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1.
Euro Surveill ; 25(24)2020 06.
Article in English | MEDLINE | ID: covidwho-605372

ABSTRACT

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Diagnostic Equipment , Pneumonia, Viral/diagnosis , Clinical Laboratory Techniques/instrumentation , Feces/virology , Germany , Humans , Laboratories , Multiplex Polymerase Chain Reaction/instrumentation , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/instrumentation , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity
2.
Exp Mol Med ; 52(6): 963-977, 2020 06.
Article in English | MEDLINE | ID: covidwho-601243

ABSTRACT

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , DNA Primers , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , Coronavirus Infections/diagnosis , HEK293 Cells , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Pharynx/virology , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/methods , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
3.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-361278

ABSTRACT

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Multiplex Polymerase Chain Reaction , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction , DNA Primers , DNA Probes , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Viral Load
4.
J Clin Virol ; 128: 104448, 2020 07.
Article in English | MEDLINE | ID: covidwho-306323

ABSTRACT

BACKGROUND: In the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays. AIMS: To evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2. METHODS: We reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory. RESULTS: Initial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results. CONCLUSION: The AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Multiplex Polymerase Chain Reaction/methods , Pandemics , Pneumonia, Viral/diagnosis , Adult , Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Oropharynx/virology , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Reproducibility of Results , Sensitivity and Specificity
5.
Emerg Infect Dis ; 26(7): 1633-1635, 2020 07.
Article in English | MEDLINE | ID: covidwho-66389

ABSTRACT

Most reverse transcription PCR protocols for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 2-3 targets for detection. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with singleplex assays. This protocol could streamline detection and decrease reagent use during current high SARS-CoV-2 testing demands.


Subject(s)
Betacoronavirus/isolation & purification , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Humans , Nasopharynx/virology
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