Molecular Biology Laboratory, OMC Healthcare (Pvt.) Limited, Rupnagar, Dhaka, Bangladesh.
Infectious Disease Laboratory, Institute for Developing Science & Health Initiatives, Dhaka, Bangladesh.
PLoS One. 2022 Oct 20;17(10):e0276464. doi: 10.1371/journal.pone.0276464. eCollection 2022.
The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19.
本研究旨在通过使用常规和指定的实时热循环仪系统，缩短用于检测 SARS-CoV-2 N1 和 N2 序列以及人 RNase P（RP）序列作为内部 mRNA 对照的多重 RT-PCR 运行时间。使用基于质粒的 N1 和 N2 阳性对照和人 RP 的合成版本，对基于Applied Biosystems（ABI）QuantStudioTM5（常规）、ABI 7500 Fast Dx（指定）和 CFX96 Touch Real Time Detection System、Bio-Rad（常规）的 Fast PCR 方案进行优化。最后，根据灵敏度、特异性和精密度对 Fast PCR 的性能进行评估。对于在 QuantStudioTM5、ABI 7500 Fast Dx 和 CFX96 Touch（常规）上进行优化的 Fast PCR 方案的 40 个循环 PCR，标准/常规与 Fast PCR 运行时间（分钟）分别为 84 比 49、96 比 48 和 103 比 61，从而分别节省 35、48 和 43 分钟。对于每个热循环仪，标准和 Fast PCR 生成了相同形状的荧光曲线、Ct 值和（3）R2（0.95 到 0.99），对于每个阳性对照的 5 个 10 对数稀释板。快速 PCR 方法产生了 100%的灵敏度和特异性的结果。COVID-19 样本的标准 PCR 和 Fast PCR Cts 的中位数测试比较没有产生显著性（p>0.5），表明 Fast PCR 和标准 PCR 是可比的。此外，每个靶标的中位数和平均值具有密切相关的值，进一步表明这两种方法是可比的。也就是说，对于 COVID-19 的检测，常规和快速 PCR 仪器之间存在等效性。
