scholarly journals A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains

2013 ◽  
Vol 61 (6) ◽  
pp. 1785-1792 ◽  
Author(s):  
Ivan Manga ◽  
Marcela Vyletělová
Keyword(s):  
Environmental Samples ◽  
Diagnostic Method ◽  
Gene Sequence ◽  
Limit Of Detection ◽  
Reference Method ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Disk Diffusion Test ◽  
Reference Strains ◽  
Reliable Diagnostic Method

The Methicillin-resistant Staphylococcus aureus (MRSA) with the livestock-associated MRSA (LA-MRSA) are of great interest to scientists and general public. The aim of our study was to present a new more rapid and reliable diagnostic method working on the RT-PCR platform applicable for monitoring of MRSA/S. aureus. The parallel testing of the S. aureus specific nuc gene sequence and the mecA gene sequence was utilised for this purpose. A collection of ten S. aureus/MRSA reference strains, fifteen genetically related non S. aureus reference strains and fifty-six environmental samples was employed for estimation of the assay performance and parameters. The environmental samples acquired in the Czech livestock farms were represented with the livestock and human nasal mucosae or skin swabs, the slaughter meat swabs and were chosen preferentially from individuals with previously confi rmed or suspected positive MRSA/S. aureus cases. The classic selective cultivation approach with the biochemical test and agar disk diffusion test was accepted as reference diagnostic method. As there were no culture positive samples that were negative using RT-PCR, our method featured with 100% sensitivity in comparison to reference method. The limit of detection allowed to identify from tens to hundreds copies of S. aureus/MRSA genome. Further, the RT-PCR assay featured with 100% inclusivity and 95% exclusivity at Cq value below 30. These parameters suggested on powerful and reliable diagnostic method with real potential of practical utilisation. We consider our method as ideal for testing of individual suspected colonies, when the results can be acquired in less than 1.5 hour.

Polymerase chain reaction amplification and typing of rotavirus in environmental samples

1995 ◽  
Vol 31 (5-6) ◽  
pp. 371-374 ◽  
Author(s):  
R. Gajardo ◽  
R. M. Pintó ◽  
A. Bosch
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Group A ◽  
Reaction Amplification ◽  
Stool Specimens ◽  
Polymerase Chain

A reverse transcription polymerase chain reaction (RT-PCR) assay is described that has been developed for the detection and serotyping of group A rotavirus in stool specimens and concentrated and non-concentrated sewage specimens.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

scholarly journals A multi-laboratory comparison of two molecular methods for the detection of toxigenic Clostridium difficile

2016 ◽  
Vol 10 (01) ◽  
pp. 62-67 ◽  
Author(s):  
Diane C Halstead ◽  
Joan Abid ◽  
Lynne Sloan ◽  
Diana Meza ◽  
Daphne Ramsey-Walker ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Molecular Characterization ◽  
Regulatory Gene ◽  
Reference Method ◽  
Molecular Methods ◽  
Detection Methods ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Toxigenic Culture

Introduction: Diarrheal disease due to toxigenic Clostridium difficile (CD) accounts for an increased number of hospitalizations and deaths each year. Published guidelines recommend reflex testing of CD antigen-positive samples to molecular testing or testing samples directly by a molecular assay. This multicenter study was designed to compare the accuracy of two different molecular methods targeting different CD genes: Xpert C. difficile Epi RUO RT-PCR assay (XPCR) which targets toxin B (Cepheid, Sunnyvale, CA) and a laboratory-developed PCR (LDPCR) which targets mutations in the tcdC regulatory gene. Methodology: Two molecular methods for toxigenic CD detection, the Xpert C. difficile Epi RUO RT-PCR assay (XPCR) [Cepheid, Sunnyvale, CA] and a laboratory-developed PCR assay (LDPCR) were compared to a consensus gold standard (CGS) or toxigenic culture (TC) as the reference method. A subset of specimens was subjected to additional molecular characterization of toxigenic CD. Results: Both molecular methods were >90% sensitive for CD detection. Discordant results were noted when molecular test results were compared to non-molecular methods. Supplemental molecular characterization illustrated inherent difficulties in comparisons using different molecular methods for CD. Conclusion: Laboratories may consider using multiple CD detection methods or combinations of methods, including molecular detection for rapid and accurate diagnosis of CD, as driven by best practices for the respective healthcare environment. Laboratories must be aware of intrinsic differences when comparing performance characteristics of different molecular assays.

Comparison of the BAX System PCR Method to Brazil's Official Method for the Detection of Salmonella in Food, Water, and Environmental Samples

2008 ◽  
Vol 71 (12) ◽  
pp. 2442-2447 ◽  
Author(s):  
INGRID BOESCHE TOMAZELLI ◽  
JOSINETE BARROS de FREITAS ◽  
LEANIA MARIA FABBI ◽  
TEREZINHA AGNESE FILIPINI ◽  
CLÁUDIA MARIA da SILVA ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Limit Of Detection ◽  
False Positive Rate ◽  
False Negative ◽  
False Negative Rate ◽  
Reference Method ◽  
Culture Method ◽  
Official Method ◽  
Positive Rate ◽  
Pcr Method

A two-stage study compared the BAX system PCR method with the reference culture method used by the Brazilian Ministry of Agriculture and Food Supply for the detection of Salmonella in food, water, and environmental samples. In stage 1, fish matrix samples (n = 258) were spiked at several levels with Salmonella and a combination of Salmonella and non-Salmonella competitive organisms. Replicates were analyzed by the BAX system PCR method and the reference method with comparable results (sensitivity ≥ 97.5%, specificity ≥ 83.3%) from both methods at the limit of detection. In stage 2, a total of 1,988 samples with 70 product types were analyzed with both methods. Five laboratories were involved in this study, and the samples used were from routine analyses. The BAX system PCR method was shown to be comparable to the reference method, with a limit of detection of 1.0 to 2.0 CFU/25 g of sample. Analysis of the results obtained in stage 2 and in the combination of stages 1 and 2 for the BAX system showed the following performance: sensitivity ≥ 99.0%, specificity ≥ 97.2%, false-negative rate ≤ 1.1%, and false-positive rate ≤ 2.8%. Therefore, the BAX system appears to be equivalent to the reference method, with ≥ 97.3% agreement.

UK Variant COVID-19

2021 ◽  
pp. 601-604
Author(s):  
Manish Kumar Balai
Keyword(s):  
Diagnostic Method ◽  
Warning Signs ◽  
Antigenic Drift ◽  
Receptor Binding Domain ◽  
Rt Pcr ◽  
Genetic Sequencing ◽  
New Variant ◽  
Control And Prevention ◽  
Reliable Diagnostic Method ◽  
Breathing Problem

Virus has a nature of antigenic drift and shift so over time their genetic make-up changes through the mutation and emerged a new strain of virus. Six type of coronavirus are known to infect the human being till now. The latest one and seventh number coronavirus is COVID-19. Diseases caused by coronavirus ranging from common cold to severe acute respiratory syndrome (SARS-CoV). One more strain or mutant COVID-19 identified at UK in September 2020 and this strain is fast and quick spreading that is 70% more transmissible than the old strain. The first “Variant Under Investigation” in December -2020 in which most significant mutation known as N501Y change the protein spike (S) of the virus that is the receptor binding domain, used to bind the human ACE2 receptors. Five alarming warning signs of new strain of coronavirus are Breathing problem, Confusion, Persistent chest pain, Tired and unable to stay awake, Bluish lips or face. RT-PCR and genetic sequencing still reliable diagnostic method for new variant of corona. Control and prevention need to follow all guidelines which are following previously and till now many vaccines developed but scientist need more research and data for its efficacy on new variant of corona.

scholarly journals Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Lei Ma ◽  
Fanwen Zeng ◽  
Bihong Huang ◽  
Feng Cong ◽  
Ren Huang ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Watery Diarrhea ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Molecules ◽  
Positive Rate

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

scholarly journals Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens

2020 ◽  
Vol 21 (7) ◽  
pp. 2574 ◽  
Author(s):  
Cyril Chik-Yan Yip ◽  
Chi-Chun Ho ◽  
Jasper Fuk-Woo Chan ◽  
Kelvin Kai-Wang To ◽  
Helen Shuk-Ying Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Respiratory Viruses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Coronavirus Infection ◽  
Polymerase Chain ◽  
Novel Coronavirus

The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.

scholarly journals Detection of Polioviruses in Sewage Using Cell Culture and Molecular Methods

2026 ◽  
Vol 65 (4) ◽  
pp. 479-483
Author(s):  
Agnieszka Figas ◽  
Magdalena Wieczorek ◽  
Bogumiła Litwińska ◽  
Włodzimierz Gut
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Cell Cultures ◽  
Environmental Samples ◽  
Genetic Material ◽  
Culture Technique ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Step Algorithm ◽  
Cell Culture Technique

The work presented here demonstrates the utility of a two-step algorithm for environmental poliovirus surveillance based on: preselection of sewage samples tested for the presence of enteroviral genetic material-RT-PCR assay and detection of infectious viruses by cell culture technique (L20B for polioviruses and RD for polio and other non-polio enteroviruses). RD and L20B cell lines were tested to determine their sensitivity for isolation of viruses from environmental samples (sewage). Finally, we wanted to determine if sewage concentration affects the results obtained for RT-PCR and cell cultures.

scholarly journals One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1560 ◽  
Author(s):  
Rashi Gautam ◽  
Slavica Mijatovic-Rustempasic ◽  
Mathew D. Esona ◽  
Ka Ian Tam ◽  
Osbourne Quaye ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Group A ◽  
One Step ◽  
Stool Samples ◽  
Pcr Assays ◽  
Type Strains

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

scholarly journals Sensitive and specific qPCR and nested RT-PCR assays for the detection of Tobacco streak virus in soybean

2021 ◽  
Author(s):  
Cristina Zambrana-Echevarria ◽  
Mitchell Roth ◽  
Ranjit Dasgupta ◽  
Thomas German ◽  
Carol Groves ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Management Strategies ◽  
Absolute Quantification ◽  
Qpcr Assay ◽  
Diagnostic Tools ◽  
Tobacco Streak Virus ◽  
Streak Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Research Fields

Tobacco streak virus (TSV) is a re-emerging and understudied pathogen of soybean (Glycine max). Management of TSV is challenging due to the multiple modes of transmission, widespread susceptibility of commercial soybean, and lack of reliable diagnostic tests for the virus. Soybean plants with TSV-like, virus-like, or no symptoms were collected from commercial and research fields in seven counties in Wisconsin. Two sensitive assays were developed for the detection of TSV: a fluorescent dye-based quantitative RT-PCR (qPCR) assay and a nested RT-PCR (nRT-PCR). Tobacco streak virus was detected in 47% and 91% of symptomatic samples using the qPCR assay and the nRT-PCR assay, respectively, suggesting that the nRT-PCR assay has higher sensitivity for detecting TSV. The qPCR assay’s limit of detection was determined at 10 fg and the assay was used to estimate the viral load in TSV-symptomatic samples. The titer of TSV in these samples was determined by absolute quantification and ranged from 15 fg to 0.796 ng. The two assays reported here provide diagnostic tools for the rapid and accurate detection of TSV that can aid in monitoring outbreaks, assessing management strategies, or screening soybean cultivars/accessions for resistance to the virus.

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