collins, Nicola E; Geysen, Dirk

2013-01-01

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products

PubMed Central

Carballo, José Luis; Hernández-Inda, Zaira L; Pérez, Pilar; García-Grávalos, María D

2002-01-01

Background The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity. Material and Methods The bioactivity of the isopropanolic (2-PrOH) extracts of 14 species of marine invertebrates and 6 species of macroalgae was evaluated with the shrimp lethality assay (lethality assay), as well as with another assay based on the inhibition of hatching of the cyst (hatchability assay). The extracts were also assayed for cytotoxicity against two human cell lines, lung carcinoma A-549 and colon carcinoma HT-29, in order to assess the sensitivity of the shrimp assays to detect cytotoxic activity. Results Two sponges (Hyatella sp, Dysidea sp.), two gorgonians (Pacifigorgia adamsii, Muricea sp.), one tunicate (Polyclinum laxum), and three echinoderms (Holothuria impatiens, Pseudoconus californica and Pharia pyramidata) showed a strong cytostatic (growth inhibition) and cytotoxic effect. The hatchability assay showed a strong activity in 4 of the species active against the two human cell lines tested (Hyatella sp, Dysidea sp., Pacifigorgia adamsii and Muricea sp.), and the lethality assay also showed a high lethality in 4 of them (Pacifigorgia adamsii, Muricea sp., Polyclinum laxum, and Pharia pyramidata). Each bioassay detected activity in 50% of the species that were considered active against the two human cell lines tested. However, the simultaneous use of both bioassays increased the percentage to 75%. Conclusions Our results seem consistent with the correlation previously established between cytotoxicity and brine shrimp lethality in plant extracts. We suggest using both bioassays simultaneously to test natural marine products for pharmacological activity. PMID:12270067

Automated Microfluidic Filtration and Immunocytochemistry Detection System for Capture and Enumeration of Circulating Tumor Cells and Other Rare Cell Populations in Blood.

PubMed

Pugia, Michael; Magbanua, Mark Jesus M; Park, John W

2017-01-01

Isolation by size using a filter membrane offers an antigen-independent method for capturing rare cells present in blood of cancer patients. Multiple cell types, including circulating tumor cells (CTCs), captured on the filter membrane can be simultaneously identified via immunocytochemistry (ICC) analysis of specific cellular biomarkers. Here, we describe an automated microfluidic filtration method combined with a liquid handling system for sequential ICC assays to detect and enumerate non-hematologic rare cells in blood.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

PubMed

Osman, Fatima; Dang, Tyler; Bodaghi, Sohrab; Vidalakis, Georgios

2017-07-01

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs. Copyright © 2017. Published by Elsevier B.V.

Simultaneous total antioxidant capacity assay of lipophilic and hydrophilic antioxidants in the same acetone-water solution containing 2% methyl-beta-cyclodextrin using the cupric reducing antioxidant capacity (CUPRAC) method.

PubMed

Ozyürek, Mustafa; Bektaşoğlu, Burcu; Güçlü, Kubilay; Güngör, Nilay; Apak, Reşat

2008-12-07

Antioxidants are health beneficial compounds that can protect cells from the damage caused by unstable molecules known as reactive oxygen species (ROS). This work reports the capacity assay of both lipophilic and hydrophilic antioxidants simultaneously, by making use of their 'host-guest' complexes with methyl-beta-cyclodextrin (M-beta-CD), a cyclic oligosaccharide, in acetonated aqueous medium using the cupric reducing antioxidant capacity (CUPRAC) method. Thus the order of antioxidant potency of various compounds irrespective of their lipophilicity could be established in the same solvent medium. M-beta-CD was introduced as the water solubility enhancer for lipophilic antioxidants. Two percent M-beta-CD (w/v) in an acetone-H(2)O (9:1, v/v) mixture was found to sufficiently solubilize beta-carotene, lycopene, vitamin E, vitamin C, synthetic antioxidants and other phenolic antioxidants. This assay was validated through linearity, additivity, precision, and recovery. The validation results demonstrate that the CUPRAC assay is reliable and robust. In acetonated aqueous solution of M-beta-CD, only CUPRAC and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays were capable of measuring carotenoids together with hydrophilic antioxidants. The CUPRAC antioxidant capacities of a wide range of polyphenolics and flavonoids were experimentally reported in this work as trolox equivalent antioxidant capacity (TEAC) in the CUPRAC assay, and compared to those found by reference methods, ABTS/horseradish peroxidase (HRP)-H(2)O(2) and ferric reducing antioxidant power (FRAP) assays.

Simultaneous direct detection of Shiga-toxin producing Escherichia coli (STEC) strains by gold nanoparticle optical sensing

USDA-ARS?s Scientific Manuscript database

Shiga-toxin producing Escherichia coli (STEC) strains (“Big Six” – O26, O45, O103, O111, O121, O145, and O157) represent significant groups of pathogens responsible for foodborne diseases. The objective of this study was to develop a colorimetric optical sensing assay that can simultaneously detect ...

Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry.

PubMed

Rodrigues, Valérie; Baudier, Jean Baptiste; Chantal, Isabelle

2017-09-01

Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R 2  > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting luciferases

PubMed Central

Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

2008-01-01

Background Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time. Results Two Rat-1 cell lines were established that stably express either green- or red-emitting luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus. Conclusion Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts. PMID:18416852

A real-time PCR approach to detect predation on anchovy and sardine early life stages

NASA Astrophysics Data System (ADS)

Cuende, Elsa; Mendibil, Iñaki; Bachiller, Eneko; Álvarez, Paula; Cotano, Unai; Rodriguez-Ezpeleta, Naiara

2017-12-01

Recruitment of sardine (Sardina pilchardus Walbaum, 1792) and anchovy (Engraulis encrasicolus Linnaeus, 1758) is thought to be regulated by predation of their eggs and larvae. Predators of sardine and anchovy can be identified by visual taxonomic identification of stomach contents, but this method is time consuming, tedious and may underestimate predation, especially in small predators such as fish larvae. Alternatively, genetic tools may offer a more cost-effective and accurate alternative. Here, we have developed a multiplex real-time polymerase chain reaction (RT-PCR) assay based on TaqMan probes to simultaneously detect sardine and anchovy remains in gut contents of potential predators. The assay combines previously described and newly generated species-specific primers and probes for anchovy and sardine detection respectively, and allows the detection of 0,001 ng of target DNA (which corresponds to about one hundredth of the total DNA present in a single egg). We applied the method to candidate anchovy and sardine egg predators in the Bay of Biscay, Atlantic Mackerel (Scomber scombrus) larvae. Egg predation observed was limited primarily to those stations where sardine and/or anchovy eggs were present. Our developed assay offers a suitable tool to understand the effects of predation on the survival of anchovy and sardine early life stages.

Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling.

PubMed

Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

2013-08-06

Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred(2) for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase, respectively. The retrieved models are compared and prediction test sets show that especially TUCKER3 performs well, even in comparison to the supervised regression method N-PLS. Copyright © 2013 Elsevier B.V. All rights reserved.

Simultaneous detection and differentiation of three Potyviridae viruses by a multiplex TaqMan real time RT-PCR assay

USDA-ARS?s Scientific Manuscript database

A multiplex TaqMan real time RT-PCR was developed for detection and differentiation of Sweet potato virus G, Sweet potato latent virus and Sweet potato mild mottle virus in one tube. Amplification and detection of a fluorogenic cytochrome oxidase gene was included as an internal control. The assay w...

Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay

USDA-ARS?s Scientific Manuscript database

A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset ...

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams

PubMed Central

2013-01-01

Background Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis. PMID:23514236

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

PubMed

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

2017-06-01

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

Anti-dengue virus serotype 2 activity and mode of action of a novel peptide.

PubMed

Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H

2015-10-01

To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.

Simultaneous determination of celecoxib, hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection.

PubMed

Störmer, Elke; Bauer, Steffen; Kirchheiner, Julia; Brockmöller, Jürgen; Roots, Ivar

2003-01-05

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.

Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria.

PubMed

Zhang, D F; Zhang, Q Q; Li, A H

2014-11-01

Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (mPCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current mPCR covered much more target bacterial species compared with previously reported species-specific mPCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (mPCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875 bp), Vibrio (524 bp), Edwardsiella (302 bp) and Streptococcus (197 bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The mPCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The mPCR detection limits for each target bacterial genera were 50 colony-forming units (CFU) in pure culture and 100 CFU in fish tissue samples. In conclusion, the mPCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This mPCR assay provides a rapid, specific and sensitive tool for the detection or identification of common fish pathogenic bacteria in aquaculture practice. © 2014 The Society for Applied Microbiology.

Fast method for simultaneous quantification of tamoxifen and metabolites in dried blood spots using an entry level LC-MS/MS system.

PubMed

Tré-Hardy, Marie; Capron, Arnaud; Antunes, Marina Venzon; Linden, Rafael; Wallemacq, Pierre

2016-11-01

The purpose of this study was to develop and validate a new liquid chromatography-tandem mass spectrometric (LC-MSMS) assay for the simultaneous quantification of tamoxifen (TAM) and its main therapeutically active metabolites, N-desmethyltamoxifen (NDT), 4-hydroxytamoxifen (4HT) and endoxifen (END) in dried blood spots. Ultrasound assisted methanolic extraction was used for TAM and metabolites extraction from dried blood spot. After evaporation and methanol reconstitution, the extract was injected into a LC-MSMS system. Reversed phase chromatography was performed on a C18 grafted column in gradient mode. TAM, metabolites, and internal standard (diazepam-d 5 ; IS) were identified in positive electrospray ionization mode using m/z transition of 372.5>72.1 (TAM); 374.23>58.10 (END); 358.27>58.10 (NDT); 388.23>44.80 (4HT) and 290.00>198.00 (IS). Total analytical run time was 6.5min. Assay was linear from 1 to 500ng/mL for all substances and presented intra and inter-assay precision and accuracy <15%. TAM, NDT, 4HT and END limits of quantification and detection were of 1 and 0.5ng/mL; 1 and 3ng/mL; 1.7 and 3ng/mL; 0.6 and 2ng/mL, respectively. Recovery ranged from 83.8 to 96.3% with matrix effect ranged from 4.3 to 29.8% for TAM and its metabolites. Hematocrit value ≤40% appeared to negatively influence accuracy of the method. In conclusion, the method described here is somewhat accessible, relatively fast, sensitive and selective with no interference. This assay might be used to investigate the level of TAM and its metabolites in DBS for therapeutic drug monitoring purposes. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

PubMed

Hasanpour, Mojtaba; Najafi, Akram

2017-06-01

Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (T m ) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains. Copyright © 2017 Elsevier B.V. All rights reserved.

ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

PubMed Central

Vänelid, Johan; Siegbahn, Agneta; Ericsson, Olle; Fredriksson, Simon; Bäcklin, Christofer; Gut, Marta; Heath, Simon; Gut, Ivo Glynne; Wallentin, Lars; Gustafsson, Mats G.; Kamali-Moghaddam, Masood; Landegren, Ulf

2011-01-01

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. PMID:21980495

The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay

PubMed Central

Garrido-Bailón, Encarna; Higes, Mariano; Martínez-Salvador, Amparo; Antúnez, Karina; Botías, Cristina; Meana, Aránzazu; Prieto, Lourdes; Martín-Hernández, Raquel

2013-01-01

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied. PMID:23919248

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.

PubMed

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar; Henning, Heiko

2016-11-15

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. © 2016. Published by The Company of Biologists Ltd.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

PubMed Central

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar

2016-01-01

ABSTRACT Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. PMID:27612509

Amelioration of the cyclophosphamide induced genotoxic damage in mice by the ethanolic extract of Equisetum arvense.

PubMed

Kour, Jasbir; Ali, Md Niamat; Ganaie, Hilal Ahmad; Tabassum, Nahida

2017-01-01

In the present study, we evaluated the potential of the plant E. arvense against the cytotoxic and mutagenic effects induced by cyclophosphamide (chemotherapeutic agent) in the bone marrow cells of mice using the Chromosome assay (CA) and Mitotic index (MI) in vivo as the biomarkers. The study was performed following 3 protocols: pre-treatment, simultaneous treatment and post-treatment with the ethanolic extract of the plant. The results demonstrated that the plant extract was not cytotoxic and mutagenic and has a protective effect against the mutagenicity induced by cyclophosphamide in pre, simultaneous and post treatments and against its cytotoxicity as well. Because of its ability to prevent chromosomal damage , E. arvense is likely to open an interesting field concerning its possible use in clinical applications, most importantly in cancer as a chemopreventive agent or even as a coadjuvant to chemotherapy to reduce the side effects associated with it.

Ultrasensitive immunochromatographic assay for the simultaneous detection of five chemicals in drinking water.

PubMed

Xing, Changrui; Liu, Liqiang; Song, Shanshan; Feng, Min; Kuang, Hua; Xu, Chuanlai

2015-04-15

In this paper, we describe the development of a multicomponent lateral-flow assay based on an antibody-antigen reaction for the rapid and simultaneous detection of trace contaminants in water, including a heavy metal, algal toxin, antibiotic, hormone, and pesticide. The representative analytes chosen for the study were lead (Pb(II), microcystin-leucine-arginine (MC-LR), chloramphenicol (CAP), testosterone (T), and chlorothalonil (CTN). Five different antigens were immobilized separately in five test lines on a nitrocellulose membrane. The monoclonal antibodies specifically recognized the corresponding antigens, and there was no cross-reactivity between the antibodies in the detection assay. Samples or standards containing the five analytes were preincubated with the freeze-dried colloidal-gold-labeled monoclonal antibody conjugates to improve the sensitivity of the assay. The results were obtained within 20min with a paper-based sensor. The cut-off values for the strip test were 4ng/mL for Pb(II), 1ng/mL for MC-LR, 0.1ng/mL for CAP, 5ng/mL for T, and 5ng/mL for CTN. The assay was evaluated using spiked water samples, and the accuracy and reproducibility of the results were good. In summary, this lateral-flow device provides an effective and rapid method for the onsite detection of multiple contaminants in water samples, with no treatment or devices required. Copyright © 2014 Elsevier B.V. All rights reserved.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment.

PubMed

Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong

2017-01-01

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus ( S. aureus ), Listeria monocytogenes ( L. monocytogenes ) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 10 2 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes , and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes , and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

PubMed Central

Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong

2017-01-01

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples. PMID:28620364

Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

USDA-ARS?s Scientific Manuscript database

In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...

Valence States Modulation Strategy for Picomole Level Assay of Hg2+ in Drinking and Environmental Water by Directional Self-Assembly of Gold Nanorods.

PubMed

Chen, Lu; Lu, Linlin; Wang, Sufan; Xia, Yunsheng

2017-06-23

In this study, we present a valence states modulation strategy for picomole level assay of Hg 2+ using directional self-assembly of gold nanorods (AuNRs) as signal readout. Hg 2+ ions are first controllably reduced to Hg + ions by appropriate ascorbic acid, and the reduced Hg + ions react with the tips of the preadded AuNRs and form gold amalgam. Such Hg + decorated AuNRs then end-to-end self-assemble into one-dimensional architectures by the bridging effects of lysine based on the high affinity of NH 2 -Hg + interactions. Correspondingly, the AuNRs' longitudinal surface plasmon resonance is gradually reduced and a new broad band appears at 900-1100 nm region simultaneously. The resulting distinctly ratiometric signal output is not only favorable for Hg 2+ ions detection but competent for their quantification. Under optimal conditions, the linear range is 22.8 pM to 11.4 nM, and the detection limit is as low as 8.7 pM. Various transition/heavy metal ions, such as Pb 2+ , Ti 2+ , Co 2+ , Fe 3+ , Mn 2+ , Ba 2+ , Fe 2+ , Ni 2+ , Al 3+ , Cu 2+ , Ag + , and Au 3+ , do not interfere with the assay. Because of ultrahigh sensitivity and excellent selectivity, the proposed system can be employed for assaying ultratrace of Hg 2+ containing in drinking and commonly environmental water samples, which is difficult to be achieved by conventional colorimetric systems. These results indicate that the present platform possesses specific advantages and potential applications in the assay of ultratrace amounts of Hg 2+ ions.

Comparison of monoplex and duplex RT-PCR assays for the detection of measles virus.

PubMed

Binkhamis, Khalifa; Gillis, Hayley; Lafreniere, Joseph Daniel; Hiebert, Joanne; Mendoza, Lillian; Pettipas, Janice; Severini, Alberto; Hatchette, Todd F; LeBlanc, Jason J

2017-01-01

Rapid and accurate detection of measles virus is important for case diagnosis and public health management. This study compared the performance of two monoplex RT-PCR reactions targeting the H and N genes to a duplex RT-PCR targeting both genes simultaneously. The duplex simplified processing without compromising assay performance characteristic. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

PubMed

Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le

2017-11-01

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.

Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

PubMed Central

Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

2015-01-01

Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

PubMed Central

Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

2013-01-01

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

A fit-for-purpose LC-MS/MS method for the simultaneous quantitation of ATP and 2,3-DPG in human K2EDTA whole blood.

PubMed

Kim, Hyeryun; Kosinski, Penelope; Kung, Charles; Dang, Lenny; Chen, Yue; Yang, Hua; Chen, Yuan-Shek; Kramer, Jordyn; Liu, Guowen

2017-09-01

Many hemolytic anemias results in major metabolic abnormalities: two common metabolite abnormalities include increased levels of 2,3-diphosphoglycerate (2,3-DPG) and decreased levels of adenosine triphosphate (ATP). To better monitor the concentration changes of these metabolites, the development of a reliable LC-MS/MS method to quantitatively profile the concentrations of 2, 3-DPG and ATP in whole blood is essential to understand the effects of investigational therapeutics. Accurate quantification of both compounds imposes great challenges to bioanalytical scientists due to their polar, ionic and endogenous nature. Here we present an LC-MS/MS method for the reliable quantification of 2,3-DPG and ATP from K 2 EDTA human whole blood (WB) simultaneously. Whole blood samples were spiked with stable isotope labeled internal standards, processed by protein precipitation extraction, and analyzed using zwitterionic ion chromatography-hydrophilic interaction chromatography (ZIC-HILIC) coupled with tandem mass spectrometry. The linear analytical range of the assay was 50-3000μg/mL. The fit-for-purpose method demonstrated excellent accuracy and precision. The overall accuracy was within ±10.5% (%RE) for both analytes and the intra- and inter-assay precision (%CV) were less than 6.7% and 6.2% for both analytes, respectively. ATP and 2,3-DPG were found to be stable in human K 2 EDTA blood for at least 8h at 4°C, 96days when stored at -70°C and after three freeze/thaw cycles. The assay has been successfully applied to K 2 EDTA human whole blood samples to support clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR.

PubMed

Nielsen, Martin K; Peterson, David S; Monrad, Jesper; Thamsborg, Stig M; Olsen, Susanne N; Kaplan, Ray M

2008-03-01

Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples.

Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

PubMed

Cui, Yanyan; Zhang, Yan; Jian, Fuchun; Zhang, Longxian; Wang, Rongjun; Cao, Shuxuan; Wang, Xiaoxing; Yan, Yaqun; Ning, Changshen

2017-05-01

Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH 2 O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10 -3  ng per μl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens. Copyright © 2017 Elsevier Inc. All rights reserved.

Brief Report: A mass spectrometry assay to simultaneously analyze ROS1 and RET fusion gene expression in non-small cell lung cancer

PubMed Central

Wijesinghe, Priyanga; Bepler, Gerold

2014-01-01

Introduction ROS1 and RET gene fusions were recently discovered in non-small cell lung cancer (NSCLC) as potential therapeutic targets with small molecule kinase inhibitors. The conventional screening methods of these fusions are time consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing PCR and the sensitivity of mass spectrometry. Methods The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false negative results. To flag false positives, the system also comprises two independent assays for each fusion gene junction. Results The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using cDNA from lung tissue of healthy individuals. The system was further validated using cDNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors. Conclusion The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover. PMID:25384172

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella.

PubMed

Baschirotto, Priscila T; Krieger, Marco A; Foti, Leonardo

2017-06-01

During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

Nanozyme-based bio-barcode assay for high sensitive and logic-controlled specific detection of multiple DNAs.

PubMed

Lin, Xiaodong; Liu, Yaqing; Tao, Zhanhui; Gao, Jinting; Deng, Jiankang; Yin, Jinjin; Wang, Shuo

2017-08-15

Since HCV and HIV share a common transmission path, high sensitive detection of HIV and HCV gene is of significant importance to improve diagnosis accuracy and cure rate at early stage for HIV virus-infected patients. In our investigation, a novel nanozyme-based bio-barcode fluorescence amplified assay is successfully developed for simultaneous detection of HIV and HCV DNAs with excellent sensitivity in an enzyme-free and label-free condition. Here, bimetallic nanoparticles, PtAu NPs , present outstanding peroxidase-like activity and act as barcode to catalyze oxidation of nonfluorescent substrate of amplex red (AR) into fluorescent resorufin generating stable and sensitive "Turn On" fluorescent output signal, which is for the first time to be integrated with bio-barcode strategy for fluorescence detection DNA. Furthermore, the provided strategy presents excellent specificity and can distinguish single-base mismatched mutant from target DNA. What interesting is that cascaded INHIBIT-OR logic gate is integrated with biosensors for the first time to distinguish individual target DNA from each other under logic function control, which presents great application in development of rapid and intelligent detection. Copyright © 2017. Published by Elsevier B.V.

Rapid and simple method by combining FTA™ card DNA extraction with two set multiplex PCR for simultaneous detection of non-O157 Shiga toxin-producing Escherichia coli strains and virulence genes in food samples.

PubMed

Kim, S A; Park, S H; Lee, S I; Ricke, S C

2017-12-01

The aim of this research was to optimize two multiplex polymerase chain reaction (PCR) assays that could simultaneously detect six non-O157 Shiga toxin-producing Escherichia coli (STEC) as well as the three virulence genes. We also investigated the potential of combining the FTA™ card-based DNA extraction with the multiplex PCR assays. Two multiplex PCR assays were optimized using six primer pairs for each non-O157 STEC serogroup and three primer pairs for virulence genes respectively. Each STEC strain specific primer pair only amplified 155, 238, 321, 438, 587 and 750 bp product for O26, O45, O103, O111, O121 and O145 respectively. Three virulence genes were successfully multiplexed: 375 bp for eae, 655 bp for stx1 and 477 bp for stx2. When two multiplex PCR assays were validated with ground beef samples, distinctive bands were also successfully produced. Since the two multiplex PCR examined here can be conducted under the same PCR conditions, the six non-O157 STEC and their virulence genes could be concurrently detected with one run on the thermocycler. In addition, all bands clearly appeared to be amplified by FTA card DNA extraction in the multiplex PCR assay from the ground beef sample, suggesting that an FTA card could be a viable sampling approach for rapid and simple DNA extraction to reduce time and labour and therefore may have practical use for the food industry. Two multiplex polymerase chain reaction (PCR) assays were optimized for discrimination of six non-O157 Shiga toxin-producing Escherichia coli (STEC) and identification of their major virulence genes within a single reaction, simultaneously. This study also determined the successful ability of the FTA™ card as an alternative to commercial DNA extraction method for conducting multiplex STEC PCR assays. The FTA™ card combined with multiplex PCR holds promise for the food industry by offering a simple and rapid DNA sample method for reducing time, cost and labour for detection of STEC in food and environmental samples. © 2017 The Society for Applied Microbiology.

Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

PubMed Central

Gubbels, J. M.; de Vos, A. P.; van der Weide, M.; Viseras, J.; Schouls, L. M.; de Vries, E.; Jongejan, F.

1999-01-01

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species. PMID:10325324

Multi-objective optimization strategy based on desirability functions used for electrophoratic separation and quantification of rosiglitazone and glimepiride in plasma and formulations.

PubMed

Hefnawy, Mohamed M; Sultan, Maha A; Al-Johar, Haya I; Kassem, Mohamed G; Aboul-Enein, Hassan Y

2012-01-01

Multiple response simultaneous optimization employing Derringer's desirability function was used for the development of a capillary electrophoresis method for the simultaneous determination of rosiglitazone (RSG) and glimepiride (GLM) in plasma and formulations. Twenty experiments, taking the two resolutions, the analysis time, and the capillary current as the responses with three important factors--buffer morality, volte and column temperature--were used to design mathematical models. The experimental responses were fitted into a second order polynomial and the six responses were simultaneously optimized to predict the optimum conditions for the effective separation of the studied compounds. The separation was carried out by using capillary zone electrophoresis (CZE) with a silica capillary column and diode array detector at 210 nm. The optimum assay conditions were 52 mmol l⁻¹ phosphate buffer, pH 7, and voltage of 22 kV at 29 °C. The method showed good agreement between the experimental data and predictive value throughout the studied parameter space. The assay limit of detection was 0.02 µg ml⁻¹ and the effective working range at relative standard deviation (RSD) of ≤ 5% was 0.05-16 µg ml⁻¹ (r = 0.999) for both drugs. Analytical recoveries of the studied drugs from spiked plasma were 97.2-101.9 ± 0.31-3.0%. The precision of the assay was satisfactory; RSD was 1.07 and 1.14 for intra- and inter-assay precision, respectively. The proposed method has a great value in routine analysis of RSG and GLM for its therapeutic monitoring and pharmacokinetic studies. Copyright © 2011 John Wiley & Sons, Ltd.

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

PubMed Central

Worrell, V E; Nagle, D P

1990-01-01

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines. PMID:2345148

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

PubMed Central

Hanson, Erin K.; Ballantyne, Jack

2014-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence. PMID:24715968

Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

PubMed

Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

2017-09-01

The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=-0.435, r=-0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=-0.319, r=-0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=-0.717, r=-0.688; P<0.0001, respectively). Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. © The Korean Society for Laboratory Medicine

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

PubMed

Hanson, Erin K; Ballantyne, Jack

2013-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

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

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

High-throughput single-molecule telomere characterization.

PubMed

McCaffrey, Jennifer; Young, Eleanor; Lassahn, Katy; Sibert, Justin; Pastor, Steven; Riethman, Harold; Xiao, Ming

2017-11-01

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines. © 2017 McCaffrey et al.; Published by Cold Spring Harbor Laboratory Press.

Multiplex PCR Assay for Identification of Six Different Staphylococcus spp. and Simultaneous Detection of Methicillin and Mupirocin Resistance

PubMed Central

Campos-Peña, E.; Martín-Nuñez, E.; Pulido-Reyes, G.; Martín-Padrón, J.; Caro-Carrillo, E.; Donate-Correa, J.; Lorenzo-Castrillejo, I.; Alcoba-Flórez, J.; Machín, F.

2014-01-01

We describe a new, efficient, sensitive, and fast single-tube multiple-PCR protocol for the identification of the most clinically significant Staphylococcus spp. and the simultaneous detection of the methicillin and mupirocin resistance loci. The protocol identifies at the species level isolates belonging to S. aureus, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, and S. saprophyticus. PMID:24829244

The future of targeted peptidomics.

PubMed

Findeisen, Peter

2013-12-01

Targeted MS is becoming increasingly important for sensitive and specific quantitative detection of proteins and respective PTMs. In this article, Ceglarek et al. [Proteomics Clin. Appl. 2013, 7, 794-801] present an LC-MS-based method for simultaneous quantitation of seven apolipoproteins in serum specimens. The assay fulfills many necessities of routine diagnostic applications, namely, low cost, high throughput, and good reproducibility. We anticipate that validation of new biomarkers will speed up with this technology and the palette of laboratory-based diagnostic tools will hopefully be augmented significantly in the near future. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and validation of a colorimetric assay for simultaneous quantification of neutral and uronic sugars.

PubMed

Rondel, Caroline; Marcato-Romain, Claire-Emmanuelle; Girbal-Neuhauser, Elisabeth

2013-05-15

A colorimetric assay based on the conventional anthrone reaction was investigated for specific quantification of uronic acids (UA) in the presence of neutral sugars and/or proteins. Scanning of glucose (Glu) and glucuronic acid (GlA) was performed after the reaction with anthrone and a double absorbance reading was made, at 560 nm and at 620 nm, in order to quantify the UA and neutral sugars separately. The assay was implemented on binary or ternary solutions containing Glu, GlA and bovine serum albumin (BSA) in order to validate its specificity towards sugars and check possible interference with other biochemical components such as proteins. Statistical analysis indicated that this assay provided correct quantification of uronic sugars from 50 to 400 mg/l and of neutral sugars from 20 to 80 mg/l, in the presence of proteins with concentrations reaching 600 mg/l. The proposed protocol can be of great interest for simultaneous determination of uronic and neutral sugars in complex biological samples. In particular, it can be used to correctly quantify the Extracellular Polymeric Substances (EPS) isolated from the biological matrix of many bacterial aggregates, even in the presence of EPS extractant such as EDTA. Copyright © 2013 Elsevier Ltd. All rights reserved.

Multicolor-FICTION

PubMed Central

Martín-Subero, José Ignacio; Chudoba, Ilse; Harder, Lana; Gesk, Stefan; Grote, Werner; Novo, Francisco Javier; Calasanz, María José; Siebert, Reiner

2002-01-01

Phenotypic and genotypic analyses of cells are increasingly essential for understanding pathogenetic mechanisms as well as for diagnosing and classifying malignancies and other diseases. We report a novel multicolor approach based on the FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) technique, which enables the simultaneous detection of morphological, immunophenotypic, and genetic characteristics of single cells. As prerequisite, multicolor interphase fluorescence in situ hybridization assays for B-cell non-Hodgkin’s lymphoma and anaplastic large-cell lymphoma have been developed. These assays allow the simultaneous detection of the most frequent primary chromosomal aberrations in these neoplasms, such as t(8;14), t(11;14), t(14;18), and t(3;14), and the various rearrangements of the ALK gene, respectively. To establish the multicolor FICTION technique, these assays were combined with the immunophenotypic detection of lineage- or tumor-specific antigens, namely CD20 and ALK, respectively. For evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes. Thus, three-dimensional information was obtained. The multicolor FICTION assays were applied to well-characterized lymphoma samples, proving the performance, validity, and diagnostic power of the technique. Future multicolor FICTION applications include the detection of preneoplastic lesions, early stage and minimal residual diseases, or micrometastases. PMID:12163366

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

PubMed

Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

2016-04-15

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of four important Eimeria species by multiplex PCR in a single assay.

PubMed

You, Myung-Jo

2014-06-01

The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay.

PubMed

Tompkins, Elaine M; Jones, Donald J L; Lamb, John H; Marsden, Debbie A; Farmer, Peter B; Brown, Karen

2008-01-01

A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.

Fate of wastewater effluent hER-agonists and hER-antagonists during soil aquifer treatment.

PubMed

Otakuye, Conroy; Quanrud, David M; Ela, Wendell P; Wicke, Daniel; Lansey, Kevin E; Arnold, Robert G

2005-04-01

Estrogen activity was measured in wastewater effluent before and after polishing via soil-aquifer treatment (SAT) using both a (hER-beta) competitive binding assay and a transcriptional activation (yeast estrogen screen, YES) assay. From the competitive binding assay, the equivalent 17alpha-ethinylestradiol (EE2) concentration in secondary effluent was 4.7 nM but decreased to 0.22 nM following SAT. The YES assay indicated that the equivalent EE2 concentration in the same effluent sample was below the method-detection limit (<2.5 x 10(-3) nM) but increased to 0.68 nM in effluent polished via SAT processes. It was hypothesized thattest-dependent differences arose because the competitive binding assay responds positively to both estrogen mimics and anti-estrogens; the YES assay responds to estrogen mimics, but test response is inhibited by anti-estrogens. The hypothesis was supported when organics extracted from wastewater effluent inhibited the YES test response to EE2 (anti-estrogenic effect). A similar extract prepared from SAT-polished effluent augmented the EE2 curve (agonist response). When hydrophobic organics in secondary effluent were fractionated, assay results indicated that several physically distinct anti-estrogens were present in the sample. From this work, it is evident that transcription-activation bioassays alone should not be relied upon to measure estrogenic activity in complex environmental samples because the simultaneous presence of both agonists and antagonist compounds can yield false negatives. Multiple in vitro bioassays, sample fractionation or tests designed to measure anti-estrogenic activity can be used to overcome this problem. It is also clear that there are circumstances under which SAT does not completely remove estrogenic activity during municipal wastewater effluent polishing.

The Antiangiogenesis Effect of Pirfenidone in Wound Healing In Vitro.

PubMed

Liu, Xiao'an; Yang, Yangfan; Guo, Xiujuan; Liu, Liling; Wu, Kaili; Yu, Minbin

2017-11-01

Abstracts Purpose: Pirfenidone is mostly used in antifibrotic and anti-inflammatory therapies. We have previously demonstrated that pirfenidone had antifibrotic and anti-inflammatory effects on the wound healing process after glaucoma filtration surgery in vitro and in vivo. Since the wound healing and reactive scarring process simultaneously involves inflammation, fibrosis, and angiogenesis, and angiogenesis plays a more important role in chronic or prolonged wound healing, we tried to explore the antiangiogenesis effect in pirfenidone and its potential multitarget function in regulating excessive scarring. The aim of the present study was to investigate the antiangiogenesis effect of pirfenidone. The proliferation of human umbilical vein endothelial cells (HUVECs) and human Tenon's fibroblasts (HTFs) were detected by WST-1 assay. The cell viability of HUVECs was measured by Trypan Blue together with lactate dehydrogenase, Annexin 5 experiment, and Ki-67 immunofluorescence assay. The functions of HUVECs and HTFs were demonstrated using cell migration assay, transwell invasion assay, and tube formation assay. The expression levels of vascular endothelial growth factor-A (VEGF-A), VEGF receptor-2 (VEGFR-2), neuropilin-1(NRP-1), and their downstream signaling proteins p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mechanistic target of rapamycin (mTOR) were indicated by western blot assay. The secretion of VEGF-A was detected by enzyme-linked immunosorbent assay. Pirfenidone inhibited proliferation, migration, invasion, and tube formation of HUVECs in vitro, and had an equivalent antiangiogenesis effect when compared with Ranibizumab in HUVECs and HTFs. Pirfenidone downregulated VEGF-A/VEGFR-2, VEGF-A/NRP-1, and its downstream signaling pathway protein expression. Pirfenidone has an antiangiogenesis effect in the wound healing process and may become an ideal multitarget antiscarring agent after glaucoma filtration surgery.

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

PubMed Central

Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

2013-01-01

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

Sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of paracetamol and guaifenesin in human plasma.

PubMed

Chen, Xiaoyan; Huang, Jia; Kong, Zhang; Zhong, Dafang

2005-03-25

A rapid and sensitive method for the simultaneous determination of paracetamol and guaifenesin in human plasma was developed and validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. After extracted from plasma samples by diethyl ether-dichloromethane (3:2, v/v), the analytes and internal standard osalmide were chromatographed on a C18 column. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method was linear in the concentration range of 0.05-20.0 microg/ml for paracetamol and 5.0-2000.0 ng/ml for guaifenesin. The intra- and inter-day precision was within 14% for both paracetamol and guaifenesin. The assay accuracy was within +/-2.4% for the analytes. This is the first assay method described for the simultaneous determination of paracetamol and guaifenesin in plasma using one chromatographic run. The method was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation, containing 650 mg paracetamol, 200 mg guaifenesin, 60 mg pseudoephedrine and 20 mg dextrorphan.

Correlation between skin-prick testing, individual specific IgE tests, and a multiallergen IgE assay for allergy detection in patients with chronic rhinitis.

PubMed

Cho, Jae Hoon; Suh, Jeffrey D; Kim, Jin Kook; Hong, Seok-Chan; Park, Il-Ho; Lee, Heung-Man

2014-01-01

Allergy test results can differ based on the method used. The most common tests include skin-prick testing (SPT) and in vitro tests to detect allergen-specific IgE. This study was designed to assess allergy test results using SPT, individual specific IgE tests, and a multiallergen IgE assay (multiple allergen simultaneous test) in patients with chronic rhinitis and controls. One hundred forty total patients were prospectively enrolled in the study, including 100 patients with chronic rhinitis and 40 control patients without atopy. All eligible patients underwent SPT, serum analysis using individual specific IgE test, and multiple allergen simultaneous test against 10 common allergens. Allergy test results were then compared to identify correlation and interest agreement. There was an 81-97% agreement between SPT and individual specific IgE test in allergen detection and an 80-98% agreement between SPT and multiple allergen simultaneous test. Individual specific IgE test and multiple allergen simultaneous test allergy detection prevalence was generally similar to SPT in patients with chronic rhinitis. All control patients had negative SPT (0/40), but low positive results were found with both individual specific IgE test (5-12.5%) and multiple allergen simultaneous test (2.5-7.5%) to some allergens, especially cockroach, Dermatophagoides farina, and ragweed. Agreement and correlation between individual specific IgE test and multiple allergen simultaneous test were good to excellent for a majority of tested allergens. This study shows good agreement and correlation between SPT with individual specific IgE test and multiple allergen simultaneous test on a majority of the tested allergens for patients with chronic rhinitis. Comparing the two in vitro tests, individual specific IgE test agrees with SPT better than multiple allergen simultaneous test.

A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

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

Letant, S E; .Ortiz, J I; Tammero, L

2007-04-11

We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. Thismore » article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.« less

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization▿

PubMed Central

Traversa, Donato; Iorio, Raffaella; Klei, Thomas R.; Kharchenko, Vitaliy A.; Gawor, Jakub; Otranto, Domenico; Sparagano, Olivier A. E.

2007-01-01

The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species. PMID:17626168

Simultaneous quantification of reparixin and paclitaxel in plasma and urine using ultra performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS): Application to a preclinical pharmacokinetic study in rats.

PubMed

Malhi, Sarandeep; Stesco, Nicholas; Alrushaid, Samaa; Lakowski, Ted M; Davies, Neal M; Gu, Xiaochen

2017-03-01

A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay was developed and validated to simultaneously quantify anticancer drugs reparixin and paclitaxel in this study. The compounds were extracted from plasma and urine samples by protein precipitation with acetone (supplemented with 0.1% formic acid). Chromatographic separation was achieved using a C18 column, and drug molecules were ionized using dual ion source electrospray and atmospheric pressure chemical ionization (DUIS: ESI-APCI). Reparixin and paclitaxel were quantified using negative and positive multiple reaction monitoring (MRM) mode, respectively. Stable isotope palcitaxel-D5 was used as the internal standard (IS). The assay was validated for specificity, recovery, carryover and sample stability under various storage conditions; it was also successfully applied to measure drug concentrations collected from a pharmacokinetic study in rats. The results confirmed that the assay was accurate and simple in quantifying both reparixin and paclitaxel in plasma and urine with minimal sample pretreatment. Copyright © 2016 Elsevier B.V. All rights reserved.

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

PubMed Central

Wagenmakers, A J; Schepens, J T; Veldhuizen, J A; Veerkamp, J H

1984-01-01

An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state. PMID:6430280

The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

PubMed

Wagenmakers, A J; Schepens, J T; Veldhuizen, J A; Veerkamp, J H

1984-05-15

An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state.

Assessment of biological activity and UPLC-MS based chromatographic profiling of ethanolic extract of Ochradenus arabicus.

PubMed

Ali, M Ajmal; Farah, M Abul; Al-Hemaid, Fahad M; Abou-Tarboush, Faisal M; Al-Anazi, Khaled M; Wabaidur, S M; Alothman, Z A; Lee, Joongku

2016-03-01

Natural products from wild and medicinal plants, either in the form of crude extracts or pure compounds provide unlimited opportunities for new drug leads owing to the unmatched availability of chemical diversity. In the present study, the cytotoxic potential of crude ethanolic extract of Ochradenus arabicus was analyzed by MTT cell viability assay in MCF-7 adenocarcinoma breast cancer cells. We further investigated its effect against oxidative stress induced by anticancer drug doxorubicin. In addition, Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) based chromatographic profiling of crude extract of O. arabicus was performed. The MTT assay data showed that the extract is moderately toxic to the MCF-7 cells. However, its treatment alone does not induce oxidative stress while doxorubicin increases the level of oxidative stress in MCF-7 cells. Whereas, simultaneous treatment of plant extract and doxorubicin significantly (p < 0.05) decreased the level of intracellular reactive oxygen species (ROS) and lipid peroxidation while an increase in the reduced glutathione and superoxide dismutase activity was observed in time and dose dependent manner. Hence, our finding confirmed cytotoxic and antioxidant potential of crude extract of O. arabicus in MCF-7 cells. However, further investigations on O. arabicus as a potential chemotherapeutic agent are needed. The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays such as chromatographic techniques is discussed.

Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

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

Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.

ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 formore » both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection« less

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction.

PubMed

Kim, Ji Yeun; Lee, Jung-Lim

2014-10-01

This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL⁻¹ in pure culture and seawater, and 10 CFU g⁻¹ in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method. © 2014 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction

PubMed Central

Kim, Ji Yeun; Lee, Jung-Lim

2014-01-01

Background This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. Results The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL−1 in pure culture and seawater, and 10 CFU g−1 in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. Conclusion Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method. © 2014 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24752974

Masking agent-free and channel-switch-mode simultaneous sensing of Fe(3+) and Hg(2+) using dual-excitation graphene quantum dots.

PubMed

Xu, Fengzhou; Shi, Hui; He, Xiaoxiao; Wang, Kemin; He, Dinggeng; Yan, Lv'an; Ye, Xiaosheng; Tang, Jinlu; Shangguan, Jingfang; Luo, Lan

2015-06-21

A novel channel-switch-mode strategy for simultaneous sensing of Fe(3+) and Hg(2+) is developed with dual-excitation single-emission graphene quantum dots (GQDs). By utilizing the dual-channel fluorescence response performance of GQDs, this strategy achieved a facile, low-cost, masking agent-free, quantitative and selective dual-ion assay even in mixed ion samples and practical water samples.

Plasma creatinine and creatine quantification by capillary electrophoresis diode array detector.

PubMed

Zinellu, Angelo; Caria, Marcello A; Tavera, Claudio; Sotgia, Salvatore; Chessa, Roberto; Deiana, Luca; Carru, Ciriaco

2005-07-15

Traditional clinical assays for nonprotein nitrogen compounds, such as creatine and creatinine, have focused on the use of enzymes or chemical reactions that allow measurement of each analyte separately. Most of these assays are mainly directed to urine quantification, so that their applicability on plasma samples is frequently hard to perform. This work describes a simple free zone capillary electrophoresis method for the simultaneous measurement of creatinine and creatine in human plasma. The effect of analytical parameters such as concentration and pH of Tris-phosphate running buffer and cartridge temperature on resolution, migration times, peak areas, and efficiency was investigated. Good separation was achieved using a 60.2-cm x 75-microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer, pH 2.25, at 15 degrees C, in less than 8 min. We compared the present method to a validated capillary electrophoresis assay, by measuring plasma creatinine in 120 normal subjects. The obtained data were compared by the Passing-Bablok regression and the Bland-Altman test. Moreover the performance of the developed method was assessed by measuring creatine and creatinine in 16 volunteers prior to and after a moderate physical exercise.

Microarray-integrated optoelectrofluidic immunoassay system

PubMed Central

Han, Dongsik

2016-01-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection. PMID:27190571

Microarray-integrated optoelectrofluidic immunoassay system.

PubMed

Han, Dongsik; Park, Je-Kyun

2016-05-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

Development and Application of a Multiplex Real-Time PCR Assay as an Indicator of Potential Allergenicity in Citrus Fruits.

PubMed

Wu, Jinlong; Chen, Lin; Lin, Dingbo; Ma, Zhaocheng; Deng, Xiuxin

2016-11-30

The effects of tissue type, harvest maturity, and genetic factors on the expression of genes that related to citrus fruit allergies remain poorly understood. In the present study, a multiplex real-time PCR assay was developed to monitor the expression of citrus allergen genes individually with the advantages of much fewer sample requirements and simultaneously multiple target genes detection. Gene specific primer pairs and Taqman probes of three citrus allergen genes Cit s 1.01, Cit s 2.01, and Cit s 3.01 and the house-keeping gene β-actin were designed based on gene sequence differences. The PCR results showed that differential expression patterns were found during the ripening process. The expression levels of Cit s 3.01 were much higher than those of Cit s 1.01 and Cit s 2.01 in both peel and pulp tissues among 10 citrus cultivars. Data suggested that Kao Phuang Pummelo could be safely consumed with a potential low risk in allergenicity. Considering that assessing allergenicity is one of the tests in food safety, this assay might also facilitate the breeding and production of "allergy-friendly" citrus fruits.

Simultaneous determination of 11 designated hallucinogenic phenethylamines by ultra-fast liquid chromatography with fluorescence detection.

PubMed

Min, Jun Zhe; Shimizu, Yoshiha; Toyo'oka, Toshimasa; Inagaki, Shinsuke; Kikura-Hanajiri, Ruri; Goda, Yukihiro

2008-10-01

To avoid the spreading of illegal drugs, a designated drug regulation system was introduced along with revision of the Pharmaceutical Affairs Law in Japan in 2006, and 32 substances including phenethylamine-type drugs were listed in April 2007. In this study, a new simultaneous determination method, based on ultra-fast liquid chromatography coupled with fluorescence detection (UFLC-FL), was developed for the 11 designated phenethylamine drugs. The phenethylamines were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 2h in 0.1M borax (pH 9.3). The resulting 11 fluorophores were completely separated by reversed-phase chromatography using an ACQUITY UPLC BEH C(18) column (2.1 mm x 100 mm 1.7 microm) and fluorometrically detected at 550 nm (excitation at 450 nm). The calibration curves obtained from the peak areas versus the injection amounts of the phenethylamines showed a good linearity. The limits of detection (signal-to-noise ratio of 3: S/N=3) on the chromatogram were in the range from 10 fmol (PMMA) to 2.5pmol (MMDA-2). Good accuracy (%) and precision (CV) by intra-day assay and inter-day assay were also obtained using the present procedure. The method was applied to the qualitative and quantitative analyses of phenethylamine in real products obtained from the Japanese market. As the results, BDB (0.24 mg/mg), MMDA-2 (0.98 mg/mL) and 2C-I (0.016 mg/mg) were identified from the different products (powder, liquid and mushroom like). Because the procedure is simple, selective and sensitive, the present method seems to be useful for the qualitative and quantitative analyses of the designated phenethylamines in various samples including biological specimens.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Simultaneous and quantitative monitoring of co-cultured Pseudomonas aeruginosa and Staphylococcus aureus with antibiotics on a diffusometric platform

NASA Astrophysics Data System (ADS)

Chung, Chih-Yao; Wang, Jhih-Cheng; Chuang, Han-Sheng

2017-04-01

Successful treatments against bacterial infections depend on antimicrobial susceptibility testing (AST). However, conventional AST requires more than 24 h to obtain an outcome, thereby contributing to high patient mortality. An antibiotic therapy based on experiences is therefore necessary for saving lives and escalating the emergence of multidrug-resistant pathogens. Accordingly, a fast and effective drug screen is necessary for the appropriate administration of antibiotics. The mixed pathogenic nature of infectious diseases emphasizes the need to develop an assay system for polymicrobial infections. On this basis, we present a novel technique for simultaneous and quantitative monitoring of co-cultured microorganisms by coupling optical diffusometry with bead-based immunoassays. This simple integration simultaneously achieves a rapid AST analysis for two pathogens. Triple color particles were simultaneously recorded and subsequently analyzed by functionalizing different fluorescent color particles with dissimilar pathogen-specific antibodies. Results suggested that the effect of the antibiotic, gentamicin, on co-cultured Pseudomonas aeruginosa and Staphylococcus aureus was effectively distinguished by the proposed technique. This study revealed a multiplexed and time-saving (within 2 h) platform with a small sample volume (~0.5 μL) and a low initial bacterial count (50 CFU per droplet, ~105 CFU/mL) for continuously monitoring the growth of co-cultured microorganisms. This technique provides insights into timely therapies against polymicrobial diseases in the near future.

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

PubMed

Murphy, Brian M; Dandy, David S; Henry, Charles S

2009-04-27

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

PubMed

Yamaguchi, Kiyoshi; Zhu, Chi; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

2017-12-01

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

A cooled CCD camera-based protocol provides an effective solution for in vitro monitoring of luciferase.

PubMed

Afshari, Amirali; Uhde-Stone, Claudia; Lu, Biao

2015-03-13

Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function.

PubMed

Inoue, Takeshi; Hoshino, Hajime; Yamashita, Taiga; Shimoyama, Seira; Agata, Kiyokazu

2015-01-01

Planarians belong to an evolutionarily early group of organisms that possess a central nervous system including a well-organized brain with a simple architecture but many types of neurons. Planarians display a number of behaviors, such as phototaxis and thermotaxis, in response to external stimuli, and it has been shown that various molecules and neural pathways in the brain are involved in controlling these behaviors. However, due to the lack of combinatorial assay methods, it remains obscure whether planarians possess higher brain functions, including integration in the brain, in which multiple signals coming from outside are coordinated and used in determining behavioral strategies. In the present study, we designed chemotaxis and thigmotaxis/kinesis tracking assays to measure several planarian behaviors in addition to those measured by phototaxis and thermotaxis assays previously established by our group, and used these tests to analyze planarian chemotactic and thigmotactic/kinetic behaviors. We found that headless planarian body fragments and planarians that had specifically lost neural activity following regeneration-dependent conditional gene knockdown (Readyknock) of synaptotagmin in the brain lost both chemotactic and thigmotactic behaviors, suggesting that neural activity in the brain is required for the planarian's chemotactic and thigmotactic behaviors. Furthermore, we compared the strength of phototaxis, chemotaxis, thigmotaxis/kinesis, and thermotaxis by presenting simultaneous binary stimuli to planarians. We found that planarians showed a clear order of predominance of these behaviors. For example, when planarians were simultaneously exposed to 400 lux of light and a chemoattractant, they showed chemoattractive behavior irrespective of the direction of the light source, although exposure to light of this intensity alone induces evasive behavior away from the light source. In contrast, when the light intensity was increased to 800 or 1600 lux and the same dose of chemoattractant was presented, planarian behaviors were gradually shifted to negative phototaxis rather than chemoattraction. These results suggest that planarians may be capable of selecting behavioral strategies via the integration of discrete brain functions when exposed to multiple stimuli. The planarian brain processes external signals received through the respective sensory neurons, thereby resulting in the production of appropriate behaviors. In addition, planarians can adjust behavioral features in response to stimulus conditions by integrating multiple external signals in the brain.

Early steps of supported bilayer formation probed by single vesicle fluorescence assays.

PubMed Central

Johnson, Joseph M; Ha, Taekjip; Chu, Steve; Boxer, Steven G

2002-01-01

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles. PMID:12496104

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

PubMed

Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

2015-03-01

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

PubMed

Van Dolah, F M; Finley, E L; Haynes, B L; Doucette, G J; Moeller, P D; Ramsdell, J S

1994-01-01

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.

Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation

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

Daly, Don S.; Anderson, Kevin K.; White, Amanda M.

Background: A microarray of enzyme-linked immunosorbent assays, or ELISA microarray, predicts simultaneously the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Making sound biological inferences as well as improving the ELISA microarray process require require both concentration predictions and creditable estimates of their errors. Methods: We present a statistical method based on monotonic spline statistical models, penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict concentrations and estimate prediction errors in ELISA microarray. PCLS restrains the flexible spline to a fit of assay intensitymore » that is a monotone function of protein concentration. With MC, both modeling and measurement errors are combined to estimate prediction error. The spline/PCLS/MC method is compared to a common method using simulated and real ELISA microarray data sets. Results: In contrast to the rigid logistic model, the flexible spline model gave credible fits in almost all test cases including troublesome cases with left and/or right censoring, or other asymmetries. For the real data sets, 61% of the spline predictions were more accurate than their comparable logistic predictions; especially the spline predictions at the extremes of the prediction curve. The relative errors of 50% of comparable spline and logistic predictions differed by less than 20%. Monte Carlo simulation rendered acceptable asymmetric prediction intervals for both spline and logistic models while propagation of error produced symmetric intervals that diverged unrealistically as the standard curves approached horizontal asymptotes. Conclusions: The spline/PCLS/MC method is a flexible, robust alternative to a logistic/NLS/propagation-of-error method to reliably predict protein concentrations and estimate their errors. The spline method simplifies model selection and fitting, and reliably estimates believable prediction errors. For the 50% of the real data sets fit well by both methods, spline and logistic predictions are practically indistinguishable, varying in accuracy by less than 15%. The spline method may be useful when automated prediction across simultaneous assays of numerous proteins must be applied routinely with minimal user intervention.« less

Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

DTIC Science & Technology

2010-02-01

specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents . Citation...Agriculture, Agriculture Research Service (USDA-ARS) Southeast Poultry Research Laboratory (SEPRL, Athens, GA) selected specimens from its reference...Flu assay as agents of flu-like illness are identified in Supplemental Information Figure S1. A single total nucleic acid preparation from a single

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay.

PubMed

Nishiwaki, Morie; Fujimuro, Masahiro; Teishikata, Yasuhiro; Inoue, Hisanori; Sasajima, Hitoshi; Nakaso, Kazuhiro; Nakashima, Kenji; Sadanari, Hidetaka; Yamamoto, Tomohiro; Fujiwara, Yoshie; Ogawa, Naoki; Yokosawa, Hideyoshi

2006-12-01

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors. (c) 2006 Wiley-Liss, Inc.

Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers

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

Boswell, G.S.; Dimitrijevich, S.D.; Gracy, R.W.

1989-10-01

A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developedmore » an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ((3H)thymidine, (14C)leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.« less

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography-mass spectrometry.

PubMed

Wang, W L; Darwin, W D; Cone, E J

1994-10-14

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester;. ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40 degrees C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC-MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

PubMed Central

Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

2016-01-01

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

Quality by Design: Multidimensional exploration of the design space in high performance liquid chromatography method development for better robustness before validation.

PubMed

Monks, K; Molnár, I; Rieger, H-J; Bogáti, B; Szabó, E

2012-04-06

Robust HPLC separations lead to fewer analysis failures and better method transfer as well as providing an assurance of quality. This work presents the systematic development of an optimal, robust, fast UHPLC method for the simultaneous assay of two APIs of an eye drop sample and their impurities, in accordance with Quality by Design principles. Chromatography software is employed to effectively generate design spaces (Method Operable Design Regions), which are subsequently employed to determine the final method conditions and to evaluate robustness prior to validation. Copyright © 2011 Elsevier B.V. All rights reserved.

Functionalized xenon as a biosensor

PubMed Central

Spence, Megan M.; Rubin, Seth M.; Dimitrov, Ivan E.; Ruiz, E. Janette; Wemmer, David E.; Pines, Alexander; Yao, Shao Qin; Tian, Feng; Schultz, Peter G.

2001-01-01

The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. We have developed an NMR-based xenon biosensor that capitalizes on the enhanced signal-to-noise, spectral simplicity, and chemical-shift sensitivity of laser-polarized xenon to detect specific biomolecules at the level of tens of nanomoles. We present results using xenon “functionalized” by a biotin-modified supramolecular cage to detect biotin–avidin binding. This biosensor methodology can be extended to a multiplexing assay for multiple analytes. PMID:11535830

Rapid Detection of Dendritic Cell and Monocyte Disorders Using CD4 as a Lineage Marker of the Human Peripheral Blood Antigen-Presenting Cell Compartment

PubMed Central

Jardine, Laura; Barge, Dawn; Ames-Draycott, Ashley; Pagan, Sarah; Cookson, Sharon; Spickett, Gavin; Haniffa, Muzlifah; Collin, Matthew; Bigley, Venetia

2013-01-01

Dendritic cells (DCs) and monocytes are critical regulators and effectors of innate and adaptive immune responses. Monocyte expansion has been described in many pathological states while monocyte and DC deficiency syndromes are relatively recent additions to the catalog of human primary immunodeficiency disorders. Clinically applicable screening tests to diagnose and monitor these conditions are lacking. Conventional strategies for identifying human DCs and monocytes have been based on the use of a lineage gate to exclude lymphocytes, thus preventing simultaneous detection of DCs, monocytes, and lymphocyte subsets. Here we demonstrate that CD4 is a reliable lineage marker for the human peripheral blood antigen-presenting cell compartment that can be used to identify DCs and monocytes in parallel with lymphocytes. Based on this principle, simple modification of a standard lymphocyte phenotyping assay permits simultaneous enumeration of four lymphocyte and five DC/monocyte populations from a single sample. This approach is applicable to clinical samples and facilitates the diagnosis of DC and monocyte disorders in a wide range of clinical settings, including genetic deficiency, neoplasia, and inflammation. PMID:24416034

Simultaneous determination of the HIV nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography.

PubMed

Verweij-van Wissen, C P W G M; Aarnoutse, R E; Burger, D M

2005-02-25

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracies for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

[Simultaneous quantitative analysis of five alkaloids in Sophora flavescens by multi-components assay by single marker].

PubMed

Chen, Jing; Wang, Shu-Mei; Meng, Jiang; Sun, Fei; Liang, Sheng-Wang

2013-05-01

To establish a new method for quality evaluation and validate its feasibilities by simultaneous quantitative assay of five alkaloids in Sophora flavescens. The new quality evaluation method, quantitative analysis of multi-components by single marker (QAMS), was established and validated with S. flavescens. Five main alkaloids, oxymatrine, sophocarpine, matrine, oxysophocarpine and sophoridine, were selected as analytes to evaluate the quality of rhizome of S. flavescens, and the relative correction factor has good repeatibility. Their contents in 21 batches of samples, collected from different areas, were determined by both external standard method and QAMS. The method was evaluated by comparison of the quantitative results between external standard method and QAMS. No significant differences were found in the quantitative results of five alkaloids in 21 batches of S. flavescens determined by external standard method and QAMS. It is feasible and suitable to evaluate the quality of rhizome of S. flavescens by QAMS.

Mu/Kappa Opioid Interactions in Rhesus Monkeys: Implications for Analgesia and Abuse Liability

PubMed Central

Negus, S. Stevens; Katrina Schrode, KA; Stevenson, Glenn W.

2008-01-01

Mu opioid receptor agonists are clinically valuable as analgesics; however, their use is limited by high abuse liability. Kappa opioid agonists also produce antinociception, but they do not produce mu agonist-like abuse-related effects, suggesting that they may enhance the antinociceptive effects and/or attenuate the abuse-related effects of mu agonists. To evaluate this hypothesis, the present study examined interactions between the mu agonist fentanyl and the kappa agonist U69,593 in three behavioral assays in rhesus monkeys. In an assay of schedule-controlled responding, monkeys responded under a fixed-ratio 30 (FR 30) schedule of food presentation. Fentanyl and U69,593 each produced rate-decreasing effects when administered alone, and mixtures of 0.22:1, 0.65:1 and 1.96:1 U69,593/fentanyl usually produced subadditive effects. In an assay of thermal nociception, tail withdrawal latencies were measured from water heated to 50°C. Fentanyl and U69,593 each produced dose-dependent antinociception, and effects were additive for all mixtures. In an assay of drug self-administration, rhesus monkeys responded for i.v. drug injection, and both dose and FR values were manipulated. Fentanyl maintained self-administration, whereas U69,593 did not. Addition of U69,593 to fentanyl produced a proportion-dependent decrease in both rates of fentanyl self-administration and behavioral economic measures of the reinforcing efficacy of fentanyl. Taken together, these results suggest that simultaneous activation of mu and kappa receptors, either with a mixture of selective drugs or with a single drug that targets both receptors, may reduce abuse liability without reducing analgesic effects relative to selective mu agonists administered alone. PMID:18837635

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

NASA Astrophysics Data System (ADS)

Bilan, Regina; Ametzazurra, Amagoia; Brazhnik, Kristina; Escorza, Sergio; Fernández, David; Uríbarri, María; Nabiev, Igor; Sukhanova, Alyona

2017-03-01

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

PubMed Central

Bilan, Regina; Ametzazurra, Amagoia; Brazhnik, Kristina; Escorza, Sergio; Fernández, David; Uríbarri, María; Nabiev, Igor; Sukhanova, Alyona

2017-01-01

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers. PMID:28300171

A highly relevant and efficient single step method for simultaneous depletion and isolation of human regulatory T cells in a clinical setting.

PubMed

Brezar, Vedran; Ruffin, Nicolas; Lévy, Yves; Seddiki, Nabila

2014-09-01

Regulatory T cells (Tregs) are pivotal in preventing autoimmunity. They play a major but still ambiguous role in cancer and viral infections. Functional studies of human Tregs are often hampered by numerous technical difficulties arising from imperfections in isolating and depleting protocols, together with the usual low cell number available from clinical samples. We standardized a simple procedure (Single Step Method, SSM), based on magnetic beads technology, in which both depletion and isolation of human Tregs with high purities are simultaneously achieved. SSM is suitable when using low cell numbers either fresh or frozen from both patients and healthy individuals. It allows simultaneous Tregs isolation and depletion that can be used for further functional work to monitor suppressive function of isolated Tregs (in vitro suppression assay) and also effector IFN-γ responses of Tregs-depleted cell fraction (OX40 assay). To our knowledge, there is no accurate standardized method for Tregs isolation and depletion in a clinical context. SSM could thus be used and easily standardized across different laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.

PubMed

Krumschnabel, Gerhard; Fontana-Ayoub, Mona; Sumbalova, Zuzana; Heidler, Juliana; Gauper, Kathrin; Fasching, Mario; Gnaiger, Erich

2015-01-01

Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2 (•-)) and particularly hydrogen peroxide (H2O2). Since H2O2 plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H2O2 production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler, and inhibitor titrations (SUIT) on respiration versus H2O2 production. The sensitivity of the H2O2 assay was strongly influenced by compounds contained in different mitochondrial respiration media, which also exerted significant effects on chemical background fluorescence changes. Near linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H2O2 production in SUIT protocols.

Spectra resolution for simultaneous spectrophotometric determination of lamivudine and zidovudine components in pharmaceutical formulation of human immunodeficiency virus drug based on using continuous wavelet transform and derivative transform techniques.

PubMed

Sohrabi, Mahmoud Reza; Tayefeh Zarkesh, Mahshid

2014-05-01

In the present paper, two spectrophotometric methods based on signal processing are proposed for the simultaneous determination of two components of an anti-HIV drug called lamivudine (LMV) and zidovudine (ZDV). The proposed methods are applied to synthetic binary mixtures and commercial pharmaceutical tablets without the need for any chemical separation procedures. The developed methods are based on the application of Continuous Wavelet Transform (CWT) and Derivative Spectrophotometry (DS) combined with the zero cross point technique. The Daubechies (db5) wavelet family (242 nm) and Dmey wavelet family (236 nm) were found to give the best results under optimum conditions for simultaneous analysis of lamivudine and zidovudine, respectively. In addition, the first derivative absorption spectra were selected for the determination of lamivudine and zidovudine at 266 nm and 248 nm, respectively. Assaying various synthetic mixtures of the components validated the presented methods. Mean recovery values were found to be between 100.31% and 100.2% for CWT and 99.42% and 97.37% for DS, respectively for determination of LMV and ZDV. The results obtained from analyzing the real samples by the proposed methods were compared to the HPLC reference method. One-way ANOVA test at 95% confidence level was applied to the results. The statistical data from comparing the proposed methods with the reference method showed no significant differences. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.

PubMed

Cheow, Lih Feng; Viswanathan, Ramya; Chin, Chee-Sing; Jennifer, Nancy; Jones, Robert C; Guccione, Ernesto; Quake, Stephen R; Burkholder, William F

2014-10-07

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.

Inhibition of mycotoxin-producing Aspergillus nomius vsc 23 by lactic acid bacteria and Saccharomyces cerevisiae

PubMed Central

Muñoz, R; Arena, M.E.; Silva, J.; González, S.N.

2010-01-01

The effect of different fermenting microorganisms on growth of a mycotoxin- producing Aspergillus nomius was assayed. Two lactic acid bacteria, Lactobacillus fermentum and Lactobacillus rhamnosus, and Saccharomyces cerevisiae, all of which are widely used in fermentation and preservation of food, were assayed on their fungus inhibitory properties. Assays were carried out by simultaneous inoculation of one of the possible inhibiting microorganisms and the fungus or subsequent inoculation of one of the microorganisms followed by the fungus. All three microorganisms assayed showed growth inhibition of the mycotoxin-producing Aspergillus strain. L. rhamnosus O236, isolated from sheep milk and selected for its technological properties, showed highest fungal inhibition of the microorganisms assayed. The use of antifungal LAB with excellent technological properties rather than chemical preservatives would enable the food industry to produce organic food without addition of chemical substances. PMID:24031582

Inhibition of mycotoxin-producing Aspergillus nomius vsc 23 by lactic acid bacteria and Saccharomyces cerevisiae.

PubMed

Muñoz, R; Arena, M E; Silva, J; González, S N

2010-10-01

The effect of different fermenting microorganisms on growth of a mycotoxin- producing Aspergillus nomius was assayed. Two lactic acid bacteria, Lactobacillus fermentum and Lactobacillus rhamnosus, and Saccharomyces cerevisiae, all of which are widely used in fermentation and preservation of food, were assayed on their fungus inhibitory properties. Assays were carried out by simultaneous inoculation of one of the possible inhibiting microorganisms and the fungus or subsequent inoculation of one of the microorganisms followed by the fungus. All three microorganisms assayed showed growth inhibition of the mycotoxin-producing Aspergillus strain. L. rhamnosus O236, isolated from sheep milk and selected for its technological properties, showed highest fungal inhibition of the microorganisms assayed. The use of antifungal LAB with excellent technological properties rather than chemical preservatives would enable the food industry to produce organic food without addition of chemical substances.

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

PubMed Central

Del Portillo, P; Thomas, M C; Martínez, E; Marañón, C; Valladares, B; Patarroyo, M E; Carlos López, M

1996-01-01

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay. PMID:8789008

Simultaneous determination of levocetirizine and pseudoephedrine in dog plasma by liquid chromatography-mass spectrometry in the presence of dextrocetirizine.

PubMed

Ryu, Jae Kuk; Yoo, Sun Dong

2012-01-01

This study describes the development of a rapid and sensitive LC-ESI-MS assay for simultaneous enantioselective determination of levocetirizine and pseudoephedrine in dog plasma in the presence of dextrocetirizine. Separations were achieved on an Ultron ES-OVM chiral column using the mobile phase consisting of 10 mM aqueous NH4OAc (pH 6.6) and acetonitrile (9:1 v/v). The retention times of pseudoephedrine, dextrocetirizine, levocetirizine and diazepam (internal standard) were 5.2, 8.3, 9.6 and 11.6 min, respectively, and the total run time was less than 15 min. The assay was validated to demonstrate the linearity, accuracy and precision, recovery and stability. The calibration curves were linear over the concentration range from 1 - 200 ng/mL for levocetirizine and from 5 - 1000 ng/mL for pseudoephedrine. The developed assay was successfully applied to a pharmacokinetic study after oral administration of the racemic cetirizine (0.5 mg/kg, or 0.25 mg/kg as levocetirizine) and pseudoephedrine (12 mg/kg) in the dog. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

PubMed

de Juan-Franco, Elena; Rodríguez-Frade, J M; Mellado, M; Lechuga, Laura M

2013-09-30

We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a polyprobe to detect six viroids of pome and stone fruit trees.

PubMed

Lin, Liming; Li, Ruhui; Mock, Ray; Kinard, Gary

2011-01-01

A simple and sensitive dot blot hybridization assay using a digoxigenin-labeled cRNA polyprobe was developed for the simultaneous detection of six viroids that infect pome and stone fruit trees. The polyprobe was constructed by cloning sequentially partial sequences of each viroid into a single vector, with run-off transcription driven by the T7 promoter. All six viroids were detectable within a dilution range of 5(-3) to 5(-4) in total nucleic acid extracts from infected trees. Individual trees were co-inoculated to create mixed infections and all four pome fruit viroids and both stone fruit viroids could be detected in pear and peach trees, respectively, using the polyprobe. The results of the assays using the polyprobe were comparable to those using single probes. The methods were validated by testing geographically diverse isolates of viroids, as well as field samples from several collections in the US. The assay offers a rapid, reliable and cost-effective approach to the simultaneous detection of six fruit trees viroids and has the potential for routine use in quarantine, certification, and plant genebank programs where many samples are tested and distributed worldwide. Published by Elsevier B.V.

HPLC analysis of 6-mercaptopurine and metabolites in extracellular body fluids.

PubMed

Rudy, J L; Argyle, J C; Winick, N; Van Dreal, P

1988-09-01

A convenient HPLC assay, which allows for the simultaneous measurement in extracellular fluids of 6-mercaptopurine and four of its metabolites, 6-thioguanine, 6-mercaptopurine riboside, 6-thioxanthine and 6-thiouric acid is described. Solid phase extraction allows for the clean isolation of analytes from plasma, urine or cerebrospinal fluid. The simultaneous determination of 6-mercaptopurine and some of its major metabolites in extracellular fluids may contribute to the monitoring of patient compliance, bioavailability, and individual variation in metabolism and absorption.

Scavenging of long-lived radicals by (-)-epigallocatechin-3- O-gallate and simultaneous suppression of mutation in irradiated mammalian cells

NASA Astrophysics Data System (ADS)

Kumagai, Jun; Nakama, Mitsuo; Miyazaki, Tetsuo; Ise, Tamaki; Kodama, Seiji; Watanabe, Masami

2002-07-01

Effect of (-)-epigallocatechin-3- O-gallate (EGCg) on scavenging long-lived radicals and its biological significance were investigated using electron-spin-resonance spectroscopy and mutation assay in cultured human embryo cells. EGCg scavenged long-lived radicals in irradiated golden hamster embryo cells and albumin solution, and simultaneously reduced mutation frequency in the irradiated human embryo cells. These results indicate that long-lived radials are involved in the induction of mutation by radiation.

Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing1

PubMed Central

Judson, Richard; Kavlock, Robert; Martin, Matt; Reif, David; Houck, Keith; Knudsen, Thomas; Richard, Ann; Tice, Raymond R.; Whelan, Maurice; Xia, Menghang; Huang, Ruili; Austin, Christopher; Daston, George; Hartung, Thomas; Fowle, John R.; Wooge, William; Tong, Weida; Dix, David

2014-01-01

Summary In vitro, high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals, but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications in which HTS assays are used to identify a high-concern subset of a collection of chemicals. The high-concern chemicals could then be tested sooner rather than later in standard guideline bioassays. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) deemphasize the need for cross-laboratory testing, and; (4) implement a web-based, transparent and expedited peer review process. PMID:23338806

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

PubMed

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

PubMed

Aitichou, Mohamed; Saleh, Sharron; Kyusung, Park; Huggins, John; O'Guinn, Monica; Jahrling, Peter; Ibrahim, Sofi

2008-11-01

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

PubMed

Hao, Lu; Xie, Jipeng; Chen, Shanyi; Wang, Shaojie; Gong, Zhuoqun; Ling, Kai-Shu; Guo, Liyun; Fan, Zaifeng; Zhou, Tao

2016-08-01

Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs. Copyright © 2016 Elsevier B.V. All rights reserved.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of salivary testosterone and cortisol in healthy men for utilization in the diagnosis of late-onset hypogonadism in males.

PubMed

Matsui, Futoshi; Koh, Eitetsu; Yamamoto, Kenrou; Sugimoto, Kazuhiro; Sin, Ho-Su; Maeda, Yuji; Honma, Seijiro; Namiki, Mikio

2009-01-01

It is well known that late-onset hypogonadism in males can cause a variety of symptoms, and the differential diagnosis is relatively difficult, including psychological disorders, stress, and mood disturbances. The level of serum cortisol can be measured to reflect a patient's level of stress. Salivary hormones facilitate the evaluation of physiological hormonal actions based on free hormone assay. For the simultaneous measurement of testosterone and cortisol levels in saliva, we validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Concerning accuracy and precision, the lower limit of quantification of salivary testosterone and cortisol were established as 5 and 10 pg/mL, respectively. Testosterone and cortisol in saliva is stable for 2 days, 14 days, and 28 days at room temperature, refrigeration and frozen, respectively. Freezing and thawing for 3 cycles and stimulation of salivation with gum chewing do not alter the measured values of testosterone and cortisol. Total, bioavailable, and free serum testosterone showed slight diurnal changes, but total and bioavailable serum cortisol showed marked diurnal changes. Salivary testosterone levels negatively correlate with age, regardless of the time of saliva collection (r=0.64, p<0.05). However, there is no relationship between salivary cortisol and age (r=0033, p>0.05). LC-MS/MS allows rapid, simultaneous, sensitive, and accurate quantification of testosterone and cortisol in saliva for the diagnosis late-onset hypogonadism or other hormone related disease.

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).

PubMed

Hornyák, Akos; Bálint, Adám; Farsang, Attila; Balka, Gyula; Hakhverdyan, Mikhayil; Rasmussen, Thomas Bruun; Blomberg, Jonas; Belák, Sándor

2012-05-01

Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale. Published by Elsevier B.V.

Multiplexing a high-throughput liability assay to leverage efficiencies.

PubMed

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

A gold nanoparticle-based semi-quantitative and quantitative ultrasensitive paper sensor for the detection of twenty mycotoxins

NASA Astrophysics Data System (ADS)

Kong, Dezhao; Liu, Liqiang; Song, Shanshan; Suryoprabowo, Steven; Li, Aike; Kuang, Hua; Wang, Libing; Xu, Chuanlai

2016-02-01

A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan reader, with the calculated limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.04-0.17, 0.06-49, 0.15-0.22, 0.056-0.49 and 0.53-1.05 μg kg-1, respectively. The analytical results of spiked samples were in accordance with the accurate content in the simultaneous detection analysis. This newly developed ICA strip assay is suitable for the on-site detection and rapid initial screening of mycotoxins in cereal samples, facilitating both semi-quantitative and quantitative determination.A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan reader, with the calculated limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.04-0.17, 0.06-49, 0.15-0.22, 0.056-0.49 and 0.53-1.05 μg kg-1, respectively. The analytical results of spiked samples were in accordance with the accurate content in the simultaneous detection analysis. This newly developed ICA strip assay is suitable for the on-site detection and rapid initial screening of mycotoxins in cereal samples, facilitating both semi-quantitative and quantitative determination. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09171c

A PDMS Device Coupled with Culture Dish for In Vitro Cell Migration Assay.

PubMed

Lv, Xiaoqing; Geng, Zhaoxin; Fan, Zhiyuan; Wang, Shicai; Pei, WeiHua; Chen, Hongda

2018-04-30

Cell migration and invasion are important factors during tumor progression and metastasis. Wound-healing assay and the Boyden chamber assay are efficient tools to investigate tumor development because both of them could be applied to measure cell migration rate. Therefore, a simple and integrated polydimethylsiloxane (PDMS) device was developed for cell migration assay, which could perform quantitative evaluation of cell migration behaviors, especially for the wound-healing assay. The integrated device was composed of three units, which included cell culture dish, PDMS chamber, and wound generation mold. The PDMS chamber was integrated with cell culture chamber and could perform six experiments under different conditions of stimuli simultaneously. To verify the function of this device, it was utilized to explore the tumor cell migration behaviors under different concentrations of fetal bovine serum (FBS) and transforming growth factor (TGF-β) at different time points. This device has the unique capability to create the "wound" area in parallel during cell migration assay and provides a simple and efficient platform for investigating cell migration assay in biomedical application.

Validation of a two-dimensional gas chromatography mass spectrometry method for the simultaneous quantification of cannabidiol, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in plasma.

PubMed

Karschner, Erin L; Barnes, Allan J; Lowe, Ross H; Scheidweiler, Karl B; Huestis, Marilyn A

2010-05-01

A sensitive analytical method for simultaneous quantification of sub-nanogram concentrations of cannabidiol (CBD), Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma is presented for monitoring cannabinoid pharmacotherapy and illicit cannabis use. Analytes were extracted from 1 mL plasma by solid-phase extraction, derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, and analyzed by two-dimensional gas chromatography mass spectrometry (2D-GCMS) with cryofocusing. The lower calibration curve was linear from 0.25-25 ng/mL for CBD and THC, 0.125-25 ng/mL for 11-OH-THC and 0.25-50 ng/mL for THCCOOH. A second higher linear range from 5-100 ng/mL, achieved through modification of injection parameters, was validated for THC, 11-OH-THC, and THCCOOH and was only implemented if concentrations exceeded the lower curve upper limit of linearity. This procedure prevented laborious re-extraction by allowing the same specimen to be re-injected for quantification on the high calibration curve. Intra- and inter-assay imprecision, determined at four quality control concentrations, were or=72.9% for all analytes. Analytes were stable when stored at 22 degrees C for 16 h, 4 degrees C for 48 h, after three freeze-thaw cycles at -20 degrees C and when stored on the autosampler for 48 h. This sensitive and specific 2D-GCMS assay provides a new means of simultaneously quantifying CBD, THC and metabolite biomarkers in clinical medicine, forensic toxicology, workplace drug testing, and driving under the influence of drugs programs.

Simple and simultaneous determination of the hiv-protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir plus M8 nelfinavir metabolite and the nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma by reversed-phase liquid chromatography.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2005-04-01

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

Haemoglobin variants may cause significant differences in haemoglobin A1c as measured by high-performance liquid chromatography and enzymatic methods in diabetic patients: a cross-sectional study.

PubMed

Otabe, Shuichi; Nakayama, Hitomi; Ohki, Tsuyoshi; Soejima, Eri; Tajiri, Yuji; Yamada, Kentaro

2017-07-01

Background We aimed to determine whether the discrepancy between haemoglobin A1c values determined by high-performance liquid chromatography and enzymatic haemoglobin A1c measurements in diabetic patients was clinically relevant. Methods We randomly recruited 1421 outpatients undergoing diabetic treatment and follow-up who underwent at least three haemoglobin A1c measurements between April 2014 and March 2015 at our clinic. In 6369 samples, haemoglobin A1c was simultaneously measured by HA-8160 and MetaboLead (enzymatic assay), and the values were compared. Results haemoglobin A1c measurements by high-performance liquid chromatography and enzymatic assay were strongly correlated (correlation coefficient: 0.9828, linear approximation curve y = 0.9986x - 0.2507). Mean haemoglobin A1c (6.8 ± 1.0%) measured by high-performance liquid chromatography was significantly higher than that measured by enzymatic assay (6.5 ± 1.0%, P < 0.0001). During the sample processing, four (0.3%) subjects presented consistently lower haemoglobin A1c values (<0.7%) by high-performance liquid chromatography than those from enzymatic assay. Of these, three had Hb Toranomon [β112 (G14) Cys→Trp]. The fourth had Hb Ube-2 [α68 (E17) Asn→Asp]. One other subject presented consistently higher haemoglobin A1c values (>1%) by high-performance liquid chromatography than those from enzymatic assay and was diagnosed with a -77 (T > C) mutation in the δ-globin gene. These unrelated asymptomatic subjects had normal erythrocyte profiles, without anaemia. Conclusions We showed that haemoglobin A1c values measured by high-performance liquid chromatography were significantly higher than those measured by enzymatic assay in diabetic subjects. However, when an oversized deviation (>0.7%) between glycaemic control status and haemoglobin A1c is apparent, clinicians should check the methods used to measure haemoglobin A1c and consider the possible presence of a haemoglobin variant.

A bioanalytical platform for simultaneous detection and quantification of biological toxins.

PubMed

Weingart, Oliver G; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin's identity and concentration. The system's performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5-1 ng · mL(-1) in buffer or in raw milk.

A Bioanalytical Platform for Simultaneous Detection and Quantification of Biological Toxins

PubMed Central

Weingart, Oliver G.; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin’s identity and concentration. The system’s performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5–1 ng·mL−1 in buffer or in raw milk. PMID:22438766

A novel multiplexed fluorescence polarisation immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk.

PubMed

Chen, Min; Wen, Kai; Tao, Xiaoqi; Ding, Shuangyang; Xie, Jie; Yu, Xuezhi; Li, Jiancheng; Xia, Xi; Wang, Yang; Xie, Sanlei; Jiang, Haiyang

2014-01-01

Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarisation immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labelled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR)) with different excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits in milk samples. Recoveries for spiked milk samples were from 76.4% to 128.4%, with a relative standard deviation lower than 13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC-MS/MS. All three instances in which FQs and SAs were present at concentrations near or above the assay limit of detection were identified as positive by the developed DB-FPIA, demonstrating that the method is suitable for rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for fast multi-analyte screening of low-molecular weight chemical residues in food samples.

Tumor suppressor miR-1 inhibits tumor growth and metastasis by simultaneously targeting multiple genes

PubMed Central

Liu, Cuilian; Zhang, Song; Wang, Qizhi; Zhang, Xiaobo

2017-01-01

Cancer progression depends on tumor growth and metastasis, which are activated or suppressed by multiple genes. An individual microRNA may target multiple genes, suggesting that a miRNA may suppress tumor growth and metastasis via simultaneously targeting different genes. However, thus far, this issue has not been explored. In the present study, the findings showed that miR-1 could simultaneously inhibit tumor growth and metastasis of gastric and breast cancers by targeting multiple genes. The results indicated that miR-1 was significantly downregulated in cancer tissues compared with normal tissues. The miR-1 overexpression led to cell cycle arrest in the G1 phase in gastric and breast cancer cells but not in normal cells. Furthermore, the miR-1 overexpression significantly inhibited the metastasis of gastric and breast cancer cells. An analysis of the underlying mechanism revealed that the simultaneous inhibition of tumor growth and metastasis mediated by miR-1 was due to the synchronous targeting of 6 miR-1 target genes encoding cyclin dependent kinase 4, twinfilin actin binding protein 1, calponin 3, coronin 1C, WAS protein family member 2 and thymosin beta 4, X-linked. In vivo assays demonstrated that miR-1 efficiently inhibited tumor growth and metastasis of gastric and breast cancers in nude mice. Therefore, our study contributed novel insights into the miR-1′s roles in tumorigenesis of gastric and breast cancers. PMID:28159933

Tumor suppressor miR-1 inhibits tumor growth and metastasis by simultaneously targeting multiple genes.

PubMed

Liu, Cuilian; Zhang, Song; Wang, Qizhi; Zhang, Xiaobo

2017-06-27

Cancer progression depends on tumor growth and metastasis, which are activated or suppressed by multiple genes. An individual microRNA may target multiple genes, suggesting that a miRNA may suppress tumor growth and metastasis via simultaneously targeting different genes. However, thus far, this issue has not been explored. In the present study, the findings showed that miR-1 could simultaneously inhibit tumor growth and metastasis of gastric and breast cancers by targeting multiple genes. The results indicated that miR-1 was significantly downregulated in cancer tissues compared with normal tissues. The miR-1 overexpression led to cell cycle arrest in the G1 phase in gastric and breast cancer cells but not in normal cells. Furthermore, the miR-1 overexpression significantly inhibited the metastasis of gastric and breast cancer cells. An analysis of the underlying mechanism revealed that the simultaneous inhibition of tumor growth and metastasis mediated by miR-1 was due to the synchronous targeting of 6 miR-1 target genes encoding cyclin dependent kinase 4, twinfilin actin binding protein 1, calponin 3, coronin 1C, WAS protein family member 2 and thymosin beta 4, X-linked. In vivo assays demonstrated that miR-1 efficiently inhibited tumor growth and metastasis of gastric and breast cancers in nude mice. Therefore, our study contributed novel insights into the miR-1's roles in tumorigenesis of gastric and breast cancers.

Constructing STR multiplex assays.

PubMed

Butler, John M

2005-01-01

Multiplex polymerase chain reaction (PCR) refers to the simultaneous amplification of multiple regions of deoxyribonucleic acid (DNA) using PCR. Commercial short tandem repeat (STR) assays that can coamplify as many as 16 different loci have become widely used in forensic DNA typing. This chapter will focus on some of the aspects of constructing robust STR multiplex assays, including careful design and quality control of PCR primers. Examples from the development of a cat STR 12plex and a human Y chromosome STR 20plex are used to illustrate the importance of various parts of the protocol. Primer design parameters and Internet-accessible resources are discussed, as are solutions to problems with residual dye artifacts that result from impure primers.

Discriminative feeding behaviour of Anopheles gambiae s.s. on endemic plants in western Kenya

PubMed Central

Manda, H.; Gouagna, L. C.; Nyandat, E.; Kabiru, E. W.; Jackson, R. R.; Foster, W. A.; Githure, J. I.; Beier, J. C.; Hassanali, A.

2009-01-01

Anopheles gambiae Giles s.s. (Diptera: Culicidae) is known to feed on plant sugars, but this is the first experimental study to consider whether it discriminates between plant species. Thirteen perennial plant species were selected on the basis of their local availability within the vicinity of human dwellings and larval habitats of An. gambiae s.s. in western Kenya. Groups of 100 or 200 mosquitoes were released into cages either with a cutting of one plant type at a time (single-plant assay) or with cuttings of all 13 plants simultaneously (choice assay), respectively, and left overnight. In the choice assay, direct observations of the percentages of mosquitoes perching or feeding on each plant were recorded over four 1-h periods each night. For both types of assay, mosquitoes were recaptured and the percentage that had fed on plants was assessed by testing them individually for the presence of fructose. To identify which plants the choice-assay mosquitoes had fed on, gas chromatography (GC) profiles of samples of mosquito homogenates were compared with GC profiles of extracts from relevant parts of each plant. Four of the plants that were observed to have been fed on most frequently in the choice assay (Parthenium hysterophorus L., Tecoma stans L., Ricinus communis L., and Senna didymobotrya Fresen) were also shown to have been ingested most often by mosquitoes in both types of assay, suggesting that An. gambiae is differentially responsive to this range of plants, regardless of whether the plants were presented singly or mixed together. Significantly more females than males fed on plants, with the exception of P. hysterophorus L., one of the plants most frequently fed on. For most plant species (ten of 13), GC profiles indicated that An. gambiae obtained sugars primarily from flowers. The exceptions were P. hysterophorus L., Lantana camara L. and R. communis L., on which An. gambiae fed more often from leaves and stems than from flowers. PMID:17373953

Anti-DNA antibodies--quintessential biomarkers of SLE.

PubMed

Pisetsky, David S

2016-02-01

Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

Treatment of in vitro enterohemorrhagic Escherichia coli infection using phage and probiotics.

PubMed

Dini, C; Bolla, P A; de Urraza, P J

2016-07-01

To assay the combination of phage and probiotics against EHEC in vitro on infected Hep-2 cells. Phage and probiotics treatments on EHEC O157:H7-infected Hep-2 cells were assayed individually or combined. The effect of freeze-drying on phage and probiotic antimicrobial activity was also studied. While treatment with phage alone increased cell detachment caused by EHEC infection, the treatments with MM alone or in combination with phage proved to effectively diminish cell damage caused by EHEC infection. Combined treatment showed a decrease in apoptotic cell count of 57·3% and a reduction in EHEC adhesion to cell monolayer of 1·2 log CFU. The simultaneous use of phage and probiotics showed no antagonistic effect, and freeze-drying did not affect their antipathogenic activity. The combination of phage and probiotics has great potential for reducing the number of pathogens adhered to epithelial cells during EHEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Further in vivo assays are needed for assessing the actual effectiveness of the treatment. This study presents a freeze-dried formulation of phage and probiotics capable of controlling EHEC infections and reducing epithelial cell damage in vitro. © 2016 The Society for Applied Microbiology.

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing bacterium in citrus trees.

PubMed

Meena, Ram Prasnna; Baranwal, V K

2016-09-01

Citrus trees harbor a large number of viral and bacterial pathogens. Citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), Citrus yellow mosaic virus (CYMV), Citrus tristeza virus (CTV) and a bacterium, Candidatus Liberibacter asiaticus (CLa) associated with huanglongbing (HLB) disease, the most prevalent pathogens in citrus orchards of different regions in India and are responsible for debilitating citriculture. For detection of these viral and bacterial pathogens a quick, sensitive and cost effective detection method is required. With this objective a multiplex polymerase chain reaction (mPCR) assay was developed for simultaneous detection of four viruses and a bacterium in citrus. Several sets of primers were designed for each virus based on the retrieved reference sequences from the GenBank. A primer pair published previously was used for greening bacterium. Each pair of primers was evaluated for their sensitivity and differentiation by simplex and mPCR. The constant amplified products were identified on the basis of molecular size in mPCR and were compared with standard PCR. The amplicons were cloned and results were confirmed with sequencing analysis. The mPCR assay was validated using naturally infected field samples for one or more citrus viruses and the huanglongbing bacterium. The mPCR assay developed here will aid in the production of virus free planting materials and rapid indexing for certification of citrus budwood programme. Copyright © 2016 Elsevier B.V. All rights reserved.

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

PubMed

Kawase, Jun; Etoh, Yoshiki; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

2016-05-20

Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2.

PubMed

Tardif, Keith D; Jorgensen, Shane; Langer, Janine; Prichard, Mark; Schlaberg, Robert

2014-11-01

Most herpes simplex virus (HSV) isolates from treatment-naïve patients are susceptible to antivirals. However, prolonged antiviral therapy can select for drug-resistant strains, especially in immunocompromised patients. Standard phenotypic methods for antiviral resistance testing are labor and time-intense and molecular resistance determinants are insufficiently understood for routine diagnostic use of genotypic resistance testing. To enable rapid, scalable antiviral susceptibility testing and minimize viral passage, we developed a 7-day, 96-well assay for simultaneous HSV 1/2 titration and phenotypic resistance testing for acyclovir and foscarnet. The assay was optimized and validated by testing clinical isolates and laboratory strains (n=39) with known IC50 for acyclovir (23 resistant) and foscarnet (1 resistant) based on plaque reduction or dye-uptake assays. A chemiluminescent detection reagent is used for quantification of cytopathic effect instead of plaque counting or measuring dye-uptake. Drug concentrations inhibiting 50% of chemiluminescent signal reduction (IC50) were determined concurrently at each of three virus dilutions. Results agree for 92.3% (acyclovir) and 100% (foscarnet) of isolates. For all three discordant samples, results of reference testing by plaque reduction agreed with the chemiluminescent assay. Reproducibility studies showed 100% qualitative agreement and 3-37% coefficient of variation based on IC50. Chemiluminescence detection as a surrogate for cellular viability with an automated plate reader provides improved throughput and workflow, as well as high accuracy and reproducibility for antiviral drug susceptibility testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

PubMed

Zhou, Qian-Jin; Wang, Lei; Chen, Jiong; Wang, Rui-Na; Shi, Yu-Hong; Li, Chang-Hong; Zhang, De-Min; Yan, Xiao-Jun; Zhang, Yan-Jun

2014-09-01

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

PubMed Central

McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M.

2017-01-01

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. PMID:28381601

Interlaboratory Evaluation of a Multiplexed High Information Content In Vitro Genotoxicity Assay

PubMed Central

Bryce, Steven M.; Bernacki, Derek T.; Bemis, Jeffrey C.; Spellman, Richard A.; Engel, Maria E.; Schuler, Maik; Lorge, Elisabeth; Heikkinen, Pekka T.; Hemmann, Ulrike; Thybaud, Véronique; Wilde, Sabrina; Queisser, Nina; Sutter, Andreas; Zeller, Andreas; Guérard, Melanie; Kirkland, David; Dertinger, Stephen D.

2017-01-01

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. PMID:28370322

Validation of a patch clamp screening protocol that simultaneously measures compound activity in multiple states of the voltage-gated sodium channel Nav1.2.

PubMed

Liu, Yi; Beck, Edward J; Flores, Christopher M

2011-12-01

Hyperactivity of voltage-gated sodium channels underlies, at least in part, a range of pathological states, including pain and epilepsy. Selective blockers of these channels may offer effective treatment of such disorders. Currently employed methods to screen for sodium channel blockers, however, are inadequate to rationally identify mechanistically diverse blockers, limiting the potential range of indications that may be treated by such agents. Here, we describe an improved patch clamp screening assay that increases the mechanistic diversity of sodium channel blockers being identified. Using QPatch HT, a medium-throughput, automated patch clamp system, we tested three common sodium channel blockers (phenytoin, lidocaine, and tetrodotoxin) with distinct mechanistic profiles at Nav1.2. The single-voltage protocol employed in this assay simultaneously measured the compound activity in multiple states, including the slow inactivated state, of the channel. A long compound incubation period (10 s) was introduced during channel inactivation to increase the probability of identifying "slow binders." As such, phenytoin, which preferentially binds with slow kinetics to the fast inactivated state, exhibited significantly higher potency than that obtained from a brief exposure (100 ms) used in typical assays. This assay also successfully detected the use-dependent block of tetrodotoxin, a well-documented property of this molecule yet unobserved in typical patch clamp protocols. These results indicate that the assay described here can increase the likelihood of identification and mechanistic diversity of sodium channel blockers from a primary screen. It can also be used to efficiently guide the in vitro optimization of leads that retain the desired mechanistic properties. © MARY ANN LIEBERT, INC.

Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

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

Kwon, Y; Hara, C A; Knize, M G

2008-05-01

As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubatedmore » with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.« less

Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci.

PubMed

McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M; Zhang, Kunyan

2017-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus ), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. Copyright © 2017 American Society for Microbiology.

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

PubMed Central

Rodríguez-Lázaro, David; D'Agostino, Martin; Pla, Maria; Cook, Nigel

2004-01-01

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5′ end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results. PMID:15583319

Liquid chromatographic tandem mass spectrometric assay for quantification of 97/78 and its metabolite 97/63: a promising trioxane antimalarial in monkey plasma.

PubMed

Singh, R P; Sabarinath, S; Gautam, N; Gupta, R C; Singh, S K

2009-07-15

The present manuscript describes development and validation of LC-MS/MS assay for the simultaneous quantitation of 97/78 and its active in-vivo metabolite 97/63 in monkey plasma using alpha-arteether as internal standard (IS). The method involves a single step protein precipitation using acetonitrile as extraction method. The analytes were separated on a Columbus C(18) (50 mm x 2 mm i.d., 5 microm particle size) column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 4, 10 mM) (80:20 v/v) at a flow rate of 0.45 mL/min, and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min and the weighted (1/x(2)) calibration curves were linear over a range of 1.56-200 ng/mL. The method was linear for both the analytes with correlation coefficients >0.995. The intra-day and inter-day accuracy (% bias) and precisions (% RSD) of the assay were less than 6.27%. Both analytes were stable after three freeze-thaw cycles (% deviation <8.2) and also for 30 days in plasma (% deviation <6.7). The absolute recoveries of 97/78, 97/63 and internal standard (IS), from spiked plasma samples were >90%. The validated assay method, described here, was successfully applied to the pharmacokinetic study of 97/78 and its active in-vivo metabolite 97/63 in Rhesus monkeys.

MUTAGENICITY OF TEFLON-COATED GLASS FIBER FILTERS: A POTENTIAL PROBLEM AND SOLUTIONS

EPA Science Inventory

Teflon-coated glass fiber filters, used in studies of airborne particulate matter, were tested for mutagenic activity using the Salmonella/mammalian-microsome (Ames) assay. For each sample, eight blank filters were simultaneously extracted with dichloromethane (DCM), and the extr...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

Serotype determination of Salmonella by xTAG assay.

PubMed

Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

2017-10-01

Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139

PubMed Central

Li, Baisheng; Liu, Xiao; Zhao, Yong; Tan, Hailing; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Qiu, Haiyan; Wang, Duochun; Diao, Baowei; Jing, Huaiqi; Yang, Ruifu; Kan, Biao

2017-01-01

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL−1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL−1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera. PMID:28662147

Anatomic viral detection is automated: the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques.

PubMed Central

Montone, K. T.; Brigati, D. J.; Budgeon, L. R.

1989-01-01

This paper presents the first automated system for simultaneously detecting human papilloma, herpes simplex, adenovirus, or cytomegalovirus viral antigens and gene sequences in standard formalin-fixed, paraffin-embedded tissue substrates and tissue culture. These viruses can be detected by colorimetric in situ nucleic acid hybridization, using biotinylated DNA probes, or by indirect immunoperoxidase techniques, using polyclonal or monoclonal antibodies, in a 2.0-hour assay performed at a single automated robotic workstation. Images FIG. 1 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 FIG. 11 PMID:2773514

Fluorescent "on-off-on" switching sensor based on CdTe quantum dots coupled with multiwalled carbon nanotubes@graphene oxide nanoribbons for simultaneous monitoring of dual foreign DNAs in transgenic soybean.

PubMed

Li, Yaqi; Sun, Li; Qian, Jing; Long, Lingliang; Li, Henan; Liu, Qian; Cai, Jianrong; Wang, Kun

2017-06-15

With the increasing concern of potential health and environmental risk, it is essential to develop reliable methods for transgenic soybean detection. Herein, a simple, sensitive and selective assay was constructed based on homogeneous fluorescence resonance energy transfer (FRET) between CdTe quantum dots (QDs) and multiwalled carbon nanotubes@graphene oxide nanoribbons (MWCNTs@GONRs) to form the fluorescent "on-off-on" switching for simultaneous monitoring dual target DNAs of promoter cauliflower mosaic virus 35s (P35s) and terminator nopaline synthase (TNOS) from transgenic soybean. The capture DNAs were immobilized with corresponding QDs to obtain strong fluorescent signals (turning on). The strong π-π stacking interaction between single-stranded DNA (ssDNA) probes and MWCNTs@GONRs led to minimal background fluorescence due to the FRET process (turning off). The targets of P35s and TNOS were recognized by dual fluorescent probes to form double-stranded DNA (dsDNA) through the specific hybridization between target DNAs and ssDNA probes. And the dsDNA were released from the surface of MWCNTs@GONRs, which leaded the dual fluorescent probes to generate the strong fluorescent emissions (turning on). Therefore, this proposed homogeneous assay can be achieved to detect P35s and TNOS simultaneously by monitoring the relevant fluorescent emissions. Moreover, this assay can distinguish complementary and mismatched nucleic acid sequences with high sensitivity. The constructed approach has the potential to be a tool for daily detection of genetically modified organism with the merits of feasibility and reliability. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles.

PubMed

Markwalter, Christine F; Ricks, Keersten M; Bitting, Anna L; Mudenda, Lwiindi; Wright, David W

2016-12-01

We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5-8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Simultaneous determination of phosphatidylcholine-derived quaternary ammonium compounds by a LC-MS/MS method in human blood plasma, serum and urine samples.

PubMed

Steuer, Christian; Schütz, Philipp; Bernasconi, Luca; Huber, Andreas R

2016-01-01

The determination of circulating trimethylamine-N-oxide (TMAO), choline, betaine, l-carnitine and O-acetyl-l-carnitine concentration in different human matrices is of great clinical interest. Recent results highlighted the prognostic value of TMAO and quaternary ammonium containing metabolites in the field of cardiovascular and kidney diseases. Herein, we report a method for the rapid and simultaneous measurement of closely related phosphatidylcholine-derived metabolites in three different biological matrices by stable isotope dilution assay. Plasma, serum and urine samples were simply deproteinized and separated by HILIC-chromatography. Detection and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed covering more than the full reference range. Assay performance metrics include intra- and interday imprecision were below 10% for all analytes. To exclude matrix effects standard addition methods were applied for all matrices. It was shown that calibration standards and quality control prepared in water can be used instead of matrix-matched calibration and controls. The LC/MS/MS-based assay described in this article may improve future clinical studies evaluating TMAO and related substances as prognostic markers for cardiovascular risk and all-cause mortality in different patient populations. Copyright © 2015 Elsevier B.V. All rights reserved.

Monitoring ASA and P2Y12-specific platelet inhibition--comparison of conventional (single) and multiple electrode aggregometry.

PubMed

Krüger, Jan-Christopher; Meves, Saskia H; Kara, Kaffer; Mügge, Andreas; Neubauer, Horst

2014-10-01

Several platelet function test systems exist for the evaluation of the platelet inhibitory effect in patients on P2Y12 inhibitors and/or acetylsalicylic acid (ASA, aspirin) therapy. Studies comparing different available assays found only a poor correlation. The objective of the present study was to evaluate the correlation and agreement between single electrode (SEA) and multiple electrode (MEA) aggregometry. In whole blood arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation was measured simultaneously using SEA (Chrono-Log) and MEA (Multiplate). We analyzed a total of 226 measurements taken from 58 patients on single ASA therapy or dual antiplatelet therapy with ASA and a thienopyridine. A cut-off value for clopidogrel/prasugrel high on-treatment platelet reactivity (HPR) of > 47 units (U) was chosen for MEA testing using hirudin and > 5 Ohm for SEA with citrate anticoagulated blood samples. The respective cut-off values for ASA HPR were > 30 U for the MEA assay and > 1 Ohm for SEA testing. There was a good correlation of the prevalence of thienopyridine-HPR in both whole blood assays (Spearman rank correlation coefficient r = 0.698) and a good inter-rate accordance (Cohen's Kappa statistic κ = 0.648). For AA-induced aggregation, the correlation of the results obtained was significant (r = 0.536; p < 0.001) and detecting ASA-HPR revealed a moderate (κ = 0.482) correlation between both impedance aggregometry assays. Platelet function testing using SEA and MEA provided both good accordance and correlation and therefore study results obtained by these two assays similarly enabled the detection of HPR of thienopyridine (and ASA) therapy.

A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A

PubMed Central

Song, Yajun; Roumagnac, Philippe; Weill, François-Xavier; Wain, John; Dolecek, Christiane; Mazzoni, Camila J.; Holt, Kathryn E.; Achtman, Mark

2010-01-01

Objectives Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. Methods By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (NalR) and/or decreased susceptibility to fluoroquinolones. Results This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (NalR = 223 and NalS = 69) and 106 isolates of Salmonella Paratyphi A (NalR = 24 and NalS = 82). All of the 247 NalR Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143/223 for Salmonella Typhi and 18/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight NalS Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. Conclusions The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes. PMID:20511368

Simultaneous occurrence of multiple aetiologies of polycythaemia: renal cell carcinoma, sleep apnoea syndrome, and relative polycythaemia in a smoker with masked polycythaemia rubra vera

PubMed Central

Shih, L.; Wang, M.; Fu, J.

2000-01-01

A 58 year old male heavy smoker presented with intracranial haemorrhage and erythrocytosis. Four aetiologies of polycythaemia—polycythaemia rubra vera (PRV), renal cell carcinoma, sleep apnoea syndrome, and relative polycythaemia—were found to be associated with the underlying causes of erythrocytosis. He did not fulfill the diagnostic criteria for PRV at initial presentation, but an erythropoietin independent erythroid progenitor assay identified the masked PRV, and the low post-phlebotomy erythropoietin concentration also suggested the likelihood of PRV evolution. This case demonstrates that a search for all the possible causes of erythrocytosis is warranted in patients who already have one aetiology of polycythaemia. Key Words: erythrocytosis • polycythaemia rubra vera • renal cell carcinoma • sleep apnoea syndrome • relative polycythaemia • endogenous erythroid colony PMID:10961184

Detecting peroxiredoxin hyperoxidation by one-dimensional isoelectric focusing.

PubMed

Cao, Zhenbo; Bulleid, Neil J

The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation.

Drug-device trials for infectious diseases: CDRH perspective.

PubMed

Meier, Kristen L; Gitterman, Steven

2011-05-01

Assessing the performance of new diagnostic tests for infectious diseases has traditionally focused on comparing the new assay against a reference standard such as culture. In this paper, we suggest that clinical trial designs with both a diagnostic and therapeutic component may be necessary to evaluate the safety and effectiveness of nonmicrobiologically based assays, with a specific emphasis on the test/marker-stratified design. General design challenges for trials of infectious diseases that simultaneously study both diagnostic and therapeutic components (eg, both devices and drugs) are also discussed.

One-week 96-well soft agar growth assay for cancer target validation.

PubMed

Ke, Ning; Albers, Aaron; Claassen, Gisela; Yu, De-hua; Chatterton, Jon E; Hu, Xiuyuan; Meyhack, Bernd; Wong-Staal, Flossie; Li, Qi-Xiang

2004-05-01

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

Evaluation of a dual-probe real time PCR system for detection of mandarin in commercial orange juice.

PubMed

Pardo, Miguel Angel

2015-04-01

A dual-probe real time PCR assay, based on the simultaneous detection of two TaqMan® probes, was evaluated for the detection of mandarin in orange juice. A single conserved polymorphism, located at the 314 position of intron belongs to chloroplast trnL gene, was confirmed by sequencing in 30 mandarin, 28 orange cultivars and 13 hybrids. The assay was also successfully evaluated in a blind trial against analysing 60 samples from different industrial processes in different countries around the world. The detection limit of the assay was established in 1% presence of mandarin detectable in processed orange juice and with a 100% precision. The quantitative application of the assay on citrus mixtures was also investigated, pointing out that the number of chloroplast DNA copies is too variable for its possible use as quantitative analysis. This assay can be employed as a routine methodology to control the accidental mixing during industrial processes and to deter intentional fraud. Copyright © 2014 Elsevier Ltd. All rights reserved.

Beyond conventional dose-response curves: Sensorgram comparison in SPR allows single concentration activity and similarity assessment.

PubMed

Gassner, C; Karlsson, R; Lipsmeier, F; Moelleken, J

2018-05-30

Previously we have introduced two SPR-based assay principles (dual-binding assay and bridging assay), which allow the determination of two out of three possible interaction parameters for bispecific molecules within one assay setup: two individual interactions to both targets, and/or one simultaneous/overall interaction, which potentially reflects the inter-dependency of both individual binding events. However, activity and similarity are determined by comparing report points over a concentration range, which also mirrors the way data is generated by conventional ELISA-based methods So far, binding kinetics have not been specifically considered in generic approaches for activity assessment. Here, we introduce an improved slope-ratio model which, together with a sensorgram comparison based similarity assessment, allows the development of a detailed, USP-conformal ligand binding assay using only a single sample concentration. We compare this novel analysis method to the usual concentration-range approach for both SPR-based assay principles and discuss its impact on data quality and increased sample throughput. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a recombinase polymerase amplification assay for Vibrio parahaemolyticus detection with an internal amplification control.

PubMed

Yang, Huan-Lan; Wei, Shuang; Gooneratne, Ravi; Mutukumira, Anthony N; Ma, Xue-Jun; Tang, Shu-Ze; Wu, Xi-Yang

2018-04-01

A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 10 3 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

Comparison of the fluorescent polarization (TDx) and the enzymatic competitive (EMIT 2000) immune assays for the measurement of cyclosporin A blood concentration.

PubMed

David-Neto, E; Ballarati, C A; Freitas, O J; Lemos, F C; Nahas, W C; Arap, S; Kalil, J

2000-01-01

Evaluation of Cyclosporin A (CyA) blood concentration is imperative in solid organ transplantation in order to achieve maximal immunosuppression with the least side effects. We compared the results of whole blood concentrations of CyA in 50 blood samples simultaneously evaluated by the fluorescent polarization immune assay (TDx) and the enzymatic competitive immune assay (EMIT 2000). There was a strong correlation between both kits for any range of CyA blood concentration (R=0.99, p<0.001). The within-run and between-days coefficient of variation were less than 4% for both assays. The cost for each CyA measurement was 50% lower for the EMIT assay when compared to the TDx assay. We concluded that the EMIT is as accurate as the TDx in measuring CyA blood concentration and has the advantage of a lower cost, as well as the possibility of widespread access to the EMIT methodology in contrast to the TDx equipment, allowing the laboratory to perform several routines within a working day.

A Rapid Survival Assay to Measure Drug-Induced Cytotoxicity and Cell Cycle Effects

PubMed Central

Valiathan, Chandni; McFaline, Jose L.

2012-01-01

We describe a rapid method to accurately measure the cytotoxicity of mammalian cells upon exposure to various drugs. Using this assay, we obtain survival data in a fraction of the time required to perform the traditional clonogenic survival assay, considered the gold standard. The dynamic range of the assay allows sensitivity measurements on a multi-log scale allowing better resolution of comparative sensitivities. Moreover, the results obtained contain additional information on cell cycle effects of the drug treatment. Cell survival is obtained from a quantitative comparison of proliferation between drug-treated and untreated cells. During the assay, cells are treated with a drug and, following a recovery period, allowed to proliferate in the presence of BrdU. Cells that synthesize DNA in the presence of bromodeoxyuridine (BrdU) exhibit quenched Hoechst fluorescence easily detected by flow cytometry; quenching is used to determine relative proliferation in treated versus untreated cells. Finally, the multi-well setup of this assay allows the simultaneous screening of multiple cell lines, multiple doses, or multiple drugs to accurately measure cell survival and cell cycle changes after drug treatment. PMID:22133811

Simultaneous isocratic HPLC determination of vigabatrin and gabapentin in human plasma by dansyl derivatization.

PubMed

Krivanek, Peter; Koppatz, Karl; Turnheim, Klaus

2003-06-01

A rapid and low-cost assay for simultaneous vigabatrin (VGA) and gabapentin (GBP) determination is described that can be performed with simple HPLC instrumentation. The method involves derivatization of the primary amine group of VGA and GBP with dansyl chloride followed by isocratic separation (column: microBondapak C-18, 10 microm, 300 x 3.9 mm; mobile phase: 50 mmol/L NaH(2)PO(4) in 40% acetonitrile) at 50 degrees C and fluorometric detection (excitation and emission wavelength: 318 and 510 nm, respectively) of the fluorescent product, which is stable for at least 7 days. Correlation coefficients of the calibration curves are >0.999 with a lower limit of detection of 0.3 microg/mL. Between- and within-run coefficients of variation are below 4.5%, and assay time is 15 minutes. This method may be used for therapeutic drug monitoring in the case of GBP and to control patient compliance in the case of VGA.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

PubMed

Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

2014-01-01

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

PubMed

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry

PubMed Central

Milman, Garry; Barnes, Allan J.; Lowe, Ross H.; Huestis, Marilyn A.

2010-01-01

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25mL oral fluid and 0.75mL buffer) was applied to conditioned CEREX® Polycrom™ THC solid phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N, O-bis-(trimethylsilyl) trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS–electron capture negative chemical ionization (NCI-MS). Linearity was 0.5-50ng/mL for THC, 11-OH-THC, CBD and 1-50ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5–500pg/mL. Intra-and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3%-6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal. PMID:20083251

Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

PubMed

Maksyutov, Rinat A; Gavrilova, Elena V; Shchelkunov, Sergei N

2016-10-01

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species. Copyright © 2016 Elsevier B.V. All rights reserved.

A TaqMan-based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander.

PubMed

Guan, Wei; Shao, Jonathan; Singh, Raghuwinder; Davis, Robert E; Zhao, Tingchang; Huang, Qi

2013-02-15

A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs. Published by Elsevier B.V.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

PubMed

Mertsch, Sonja; Thanos, Solon

2017-01-01

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Development of a Multi-Point Quantitation Method to Simultaneously Measure Enzymatic and Structural Components of the Clostridium thermocellum Cellulosome Protein Complex

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

Dykstra, Andrew B; St. Brice, Lois; Rodriguez, Jr., Miguel

2014-01-01

Clostridium thermocellum has emerged as a leading bioenergy-relevant microbe due to its ability to solubilize cellulose into carbohydrates, mediated by multi-component membrane-attached complexes termed cellulosomes. To probe microbial cellulose utilization rates, it is desirable to be able to measure the concentrations of saccharolytic enzymes and estimate the total amount of cellulosome present on a mass basis. Current cellulase determination methodologies involve labor-intensive purification procedures and only allow for indirect determination of abundance. We have developed a method using multiple reaction monitoring (MRM-MS) to simultaneously quantitate both enzymatic and structural components of the cellulosome protein complex in samples ranging in complexitymore » from purified cellulosomes to whole cell lysates, as an alternative to a previously-developed enzyme-linked immunosorbent assay (ELISA) method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision for determination of the mass concentration of cellulosome components.« less

Fast and simultaneous detection of prominent natural antioxidants using analytical microsystems for capillary electrophoresis with a glassy carbon electrode: a new gateway to food environments.

PubMed

Blasco, Antonio Javier; Barrigas, Inés; González, María Cristina; Escarpa, Alberto

2005-12-01

This paper examines for the first time the analytical possibilities of fast and simultaneous detection of prominent natural antioxidants including examples of flavonoids and vitamins using a CE microchip with electrochemical detection (ED). Unpinched injection conditions, zone electrophoretic separation and amperometric detection were carefully assayed and optimised. Analysis involved the zone electrophoretic separation of arbutin, (+)-catechin and ascorbic acid in less than 4 min using a borate buffer (pH 9.0, 50 mM), employing 2 kV as the separation voltage and +1.0 V as the detection potential. In addition, the separation of different 'couples' of natural antioxidants of food significance including (+)-catechin and ascorbic acid, (+)-catechin and rutin, as well as arbutin and phlorizdin is proposed. To demonstrate the potential and future role of CE microsystems, analytical possibilities and a new route in the raw sample analysis are presented. The preliminary results obtained allow the proposal of CE-ED microchips as a real gateway to microanalysis in foods.

Multiplex PCR assay to identify methicillin-resistant Staphylococcus haemolyticus.

PubMed

Schuenck, Ricardo P; Pereira, Eliezer M; Iorio, Natalia L P; Dos Santos, Kátia R N

2008-04-01

Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

PubMed

Krungkrai, J; Wutipraditkul, N; Prapunwattana, P; Krungkrai, S R; Rochanakij, S

2001-12-15

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes. (c)2001 Elsevier Science.

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

PubMed

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

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

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminatedmore » the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.« less

Surface enhanced Raman spectroscopy based nanoparticle assays for rapid, point-of-care diagnostics

NASA Astrophysics Data System (ADS)

Driscoll, Ashley J.

Nucleotide and immunoassays are important tools for disease diagnostics. Many of the current laboratory-based analytical diagnostic techniques require multiple assay steps and long incubation times before results are acquired. In the development of bioassays designed for detecting the emergence and spread of diseases in point-of-care (POC) and remote settings, more rapid and portable analytical methods are necessary. Nanoparticles provide simple and reproducible synthetic methods for the preparation of substrates that can be applied in colloidal assays, providing gains in kinetics due to miniaturization and plasmonic substrates for surface enhanced spectroscopies. Specifically, surface enhanced Raman spectroscopy (SERS) is finding broad application as a signal transduction method in immunological and nucleotide assays due to the production of narrow spectral peaks from the scattering molecules and the potential for simultaneous multiple analyte detection. The application of SERS to a no-wash, magnetic capture assay for the detection of West Nile Virus Envelope and Rift Valley Fever Virus N antigens is described. The platform utilizes colloid based capture of the target antigen in solution, magnetic collection of the immunocomplexes and acquisition of SERS spectra by a handheld Raman spectrometer. The reagents for a core-shell nanoparticle, SERS based assay designed for the capture of target microRNA implicated in acute myocardial infarction are also characterized. Several new, small molecule Raman scatterers are introduced and used to analyze the enhancing properties of the synthesized gold coated-magnetic nanoparticles. Nucleotide and immunoassay platforms have shown improvements in speed and analyte capture through the miniaturization of the capture surface and particle-based capture systems can provide a route to further surface miniaturization. A reaction-diffusion model of the colloidal assay platform is presented to understand the interplay of system parameters such as particle diameter, initial analyte concentration and dissociation constants. The projected sensitivities over a broad range of assay conditions are examined and the governing regime of particle systems reported. The results provide metrics in the design of more robust analytics that are of particular interest for POC diagnostics.

Assessment of relative bioavailability of heavy metals in soil using in vivo mouse model and its implication for risk assessment compared with bioaccessibility using in vitro assay.

PubMed

Kang, Yuan; Pan, Weijian; Liang, Siyun; Li, Ning; Zeng, Lixuan; Zhang, Qiuyun; Luo, Jiwen

2016-10-01

There is limited study to simultaneously determine the relative bioavailability of heavy metals such as Cd, Pb, Cu, Cr(VI), and Ni in soil samples. In the present study, the bioaccessibility of heavy metals using in vitro assay was compared with the relative bioavailability of heavy metals using in vivo mouse model. The bioaccessibility of heavy metals ranged from 9.05 ± 0.97 % (Cr) to 42.8 ± 3.52 % (Cd). The uptake profile of heavy metals in soil and solution samples in mouse revealed that the uptake kinetics could be fitted to a two-compartment model. The relative bioavailability of heavy meals ranged from 34.8 ± 7.0 % (Ni) to 131 ± 20.3 % (Cu). Poor correlation between bioaccessibility and relative bioavailability of heavy metals was observed (r (2) = 0.11, p > 0.05). The relative bioavailability of heavy metals was significantly higher than the bioaccessibility of heavy metals (p < 0.05). The present study indicated that the in vitro digestion method should be carefully employed in risk assessment.

Evaluation of the genotoxic and antigenotoxic potential of Baccharis dracunculifolia extract on V79 cells by the comet assay.

PubMed

Munari, Carla Carolina; Alves, Jacqueline Morais; Bastos, Jairo Kenupp; Tavares, Denise Crispim

2010-01-01

Baccharis dracunculifolia (Asteraceae), the main botanical source of green propolis, is a shrub of the Brazilian 'cerrado'. In folk medicine it is used as an anti-inflammatory agent, mainly for the treatment of gastrointestinal diseases. The aim of the present study was to evaluate the genotoxic and antigenotoxic effects of B. dracunculifolia ethyl acetate extract (Bd-EAE) on Chinese hamster lung fibroblasts (V79 cells) by the comet assay. Methyl methanesulfonate (MMS; 200 microM) was used as an inducer of DNA damage. Genotoxicity was evaluated using four different concentrations of Bd-EAE: 12.5, 25.0, 50.0 and 100.0 microg ml(-1). Antigenotoxicity was assessed before, simultaneously, and after treatment with the mutagen. The results showed a significant increase in the frequency of DNA damage in cultures treated with 50.0 and 100.0 microg ml(-1) Bd-EAE. Regarding its antigenotoxic potential, Bd-EAE reduced the frequency of DNA damage induced by MMS. However, this chemopreventive activity depended on the concentrations and treatment regimens used. The antioxidant activity of phenolic components present in Bd-EAE may contribute to reduce the alkylation damage induced by MMS. In conclusion, our findings confirmed the chemopreventive activity of Bd-EAE and showed that this effect occurs under different mechanism.

High-throughput behavioral screening method for detecting auditory response defects in zebrafish.

PubMed

Bang, Pascal I; Yelick, Pamela C; Malicki, Jarema J; Sewell, William F

2002-08-30

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles. Copyright 2002 Elsevier Science B.V.

Resequencing Pathogen Microarray (RPM) for prospective detection and identification of emergent pathogen strains and variants

NASA Astrophysics Data System (ADS)

Tibbetts, Clark; Lichanska, Agnieszka M.; Borsuk, Lisa A.; Weslowski, Brian; Morris, Leah M.; Lorence, Matthew C.; Schafer, Klaus O.; Campos, Joseph; Sene, Mohamadou; Myers, Christopher A.; Faix, Dennis; Blair, Patrick J.; Brown, Jason; Metzgar, David

2010-04-01

High-density resequencing microarrays support simultaneous detection and identification of multiple viral and bacterial pathogens. Because detection and identification using RPM is based upon multiple specimen-specific target pathogen gene sequences generated in the individual test, the test results enable both a differential diagnostic analysis and epidemiological tracking of detected pathogen strains and variants from one specimen to the next. The RPM assay enables detection and identification of pathogen sequences that share as little as 80% sequence similarity to prototype target gene sequences represented as detector tiles on the array. This capability enables the RPM to detect and identify previously unknown strains and variants of a detected pathogen, as in sentinel cases associated with an infectious disease outbreak. We illustrate this capability using assay results from testing influenza A virus vaccines configured with strains that were first defined years after the design of the RPM microarray. Results are also presented from RPM-Flu testing of three specimens independently confirmed to the positive for the 2009 Novel H1N1 outbreak strain of influenza virus.

LC-MS/MS method for the simultaneous quantification of luteolin, wedelolactone and apigenin in mice plasma using hansen solubility parameters for liquid-liquid extraction: Application to pharmacokinetics of Eclipta alba chloroform fraction.

PubMed

Cheruvu, Hanumanth Srikanth; Yadav, Navneet K; Valicherla, Guru R; Arya, Rakesh K; Hussain, Zakir; Sharma, Chetan; Arya, Kamal R; Singh, Rama K; Datta, Dipak; Gayen, Jiaur R

2018-04-01

Eclipta alba (Bhringraj) in ayurveda has been widely used as a traditional medicine for its multi-therapeutic properties for ages. Luteolin (LTL), wedelolactone (WDL) and apigenin (APG) are the three main bioactive phytochemicals present in Eclipta alba extract. However there was a lack of sensitive bioanalytical method for the pharmacokinetics of these free compounds in plasma which majorly contributes for their activities after oral administration of Eclipta alba. The present study aims to develop a sensitive, rapid and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of mice plasma concentrations of LTL, WDL and APG using quercetin as an internal standard for the pharmacokinetic analysis. Analytes were separated on Phenomenex Luna C18 (150 × 4.6 mm, 3.0 μm) column with mobile phase containing methanol: acetonitrile (90: 10, v/v) and 0.1% formic acid in 10 mM ammonium formate buffer in the ratio of 70: 30 (v/v) in isocratic mode. Liquid-liquid extraction was optimized using Hansen solubility parameters and diethyl ether finalized as an extraction solvent for the recovery ranging from 61 to 76% for all analytes in mice plasma. The validated method has an accuracy and precision over the linearity range of 0.1-200 ng/mL with a correlation coefficient (r 2 ) of ≥0.997. The intra and inter-day assay accuracy was between 98.17 and 107% and 95.83-107.89% respectively and the intra and inter day assay precision ranged from 0.37-6.05% and 1.85-10.76%, respectively for all the analytes. This validated method can be used for future clinical investigation studies of Eclipta alba extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

Biochemistry of Cells.

ERIC Educational Resources Information Center

McIntosh, Elizabeth; Moss, Robert

1995-01-01

While other lab exercises allow the student to isolate and study one component of the cell, the purpose of this lab is to break down the cell into several components and perform simultaneous assays to determine the constituents. Centrifugation is used as a separation technique. Provides procedure and expected results. (LZ)

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

A new metabolomic assay to examine inflammation and redox pathways following LPS challenge

PubMed Central

2012-01-01

Background Shifts in intracellular arginine (Arg) and sulfur amino acid (SAA) redox metabolism modulate macrophage activation, polarization and phenotype. Despite their importance in inflammation and redox regulatory pathways, comprehensive analysis of these metabolic networks was not previously possible with existing analytical methods. Methods The Arg/thiol redox LC-MS/MS metabolomics assay permits simultaneous assessment of amino acids and derivative products generated from Arg and SAA metabolism. Using this assay, LPS-induced changes in macrophage amino acid metabolism were monitored to identify pathway shifts during activation and their linkage to cellular redox regulation. Results Metabolite concentrations most significantly changed after treatment of a macrophage-like cell line (RAW) with LPS for 24 hrs were citrulline (Cit) (48-fold increase), ornithine (Orn) (8.5-fold increase), arginine (Arg) (66% decrease), and aspartic acid (Asp) (73% decrease). The ratio Cit + Orn/Arg + Asp (CO/AA) was more sensitive to LPS stimulation than other amino acid ratios commonly used to measure LPS-dependent inflammation (e.g., SAM/SAH, GSH/GSSG) and total media NOx. The CO/AA ratio was also the first ratio to change significantly after LPS treatment (4 hrs). Changes in the overall metabolomic profile over time indicated that metabolic pathways shifted from Arg catabolism to thiol oxidation. Conclusions Simultaneous quantification of Arg and SAA metabolic pathway shifts following LPS challenge of macrophage indicate that, in this system, the Arg-Citrulline/NO cycle and arginase pathways are the amino acid metabolic pathways most sensitive to LPS-challenge. The cellular (Cit + Orn)/(Arg + Asp) ratio, which summarizes this pathway, was more responsive to lower concentrations of LPS and responded earlier than other metabolic biomarkers of macrophage activation including GSH redox. It is suggested that the CO/AA ratio is a redox- independent early biomarker of macrophage activation. The ability to measure both the CO/AA and GSH-redox ratios simultaneously permits quantification of the relative effects of LPS challenge on macrophage inflammation and oxidative stress pathways. The use of this assay in humans is discussed, as are clinical implications. PMID:23036094

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

PubMed Central

Wernike, Kerstin; Hoffmann, Bernd

2013-01-01

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance. PMID:23303496

miR-107 and miR-25 simultaneously target LATS2 and regulate proliferation and invasion of gastric adenocarcinoma (GAC) cells

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

Zhang, Mingjun; Wang, Xiaolei; Li, Wanhu

Although a series of oncogenes and tumor suppressors were identified in the pathological development of gastric adenocarcinoma (GAC), the underlying molecule mechanism were still not fully understood. The current study explored the expression profile of miR-107 and miR-25 in GAC patients and their downstream regulative network. qRT-PCR analysis was performed to quantify the expression of these two miRNAs in serum samples from both patients and healthy controls. Dual luciferase assay was conducted to verify their putative bindings with LATS2. MTT assay, cell cycle assay and transwell assay were performed to explore how miR-107 and miR-25 regulate proliferation and invasion ofmore » gastric cancer cells. Findings of this study demonstrated that total miR-107 or miR-25 expression might be overexpressed in gastric cancer patients and they can simultaneously and synchronically regulate LATS2 expression, thereby affecting gastric cancer cell growth and invasion. Therefore, the miR-25/miR-107-LATS2 axis might play an important role in proliferation and invasion of the gastric cancer cells. - Highlights: • Total miR-107 and miR-25 expression is significantly increased in GAC patients. • Both miR-107 and miR-25 can promote proliferation and invasion of GAC cells. • Both miR-107 and miR-25 can target LATS2 and regulate its expression. • miR-107 and miR-25 regulate proliferation and invasion of GAC cells though LATS2.« less

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay▿ †

PubMed Central

Das, S.; Pingle, M. R.; Muñoz-Jordán, J.; Rundell, M. S.; Rondini, S.; Granger, K.; Chang, G.-J. J.; Kelly, E.; Spier, E. G.; Larone, D.; Spitzer, E.; Barany, F.; Golightly, L. M.

2008-01-01

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV. PMID:18685000

Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging.

PubMed

Dempsey, Graham T; Chaudhary, Khuram W; Atwater, Nicholas; Nguyen, Cuong; Brown, Barry S; McNeish, John D; Cohen, Adam E; Kralj, Joel M

2016-01-01

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative seeks an in vitro test to accurately predict clinical Torsades de Pointes (TdP). We developed a cardiotoxicity assay incorporating simultaneous measurement of the action potential (AP) waveform and Ca(2+) transient (CT) in human iPSC-derived cardiomyocytes (CMs). Concurrent optogenetic pacing provided a well-controlled electrophysiological background. We used the Optopatch platform for all-optical electrophysiology (Hochbaum et al., 2014). In a monolayer culture, a subset of cells expressed a genetically encoded, calcium and voltage reporter, CaViar (Hou, Kralj, Douglass, Engert, & Cohen, 2014), while others expressed a channelrhodopsin variant, CheRiff. Optical pacing of CheRiff-expressing cells synchronized the syncytium. We screened 12 compounds (11 acute, 1 chronic) to identify electrophysiological (AP rise time, AP50, AP90, beat rate) and CT effects in spontaneously beating and paced cultures (1Hz, 2Hz). CaViar reported spontaneous and paced APs and CTs with high signal-to-noise ratio and low phototoxicity. Quinidine, flecainide, E-4031, digoxin and cisapride prolonged APs, while verapamil and nifedipine shortened APs. Early after depolarizations (EADs) were elicited by quinidine, flecainide and cisapride. All but four compounds (amiodarone, chromanol, nifedipine, verapamil) prolonged AP rise time. Nifedipine and verapamil decreased CT amplitude, while digoxin increased CT amplitude. Pentamidine prolonged APs after chronic exposure. The Optopatch platform provides a robust assay to measure APs and CTs in hiPSC-CMs. This addresses the CiPA mandate and will facilitate comparisons of cell-based assays to human clinical data. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.

PubMed

Zheng, Naiyu; Zeng, Jianing; Manney, Amy; Williams, Lakenya; Aubry, Anne-Françoise; Voronin, Kimberly; Buzescu, Adela; Zhang, Yan J; Allentoff, Alban; Xu, Carrie; Shen, Hongwu; Warner, William; Arnold, Mark E

2016-04-15

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein. Copyright © 2016 Elsevier B.V. All rights reserved.

Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites

PubMed Central

2015-01-01

New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. PMID:25102353

Simultaneous chiral discrimination of multiple profens by cyclodextrin-modified capillary electrophoresis in normal and reversed polarity modes.

PubMed

La, Sookie; Kim, Jiyung; Kim, Jung-Han; Goto, Junichi; Kim, Kyoung-Rae

2003-08-01

Simultaneous enantioseparations of nine profens for their accurate chiral discrimination were achieved by capillary electrophoresis (CE) in the normal polarity (NP) mode with a single cyclodextrin (CD) system and in the reversed polarity (RP) mode with a dual CD system. The single CD system in the NP mode employed heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) added at 75 mM-100 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) as the optimum run buffer. The dual CD system operated in the RP mode used 30 mM TMbetaCD and 1.0% anionic carboxymethyl-beta-cyclodextrin dissolved in pH 3.0, 100 mM phosphoric acid-triethanolamine buffer containing 0.01% hexadimethrine bromide added to reverse the electroosmotic flow. Fairly good enantiomeric resolutions and the opposite enantiomer migration orders were achieved in the two modes. Relative migration times to internal standard under respective optimum conditions were characteristic of each enantiomer with good precision (< 2% relative standard deviation, RSD), thereby enabling to crosscheck the chemical identification of profens and also their accurate chiralities. The method linearity in the two modes was found to be adequate (r > or = 0.9991) for the chiral assay of the profens investigated. Simultaneous enantiomeric purity test of ibuprofen, ketoprofen and flurbiprofen in a mixture was feasible in a single analysis by the present method.

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.

PubMed

Huy, Nguyen Tien; Hang, Le Thi Thuy; Boamah, Daniel; Lan, Nguyen Thi Phuong; Van Thanh, Phan; Watanabe, Kiwao; Huong, Vu Thi Thu; Kikuchi, Mihoko; Ariyoshi, Koya; Morita, Kouichi; Hirayama, Kenji

2012-12-01

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

[Comparison of the clinical performance of the ECLusys HIV combi assay with the Lumipulse f and HISCL 2000-i HIV-1/2 ab screening assays].

PubMed

Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

2012-04-01

We compared the ECLusys HIV combi assay (ECL HIV Ag/Ab) to the Lumipulse Forte (LPf HIV 1/2 Ab) and HISCL (HIS HIV 1/2 Ab) assays. In a dilution sensitivity test using dilution panels of WHO HIV antibody international reference panel (HIV-1 Subtype A, B, C, E, HIV-1 Group O, HIV-2) and HIV-1/2 Ab CE marked material(HIV-1, HIV-2) parent specimens, the ECL assay enabled detection at a higher level of sensitivity than either the LPf assay or the HIS assay for all dilution panels. In an early detection test in the early phase of infection in which a BBI HIV seroconversion panel was used, the ECL assay enabled detection 7 days after initial blood sample collection, whereas the LPf and HIS assays enabled detection after 27 days. In a specificity test using high RF positive specimens (n=33), pregnancy specimens (n=35), cytomegalovirus antibody positive specimens (n=36), and high M protein positive specimens (n=21) that were confirmed negative for HIV-1/2 antibodies by the LPf assay, negative results were obtained for all specimens on both the ECL assay and the HIS assay. In a correlation test using routinely collected clinical specimens (n=121), including positive stock specimens, the ECL and HIS assays demonstrated the highest agreement rate 98.3%. The above results confirmed that the fourth-generation reagent ECL assay, which simultaneously detects both HIV-1/2 antibodies and p24 antigens, is both highly sensitive and specific, and is a suitable assay for use in routine testing.

Effect of genotypes on the quantification of hepatitis C virus (HCV) RNA in clinical samples using the Amplicor HCV Monitor Test and the Quantiplex HCV RNA 2.0 assay (bDNA).

PubMed

Tong, C Y; Hollingsworth, R C; Williams, H; Irving, W L; Gilmore, I T

1998-07-01

The Amplicor HCV Monitor test and the Quantiplex HCV RNA 2.0 (bDNA) assay are two commercially available assays for the quantification of hepatitis C virus (HCV) RNA in clinical samples. A direct comparison of the two assays was carried out using sera frozen previously from patients known to be chronically infected with HCV. Overall, 61 samples from 51 patients were tested simultaneously by the two methods: 67% (28/42) of the patients were infected by HCV genotype/serotype 1, 10% (4/42) with type 2, and 24% (10/42) with type 3. When the absolute value from each assay was examined, the Quantiplex assay gave a consistently higher reading and the mean logarithmic difference between the two assays was 1.4 (1.0 in type 1, 2.0 in type 2, and 2.2 in type 3). When analyzed according to genotype, strong correlation was observed between the two assays for type 1 (r = 0.83, 95% CI 0.63-0.93, P < 0.01), but not for nontype 1 samples. Despite the difference in absolute level reported by the two assays, there was a consistent trend of change in HCV RNA concentration by both assays in patients whose consecutive samples were analyzed and the differences between the two assays in consecutive samples were within 0.4 log of each other. The results suggested that with samples containing genotype 1, the Amplicor assay was more sensitive than the Quantiplex assay by about one log. However, the sensitivities of the two assays with nontype 1 samples were much closer probably due to the failure of the Amplicor assay to quantify nontype 1 genotypes effectively.

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

PubMed Central

Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

2002-01-01

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

Optimized separation procedures for the simultaneous assay of three plant hormones in liquid biofertilizers

USDA-ARS?s Scientific Manuscript database

The overuse of petrochemical-based synthetic fertilizers has caused detrimental effects to soil, water supplies, foods, and animal health. This, in addition to increased awareness of organic farming, has generated considerable interest in the evaluation of renewable biofertilizers. In order to stu...

Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens.

PubMed Central

Tinwell, Helen; Ashby, John

2004-01-01

We have evaluated whether mixtures of estrogens, present in the mix at doses that are individually inactive in the immature rat uterotrophic assay, can give a uterotrophic response. Seven chemicals were evaluated: nonylphenol, bisphenol A (BPA), methoxychlor, genistein (GEN), estradiol, diethylstilbestrol, and ethinyl estradiol. Dose responses in the uterotrophic assay were constructed for each chemical. The first series of experiments involved evaluating binary mixtures of BPA and GEN at dose levels that gave moderate uterotrophic responses when tested individually. The mixtures generally showed an intermediate or reduced uterotrophic effect compared with when the components of the mixture were tested alone at the dose used in the mixture. The next series of experiments used a multicomponent (complex) mixture of all seven chemicals evaluated at doses that gave either weakly positive or inactive uterotrophic responses when tested individually in the assay. Doses that were nominally equi-uterotrophic ranged over approximately six orders of magnitude for the seven chemicals. Doses of agents that gave a weak uterotrophic response when tested individually gave a marginally enhanced positive response in the assay when tested combined as a mixture. Doses of agents that gave a negative uterotrophic response when tested individually gave a positive response when tested as a mixture. These data indicate that a variety of different estrogen receptor (ER) agonists, present individually at subeffective doses, can act simultaneously to evoke an ER-regulated response. However, translating these findings into the process of environmental hazard assessment will be difficult. The simple addition of the observed, or predicted, activities for the components of a mixture is confirmed here to be inappropriate and to overestimate the actual effect induced by the mixture. Equally, isobole analysis is only suitable for two- or three-component mixtures, and concentration addition requires access to dose-response data and EC50 values (concentration giving 50% of the maximum response) for the individual components of the mixture--requirements that will rarely be fulfilled for complex environmental samples. Given these uncertainties, we conclude that it may be most expedient to select and bioassay whole environmental mixtures of potential concern. PMID:15064164

Large-Scale in-House Cell-Based Assay for Evaluating the Serostatus in Patients with Neuromyelitis Optica Spectrum Disorder Based on New Diagnostic Criteria.

PubMed

Kim, Yeseul; Kim, Gayoung; Kong, Byung Soo; Lee, Ji Eun; Oh, Yu Mi; Hyun, Jae Won; Kim, Su Hyun; Joung, AeRan; Kim, Byoung Joon; Choi, Kyungho; Kim, Ho Jin

2017-04-01

The detection of aquaporin 4-IgG (AQP4-IgG) is now a critical diagnostic criterion for neuromyelitis optica spectrum disorder (NMOSD). To evaluate the serostatus of NMOSD patients based on the 2015 new diagnostic criteria using a new in-house cell-based assay (CBA). We generated a stable cell line using internal ribosome entry site-containing bicistronic vectors, which allow the simultaneous expression of two proteins (AQP4 and green fluorescent protein) separately from the same RNA transcript. We performed in-house CBA using serum from 386 patients: 178 NMOSD patients diagnosed according to the new diagnostic criteria without AQP4-IgG, 63 high risk NMOSD patients presenting 1 of the 6 core clinical characteristics of NMOSD but not fulfilling dissemination in space, and 145 patients with other neurological diseases, including 66 with multiple sclerosis. The serostatus of 111 definite and high risk NMOSD patients were also tested using a commercial CBA kit with identical serum to evaluate the correlation between the 2 methods. All assays were performed by two independent and blinded investigators. Our in-house assay yielded a specificity of 100% and sensitivities of 80% (142 of 178) and 76% (48 of 63) when detecting definite- and high risk NMOSD patients, respectively. The comparison with the commercial CBA kit revealed a correlation for 102 of the 111 patients: no correlation was present in 7 patients who were seronegative using the commercial method but seropositive using the in-house method, and in 2 patients who were seropositive using the commercial method but seronegative using the in-house method. These results demonstrate that our in-house CBA is a highly specific and sensitive method for detecting AQP4-IgG in NMOSD patients. Copyright © 2017 Korean Neurological Association

Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis.

PubMed

Laopajon, Witida; Takheaw, Nuchjira; Kasinrerk, Watchara; Pata, Supansa

2016-01-01

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.

A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma.

PubMed

Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John; Theander, Thor G; Jensen, Anja T R; Turner, Louise

2008-06-12

The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlex100 (BioRad, Hercules, CA, USA) system which can quantify multiple antibodies simultaneously in a small plasma volume. A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex100 system against pooled human hyper-immune plasma before and after lyophilization. The bead-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were > or = 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether they were performed on individual beads or on multiplexed beads; lyophilization had no impact on the assay performance. Spearman's rank correlation coefficients (Rho) were > or = 0.97, (P < 0.0001) for all comparisons. Importantly, the reactivity of protein-coupled non-lyophilized beads decreased with long term storage at 4 degrees C in the dark. Using this lyophilized multiplex assay, antibody reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma.

A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams

NASA Astrophysics Data System (ADS)

Chen, Yanni; Wang, Yongwei; Liu, Liqiang; Wu, Xiaoling; Xu, Liguang; Kuang, Hua; Li, Aike; Xu, Chuanlai

2015-10-01

A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples.A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04987c

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

PubMed

Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

2016-07-01

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Evaluation of the Triage TOX Drug Screen Assay for Detection of 11 Drugs of Abuse and Therapeutic Drugs.

PubMed

Bang, Hae In; Jang, Mi Ae; Lee, Yong Wha

2017-11-01

The demand for rapid and broad clinical toxicology screens is on the rise. Recently, a new rapid toxicology screening test, the Triage TOX Drug Screen (Alere Inc., USA), which can simultaneously detect 11 drugs of abuse and therapeutic drugs with an instrument-read cartridge, was developed. In the present study, we evaluated the efficacy of this new on-site immunoassay using 105 urine specimens; the results were compared with those obtained by using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-TMS). Precision was evaluated according to the CLSI EP12-A2 for analyte concentrations near the cutoff, including C₅₀ and±30% of C₅₀, for each drug using standard materials. The C₅₀ specimens yielded 35-65% positive results and the±30% concentration range of all evaluated drugs encompassed the C₅-C₉₅ interval. The overall percent agreement of the Triage TOX Drug Screen was 92.4-100% compared with UPLC-TMS; however, the Triage TOX Drug Screen results showed some discordant cases including acetaminophen, amphetamine, benzodiazepine, opiates, and tricyclic antidepressants. The overall performance of the Triage TOX Drug Screen assay was comparable to that of UPLC-TMS for screening of drug intoxication in hospitals. This assay could constitute a useful screening method for drugs of abuse and therapeutic drugs in urine. © The Korean Society for Laboratory Medicine.

Disposable inkjet-printed electrochemical platform for detection of clinically relevant HER-2 breast cancer biomarker.

PubMed

Carvajal, Susanita; Fera, Samantha N; Jones, Abby L; Baldo, Thaisa A; Mosa, Islam M; Rusling, James F; Krause, Colleen E

2018-05-01

Rapidly fabricated, disposable sensor platforms hold tremendous promise for point-of-care detection. Here, we present an inexpensive (< $0.25) fully inkjet printed electrochemical sensor with integrated counter, reference, and working electrodes that is easily scalable for commercial fabrication. The electrochemical sensor platform featured an inkjet printed gold working 8-electrode array (WEA) and counter electrode (CE), along with an inkjet -printed silver electrode that was chlorinated with bleach to produce a Ag/AgCl quasi-reference electrode (RE). As proof of concept, the electrochemical sensor was successfully applied for detection of clinically relevant breast cancer biomarker Human Epidermal Growth Factor Receptor 2 (HER-2). Capture antibodies were bound to a chemically modified surface on the WEA and placed into a microfluidic device. A full sandwich immunoassay was constructed following a simultaneous injection of target protein, biotinylated antibody, and polymerized horseradish peroxide labels into the microfluidic device housing the WEA. With an ultra fast assay time, of only 15mins a clinically relevant limit of detection of 12pgmL -1 was achieved. Excellent reproducibility and sensitivity were observed through recovery assays preformed in human serum with recoveries ranging from 76% to 103%. These easily fabricated and scalable electrochemical sensor platforms can be readily adapted for multiplex detection following this rapid assay protocol for cancer diagnostics. Copyright © 2018 Elsevier B.V. All rights reserved.

A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

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

McNally, N.; Liu, Xiang Yang; Choudary, P.V.

1997-01-01

The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also bemore » used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.« less

Optimized MOL-PCR for Characterization of Microbial Pathogens.

PubMed

Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

2016-01-06

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium. Copyright © 2016 John Wiley & Sons, Inc.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

PubMed

Kamei, Kazumasa; Kawabata, Hiroki; Asakura, Masahiro; Samosornsuk, Worada; Hinenoya, Atsushi; Nakagawa, Shinsaku; Yamasaki, Shinji

2016-05-20

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Lung adenocarcinoma in the era of targeted therapies: histological classification, sample prioritization, and predictive biomarkers.

PubMed

Conde, E; Angulo, B; Izquierdo, E; Paz-Ares, L; Belda-Iniesta, C; Hidalgo, M; López-Ríos, F

2013-07-01

The arrival of targeted therapies has presented both a conceptual and a practical challenge in the treatment of patients with advanced non-small cell lung carcinomas (NSCLCs). The relationship of these treatments with specific histologies and predictive biomarkers has made the handling of biopsies the key factor for success. In this study, we highlight the balance between precise histological diagnosis and the practice of conducting multiple predictive assays simultaneously. This can only be achieved where there is a commitment to multidisciplinary working by the tumor board to ensure that a sensible protocol is applied. This proposal for prioritizing samples includes both recent technological advances and the some of the latest discoveries in the molecular classification of NSCLCs.

Hybrid nanosensor for colorimetric and ultrasensitive detection of nuclease contaminations

NASA Astrophysics Data System (ADS)

Cecere, Paola; Valentini, Paola; Pompa, Pier Paolo

2016-04-01

Nucleases are ubiquitous enzymes that degrade DNA or RNA, thus they can prejudice the good outcome of molecular biology experiments involving nucleic acids. We propose a colorimetric test for the naked-eye detection of nuclease contaminations. The system uses an hybrid nanosensor, based on gold nanoparticles functionalized with DNA probes. Our assay is rapid, instrument-free, simple and low-cost. Moreover, it reaches sensitivity equal or better than those of commercial kits, and presents a lot of advantageous aspects. Therefore, it is very competitive, with a real market potential. This test will be relevant in routine process monitoring in scientific laboratories, and in quality control in clinical laboratories and industrial processes, allowing the simultaneous detection of nucleases with different substrate specificities and large-scale screening.

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

PubMed

Mekuria, Genet; Ramesh, Sunita A; Alberts, Evita; Bertozzi, Terry; Wirthensohn, Michelle; Collins, Graham; Sedgley, Margaret

2003-12-01

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

Comparison of five assays for detection of Clostridium difficile toxin.

PubMed

Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

2011-07-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Comparison of Five Assays for Detection of Clostridium difficile Toxin

PubMed Central

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay.

PubMed

Del Prete, Raffaele; Di Taranto, Anna Maria; Lipsi, Maria Rosaria; Natalicchio, Maria Iole; Antonetti, Raffaele; Miragliotta, Giuseppe

2009-04-01

The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.

Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples▿

PubMed Central

Hill, Vincent R.; Kahler, Amy M.; Jothikumar, Narayanan; Johnson, Trisha B.; Hahn, Donghyun; Cromeans, Theresa L.

2007-01-01

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for φX174 bacteriophage, MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts, respectively. Real-time PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance. PMID:17483281

Sensitivity and Specificity of Human Immunodeficiency Virus Rapid Serologic Assays and Testing Algorithms in an Antenatal Clinic in Abidjan, Ivory Coast

PubMed Central

Koblavi-Dème, Stéphania; Maurice, Chantal; Yavo, Daniel; Sibailly, Toussaint S.; N′guessan, Kabran; Kamelan-Tano, Yvonne; Wiktor, Stefan Z.; Roels, Thierry H.; Chorba, Terence; Nkengasong, John N.

2001-01-01

To evaluate serologic testing algorithms for human immunodeficiency virus (HIV) based on a combination of rapid assays among persons with HIV-1 (non-B subtypes) infection, HIV-2 infection, and HIV-1–HIV-2 dual infections in Abidjan, Ivory Coast, a total of 1,216 sera with known HIV serologic status were used to evaluate the sensitivity and specificity of four rapid assays: Determine HIV-1/2, Capillus HIV-1/HIV-2, HIV-SPOT, and Genie II HIV-1/HIV-2. Two serum panels obtained from patients recently infected with HIV-1 subtypes B and non-B were also included. Based on sensitivity and specificity, three of the four rapid assays were evaluated prospectively in parallel (serum samples tested by two simultaneous rapid assays) and serial (serum samples tested by two consecutive rapid assays) testing algorithms. All assays were 100% sensitive, and specificities ranged from 99.4 to 100%. In the prospective evaluation, both the parallel and serial algorithms were 100% sensitive and specific. Our results suggest that rapid assays have high sensitivity and specificity and, when used in parallel or serial testing algorithms, yield results similar to those of enzyme-linked immunosorbent assay-based testing strategies. HIV serodiagnosis based on rapid assays may be a valuable alternative in implementing HIV prevention and surveillance programs in areas where sophisticated laboratories are difficult to establish. PMID:11325995

Validation of an Immunodiagnostic Assay for Detection of 13 Streptococcus pneumoniae Serotype-Specific Polysaccharides in Human Urine

PubMed Central

Huijts, Susanne M.; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C.; Bonten, Marc J. M.; Jansen, Kathrin U.

2012-01-01

To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults. PMID:22675155

Validation of an immunodiagnostic assay for detection of 13 Streptococcus pneumoniae serotype-specific polysaccharides in human urine.

PubMed

Pride, Michael W; Huijts, Susanne M; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C; Bonten, Marc J M; Jansen, Kathrin U

2012-08-01

To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults.

High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions

PubMed Central

Li, Guannan; Huang, Ke; Nikolic, Dejan

2015-01-01

Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry–based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography–tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. PMID:26285764

Simultaneous measurement of proguanil and its metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography, and its preliminary application in relation to genetically determined S-mephenytoin 4'-hydroxylation status.

PubMed

Kusaka, M; Setiabudy, R; Chiba, K; Ishizaki, T

1996-02-01

A simple high-performance liquid chromatographic (HPLC) assay method was developed for the measurement of proguanil (PG) and its major metabolites, cycloguanil (CG) and 4-chlorophenyl-biguanide (CPB), in human plasma and urine. The assay allowed the simultaneous determination of all analytes in 1 ml of plasma or 0.1 ml of urine. The detection limits of PG, CG, and CPB, defined as the signal-to-noise ratio of 3, were 1 and 5 ng/ml for plasma and urine samples, respectively. Recoveries of the analytes and the internal standard (pyrimethamine) were > 62% from plasma and > 77% from urine. Intra-assay and interassay coefficients of variation for all analytes in plasma and urine were < 10% except for the values of CG and CPB, which ranged from 10% to 15% at one or two concentrations among 4-5 concentrations studied. The clinical applicability of the method was assessed by the preliminary pharmacokinetic study of PG, CG, and CPB in six healthy volunteers with the individually known phenotypes (extensive and poor metabolizers) of S-mephenytoin 4'-hydroxylation, suggesting that individuals with a poor metabolizer phenotype of S-mephenytoin have a much lower capacity to bioactivate PG to CG compared with the extensive metabolizers.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

PubMed

Miyaoka, Yuichiro; Berman, Jennifer R; Cooper, Samantha B; Mayerl, Steven J; Chan, Amanda H; Zhang, Bin; Karlin-Neumann, George A; Conklin, Bruce R

2016-03-31

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Simultaneous Assay of Isotopic Enrichment and Concentration of Guanidinoacetate and Creatine by Gas Chromatography-Mass Spectrometry

PubMed Central

Kasumov, Takhar; Gruca, Lourdes L.; Dasarathy, Srinivasan; Kalhan, Satish C.

2012-01-01

A gas chromatographic-mass spectrometric (GC-MS) method for the simultaneous measurement of isotopic enrichment and concentration of guanidinoacetic acid and creatine in plasma sample for kinetic studies is reported. The method, based on preparation of the bis(trifluoromethyl)-pyrimidine methyl ester derivatives of guanidinoacetic acid and creatine, is robust and sensitive. The lowest measurable m1 and m3 enrichment for guanidinoacetic acid and creatine, respectively, was 0.3%. The calibration curves for measurements of concentration were linear over a range of 0.5-250 μM guanidinoacetic acid and 2-500 μM for creatine. The method was reliable for inter-assay and intra-assay precision, accuracy and linearity. The technique was applied in a healthy adult to determine in vivo fractional synthesis rate of creatine using primed- constant rate infusion of [1-13C]glycine. It was found that isotopic enrichment of guanidinoacetic acid reached plateau by 30 min of infusion of [1-13C]glycine, indicating either a small pool size or a rapid turnover rate or both, of guanidinoacetic acid. In contrast, tracer appearance in creatin was slow (slope: 0.00097), suggesting a large pool size and a slow rate of synthesis of creatine. This method can be used to estimate rate of synthesis of creatine in-vivo in human and animal studies. PMID:19646413

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

PubMed

Zhang, Yuyao; Ma, Xiuli; Huang, Bing; Li, Yufeng; Yu, Kexiang; Li, Jianliang; Liu, Cunxia; Han, Hongyu; Cui, Yanshun

2015-04-04

To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

In vivo combination of misonidazole and the chemotherapeutic agent CCNU.

PubMed Central

Siemann, D. W.

1981-01-01

The response of intramuscularly growing KHT sarcomas to the chemotherapeutic agent (1-(2-cloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) alone or simultaneously with the chemical radio-sensitizer misonidazole (MISO) was assessed using either a tumour growth-delay assay or an in vivo-in vitro tumour-excision assay. Median tumour growth delay following the combination of 20 mg/kg CCNU and either 0.5 or 1.0 mg/g MISO was 19.5 and 21.5 days, compared to 10 days for this CCNU dose alone. A similar degree of enhanced tumour response by MISO (factor of approximately 2 in tumour growth delay) was seen in RIF-1 tumours treated with 20 mg/kg CCNU plus 1.0 mg/g MISO. Clonogenic cell-survival studies with KHT sarcomas demonstrated that MISO at doses of 0.25, 0.5 or 1.0 mg/g given simultaneously with a range of CCNU doses produced dose-modifying factors (DMFs) of 1.9, 2.1 and 2.4 respectively. Normal tissue toxicity assessed by an LD50/7 assay led to DMFs of 1.2 and 1.4 for CCNU doses combined with 0.5 and 1.0 mg/g MISO. Thus in this animal tumour model the combination of CCNU and MISO appears to lead to a potential gain by a factor of approximately 1.7. PMID:7225287

Anaplasma marginale and A. phagocytophilum in cattle in Tunisia.

PubMed

M'ghirbi, Youmna; Bèji, Marwa; Oporto, Beatriz; Khrouf, Fatma; Hurtado, Ana; Bouattour, Ali

2016-10-20

Tick-borne diseases caused by Anaplasma species put serious constraints on the health and production of domestic cattle in tropical and sub-tropical regions. After recovering from a primary infection, cattle typically become persistent carriers of pathogens and play a critical role in the epidemiology of the disease, acting as reservoirs of the Anaplasma spp. In this study a duplex PCR assay was used for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle using two primer pairs targeting msp4 and msp2 genes, respectively. We used this method to analyze DNA preparations derived from 328 blood cattle samples that were collected from 80 farms distributed among Tunisia's four bioclimatic zones. The prevalence of the A. marginale infection (24.7 %) was significantly higher and more widespread (in all bioclimatic areas) than that of A. phagocytophilum (0.6 %), which was found in a mixed infection with A. marginale. The duplex PCR assay used proved to be a rapid, specific and inexpensive mean for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle blood. It allowed us to report the identification of A. phagocytophilum for the first time in cattle in Tunisia and confirm the presence of A. marginale in cattle from several geographical areas of the country. Further epidemiological studies undertaken using this assay will help improve the surveillance of the associated diseases in the regions where they are endemic.

Simultaneous ultramicroanalysis of both 17-keto-and 17beta-hydroxy androgens in biological fluids.

PubMed

Ganjam, V K

1976-11-01

Sensitive methods for quantifying androgens were lacking. Therefore, a relatively simple procedure for separating steroids was combined with highly specific assay methods so that eight androgens could be measured with high accuracy, precision and sensitivity. Semi-automated separations on Sephadex LH-20 columns used heptane:methylene chloride:ethanol:water (50:50:1:0.12) and a flow rate of 17.0 min/ml. The six peaks eluted contained androstenedine; androsterone, epiandrosterone and dihydrotestosterone; testosterone and dehydroepiandrosterone; 3alpha-androstanediol; 3beta-androstanediol; and androstenediol. Androstenedione, dehydroepiandrosterone and androstenediol were quantified using specific antisera (sensitivity less than or equal to 75 pg). Testosterone and dihydrotestosterone were measured by competitive protein-binding assays using rabbit TeBG (sensitivity less than or equal to 150 pg). 3alpha- and 3beta-androstanediol were similarly assayed using human TeBG (sensitivity approximately 150 pg). Androsterone was reduced with NaBH4 and the resulting 3alpha-androstanediol was assayed using human TeBG (sensitivity approximately 200 pg). Inter- and intra-assay variations were less than 10% for radioimmunoassays and less than 16% for competitive protein-binding assays over the entire dose response curve.

Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination.

PubMed

Libert, Xavier; Packeu, Ann; Bureau, Fabrice; Roosens, Nancy H; De Keersmaecker, Sigrid C J

2017-01-01

Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.

A microfluidic device integrating dual CMOS polysilicon nanowire sensors for on-chip whole blood processing and simultaneous detection of multiple analytes.

PubMed

Kuan, Da-Han; Wang, I-Shun; Lin, Jiun-Rue; Yang, Chao-Han; Huang, Chi-Hsien; Lin, Yen-Hung; Lin, Chih-Ting; Huang, Nien-Tsu

2016-08-02

The hemoglobin-A1c test, measuring the ratio of glycated hemoglobin (HbA1c) to hemoglobin (Hb) levels, has been a standard assay in diabetes diagnosis that removes the day-to-day glucose level variation. Currently, the HbA1c test is restricted to hospitals and central laboratories due to the laborious, time-consuming whole blood processing and bulky instruments. In this paper, we have developed a microfluidic device integrating dual CMOS polysilicon nanowire sensors (MINS) for on-chip whole blood processing and simultaneous detection of multiple analytes. The micromachined polymethylmethacrylate (PMMA) microfluidic device consisted of a serpentine microchannel with multiple dam structures designed for non-lysed cells or debris trapping, uniform plasma/buffer mixing and dilution. The CMOS-fabricated polysilicon nanowire sensors integrated with the microfluidic device were designed for the simultaneous, label-free electrical detection of multiple analytes. Our study first measured the Hb and HbA1c levels in 11 clinical samples via these nanowire sensors. The results were compared with those of standard Hb and HbA1c measurement methods (Hb: the sodium lauryl sulfate hemoglobin detection method; HbA1c: cation-exchange high-performance liquid chromatography) and showed comparable outcomes. Finally, we successfully demonstrated the efficacy of the MINS device's on-chip whole blood processing followed by simultaneous Hb and HbA1c measurement in a clinical sample. Compared to current Hb and HbA1c sensing instruments, the MINS platform is compact and can simultaneously detect two analytes with only 5 μL of whole blood, which corresponds to a 300-fold blood volume reduction. The total assay time, including the in situ sample processing and analyte detection, was just 30 minutes. Based on its on-chip whole blood processing and simultaneous multiple analyte detection functionalities with a lower sample volume requirement and shorter process time, the MINS device can be effectively applied to real-time diabetes diagnostics and monitoring in point-of-care settings.

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

PubMed

Benitez, Alvaro J; Winchell, Jonas M

2016-04-01

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

Advances in tuberculosis diagnostics: the Xpert MTB/RIF assay and future prospects for a point-of-care test

PubMed Central

Lawn, Stephen D; Mwaba, Peter; Bates, Matthew; Piatek, Amy; Alexander, Heather; Marais, Ben J; Cuevas, Luis E; McHugh, Timothy D; Zijenah, Lynn; Kapata, Nathan; Abubakar, Ibrahim; McNerney, Ruth; Hoelscher, Michael; Memish, Ziad A; Migliori, Giovanni Battista; Kim, Peter; Maeurer, Markus; Schito, Marco; Zumla, Alimuddin

2015-01-01

Rapid progress has been made in the development of new diagnostic assays for tuberculosis in recent years. New technologies have been developed and assessed, and are now being implemented. The Xpert MTB/RIF assay, which enables simultaneous detection of Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance, was endorsed by WHO in December, 2010. This assay was specifically recommended for use as the initial diagnostic test for suspected drug-resistant or HIV-associated pulmonary tuberculosis. By June, 2012, two-thirds of countries with a high tuberculosis burden and half of countries with a high multidrug-resistant tuberculosis burden had incorporated the assay into their national tuberculosis programme guidelines. Although the development of the Xpert MTB/RIF assay is undoubtedly a landmark event, clinical and programmatic effects and cost-effectiveness remain to be defined. We review the rapidly growing body of scientific literature and discuss the advantages and challenges of using the Xpert MTB/RIF assay in areas where tuberculosis is endemic. We also review other prospects within the developmental pipeline. A rapid, accurate point-of-care diagnostic test that is affordable and can be readily implemented is urgently needed. Investment in the tuberculosis diagnostics pipeline should remain a major priority for funders and researchers. PMID:23531388

Novel Surface-Enhanced Raman Scattering-based Assays for Ultra-sensitive Detection of Human Pluripotent Stem Cells

PubMed Central

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O.; Nie, Shuming; Xu, Chunhui

2017-01-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 106 cells, a sensitivity (0.0001%) which was ~2,000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5+ and TRA-1-60+ cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. PMID:27509304

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

PubMed

Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

2013-10-30

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Development and Validation of Chemometric Spectrophotometric Methods for Simultaneous Determination of Simvastatin and Nicotinic Acid in Binary Combinations.

PubMed

Alahmad, Shoeb; Elfatatry, Hamed M; Mabrouk, Mokhtar M; Hammad, Sherin F; Mansour, Fotouh R

2018-01-01

The development and introduction of combined therapy represent a challenge for analysis due to severe overlapping of their UV spectra in case of spectroscopy or the requirement of a long tedious and high cost separation technique in case of chromatography. Quality control laboratories have to develop and validate suitable analytical procedures in order to assay such multi component preparations. New spectrophotometric methods for the simultaneous determination of simvastatin (SIM) and nicotinic acid (NIA) in binary combinations were developed. These methods are based on chemometric treatment of data, the applied chemometric techniques are multivariate methods including classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS). In these techniques, the concentration data matrix were prepared by using the synthetic mixtures containing SIM and NIA dissolved in ethanol. The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbance at 12 wavelengths in the range 216 - 240 nm at 2 nm intervals in the zero-order. The spectrophotometric procedures do not require any separation step. The accuracy, precision and the linearity ranges of the methods have been determined and validated by analyzing synthetic mixtures containing the studied drugs. Chemometric spectrophotometric methods have been developed in the present study for the simultaneous determination of simvastatin and nicotinic acid in their synthetic binary mixtures and in their mixtures with possible excipients present in tablet dosage form. The validation was performed successfully. The developed methods have been shown to be accurate, linear, precise, and so simple. The developed methods can be used routinely for the determination dosage form. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Evaluation of Agaricus blazei in vivo for antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities.

PubMed

Ishii, Priscila Lumi; Prado, Carolina Kato; Mauro, Mariana de Oliveira; Carreira, Clísia Mara; Mantovani, Mário Sérgio; Ribeiro, Lúcia Regina; Dichi, Jane Bandeira; Oliveira, Rodrigo Juliano

2011-04-01

The development of various types of cancer results from the interaction among endogenous, environmental and hormonal factors, where the most notable of these factors is diet. The aim of the present study was to determine the antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities of Agaricus blazei. The test antigenotoxicity (Comet Assay) and anticarcinogenic (Test of Aberrant Crypt Foci) assess changes in DNA and/or intestinal mucosa that correlate to cancer development. Tests of phagocytosis in the spleen and differential count in blood cells allow the inference of modulation of the immune system as well as to propose a way of eliminating cells with DNA damage. Supplementation with the mushroom was carried out under pre-treatment, simultaneous treatment, post-treatment and pre-treatment+continuous conditions. Statistical analysis demonstrated that the mushroom did not have genotoxic activity but showed antigenotoxic activity. Supplementation caused an increase in the number of monocytes and in phagocytic activity, suggesting that supplementation increases a proliferation of monocytes, consequently increasing phagocytic capacity especially in the groups pre-treatment, simultaneous and pre-treatment+continuous. The data suggest that A. blazei could act as a functional food capable of promoting immunomodulation which can account for the destruction of cells with DNA alterations that correlate with the development of cancer, since this mushroom was demonstrated to have a preventive effect against pre-neoplastic colorectal lesions evaluated by the aberrant crypt foci assay. According to these results and the literature, it is believed that supplementation with A. blazei can be an efficient method for the prevention of cancer as well as possibly being an important coadjuvant treatment in chemotherapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Development and validation of an HPLC-DAD method for simultaneous determination of cocaine, benzoic acid, benzoylecgonine and the main adulterants found in products based on cocaine.

PubMed

Floriani, Gisele; Gasparetto, João Cleverson; Pontarolo, Roberto; Gonçalves, Alan Guilherme

2014-02-01

Here, an HPLC-DAD method was developed and validated for simultaneous determination of cocaine, two cocaine degradation products (benzoylecgonine and benzoic acid), and the main adulterants found in products based on cocaine (caffeine, lidocaine, phenacetin, benzocaine and diltiazem). The new method was developed and validated using an XBridge C18 4.6mm×250mm, 5μm particle size column maintained at 60°C. The mobile phase consisted of a gradient of acetonitrile and ammonium formate 0.05M - pH 3.1, eluted at 1.0mL/min. The volume of injection was 10μL and the DAD detector was set at 274nm. Method validation assays demonstrated suitable sensitivity, selectivity, linearity, precision and accuracy. For selectivity assay, a MS detection system could be directly adapted to the method without the need of any change in the chromatographic conditions. The robustness study indicated that the flow rate, temperature and pH of the mobile phase are critical parameters and should not be changed considering the conditions herein determined. The new method was then successfully applied for determining cocaine, benzoylecgonine, benzoic acid, caffeine, lidocaine, phenacetin, benzocaine and diltiazem in 115 samples, seized in Brazil (2007-2012), which consisted of cocaine paste, cocaine base and salt cocaine samples. This study revealed cocaine contents that ranged from undetectable to 97.2%, with 97 samples presenting at least one of the degradation products or adulterants here evaluated. All of the studied degradation products and adulterants were observed among the seized samples, justifying the application of the method, which can be used as a screening and quantification tool in forensic analysis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Suspected hypoglycaemia in out patient practice: accuracy of dried blood spot analysis.

PubMed

Parker, D R; Bargiota, A; Cowan, F J; Corrall, R J

1997-12-01

The assay of dried blood spots on filter paper to determine blood glucose concentration has been used to detect hypoglycaemia in out patients. We assessed the accuracy of this approach in assaying blood glucose concentrations in the hypoglycaemic range. Volunteers were rendered hypoglycaemic by intravenous infusion of insulin. The glucose concentration in simultaneously taken blood samples was measured either fresh or after drying on filter paper. Twenty-four healthy young volunteers and 9 patients with insulin-dependent diabetes were studied. Plasma glucose concentrations were measured using a standard auto analyser glucose oxidase method. Whole blood taken simultaneously was placed on prepared filter paper and allowed to dry; glucose concentration was then measured using a well-established technique. A correction factor was applied to convert the glucose concentration of plasma to that of whole blood. The relationship between glucose concentrations measured by the two methods was determined by regression coefficient. In the unequivocally hypoglycaemic range (plasma < or = 2.5 mmol/l), corrected dried blood spot glucose concentrations significantly correlated with standard plasma glucose concentrations (r = 0.81; P < 0.001). The dried blood spot method had a sensitivity of 91%. In the range designated probable hypoglycaemia (plasma < or = 3.3 mmol/l), there was also significant correlation (r = 0.90; P < 0.001) and the sensitivity was 96%. The specificity of the dried blood spot method was 100% in both ranges. Measurement of glucose concentrations in dried blood spots is specific and sensitive in the hypoglycaemic range. The present study indicates that hypoglycaemia may be excluded or confirmed respectively when levels in excess of 3.7 or below 2.8 mmol/l are found in uncorrected dried blood spot analysis.

Specific targeting and noninvasive magnetic resonance imaging of an asthma biomarker in the lung using polyethylene glycol functionalized magnetic nanocarriers.

PubMed

Al Faraj, Achraf; Shaik, Asma Sultana; Afzal, Sibtain; Al-Muhsen, Saleh; Halwani, Rabih

2016-05-01

Simultaneous inhibition of IL4 and IL13 via the common receptor chain IL4Rα to block adequately their biologic effects presents a promising therapeutic approach to give the additional relief required for asthma patients. In this study, superparamagnetic iron oxide nanoparticles were conjugated with anti-IL4Rα blocking antibodies via polyethylene glycol (PEG) polymers. The delivery of these blocking antibodies to the inflammatory sites in the lung via the developed nanocarriers was assessed using noninvasive free-breathing pulmonary MRI. Biocompatibility assays confirmed the safety of the developed nanocarriers for pre-clinical investigations. For all the investigated formulations, nanocarriers were found to be very stable at neutral pH. However, the stability noticeably decreased with the PEG length in acidic environment and thus the loaded antibodies were preferentially released. Immunofluorescence and fluorimetry assays confirmed the binding of the nanocarriers to the IL4Rα asthma biomarker. Pulmonary MRI performed using an ultra-short echo time sequence allowed simultaneous noninvasive monitoring of inflammatory responses induced by ovalbumin challenge and tracking of the developed nanocarriers, which were found to colocalize with the inflammatory sites in the lung. Targeting of the developed nanocarriers to areas rich in IL4Rα positive inflammatory cells was confirmed using histological and flow cytometry analyses. The anti-IL4Rα-conjugated nanocarriers developed here have been confirmed to be efficient in targeting key inflammatory cells during chronic lung inflammation following intrapulmonary administration. Targeting efficiency was monitored using noninvasive MRI, allowing detection of the nanocarriers' colocalizations with the inflammatory sites in the lung of ovalbumin-challenged asthmatic mice. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Development of a new multi-analyte assay for the simultaneous detection of opioids in serum and other body fluids using liquid chromatography-tandem mass spectrometry.

PubMed

Eckart, K; Röhrich, J; Breitmeier, D; Ferner, M; Laufenberg-Feldmann, R; Urban, R

2015-09-15

A liquid chromatography-tandem mass spectrometry method using electrospray ionization in positive ionization mode was developed for the simultaneous detection of multiple opioid-type drugs in plasma. The presented assay allows the quantitative determination of alfentanil, buprenorphine, codeine, desomorphine, dextromethorphan, dextrorphan, dihydrocodeine, dihydromorphine, ethylmorphine, fentanyl, hydrocodone, hydromorphone, methadone, morphine, naloxone, naltrexone, oxycodone, oxymorphone, pentazocine, pethidine, pholcodine, piritramide, remifentanil, sufentanil, and tramadol as well as the metabolites 6-monoacetylmorphine, bisnortilidine, morphine-3-glucuronide, morphine-6-glucuronide, naltrexol, norbuprenorphine, norfentanyl, norpethidine, nortilidine, and O-desmethyltramadol. Serum and blood samples were purified by solid-phase extraction. The analytes were separated on a phenyl-hexyl (100mm) column by formic acid/acetonitrile gradient elution using an UPLC 1290 Infinity coupled with a 6490 Triple Quadrupole mass spectrometer. The limits of detection ranged from 0.02 to 0.6ng/mL and the lower limits of quantification ranged from 0.1 to 2.0ng/mL. The calibration curves were linear between Calibration Levels 1-6 for all 35 substances. Recovery rates ranged between 51 and 88% for all compounds except alfentanil, bisnortilidine, pethidine, and morphine-3-glucuronide. The matrix effect ranged from 86% for ethylmorphine to 105% for desomorphine. Using the validation procedure proposed by the German Society of Toxicological and Forensic Chemistry, acceptable precision and accuracy data for almost all analytes were obtained. The method was successfully applied to 206 authentic serum samples provided by the palliative and intensive care units of the University Medical Center and the police authorities. Furthermore, a suspected fatal intoxication is demonstrated by an analysis of the sufentanil in post mortem body fluids and tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

Lab-On-a-Chip for carbon nanotubes based immunoassay detection of Staphylococcal Enterotoxin B (SEB).

PubMed

Yang, Minghui; Sun, Steven; Kostov, Yordan; Rasooly, Avraham

2010-04-21

We describe a new eight channel Lab-On-a-Chip (LOC) for a Carbon Nanotube (CNT) based immunoassay with optical detection of Staphylococcal Enterotoxin B (SEB) for food safety applications. In this work, we combined four biosensing elements: (1) CNT technology for primary antibody immobilization, (2) Enhanced Chemiluminescence (ECL) for light signal generation, (3) a cooled charge-coupled device (CCD) for detection and (4) polymer lamination technology for developing a point of care immunological assay for SEB detection. Our concept for developing versatile LOCs, which can be used for many different applications, is to use a modular design with interchangeable recognition elements (e.g. various antibodies) to determine the specificity. Polymer lamination technology was used for the fabrication of a six layer, syringe operated LOC capable of analyzing eight samples simultaneously. An anti-SEB antibody-nanotube mixture was immobilized onto a polycarbonate strip, to serve as an interchangeable ligand surface that was then bonded onto the LOC. SEB samples are loaded into the device and detected by an ELISA assay using Horse Radish Peroxidase (HRP) conjugated anti-SEB IgG as a secondary antibody and ECL, with detection by a previously described portable cooled CCD detector. Eight samples of SEB in buffer or soy milk were assayed simultaneously with a limit of detection of 0.1 ng mL(-1). CNT immobilization of the antibody increased the sensitivity of detection six fold. Use of a simple interchangeable immunological surface allows this LOC to be adapted to any immunoassay by simply replacing the ligand surface. A syringe was used to move fluids for this assay so no power is needed to operate the device. Our versatile portable point-of-care CCD detector combined with the LOC immunoassay method described here can be used to reduce the exposure of users to toxins and other biohazards when working outside the lab, as well as to simplify and increase sensitivity for many other types of immunological diagnostics and detection assays.

Simultaneous determination of nikethamide and lidocaine in human blood and cerebrospinal fluid by high performance liquid chromatography.

PubMed

Chen, Lili; Liao, Linchuan; Zuo, Zhong; Yan, Youyi; Yang, Lin; Fu, Qiang; Chen, Yu; Hou, Junhong

2007-04-11

Nikethamide and lidocaine are often requested to be quantified simultaneously in forensic toxicological analysis. A simple reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for their simultaneous determination in human blood and cerebrospinal fluid. The method involves simple protein precipitation sample treatment followed by quantification of analytes using HPLC at 263 nm. Analytes were separated on a 5 microm Zorbax Dikema C18 column (150 mm x 4.60 mm, i.d.) with a mobile phase of 22:78 (v/v) mixture of methanol and a diethylamine-acetic acid buffer, pH 4.0. The mean recoveries were between 69.8 and 94.4% for nikethamide and between 78.9 and 97.2% for lidocaine. Limits of detection (LODs) for nikethamide and lidocaine were 0.008 and 0.16 microg/ml in plasma and 0.007 and 0.14 microg/ml in cerebrospinal fluid, respectively. The mean intra-assay and inter-assay coefficients of variation (CVs) for both analytes were less than 9.2 and 10.8%, respectively. The developed method was applied to blood sample analyses in eight forensic cases, where blood concentrations of lidocaine ranged from 0.68 to 34.4 microg/ml and nikethamide ranged from 1.25 to 106.8 microg/ml. In six cases cerebrospinal fluid analysis was requested. The values ranged from 20.3 to 185.6 microg/ml of lidocaine and 8.0 to 72.4 microg/ml of nikethamide. The method is simple and sensitive enough to be used in toxicological analysis for simultaneous determination of nikethamide and lidocaine in blood and cerebrospinal fluid.

Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS.

PubMed

Blue, Steven W; Winchell, Andrea J; Kaucher, Amy V; Lieberman, Rachel A; Gilles, Christopher T; Pyra, Maria N; Heffron, Renee; Hou, Xuanlin; Coombs, Robert W; Nanda, Kavita; Davis, Nicole L; Kourtis, Athena P; Herbeck, Joshua T; Baeten, Jared M; Lingappa, Jairam R; Erikson, David W

2018-04-01

The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples. We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5). Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC. We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum. This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing. Copyright © 2018 Elsevier Inc. All rights reserved.

Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis.

PubMed

Martinez, Andres W; Phillips, Scott T; Carrilho, Emanuel; Thomas, Samuel W; Sindi, Hayat; Whitesides, George M

2008-05-15

This article describes a prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial urine. The combination of patterned paper, a portable method for obtaining digital images, and a method for exchanging results of the assays with off-site diagnosticians offers new opportunities for inexpensive monitoring of health, especially in situations that require physicians to travel to patients (e.g., in the developing world, in emergency management, and during field operations by the military) to obtain diagnostic information that might be obtained more effectively by less valuable personnel.

Performance evaluation of the Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA, a 4th generation HIV assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma.

PubMed

Bentsen, Christopher; McLaughlin, Lisa; Mitchell, Elizabeth; Ferrera, Carol; Liska, Sally; Myers, Robert; Peel, Sheila; Swenson, Paul; Gadelle, Stephane; Shriver, M Kathleen

2011-12-01

A multi-center study was conducted to evaluate the Bio-Rad GS HIV Combo Ag/Ab EIA, a 4th generation HIV-1/HIV-2 assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma in adult and pediatric populations. The objectives of the study were to assess assay performance for the detection of acute HIV infections; sensitivity in known HIV positive samples; percent agreement with HIV status; specificity in low and high risk individuals of unknown HIV status; and to compare assay performance to a 3rd generation HIV assay. The evaluation included testing 9150 samples at four U.S. clinical trial sites, using three kit lots. Unlinked samples were from routine testing, repositories or purchased from vendors. GS HIV Combo Ag/Ab EIA detection in samples from individuals in two separate populations with acute HIV infection was 95.2% (20/21) and 86.4% (38/44). Sensitivity was 100% (1603/1603) in known antibody positive [HIV-1 Groups M and O, and HIV-2] samples. HIV p24 antigen detection was 100% (53/53) in HIV-1 culture supernatants. HIV-1 seroconversion panel detection improved by a range of 0-20 days compared to a 3rd generation HIV test. Specificity was 99.9% (5989/5996) in low risk, 99.9% (959/960) in high risk and 100% (100/100) in pediatric populations. The GS HIV Combo Ag/Ab EIA significantly reduced the diagnostic window when compared to the 3rd generation screening assay, enabling earlier diagnosis of HIV infection. The performance parameters of the Bio-Rad GS HIV Combo Ag/Ab EIA are well suited for use in HIV diagnostic settings. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine.

PubMed

Campbell, John P; Cobbold, Mark; Wang, Yanyun; Goodall, Margaret; Bonney, Sarah L; Chamba, Anita; Birtwistle, Jane; Plant, Timothy; Afzal, Zaheer; Jefferis, Roy; Drayson, Mark T

2013-05-31

Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated. Copyright © 2013 Elsevier B.V. All rights reserved.

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

PubMed

Erickson, A; Fisher, M; Furukawa-Stoffer, T; Ambagala, A; Hodko, D; Pasick, J; King, D P; Nfon, C; Ortega Polo, R; Lung, O

2018-04-01

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases. © 2017 Her Majesty the Queen in Right of Canada • Transboundary and Emerging Diseases.

Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.

PubMed

Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

2016-10-01

Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

A systems approach for analysis of high content screening assay data with topic modeling.

PubMed

Bisgin, Halil; Chen, Minjun; Wang, Yuping; Kelly, Reagan; Fang, Hong; Xu, Xiaowei; Tong, Weida

2013-01-01

High Content Screening (HCS) has become an important tool for toxicity assessment, partly due to its advantage of handling multiple measurements simultaneously. This approach has provided insight and contributed to the understanding of systems biology at cellular level. To fully realize this potential, the simultaneously measured multiple endpoints from a live cell should be considered in a probabilistic relationship to assess the cell's condition to response stress from a treatment, which poses a great challenge to extract hidden knowledge and relationships from these measurements. In this work, we applied a text mining method of Latent Dirichlet Allocation (LDA) to analyze cellular endpoints from in vitro HCS assays and related to the findings to in vivo histopathological observations. We measured multiple HCS assay endpoints for 122 drugs. Since LDA requires the data to be represented in document-term format, we first converted the continuous value of the measurements to the word frequency that can processed by the text mining tool. For each of the drugs, we generated a document for each of the 4 time points. Thus, we ended with 488 documents (drug-hour) each having different values for the 10 endpoints which are treated as words. We extracted three topics using LDA and examined these to identify diagnostic topics for 45 common drugs located in vivo experiments from the Japanese Toxicogenomics Project (TGP) observing their necrosis findings at 6 and 24 hours after treatment. We found that assay endpoints assigned to particular topics were in concordance with the histopathology observed. Drugs showing necrosis at 6 hour were linked to severe damage events such as Steatosis, DNA Fragmentation, Mitochondrial Potential, and Lysosome Mass. DNA Damage and Apoptosis were associated with drugs causing necrosis at 24 hours, suggesting an interplay of the two pathways in these drugs. Drugs with no sign of necrosis we related to the Cell Loss and Nuclear Size assays, which is suggestive of hepatocyte regeneration. The evidence from this study suggests that topic modeling with LDA can enable us to interpret relationships of endpoints of in vitro assays along with an in vivo histological finding, necrosis. Effectiveness of this approach may add substantially to our understanding of systems biology.

Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays

PubMed Central

Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

2012-01-01

Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

Detection of high-risk thrombophilia with an automated, global test: the Coagulation Inhibitor Potential assay.

PubMed

Réger, Barbara; Losonczy, Hajna; Nagy, Ágnes; Péterfalvi, Ágnes; Mózes, Réka; Pótó, László; Farkas, Nelli; Kovács, Gábor L; Miseta, Attila; Hussain, Alizadeh; Tóth, Orsolya

2018-05-17

: The diagnosis of thrombophilia is a cost-consuming and time-consuming process, as each defect should be separately investigated. The Coagulation Inhibitor Potential (CIP) assay is a promising new global test, sensitive for most of the hereditary thrombophilias, developed for manual methodology. We adapt the original method to an optical coagulation analyser. By this automation, the test will be easier, faster and more precise, and it also allows carrying out 18 measurements simultaneously. The CIP assay was performed in 126 healthy subjects and 193 patients with different types of hereditary thrombophilia conditions. Detected with conventional laboratory tests high-risk thrombophilia was present in 70 patients: deficiencies of antithrombin (AT) (n = 12), protein C (PC) (n = 14), protein S (PS) (n = 6), homozygous factor V Leiden (FVL) mutation (n = 9) and combined types (n = 29). Low-risk thrombophilia was present in 123 patients: heterozygous FVL (n = 115) and FII G2010A mutation (n = 8). Significantly lower median CIP values were found for AT-,PC-, PS deficiencies, homozygous and heterozygous FVL mutations and combined thrombophilias (P < 0.01) as compared with healthy controls. There was no significant difference between the heterozygous FIIG20210A (P = 0.669) thrombophilia group and the healthy controls. The best performance of the test was achieved at the cut-off value of 90.0 U (area: 0.981) with 96% sensitivity and 92% specificity in the high-risk thrombophilia group estimated by receiver operating characteristic analysis. The new method seems to be appropriate and reliable for the detection of AT-, PC- and PS deficiencies, homozygous FVL mutation and also for combined deficiencies. The automated CIP test is insensitive to FII G2010A mutation.

Sandia National Laboratories: Lighting up disease-carrying mosquitoes

Science.gov Websites

Sandia researchers added a different DNA fragment sequence called a quench probe that complements a short is so bright, QUASR can screen up to three different targets simultaneously, saving time and money international health emergency. "Conceptually, it's not difficult to adapt the assay for a different virus

Estimation of the Asymmetrical Arrangement of Plasma Membrane Aminophospholipids. An Experimental Assay for Students of Biochemistry and Molecular Biology.

ERIC Educational Resources Information Center

Sanchez-Yague, J.; And Others

1987-01-01

Describes an experiment to discover the topology of plasma membrane aminophospholipids (phosphatidylethanolamine and phosphatidylserine) using whole platelets and trinitrobenzene sulfonate (TNBS) as a probe. Reports changes in phospholipid distribution during platelet activation with simultaneous action of thrombia and collagen. Details the…

Evaluation of Fluorescence Microsphere Immunoassay for the Detection of Antibodies to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

USDA-ARS?s Scientific Manuscript database

Rift Valley Fever virus (RVFV) is a zoonotic virus that infects ruminants including cattle, sheep, goats, camels and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high throughput test for disease surveillance and vaccine eva...

Transcriptome amplification coupled with nanopore sequencing as a surveillance tool for plant pathogens in plant and insect tissues

USDA-ARS?s Scientific Manuscript database

There are many plant pathogen-specific diagnostic assays, based on PCR and immune-detection. However, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitat...

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

PubMed Central

Das, Sanchita; Rundell, Mark S.; Mirza, Aashiq H.; Pingle, Maneesh R.; Shigyo, Kristi; Garrison, Aura R.; Paragas, Jason; Smith, Scott K.; Olson, Victoria A.; Larone, Davise H.; Spitzer, Eric D.; Barany, Francis; Golightly, Linnie M.

2015-01-01

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus). PMID:26381398

A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.

PubMed

Kunthalert, Duangkamol; Henghiranyawong, Kritsada; Sistayanarain, Anchalee; Khoothiam, Krissana

2013-02-01

The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance. We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates. The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%. Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

PubMed

Das, Sanchita; Rundell, Mark S; Mirza, Aashiq H; Pingle, Maneesh R; Shigyo, Kristi; Garrison, Aura R; Paragas, Jason; Smith, Scott K; Olson, Victoria A; Larone, Davise H; Spitzer, Eric D; Barany, Francis; Golightly, Linnie M

2015-01-01

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

Development and validation of a novel stability-indicating HPLC method for the simultaneous assay of betamethasone-17-valerate, fusidic acid, potassium sorbate, methylparaben and propylparaben in a topical cream preparation.

PubMed

Byrne, Jonathan; Velasco-Torrijos, Trinidad; Reinhardt, Robert

2014-08-05

A novel stability-indicating reversed phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous assay of betamethasone-17-valerate, fusidic acid and potassium sorbate as well as methyl- and propylparaben in a topical cream preparation has been developed. A 100mm×3.0mm ID. Ascentis Express C18 column maintained at 30°C and UV detection at 240nm were used. A gradient programme was employed at a flow-rate of 0.75ml/min. Mobile phase A comprised of an 83:17 (v/v) mixture of acetonitrile and methanol and mobile phase B of a 10g/l solution of 85% phosphoric acid in purified water. The method has been validated according to current International Conference on Harmonisation (ICH) guidelines and applied during formulation development and stability studies. The procedure has been shown to be stability-indicating for the topical cream. Copyright © 2014 Elsevier B.V. All rights reserved.

High-sensitivity simultaneous liquid chromatography-tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma.

PubMed

Gandhi, Abhishek; Guttikar, Swati; Trivedi, Priti

2015-10-01

A sensitive and simultaneous liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of ethinyl estradiol and levonorgestrel. The analytes were extracted with methyl-tert-butyl ether: n-hexane (50:50, v/v) solvent mixture, followed by dansyl derivatization. The chromatographic separation was performed on a Kinetex C 18 (50 mm×4.6 mm, 2.6 µm) column with a mobile phase of 0.1% (v/v) formic acid in water and acetonitrile in gradient composition. The mass transitions were monitored in electrospray positive ionization mode. The assay exhibited a linear range of 0.100-20.0 ng/mL for levonorgestrel and 4.00-500 pg/mL for ethinyl estradiol in human plasma. A run time of 9.0 min for each sample made it possible to analyze a throughput of more than 100 samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic and bioequivalence studies.

Quantification of phenylethylamine and p-tyramine in rat tissues using a new radioenzymatic assay

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

Hamburger, S.A.; Henry, D.P.

Phenylethylamine (PEA) and p-tyramine (p-tym) are biologically active aralkylamines that are found in a number of mammalian tissues, including brain and plasma. The investigation of the biological role of these substances has been hampered by the lack of accessible assay methodology. They have developed a new radioenzymatic assay using barley root tyramine N-methyltransferase and tritiated S-adenosylmethionine. The products formed by the reaction are isolated by TLC. The assay sensitivity was 2.1 and 1.0 pg/tube for PEA and p-tym, respectively. The concentration of PEA and p-tym was determined simultaneously in tissues from Sprague-Dawley rats (280 gm). Plasma PEA and p-tym weremore » 478 +/- 66 and 309 +/- 69 pg/ml, respectively. They conclude that this new procedure is applicable to all tissues examined in that all tissues contain both PEA and p-tym and that these amines are heterogeneously distributed in rat tissues.« less

Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

PubMed Central

Olkhov-Mitsel, Ekaterina; Zdravic, Darko; Kron, Ken; van der Kwast, Theodorus; Fleshner, Neil; Bapat, Bharati

2014-01-01

Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings. PMID:24651255

Competitive fluorescent pseudo-immunoassay exploiting molecularly imprinted polymers for the detection of biogenic amines in fish matrix.

PubMed

Mattsson, Leena; Xu, Jingjing; Preininger, Claudia; Tse Sum Bui, Bernadette; Haupt, Karsten

2018-05-01

We developed a competitive fluorescent molecularly imprinted polymer (MIP) assay to detect biogenic amines in fish samples. MIPs synthesized by precipitation polymerization using histamine as template were used in a batch binding assay analogous to competitive fluoroimmunoassays. Introducing a complex sample matrix, such as fish extract, into the assay changes the environment and the binding conditions, therefore the importance of the sample preparation is extensively discussed. Several extraction and purification methods for fish were comprehensively studied, and an optimal clean-up procedure for fish samples using liquid-liquid extraction was developed. The feasibility of the competitive MIP assay was shown in the purified fish extract over a broad histamine range (1 - 430µM). The MIP had the highest affinity towards histamine, but recognized also the structurally similar biogenic amines tyramine and tryptamine, as well as spermine and spermidine, providing simultaneous analysis and assessment of the total amount of biogenic amines. Copyright © 2018 Elsevier B.V. All rights reserved.

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

PubMed

Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

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

Hornemann, Andrea, E-mail: andrea.hornemann@ptb.de; Hoehl, Arne, E-mail: arne.hoehl@ptb.de; Ulm, Gerhard, E-mail: gerhard.ulm@ptb.de

Bio-diagnostic assays of high complexity rely on nanoscaled assay recognition elements that can provide unique selectivity and design-enhanced sensitivity features. High throughput performance requires the simultaneous detection of various analytes combined with appropriate bioassay components. Nanoparticle induced sensitivity enhancement, and subsequent multiplexed capability Surface-Enhanced InfraRed Absorption (SEIRA) assay formats are fitting well these purposes. SEIRA constitutes an ideal platform to isolate the vibrational signatures of targeted bioassay and active molecules. The potential of several targeted biolabels, here fluorophore-labeled antibody conjugates, chemisorbed onto low-cost biocompatible gold nano-aggregates substrates have been explored for their use in assay platforms. Dried films were analyzedmore » by synchrotron radiation based FTIR/SEIRA spectro-microscopy and the resulting complex hyperspectral datasets were submitted to automated statistical analysis, namely Principal Components Analysis (PCA). The relationships between molecular fingerprints were put in evidence to highlight their spectral discrimination capabilities. We demonstrate that robust spectral encoding via SEIRA fingerprints opens up new opportunities for fast, reliable and multiplexed high-end screening not only in biodiagnostics but also in vitro biochemical imaging.« less

A glass fiber/diethylaminoethyl double filter binding assay that measures apoptotic internucleosomal DNA fragmentation.

PubMed

Erusalimsky, J D; John, J; Hong, Y; Moore, M

1996-11-15

A filter binding assay that measures internucleosomal DNA fragmentation associated with apoptosis is described. The assay is based on a novel principle that consists of using simultaneously two kinds of glass fiber filters to harvest [3H]thymidine-prelabeled cells following their incubation with inducers of apoptosis. One filter, which is neutral, traps intact chromatin and high-molecular-weight DNA. The other filter, which is positively charged with DEAE active groups, traps low-molecular-weight DNA fragments. DNA fragmentation is quantified by measuring the radioactivity retained by each of the filters. The assay was evaluated with the histiocytic lymphoma cell line U937 and the topoisomerase inhibitors camptothecin, etoposide, and doxorubicin. These agents caused a dose-dependent decrease of radioactivity in the neutral filter and a parallel increase of radioactivity in the DEAE filter. Irradiation-induced single strand breaks and topoisomerase-mediated primary DNA damage were not detected by this method. Consistent with the detection of internucleosomal DNA fragmentation, the effects measured by this assay were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using this assay were validated by observation of DNA ladders on agarose gels and by morphologic examination of apoptotic features. Evaluation of the assay in a mock screen demonstrated that the introduction of the DEAE filter increases the assay sensitivity and eliminates false positives. Thus, this assay may be used in high-throughput screening approaches to discover novel modulators of apoptosis.

Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1.

PubMed

Decaro, Nicola; Amorisco, Francesca; Desario, Costantina; Lorusso, Eleonora; Camero, Michele; Bellacicco, Anna Lucia; Sciarretta, Rossana; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio

2010-10-01

A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Use of the HPV MLPA assay in cervical cytology for the prediction of high grade lesions.

PubMed

Litjens, Rogier J N T M; Theelen, Wendy; van de Pas, Yvonne; Ossel, Jessica; Reijans, Martin; Simons, Guus; Speel, Ernst-Jan M; Slangen, Brigitte F M; Ramaekers, Frans C S; Kruitwagen, Roy F P M; Hopman, Anton H N

2013-08-01

Current screening methods for uterine cervical cancer such as Papanicolaou smears and/or high risk human Papillomavirus (HR-HPV) detection have a high negative predictive value but a low positive predictive value for the presence of high grade cervical lesions. Therefore, new parameters are needed to reduce the rate of unnecessary referrals for colposcopy. The predictive value of the HPV multiplex ligation-dependent probe amplification (MLPA) assay, which can assess simultaneously HPV16/18 viral load and viral integration, was evaluated. The assay was applied to 170 cervical cytological samples, and the results were correlated with the matching histological follow-up. The GP5+/6+ assay and qPCR were used as a control for HR-HPV typing. The MLPA assay classified a higher percentage of cases as high-risk (high-viral load and/or viral integration) with higher grades of dysplasia. There was a high correlation between the HPV MLPA assay and qPCR for viral load and HPV genotyping, and between the MLPA assay and the GP5+/6+ assay for HPV genotyping. The sensitivity and specificity of the HPV MLPA assay for the detection of high-grade lesions were 44% and 93%, respectively. This study demonstrates that the HPV MLPA assay can reliably detect HPV 16/18, viral load, and viral integration in cytological samples. Also, high-risk classification correlated well with the presence of high-grade dysplasia. However, for the implementation of the MLPA assay into clinical practice, additional HR-HPV types need to be included to increase the sensitivity of the assay, and thereby increase its negative predictive value. Copyright © 2013 Wiley Periodicals, Inc.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

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

Hindson, B J; Reid, S M; Baker, B R

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

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

Hindson, B J; Baker, B R; Bentley Tammero, L F

2007-09-18

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspectmore » cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

PubMed

Peng, Xiujuan; Nguyen, Alex; Ghosh, Debadyuti

2018-02-01

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*10 1 -2.75*10 8 genome copies(gc)/μL and 2.66*10 1 -2.66*10 8 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (E s ) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*10 7 gc/μL and 2.66*10 1 -2.66*10 7 gc/μL of M13 and T7 phage DNA, with an amplification efficiency E s of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS). Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous detection of dual biomarkers from humans exposed to organophosphorus pesticides by combination of immunochromatographic test strip and ellman assay.

PubMed

Yang, Mingming; Zhao, Yuting; Wang, Limin; Paulsen, Michael; Simpson, Christopher D; Liu, Fengquan; Du, Dan; Lin, Yuehe

2018-05-01

A novel sandwich immunoassay based immunochromatographic test strip (ICTS) has been developed for simultaneously measuring both butyrylcholinesterase (BChE) activity and the total amount of BChE (including inhibited and active enzyme) from 70 μLpost-exposure human plasma sample. The principle of this method is based on the BChE monoclonal antibody (MAb) capable of acting as both capture antibody and detection antibody. The BChE MAb which was immobilized on the test line was able to recognize both organophosphorus BChE adducts (OP-BChE) and BChE and provided equal binding affinity, permitting detection of the total enzyme amount in post-exposure human plasma samples. The formed immunocomplexes on the test line can further be excised from the test-strip for subsequent off-line measurement of BChE activity using the Ellman assay. Therefore, dual biomarkers of BChE activity and phosphorylation (OP-BChE) will be obtained simultaneously. The whole sandwich-immunoassay was performed on one ICTS, greatly reducing analytical time. The ICTS sensor showed excellent linear responses for assaying total amount of BChE and active BChE ranging from 0.22 to 3.58nM and 0.22-7.17nM, respectively. Both the signal detection limits are 0.10nM. We validated the practical application of the proposed method to measure 124 human plasma samples from orchard workers and cotton farmers with long-term exposure to organophosphorus pesticides (OPs). The results were in highly agreement with LC/MS/MS which verified our method is extremely accurate. Combining the portability and rapidity of test strip and the compatibility of BChE MAb as both capture antibody and detection antibody, the developed method provides a baseline-free, low-cost and rapid tool for in-field monitoring of OP exposures. Copyright © 2018 Elsevier B.V. All rights reserved.

Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS[S

PubMed Central

Sánchez-Guijo, Alberto; Oji, Vinzenz; Hartmann, Michaela F.; Traupe, Heiko; Wudy, Stefan A.

2015-01-01

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R2 > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood. PMID:26239050

Overcoming S-Phase Checkpoint-Mediated Resistance: Sequence-Dependent Synergy of Gemcitabine and SN-38 In Human Carcinoma Cell Lines*

PubMed Central

Gálvez-Peralta, Marina; Dai, Nga T.; Loegering, David A.; Flatten, Karen; Safgren, Stephanie; Wagner, Jill; Ames, Matthew M.; Karnitz, Larry M.; Kaufmann, Scott H.

2008-01-01

Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine and SN-38 (the active metabolite of irinotecan), two S phase-selective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony forming assays revealed that simultaneous treatment of Ovcar-5 ovarian cancer cells or BxPC-3 pancreatic cancer cells with gemcitabine and SN-38 resulted in antagonistic effects. In contrast, sequential treatment with the two agents in either order resulted in synergistic antiproliferative effects, although the mechanism of synergy varied with the sequence. In particular, SN-38 arrested cells in S phase, enhanced the accumulation of gemcitabine metabolites and diminished checkpoint kinase 1, thereby sensitizing cells in the SN-38 → gemcitabine sequence. Gemcitabine treatment followed by removal allowed prolonged progression through S phase, contributing to synergy of the gemcitabine → SN-38 sequence. Collectively, these results suggest that S phase selective agents might exhibit more cytotoxicity when administered sequentially rather than simultaneously. PMID:18509065

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

PubMed Central

Minguzzi, Stefano; Terlizzi, Federica; Lanzoni, Chiara; Poggi Pollini, Carlo; Ratti, Claudio

2016-01-01

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus. PMID:26742106

Development and validation of a solid-phase extraction gas chromatography–mass spectrometry method for the simultaneous quantification of methadone, heroin, cocaine and metabolites in sweat

PubMed Central

Brunet, Bertrand R.; Barnes, Allan J.; Scheidweiler, Karl B.; Mura, Patrick

2009-01-01

A sensitive and specific method is presented to simultaneously quantify methadone, heroin, cocaine and metabolites in sweat. Drugs were eluted from sweat patches with sodium acetate buffer, followed by SPE and quantification by GC/MS with electron impact ionization and selected ion monitoring. Daily calibration for anhydroecgonine methyl ester, ecgonine methyl ester, cocaine, benzoylecgonine (BE), codeine, morphine, 6-acetylcodeine, 6-acetylmorphine (6AM), heroin (5–1000 ng/patch) and methadone (10–1000 ng/patch) achieved determination coefficients of >0.995, and calibrators quantified to within ±20% of the target concentrations. Extended calibration curves (1000–10,000 ng/patch) were constructed for methadone, cocaine, BE and 6AM by modifying injection techniques. Within (N=5) and between-run (N=20) imprecisions were calculated at six control levels across the dynamic ranges with coefficients of variation of <6.5%. Accuracies at these concentrations were ±11.9% of target. Heroin hydrolysis during specimen processing was <11%. This novel assay offers effective monitoring of drug exposure during drug treatment, workplace and criminal justice monitoring programs. PMID:18607576

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay

PubMed Central

Augustine, Swinburne A. J.; Simmons, Kaneatra J.; Eason, Tarsha N.; Curioso, Clarissa L.; Griffin, Shannon M.; Wade, Timothy J.; Dufour, Alfred; Fout, G. Shay; Grimm, Ann C.; Oshima, Kevin H.; Sams, Elizabeth A.; See, Mary Jean; Wymer, Larry J.

2017-01-01

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations. PMID:28507984

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom.

PubMed

Prezoto, B C; Tanaka-Azevedo, A M; Marcelino, J R; Tashima, A K; Nishiduka, E S; Kapronezai, J; Mota, J O; Rocha, M M T; Serino-Silva, C; Oguiura, N

2018-06-15

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED 50 and LD 50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A 2 -like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED 50 ) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control. Copyright © 2018 Elsevier Ltd. All rights reserved.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

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

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

Design criteria for developing low-resource magnetic bead assays using surface tension valves

PubMed Central

Adams, Nicholas M.; Creecy, Amy E.; Majors, Catherine E.; Wariso, Bathsheba A.; Short, Philip A.; Wright, David W.; Haselton, Frederick R.

2013-01-01

Many assays for biological sample processing and diagnostics are not suitable for use in settings that lack laboratory resources. We have recently described a simple, self-contained format based on magnetic beads for extracting infectious disease biomarkers from complex biological samples, which significantly reduces the time, expertise, and infrastructure required. This self-contained format has the potential to facilitate the application of other laboratory-based sample processing assays in low-resource settings. The technology is enabled by immiscible fluid barriers, or surface tension valves, which stably separate adjacent processing solutions within millimeter-diameter tubing and simultaneously permit the transit of magnetic beads across the interfaces. In this report, we identify the physical parameters of the materials that maximize fluid stability and bead transport and minimize solution carryover. We found that fluid stability is maximized with ≤0.8 mm i.d. tubing, valve fluids of similar density to the adjacent solutions, and tubing with ≤20 dyn/cm surface energy. Maximizing bead transport was achieved using ≥2.4 mm i.d. tubing, mineral oil valve fluid, and a mass of 1-3 mg beads. The amount of solution carryover across a surface tension valve was minimized using ≤0.2 mg of beads, tubing with ≤20 dyn/cm surface energy, and air separators. The most favorable parameter space for valve stability and bead transport was identified by combining our experimental results into a single plot using two dimensionless numbers. A strategy is presented for developing additional self-contained assays based on magnetic beads and surface tension valves for low-resource diagnostic applications. PMID:24403996

In vitro biotransformation rates in fish liver S9: effect of dosing techniques.

PubMed

Lee, Yung-Shan; Lee, Danny H Y; Delafoulhouze, Maximilien; Otton, S Victoria; Moore, Margo M; Kennedy, Chris J; Gobas, Frank A P C

2014-08-01

In vitro biotransformation assays are currently being explored to improve estimates of bioconcentration factors of potentially bioaccumulative organic chemicals in fish. The present study compares thin-film and solvent-delivery dosing techniques as well as single versus multiple chemical dosing for measuring biotransformation rates of selected polycyclic aromatic hydrocarbons in rainbow trout (Oncorhynchus mykiss) liver S9. The findings show that biotransformation rates of very hydrophobic substances can be accurately measured in thin-film sorbent-dosing assays from concentration-time profiles in the incubation medium but not from those in the sorbent phase because of low chemical film-to-incubation-medium mass-transfer rates at the incubation temperature of 13.5 °C required for trout liver assays. Biotransformation rates determined by thin-film dosing were greater than those determined by solvent-delivery dosing for chrysene (octanol-water partition coefficient [KOW ] =10(5.60) ) and benzo[a]pyrene (KOW  =10(6.04) ), whereas there were no statistical differences in pyrene (KOW  =10(5.18) ) biotransformation rates between the 2 methods. In sorbent delivery-based assays, simultaneous multiple-chemical dosing produced biotransformation rates that were not statistically different from those measured in single-chemical dosing experiments for pyrene and benzo[a]pyrene but not for chrysene. In solvent-delivery experiments, multiple-chemical dosing produced biotransformation rates that were much smaller than those in single-chemical dosing experiments for all test chemicals. While thin-film sorbent-phase and solvent delivery-based dosing methods are both suitable methods for measuring biotransformation rates of substances of intermediate hydrophobicity, thin-film sorbent-phase dosing may be more suitable for superhydrophobic chemicals. © 2014 SETAC.

Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

PubMed

Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

2013-01-01

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Integrated printed circuit board device for cell lysis and nucleic acid extraction.

PubMed

Marshall, Lewis A; Wu, Liang Li; Babikian, Sarkis; Bachman, Mark; Santiago, Juan G

2012-11-06

Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 μm × 300 μm × 3.7 cm microfluidic channel connecting two 15 μL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 μL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the system's integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

BPIC: A novel anti-tumor lead capable of inhibiting inflammation and scavenging free radicals.

PubMed

Li, Shan; Wang, Yuji; Zhao, Ming; Wu, Jianhui; Peng, Shiqi

2015-03-01

Inflammation has a critical role in the tumor progression, free radical damage can worse the status of patients in cancer condition. The anti-cancer agents capable of inhibiting inflammation and scavenging free radicals attract a lot of our interest. Aimed at the discovery of such anti-tumor agent, a novel intercalator, benzyl 1-[4-hydroxy-3-(methoxycarbonyl)-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (BPIC) was presented. The docking investigation of BPIC and doxorubicin towards the DNA (PDB ID: 1NAB) gave equal score and similar feature. The anti-proliferation assay of 8 cancer cells identified S180 cells had equal sensitivity to BPIC and doxorubicin. The anti-tumor assay defined the efficacy of BPIC been 2 folds higher than that of doxorubicin. At 1μmol/kg of dose BPIC effectively inhibited xylene-induced ear edema and decreased the plasma TNF-α and IL-8 of the mice. BPIC scavenged ∙OH, ∙O2(-) and NO free radicals in a concentration dependent manner and NO free radicals had the highest sensitivity. BPIC could be a novel anti-tumor lead capable of simultaneously inhibiting inflammation and scavenging free radicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development and Application of a High-Performance Liquid Chromatography Stability-Indicating Assay for Beyond-Use Date Determination of Compounded Topical Gels Containing Multiple Active Drugs.

PubMed

Gorman, Gregory; Sokom, Simara; Coward, Lori; Arnold, John J

2017-01-01

Topical gels compounded by pharmacists are important clinical tools for the management of pain. Nevertheless, there is often a dearth of information about the chemical stability of drugs included in these topical formulations, complicating the assignment of beyond-use dating. The purpose of this study was to develop a high-performance liquid chromatography photodiode array-based stability-indicating assay that could simultaneously resolve six drugs (amitriptyline, baclofen, clonidine, gabapentin, ketoprofen, lidocaine) commonly included in topical gels for pain management and their potential degradation products. Furthermore, this method was applied to the determination of beyond-use dating of combinations of these drugs prepared in commonly utilized bases (Lipobase, Lipoderm, Pluronic organogel). Gabapentin was determined to be the least stable component in all formulations tested. Measured stability ranged between 7 to 49 days depending on the base and other active drugs present in the formulation. In the absence of gabapentin, baclofen was the next least stable component, lasting for 120 days, regardless of the type of formulating base used. Copyright© by International Journal of Pharmaceutical Compounding, Inc.

Flavonoid detection in hydroethanolic extract of Pouteria torta (Sapotaceae) leaves by HPLC-DAD and the determination of its mutagenic activity.

PubMed

Costa, Daryne L M G; Rinaldo, Daniel; Varanda, Eliana A; de Sousa, Juliana F; Nasser, Ana L M; Silva, Ana C Z; Baldoqui, Débora C; Vilegas, Wagner; dos Santos, Lourdes Campaner

2014-10-01

It is well known that phytotherapy has grown in popularity in recent years. Because a drug cannot be administered without ensuring its effectiveness and safety, the standardization and regulation of phytotherapeutic drugs are required by the global market and governmental authorities. This article describes a simple and reliable high-performance liquid chromatography-diode array detection analysis method for the simultaneous detection of myricetin-3-O-β-D-galactopyranoside, myricetin-3-O-α-L-arabinopyranoside, and myricetin-3-O-α-L-rhaminopyranoside present in the hydroethanolic extract (ethanol/H2O, 7:3, v/v) of Pouteria torta. The mutagenic activity of the extract was evaluated on Salmonella typhimurium and by an in vivo micronucleus test on the peripheral blood cells of Swiss mice. The linearity, sensitivity, selectivity, repeatability, accuracy, and precision of the assay were evaluated. The analytical curves were linear and exhibited good repeatability (with a deviation of less than 5%) and demonstrated good recovery (within the 83-107% range). The results demonstrate that the hydroethanolic extract exhibited a mutagenic activity in both assays, suggesting caution in the use of this plant in folk medicine.

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

PubMed Central

Settanni, Luca; van Sinderen, Douwe; Rossi, Jone; Corsetti, Aldo

2005-01-01

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies. PMID:15933001

Flavonoid Detection in Hydroethanolic Extract of Pouteria torta (Sapotaceae) Leaves by HPLC-DAD and the Determination of Its Mutagenic Activity

PubMed Central

Costa, Daryne L.M.G.; Rinaldo, Daniel; Varanda, Eliana A.; de Sousa, Juliana F.; Nasser, Ana L.M.; Silva, Ana C.Z.; Baldoqui, Débora C.; Vilegas, Wagner

2014-01-01

Abstract It is well known that phytotherapy has grown in popularity in recent years. Because a drug cannot be administered without ensuring its effectiveness and safety, the standardization and regulation of phytotherapeutic drugs are required by the global market and governmental authorities. This article describes a simple and reliable high-performance liquid chromatography–diode array detection analysis method for the simultaneous detection of myricetin-3-O-β-D-galactopyranoside, myricetin-3-O-α-L-arabinopyranoside, and myricetin-3-O-α-L-rhaminopyranoside present in the hydroethanolic extract (ethanol/H2O, 7:3, v/v) of Pouteria torta. The mutagenic activity of the extract was evaluated on Salmonella typhimurium and by an in vivo micronucleus test on the peripheral blood cells of Swiss mice. The linearity, sensitivity, selectivity, repeatability, accuracy, and precision of the assay were evaluated. The analytical curves were linear and exhibited good repeatability (with a deviation of less than 5%) and demonstrated good recovery (within the 83–107% range). The results demonstrate that the hydroethanolic extract exhibited a mutagenic activity in both assays, suggesting caution in the use of this plant in folk medicine. PMID:25055245

Performance of the Abbott RealTime CT/NG for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

PubMed

Gaydos, C A; Cartwright, C P; Colaninno, P; Welsch, J; Holden, J; Ho, S Y; Webb, E M; Anderson, C; Bertuzis, R; Zhang, L; Miller, T; Leckie, G; Abravaya, K; Robinson, J

2010-09-01

A multicenter clinical study was conducted to evaluate the performance characteristics of the Abbott RealTime CT/NG assay, a multiplex real-time PCR assay, for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The specimens were collected from a total of 3,832 male and female subjects at 16 geographically diverse sites. Specimens included male and female urine samples, male urethral swabs, female endocervical swabs, and self-collected and clinician-collected vaginal swabs. Specimens were tested with the automated Abbott RealTime CT/NG assay, Aptima Combo 2 assay (Gen-Probe), ProbeTec ET CT/GC assay (Becton Dickinson), and culture for N. gonorrhoeae. The Aptima Combo 2 assay, the ProbeTec assay, and the N. gonorrhoeae culture were used as the reference assays. For each subject, a patient infected status (PIS) was determined based on the combined results from the reference assays. The overall prevalence in female subjects was 8.9% for C. trachomatis and 3.8% for N. gonorrhoeae. The overall male prevalence was 18.2% for C. trachomatis and 16.7% for N. gonorrhoeae. The overall sensitivity and specificity of the Abbott RealTime CT/NG assay were 92.4% and 99.2% for C. trachomatis and 96.9% and 99.7% for N. gonorrhoeae, respectively. In comparison, the sensitivity and specificity, respectively, for the Aptima Combo 2 assay were 94.5% and 99.0% for C. trachomatis and 96.1% and 99.5% for N. gonorrhoeae, and those for the ProbeTec ET assay were 90.3% and 99.5% for C. trachomatis and 92.0% and 97.3% for N. gonorrhoeae in this study. The Abbott RealTime CT/NG assay offers C. trachomatis and N. gonorrhoeae dual detection with high sensitivity and specificity. The automated assay provides a useful alternative nucleic acid amplification assay for clinical laboratories and clinicians.

Performance of the Abbott RealTime CT/NG for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae▿

PubMed Central

Gaydos, C. A.; Cartwright, C. P.; Colaninno, P.; Welsch, J.; Holden, J.; Ho, S. Y.; Webb, E. M.; Anderson, C.; Bertuzis, R.; Zhang, L.; Miller, T.; Leckie, G.; Abravaya, K.; Robinson, J.

2010-01-01

A multicenter clinical study was conducted to evaluate the performance characteristics of the Abbott RealTime CT/NG assay, a multiplex real-time PCR assay, for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The specimens were collected from a total of 3,832 male and female subjects at 16 geographically diverse sites. Specimens included male and female urine samples, male urethral swabs, female endocervical swabs, and self-collected and clinician-collected vaginal swabs. Specimens were tested with the automated Abbott RealTime CT/NG assay, Aptima Combo 2 assay (Gen-Probe), ProbeTec ET CT/GC assay (Becton Dickinson), and culture for N. gonorrhoeae. The Aptima Combo 2 assay, the ProbeTec assay, and the N. gonorrhoeae culture were used as the reference assays. For each subject, a patient infected status (PIS) was determined based on the combined results from the reference assays. The overall prevalence in female subjects was 8.9% for C. trachomatis and 3.8% for N. gonorrhoeae. The overall male prevalence was 18.2% for C. trachomatis and 16.7% for N. gonorrhoeae. The overall sensitivity and specificity of the Abbott RealTime CT/NG assay were 92.4% and 99.2% for C. trachomatis and 96.9% and 99.7% for N. gonorrhoeae, respectively. In comparison, the sensitivity and specificity, respectively, for the Aptima Combo 2 assay were 94.5% and 99.0% for C. trachomatis and 96.1% and 99.5% for N. gonorrhoeae, and those for the ProbeTec ET assay were 90.3% and 99.5% for C. trachomatis and 92.0% and 97.3% for N. gonorrhoeae in this study. The Abbott RealTime CT/NG assay offers C. trachomatis and N. gonorrhoeae dual detection with high sensitivity and specificity. The automated assay provides a useful alternative nucleic acid amplification assay for clinical laboratories and clinicians. PMID:20668135

Application of different spectrophotometric methods for simultaneous determination of elbasvir and grazoprevir in pharmaceutical preparation

NASA Astrophysics Data System (ADS)

Attia, Khalid A. M.; El-Abasawi, Nasr M.; El-Olemy, Ahmed; Abdelazim, Ahmed H.

2018-01-01

The first three UV spectrophotometric methods have been developed of simultaneous determination of two new FDA approved drugs namely; elbasvir and grazoprevir in their combined pharmaceutical dosage form. These methods include simultaneous equation, partial least squares with and without variable selection procedure (genetic algorithm). For simultaneous equation method, the absorbance values at 369 (λmax of elbasvir) and 253 nm (λmax of grazoprevir) have been selected for the formation of two simultaneous equations required for the mathematical processing and quantitative analysis of the studied drugs. Alternatively, the partial least squares with and without variable selection procedure (genetic algorithm) have been applied in the spectra analysis because the synchronous inclusion of many unreal wavelengths rather than by using a single or dual wavelength which greatly increases the precision and predictive ability of the methods. Successfully assay of the drugs in their pharmaceutical formulation has been done by the proposed methods. Statistically comparative analysis for the obtained results with the manufacturing methods has been performed. It is noteworthy to mention that there was no significant difference between the proposed methods and the manufacturing one with respect to the validation parameters.

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. PMID:24468782

Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection

PubMed Central

Pritzkow, Sandra; Wagenführ, Katja; Daus, Martin L.; Boerner, Susann; Lemmer, Karin; Thomzig, Achim; Mielke, Martin; Beekes, Michael

2011-01-01

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants. PMID:21647368

Additive mixture effects of estrogenic chemicals in human cell-based assays can be influenced by inclusion of chemicals with differing effect profiles.

PubMed

Evans, Richard Mark; Scholze, Martin; Kortenkamp, Andreas

2012-01-01

A growing body of experimental evidence indicates that the in vitro effects of mixtures of estrogenic chemicals can be well predicted from the estrogenicity of their components by the concentration addition (CA) concept. However, some studies have observed small deviations from CA. Factors affecting the presence or observation of deviations could include: the type of chemical tested; number of mixture components; mixture design; and assay choice. We designed mixture experiments that address these factors, using mixtures with high numbers of components, chemicals from diverse chemical groups, assays with different in vitro endpoints and different mixture designs and ratios. Firstly, the effects of mixtures composed of up to 17 estrogenic chemicals were examined using estrogenicity assays with reporter-gene (ERLUX) and cell proliferation (ESCREEN) endpoints. Two mixture designs were used: 1) a 'balanced' design with components present in proportion to a common effect concentration (e.g. an EC(10)) and 2) a 'non-balanced' design with components in proportion to potential human tissue concentrations. Secondly, the individual and simultaneous ability of 16 potential modulator chemicals (each with minimal estrogenicity) to influence the assay outcome produced by a reference mixture of estrogenic chemicals was examined. Test chemicals included plasticizers, phthalates, metals, PCBs, phytoestrogens, PAHs, heterocyclic amines, antioxidants, UV filters, musks, PBDEs and parabens. In all the scenarios tested, the CA concept provided a good prediction of mixture effects. Modulation studies revealed that chemicals possessing minimal estrogenicity themselves could reduce (negatively modulate) the effect of a mixture of estrogenic chemicals. Whether the type of modulation we observed occurs in practice most likely depends on the chemical concentrations involved, and better information is required on likely human tissue concentrations of estrogens and of potential modulators. Successful prediction of the effects of diverse chemical combinations might be more likely if chemical profiling included consideration of effect modulation.

Additive Mixture Effects of Estrogenic Chemicals in Human Cell-Based Assays Can Be Influenced by Inclusion of Chemicals with Differing Effect Profiles

PubMed Central

Evans, Richard Mark; Scholze, Martin; Kortenkamp, Andreas

2012-01-01

A growing body of experimental evidence indicates that the in vitro effects of mixtures of estrogenic chemicals can be well predicted from the estrogenicity of their components by the concentration addition (CA) concept. However, some studies have observed small deviations from CA. Factors affecting the presence or observation of deviations could include: the type of chemical tested; number of mixture components; mixture design; and assay choice. We designed mixture experiments that address these factors, using mixtures with high numbers of components, chemicals from diverse chemical groups, assays with different in vitro endpoints and different mixture designs and ratios. Firstly, the effects of mixtures composed of up to 17 estrogenic chemicals were examined using estrogenicity assays with reporter-gene (ERLUX) and cell proliferation (ESCREEN) endpoints. Two mixture designs were used: 1) a ‘balanced’ design with components present in proportion to a common effect concentration (e.g. an EC10) and 2) a ‘non-balanced’ design with components in proportion to potential human tissue concentrations. Secondly, the individual and simultaneous ability of 16 potential modulator chemicals (each with minimal estrogenicity) to influence the assay outcome produced by a reference mixture of estrogenic chemicals was examined. Test chemicals included plasticizers, phthalates, metals, PCBs, phytoestrogens, PAHs, heterocyclic amines, antioxidants, UV filters, musks, PBDEs and parabens. In all the scenarios tested, the CA concept provided a good prediction of mixture effects. Modulation studies revealed that chemicals possessing minimal estrogenicity themselves could reduce (negatively modulate) the effect of a mixture of estrogenic chemicals. Whether the type of modulation we observed occurs in practice most likely depends on the chemical concentrations involved, and better information is required on likely human tissue concentrations of estrogens and of potential modulators. Successful prediction of the effects of diverse chemical combinations might be more likely if chemical profiling included consideration of effect modulation. PMID:22912892

Array biosensor: recent developments

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Rowe-Taitt, Chris A.; Feldstein, Mark J.; Ligler, Frances S.

1999-05-01

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Posaconazole and Vincristine in Rat Plasma

PubMed Central

Khalil, Hadeel A.; El-Yazbi, Ahmed F.; Belal, Tarek S.; Hamdy, Dalia A.

2015-01-01

Purpose. Developing a validated HPLC-DAD method for simultaneous determination of posaconazole (PSZ) and vincristine (VCR) in rat plasma. Methods. PSZ, VCR, and itraconazole (ITZ) were extracted from 200 μL plasma using diethyl ether in the presence of 0.1 M sodium hydroxide solution. The organic layer was evaporated in vacuo and dried residue was reconstituted and injected through HC-C18 (4.6 × 250 mm, 5 μm) column. In the mobile phase, acetonitrile and 0.015 M potassium dihydrogen orthophosphate (30 : 70 to 80 : 20, linear gradient over 7 minutes) pumped at 1.5 mL/min. VCR and PSZ were measured at 220 and 262 nm, respectively. Two Sprague Dawley rats were orally dosed PSZ followed by iv dosing of VCR and serial blood sampling was performed. Results. VCR, PSZ, and ITZ were successfully separated within 11 min. Calibration curves were linear over the range of 50–5000 ng/mL for both drugs. The CV% and % error of the mean were ≤18% and limit of quantitation was 50 ng/mL for both drugs. Rat plasma concentrations of PSZ and VCR were simultaneously measured up to 72 h and their calculated pharmacokinetics parameters were comparable to the literature. Conclusion. The assay was validated as per ICH guidelines and is appropriate for pharmacokinetics drug-drug interaction studies. PMID:27034675

Biodegradation of p-cresol and sulfide removal by a marine-denitrifying consortium.

PubMed

Meza-Escalante, Edna R; Alvarez, Luis H; Serrano, Denisse; Mendoza, Erika; Bonola, Ramsés

2015-02-01

The simultaneous removal of sulfide and p-cresol was carried out by using a marine-denitrifying consortium collected in the coastal zone of Sonora, Mexico. Different experimental conditions were used to evaluate the capacity of the consortium to simultaneously eliminate nitrate, sulfide, and p-cresol. For instance, the first set of assays was conducted at different sulfide concentrations (20, 50, and 100 mg S(2À) L(À1) ), with a fixed concentration of p-cresol (45 mg C L(À1) ). The second set of assays was developed at different concentrations of p-cresol (45, 75, and 100 mg C L(-1) ), in the presence of 20 mg S(2À) L(À1) . In all cases, the concentration of nitrate was stoichiometrically added for the complete oxidization of the substrates. The results showed removal efficiencies up to 92% for p-cresol and nitrate at 20 and 50 mg S(2À) L(À1) ; whereas at 100 mg S(2À) L(À1) removal efficiencies were 77% and 59% for p-cresol and nitrate, respectively. On the other hand, sulfide (20 mg L(À1) ) was completely removed under different concentrations of p-cresol tested, with a partial accumulation of nitrite according to the increment of p-cresol concentration. The results obtained indicate that the marine consortium was able to simultaneously remove the pollutants studied. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

PubMed

Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

2016-01-01

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

NASA Astrophysics Data System (ADS)

Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

2002-06-01

Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

Early Detection of Dengue Virus by Use of Reverse Transcription-Recombinase Polymerase Amplification

PubMed Central

Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Danlami, Mohammed Bashar; Shu, Meng-Hooi; Johari, Jefree; Hooi, Poh-Sim; Brooks, David; Piepenburg, Olaf; Nentwich, Oliver; Wilder-Smith, Annelies; Franco, Leticia; Tenorio, Antonio

2015-01-01

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue. PMID:25568438

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

PubMed Central

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun

2017-01-01

ABSTRACT The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. PMID:28659320

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

PubMed

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

USDA-ARS?s Scientific Manuscript database

Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

Non-animal sensitization testing: state-of-the-art.

PubMed

Vandebriel, Rob J; van Loveren, Henk

2010-05-01

Predictive tests to identify the sensitizing properties of chemicals are carried out using animals. In the European Union timelines for phasing out many standard animal tests were established for cosmetics. Following this policy, the new European Chemicals Legislation (REACH) favors alternative methods, if validated and appropriate. In this review the authors aim to provide a state-of-the art overview of alternative methods (in silico, in chemico, and in vitro) to identify contact and respiratory sensitizing capacity and in some occasions give a measure of potency. The past few years have seen major advances in QSAR (quantitative structure-activity relationship) models where especially mechanism-based models have great potential, peptide reactivity assays where multiple parameters can be measured simultaneously, providing a more complete reactivity profile, and cell-based assays. Several cell-based assays are in development, not only using different cell types, but also several specifically developed assays such as three-dimenionally (3D)-reconstituted skin models, an antioxidant response reporter assay, determination of signaling pathways, and gene profiling. Some of these assays show relatively high sensitivity and specificity for a large number of sensitizers and should enter validation (or are indeed entering this process). Integrating multiple assays in a decision tree or integrated testing system is a next step, but has yet to be developed. Adequate risk assessment, however, is likely to require significantly more time and efforts.

Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens.

PubMed

Yang, Peng; Peng, Xiaomin; Cui, Shujuan; Shao, Junbin; Zhu, Xuping; Zhang, Daitao; Liang, Huijie; Wang, Quanyi

2013-07-30

Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. A panel consisting of seven duplex real-time PCR assays was developed to simultaneously detect 13 streptococcal SAgs and one internal control which may be important in the control of GAS-mediated diseases. Primer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs. The reaction conditions of the duplex real-time PCR were optimized and the specificity of the duplex assays was evaluated using SAg positive strains. The limit of detection of the duplex assays was determined by using 10-fold serial dilutions of the DNA of 13 streptococcal SAgs and compared to a conventional polymerase chain reaction (PCR) method for evaluating the duplex assays sensitivity. Using the duplex assays, we were able to differentiate between 13 SAgs from Streptococcus strains and other non-Streptococcus bacteria without cross-reaction. On the other hand, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR. The panel was highly specific (100%) and the limit of detection of these duplex groups was at least ten times lower than that obtained by using a conventional PCR method.

Mini-SNaPshot multiplex assays authenticate elephant ivory and simultaneously identify the species origin.

PubMed

Kitpipit, Thitika; Thongjued, Kantima; Penchart, Kitichaya; Ouithavon, Kanita; Chotigeat, Wilaiwan

2017-03-01

Illegal trading of ivory is mainly responsible for the dramatic decline in elephant populations. Thailand is one of the largest laundering hotspots for African ivory, as the domestic Asian elephant ivory can be legally traded. So, to help combat ivory poaching and smuggling, an efficient method is needed to identify the elephant species from its ivory and ivory products. In this study, a mini-SNaPshot ® multiplex assay was developed and fully validated for the identification of confiscated ivory and low DNA template ivory products. Elephantid- and elephant species-specific mitochondrial single nucleotide polymorphisms (SNPs) were identified from 207 mammalian and 1705 elephant/mammoth cytochrome b sequence alignments. Seven informative SNPs were used for assay development. The assay unambiguously and accurately identified authentic elephant ivory and its species of origin on the basis of peak size and color observed in the haplotype profile. The assay was highly efficient for analysis of confiscated ivory and low-template ivory products with a 99.29% success rate (N=140). It was highly reproducible, exhibited no cross-reaction with eight other mammalian DNA; and had 100% identification accuracy. In addition, nested and direct PCR amplification were also compatible with the developed assay. This efficient assay should benefit wildlife forensic laboratories and aid in the prosecution of elephant-related crimes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Laboratory validation of two real-time RT-PCR methods with 5'-tailed primers for an enhanced detection of foot-and-mouth disease virus.

PubMed

Vandenbussche, Frank; Lefebvre, David J; De Leeuw, Ilse; Van Borm, Steven; De Clercq, Kris

2017-08-01

The 3D and 5UTR real-time RT-PCR assays (RT-qPCR) from Callahan et al. (2002) and Reid et al. (2002) are commonly used reference methods for the detection of foot-and-mouth disease virus (FMDV). For an optimal detection of FMDV in clinical samples, it is advised to use both assays simultaneously (King et al., 2006). Recently, Vandenbussche et al. (2016) showed that the addition of 5'-tails to the FMDV-specific primers enhances the detection of FMDV in both the 3D and the 5UTR RT-qPCR assay. To validate the 3D and 5UTR RT-qPCR assays with 5'-tailed primers for diagnostic purposes, both assays were run in parallel in a triplex one-step RT-qPCR protocol with beta-actin as an internal control and synthetic RNA as an external control. We obtained low limits of detection and high linearity's, high repeatability and reproducibility, near 100% analytical specificity and >99% diagnostic accuracy for both assays. It was concluded that the 3D and 5UTR RT-qPCR assays with 5'-tailed primers are particularly suited for the detection of FMDV as well as to exclude the presence of FMDV. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

PubMed Central

Pooler, M R; Ritchie, D F; Hartung, J S

1996-01-01

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria. PMID:8795198

Water quality monitoring using an automated portable fiber optic biosensor: RAPTOR

NASA Astrophysics Data System (ADS)

Anderson, George P.; Rowe-Taitt, Chris A.

2001-03-01

The RAPTOR is a portable, automated biosensor capable of performing rapid, ten-minute assays on a sample for four target analytes simultaneously. Samples are analyzed using a fluorescent sandwich immunoassay on the surface of short polystyrene optical probes with capture antibody adsorbed to the probe surface. Target analytes bound to the fiber by capture antibodies are detected with fluorescently labeled tracer antibodies, which are held in a separate reservoir. Since target recognition is a two-step process, selectivity is enhanced, and the optical probes can be reused up to forty times, or until a positive result is obtained. This greatly reduces the logistical burden for field operations. Numerous assays for toxins, such as SEB and ricin, and bacteria, such as Bacillus anthracis and Francisella tularensis, have been developed for the RAPTOR. An assay of particular interest for water quality monitoring and the screening of fruits and vegetables is detection of Giardia cysts. Giardia lamblia is a parasitic protozoan common in the developing world that causes severe intestinal infections. Thus, a simple field assay for screening water supplies would be highly useful. Such an assay has been developed using the RAPTOR. The detection limit for Giardia cysts was 5x104/ml for a 10-minute assay.

Impedance spectroscopy applied to the fast wounding dynamics of an electrical wound-healing assay in mammalian cells

NASA Astrophysics Data System (ADS)

Bellotti, Mariela I.; Giana, Fabián E.; Bonetto, Fabián J.

2015-08-01

Electrical wound-healing assays are often used as a means to study in vitro cell migration and proliferation. In such analysis, a cell monolayer that sits on a small electrode is electrically wounded and its spectral impedance is then continuously measured in order to monitor the healing process. The relatively slow dynamics of the cell healing have been extensively studied, while those of the much faster wounding phase have not yet been investigated. An analysis of the electrical properties of a particular cell type during this phase could give extra information about the changes in the cell membrane due to the application of the wounding current, and could also be useful to optimize the wounding regime for different cell types. The main issue when trying to register information about these dynamics is that the traditional measurement scheme employed in typical wound-healing assays doesn’t allow the simultaneous application of the wounding signal and measurement of the system’s impedance. In this paper, we overcome this limitation by implementing a measurement strategy consisting of cycles of fast alternating low- and high-voltage signals applied on electrodes covered with mammalian cells. This approach is capable of registering the fast impedance changes during the transient regime corresponding to the cell wounding process. Furthermore, these quasi-simultaneous high- and low-voltage measurements can be compared in order to obtain an empirical correlation between both quantities.

In vitro anti-reovirus activity of kuraridin isolated from Sophora flavescens against viral replication and hemagglutination.

PubMed

Kwon, Hyung-Jun; Jeong, Jae-Ho; Lee, Seung Woong; Ryu, Young Bae; Jeong, Hyung Jae; Jung, Kyungsook; Lim, Jae Sung; Cho, Kyoung-Oh; Lee, Woo Song; Rho, Mun-Chual; Park, Su-Jin

2015-08-01

In this study, we evaluated the anti-reovirus activity of kuraridin isolated from the roots of Sophora flavescens. In particular, we focused on whether this property is attributable to direct inhibition of reovirus attachment and/or inhibition of viral replication with the aid of time-of-addition (pre-treatment, simultaneous treatment, and post-treatment) experiments. No significant antiviral activity of kuraridin was detected in the pre-treatment assay. In the simultaneous assay, the 50% effective inhibitory concentrations (EC50) of kuraridin were 15.3-176.9 μM against human type 1-3 reoviruses (HRV1-3) and Korean porcine reovirus (PRV). Kuraridin completely blocked binding of viral sigma 1 protein to sialic acids at concentrations lower than 82.5 μM in the hemagglutination inhibition assay. Moreover, kuraridin inhibited HRV1-3 and PRV viral replication with EC50 values of 14.0-62.0 μM. Quantitative real-time PCR analysis disclosed strong suppression of reovirus RNA synthesis at the late stage (18 h) of virus replication by kuraridin. The viral yields of kuraridin-treated cells were significantly reduced at 24 h post-infection, compared with DMSO-treated cells. Our results collectively suggest that kuraridin inhibits virus adsorption and replication by inhibiting hemagglutination, viral RNA and protein synthesis and virus shedding, supporting its utility as a viable candidate antiviral drug against reoviruses. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

PubMed Central

Katz, David; Shi, Wei; Patrusheva, Irina; Perelygina, Ludmila; Gowda, Manjunath S; Krug, Peter W; Filfili, Chadi N; Ward, John A; Hilliard, Julia K

2012-01-01

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV. PMID:23561887

Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic-resistant staphylococci, with simultaneous identification of PVL-positive and -negative Staphylococcus aureus and coagulase negative staphylococci.

PubMed

Panda, Sasmita; Kar, Sarita; Choudhury, Ranginee; Sharma, Savitri; Singh, Durg V

2014-03-01

We developed a multiplex PCR to detect the presence of methicillin- (mecA), cadmium/zinc-(czrC) and antiseptic-resistant (qacA/B) staphylococci and to identify Panton-Valentine leukocidin (PVL)-positive and -negative Staphylococcus aureus and coagulase-negative staphylococci (CoNS) from infected and healthy eyes. The assay was validated on 177 staphylococci comprising of 55 each of S. aureus and CoNS isolated from infected eyes and five S. aureus and 62 CoNS isolated from healthy eyes and nine direct ocular samples. Nine direct ocular samples for in situ testing consisted of corneal scrapings (4), conjunctiva swabs (2) and others (3). Multiplex PCR result was correlated with genotype data obtained with single PCR and dot-blot assay. The control strains that were positive in multiplex PCR for 16S rRNA, nuc, mecA, pvl, czrC and qacA/B genes were also positive in the dot-blot assay. The specificity of amplified genes obtained with reference strains was further confirmed by DNA sequencing. The single step-hexaplex PCR method can be used for rapid detection of mecA, nuc, pvl, czrC and qacA/B genes in staphylococci with simultaneous identification of PVL-positive and -negative S. aureus and CoNS from a variety of ocular samples. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Multiplex quantification of Escherichia coli, Salmonella typhi and Vibrio cholera with three DNA targets in single reaction assay.

PubMed

Jangampalli Adi, Pradeepkiran; Naidu, Jagadish R; Matcha, Bhaskar

2017-09-01

Escherichia coli (E. coli), Salmonella typhi and Vibrio cholera harmful pathogens, which causes various diseases in humans. Rapid diagnosis of bacterial infection is an important for patient management and appropriate therapy during the early phase of the bacterial infected diseases. Among the existing techniques for identifying pathogens were less sensitive and time-consuming processes. In the present study total, 48 clinical 31 blood and 17 urine samples of patients suspected with the infections were collected from SVRR Hospital and used to detect the pathogens. Multiplex polymerase chain reaction (PCR) assay was set to design for the identification of Escherichia coli, Salmonella typhi and Vibrio cholera from the different clinical samples. Rapid diagnosis of Escherichia coli (E. coli), Salmonella and Vibrio cholera pathogens can be done with simultaneously in a single multiplex PCR assay by using specific primers with adjusted PCR conditions. Through this approach, the results represented with out of 31 blood samples 1-15 shows the positive with E. coli and remaining 14 only 11 were correlated with multiplex results of Vibrio cholera, remaining the urine samples all are positive with 17 samples correlate with the Salmonella typhi. Through the high specificity benefits of excellent sensitivity, with high resolution and reproducibility. This method of results proved and illustrates the best potential system for diagnosing the infectious disease with modern trendy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthetic (+)-antroquinonol exhibits dual actions against insulin resistance by triggering AMP kinase and inhibiting dipeptidyl peptidase IV activities.

PubMed

Hsu, C Y; Sulake, R S; Huang, P-K; Shih, H-Y; Sie, H-W; Lai, Y-K; Chen, C; Weng, C F

2015-01-01

The fungal product (+)-antroquinonol activates AMP kinase (AMPK) activity in cancer cell lines. The present study was conducted to examine whether chemically synthesized (+)-antroquinonol exhibited beneficial metabolic effects in insulin-resistant states by activating AMPK and inhibiting dipeptidyl peptidase IV (DPP IV) activity. Effects of (+)-antroquinonol on DPP IV activity were measured with a DPPIV Assay Kit and effects on GLP-1-induced PKA were measured in AR42J cells. Translocation of the glucose transporter 4, GLUT4, induced either by insulin-dependent PI3K/AKT signalling or by insulin-independent AMPK activation, was assayed in differentiated myotubes. Glucose uptake and GLUT4 translocation were assayed in L6 myocytes. Mice with diet-induced obesity were used to assess effects of acute and chronic treatment with (+)-antroquinonol on glycaemic control in vivo. The results showed that of (+)-antroquinonol (100 μM ) inhibited the DPP IV activity as effectively as the clinically used inhibitor, sitagliptin. The phosphorylation of AMPK Thr(172) in differentiated myotubes was significantly increased by (+)-antroquinonol. In cells simultaneously treated with S961 (insulin receptor antagonist), insulin and (+)-antroquinonol, the combination of (+)-antroquinonol plus insulin still increased both GLUT4 translocation and glucose uptake. Further, (+)-antroquinonol and sitagliptin reduced blood glucose, when given acutely or chronically to DIO mice. Chemically synthesized (+)-antroquinonol exhibits dual effects to ameliorate insulin resistance, by increasing AMPK activity and GLUT4 translocation, along with inhibiting DPP IV activity. © 2014 The British Pharmacological Society.