scholarly journals Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European BatLyssavirusType 1

2015 ◽  
Vol 2015 ◽  
pp. 1-18 ◽  
Author(s):  
Evelyne Picard-Meyer ◽  
Carine Peytavin de Garam ◽  
Jean Luc Schereffer ◽  
Clotilde Marchal ◽  
Emmanuelle Robardet ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Evaluation Criteria ◽  
Sybr Green ◽  
Detection Sensitivity ◽  
Analytical Sensitivity ◽  
Reaction Efficiency ◽  
Qrt Pcr ◽  
One Step ◽  
Pcr Assays

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency,R2values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

scholarly journals A Method to evaluate analytical sensitivity of qRT-PCR kits for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Di Wang ◽  
Zhidong Wang ◽  
Xiao Wu ◽  
Yongzhuo Zhang ◽  
Yunhua Gao
Keyword(s):  
Reference Material ◽  
Limit Of Detection ◽  
False Negative ◽  
Detection Sensitivity ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Limit Of Quantification ◽  
Qrt Pcr ◽  
Pcr Products ◽  
Pcr Assays

Abstract Background: The ongoing Coronavirus disease 2019 (COVID-19) pandemic has spread across the globe and is representing a huge challenge for all human population. Many commercial qRT-PCR assays have been developed to detect SARS-CoV-2, but related method validation especially the sensitivity evaluation has been insufficient, resulting in some false-negative cases have been reported. Methods: The analytical sensitivity of nine brands of qRT-PCR kits for detecting SARS-CoV-2 was evaluated in parallel based on a newly developed certified reference material, which was derived from genomic RNA of SARS-CoV-2 from clinical positive specimens. After validation of the the reference material by digital PCR, the detection sensitivity of these kits was preliminarily tested using the serially diluted reference material, resulting in three kits with two significantly different sensitivity levels were selected for further evaluation. We sequenced the qRT-PCR products for assay specificity evaluation, and used serial dilutions of the reference material to calculate amplification efficiency and estimate the limit of quantification as well as 95% limit of detection..Results: The results indicated that the analytical sensitivity varied markedly among these kits. For the three types of qRT-PCR kits (Kit-1, Kit-2 and Kit-7), specificity of the PCR products was confirmed by sequence alignment, in which the target amplicons completely matched the corresponding parts of the genome of SARS-CoV-2. The resulting limit of detection from replicate tests for the Kit-1 and Kit-2 was 5.6 copies (N), 3.5 copies (ORF 1ab), and 6.4 copies (N), 4.6 copies (ORF 1ab), respectively, at 95% probability. Compared with Kit-7, the limit of detection as well as limit of quantification of Kit-1 and Kit-2 were significantly lower, further supporting that the both kits worked well to detect low abundance of SARS-CoV-2.Conclusions: Considering that most of the tested kits have been approved for in vitro diagnostics (IVD) in China, the established method here provides a reliable tool to evaluate the sensitivity performance of various qRT-PCR kits for SARS-CoV-2 detection and thus enhance quality control of qRT-PCR assays, improving the laboratory diagnostic capability for fighting the COVID-19 pandemic.

scholarly journals Evaluation of Real-Time RT-PCR for Diagnostic Use in Detection of Puumala Virus

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 661 ◽  
Author(s):  
Silja Niskanen ◽  
Anne Jääskeläinen ◽  
Olli Vapalahti ◽  
Tarja Sironen
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Hemorrhagic Fever ◽  
Laboratory Diagnosis ◽  
Hantavirus Infection ◽  
Puumala Virus ◽  
Analytical Sensitivity ◽  
Serum Samples ◽  
Qrt Pcr ◽  
One Step

Puumala virus (PUUV) is the most common cause of hantavirus infection in Europe, with thousands of cases occurring particularly in Northern, Central and Eastern Europe and Russia. It causes a mild form of hemorrhagic fever with renal syndrome also known as nephropathia epidemica (NE) with clinical picture ranging from mild to severe. Currently, the laboratory diagnosis of NE is mainly based on serology. Here, we evaluated a real-time one-step qRT-PCR (PUUV-qRT-PCR) for detection of PUUV with 238 consecutive diagnostic serum samples from patients with suspected PUUV infection. The PUUV-qRT-PCR was both specific and sensitive for PUUV RNA. The analytical sensitivity (limit of detection) was estimated to be four copies of PUUV per reaction. Altogether 28 out of 30 (93%) PUUV IgM positive samples were positive also for PUUV RNA. No false positives were detected and the specificity was thus 100%. Interestingly, one sample was found positive in PUUV-qRT-PCR prior to subsequent IgM and IgG seroconversion. PUUV-qRT-PCR could be used for diagnostics in the early phase of NE infection and might be helpful especially in the rare severe cases when the patient’s condition may deteriorate rapidly.

Assessment of Commercial Real-Time PCR Assays for Detection of Malaria Infection in a Non-Endemic Setting

2021 ◽  
Author(s):  
Alexandra Martín Ramírez ◽  
Thuy Huong Ta Tang ◽  
Marta Lanza Suárez ◽  
Ana Álvarez Fernández ◽  
Carlota Muñoz García ◽  
...  
Keyword(s):  
Real Time ◽  
Malaria Control ◽  
Limit Of Detection ◽  
Reference Method ◽  
Malaria Diagnosis ◽  
Mean Value ◽  
Analytical Sensitivity ◽  
Accurate Treatment ◽  
Pcr Assays ◽  
Good Concordance

Malaria control and elimination require prompt diagnosis and accurate treatment. Conventional methods such as rapid diagnostic tests (RDTs) and microscopy lack the characteristics to detect low parasitemias, commonly found in asymptomatic parasitemias and/or submicroscopic malaria carriers. On the contrary, molecular methods have higher sensitivity and specificity. This study evaluated the performance of two commercial real-time polymerase chain reaction (PCR) assays, RealStar® Malaria PCR (RealStar-genus) and RealStar Malaria Screen&Type PCR (RealStar-species), compared with the reference Nested Multiplex Malaria PCR, for the detection of the main five Plasmodium species affecting humans. A total of 121 samples were evaluated. Values of sensitivity (98.9% and 97.8%) and specificity (100% and 96.7%) of the RealStar-genus and the RealStar-species assays, respectively, were very good. The limit of detection (LoD) for the RealStar-genus assay showed a mean value of 0.28 parasites/µL with Plasmodium falciparum samples; while, the LoD of the RealStar-species assay ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale. The time to complete a diagnosis was established in 4 hours. Our findings showed a very good concordance of both assays compared with the reference method, with a very good analytical sensitivity. RealStar-species assay was able to correctly characterize double and triple infections. Therefore, these RealStar assays have shown to be useful tools in malaria diagnosis in non-endemic countries and even endemic countries, and for malaria control in general, detecting low parasitemias with sensitivity similar to the most sensitive methods as nested PCR, but with lower time to get the results.

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 Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

2015 ◽  
Vol 53 (9) ◽  
pp. 2956-2960 ◽  
Author(s):  
Timothy R. Southern ◽  
Lori D. Racsa ◽  
César G. Albariño ◽  
Paul D. Fey ◽  
Steven H. Hinrichs ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Whole Blood ◽  
Limit Of Detection ◽  
Clinical Specimens ◽  
Qrt Pcr ◽  
Zaire Ebolavirus ◽  
Pcr Assays ◽  
Blood Specimens

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107to 4 × 10250% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD.

scholarly journals Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e95635 ◽  
Author(s):  
Zheng Pang ◽  
Aqian Li ◽  
Jiandong Li ◽  
Jing Qu ◽  
Chengcheng He ◽  
...  
Keyword(s):  
Real Time ◽  
Hemorrhagic Fever ◽  
Qrt Pcr ◽  
One Step ◽  
Pcr Assays ◽  
Detection And Quantification

scholarly journals A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Vanessa Suin ◽  
Florence Nazé ◽  
Aurélie Francart ◽  
Sophie Lamoral ◽  
Stéphane De Craeye ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Antigen Test ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Infectious Dose ◽  
Qrt Pcr ◽  
One Step ◽  
Polymerase Chain

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV.In silicosequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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