Strand-Specific Reverse Transcription PCR for Detection of Replicating SARS-CoV-2.
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ABSTRACT:
SUBMITTER: Hogan CA
PROVIDER: S-EPMC7853532 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The emergence of a new coronavirus in Wuhan China has triggered a global need for accurate diagnostic assays. Initially, mostly laboratory developed molecular tests were available but shortly thereafter different commercial assays started to appear and are still increasing in number. Although independent performance evaluations are ongoing, available data is still scarce. Here we provide a direct comparison of key performance characteristics of 13 commercial RT-PCR assays. Thirteen RT-PCR assays were selected based on the criteria that they can be used following generic RNA extraction protocols, on common PCR platforms and availability. Using a 10-fold and 2-fold dilution series of a quantified SARS-CoV-2 cell-cultured virus stock, performance was assessed compared to our in house validated assay. Specificity was tested by using RNA extracted from cultured common human coronaviruses. All RT-PCR kits included in this study exhibited PCR efficiencies > 90%, except for the Sentinel Diagnostics B E-gene RUO assay (80%). Analytical sensitivity varied between 3.3 RNA copies to 330 RNA copies. Only one assay cross reacted with another human coronavirus (MERS). This study provides a technical baseline of 13 different commercial PCR assays for SARS-CoV-2 detection that can be used by laboratories interested in purchasing any of these for further full clinical validation.
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Project description:Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.
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Project description:Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.
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Project description: Not available
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Project description:INTRODUCTION The Coronavirus disease 2019 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the rise of many available modalities for diagnosis. One such modality is the Reverse Transcription–Polymerase Chain Reaction (RT-PCR) kits which require evaluation amongst the many available commercial kits in the market. METHODS We conducted a performance evaluation of twelve RT‐PCR SARS‐CoV‐2 commercial kits. A total of 75 nasopharyngeal and oropharyngeal clinical samples were selected with their cycling threshold (Ct) values. Inclusion of 5 gene targets: E gene, N gene, S gene, RdRp and ORF1ab were assessed. Data was analyzed using R software version 4.1.1 and Microsoft Excel RESULTS We observe that, the positive sample's Ct values differs significantly across the 12 diagnostic kits. However, for gene-specific analysis, we observe that, positive sample's Ct values does not differ significantly across gene targets. There is significant difference in Ct values in Commercial kits targeting all genes except S-gene. All the commercial kits Altona (E and S genes), Thermo (ORF1ab and N genes), Multiplex (E, ORF1ab, RdRdp genes), Meril (N and ORF1ab genes), S D Biosensor (E and ORF1ab genes), Lab Gun (RdRp and N genes) and Lab systems (ORF1ab and E genes) scored a sensitivity of 100%. All other kits scored sensitivity above 95% and lowest sensitivity with the Genes2me (E gene) and Genes2me (RdRp) at 95.08% each. All kits were 100% specific. CONCLUSION This study provides an accurate comprehensive assessment of the different kits in the detection of SARS-CoV-2 which may promote standardization of testing across laboratories.
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Project description:Diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for patient management, infection prevention, and the public health response for coronavirus disease 2019 (COVID-19). The efficacy and reliability of these assays are of paramount importance in both tracking and controlling the spread of the virus. Real-time reverse transcription-PCR (RT-PCR) assays rely on a fixed genetic sequence for primer and probe binding. Mutations can potentially alter the accuracy of these assays and lead to unpredictable analytical performance characteristics and false-negative results. Here, we identify a G-to-U transversion (nucleotide 26372) in the SARS-CoV-2 E gene in three specimens with reduced viral detection efficiency using a widely available commercial assay. Further analysis of the public GISAID repository led to the identification of 18 additional genomes with this mutation, which reflect five independent mutational events. This work supports the use of dual-target assays to reduce the number of false-negative PCR results.
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Project description:Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.
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Project description:Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.
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