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Simultaneous detection of SARS-CoV-2 and identification of spike D614G mutation using point-of-care real-time polymerase chain reaction.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with high mortality and infectivity rates in humans since its emergence. Analysis using high-accuracy real-time polymerase chain reaction (PCR) is recommended for the detection of general respiratory viruses including SARS-CoV-2, but it takes a long time (e.g. ~ 6 h); moreover, on-site diagnosis is difficult owing to the need for skilled technicians and advanced laboratory facilities. Currently, the importance of point-of-care testing (POCT) is being emphasized for the rapid detection of SARS-CoV-2. Here, we developed a multiplex real-time reverse transcription PCR (rRT-PCR) analysis that not only detects SARS-CoV-2 but also D614G strains with higher contagiousness than wild types among SARS-CoV-2 mutants using probe-based rRT-PCR. Moreover, this method was applied to portable PCR equipment capable of POCT to confirm high detection sensitivity and specificity. Multiple assays were possible with fluorescence labeling of individual probes. Furthermore, using a microfluidic chip-based point-of-care testing rRT-PCR platform, detection time was reduced by more than half compared with the commonly used detection system. This demonstrates that our assay has 100% of high sensitivity and specificity and could thus aid in the rapid and simple screening of SARS-CoV-2 carrying the mutation. We present a rapid detection method for mutations in SARS-CoV-2.

SUBMITTER: Lee SY

PROVIDER: S-EPMC8897296 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

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Simultaneous detection of SARS-CoV-2 and identification of spike D614G mutation using point-of-care real-time polymerase chain reaction.

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