Kumar Arbind, Kumar Arun, Padwad Yogendra, Sharma Shaifali, Kumar Sanjay
COVID-19 Testing facility, CSIR-Institute of Himalayan Bioresource Technology (IHBT), Palampur, India.
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (IHBT), Palampur, India.
J Infect Dev Ctries. 2025 Jun 30;19(6):833-842. doi: 10.3855/jidc.18079.
The aim of this study is to develop a one-step real-time PCR assay for SARS-CoV-2 detection. The study was designed to circumvent the routine RNA isolation step and to optimize a lysis buffer and parameters for direct quantitative PCR.
A lysis solution was prepared using Tween-20, Triton X-100, EDTA, and Tris buffer (pH 7.4). Various parameters including the use of detergent combinations, V/V ratios and usage of carrier molecules were standardized to achieve the optimal amplification curve and Ct values of SARS-CoV-2 gene for improving the routine diagnostics procedures.
Adding carrier molecules [Poly(A), glycogen, and linear polyacrylamide] to the lysis solution significantly improved real-time reverse-transcription PCR (rtRT-PCR) efficacy. Poly(A) was the most effective of all carriers. The diagnostic potential of this Poly(A) solution was demonstrated using 150 patient swabs infected with SARS-CoV-2 and 200 uninfected swab samples, and the sensitivity of the rtRT-PCR diagnostic test was estimated to be 98.6 (95% CI: 96.0, 101.17, p < 0.001) for group 1; Ct ≤ 25 and 87.2 (95% CI: 80.2, 94.0, p < 0.001) for group 2; Ct ≥ 26-30, with excellent accuracy (0.9 < AUC < 1.0), and 100% specificity.
Our finding imply that this strategy is feasible, and it may contribute to the development of a rapid, less laborious, and economical rtRT-PCR test for dealing with the SARS-COV-2 disease in the pandemic area.
本研究的目的是开发一种用于检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的一步法实时聚合酶链反应（PCR）检测方法。该研究旨在规避常规的RNA分离步骤，并优化用于直接定量PCR的裂解缓冲液和参数。
使用吐温-20、曲拉通X-100、乙二胺四乙酸（EDTA）和三羟甲基氨基甲烷（Tris）缓冲液（pH 7.4）制备裂解溶液。对包括洗涤剂组合的使用、体积/体积比和载体分子的使用等各种参数进行标准化，以获得SARS-CoV-2基因的最佳扩增曲线和循环阈值（Ct）值，从而改进常规诊断程序。
向裂解溶液中添加载体分子[聚腺苷酸（Poly(A)）、糖原和线性聚丙烯酰胺]可显著提高实时逆转录PCR（rtRT-PCR）的效率。Poly(A)是所有载体中最有效的。使用150份感染SARS-CoV-2的患者拭子和200份未感染的拭子样本证明了这种Poly(A)溶液的诊断潜力，rtRT-PCR诊断试验的灵敏度估计对于第1组为98.6（95%置信区间：96.0，101.17，p<0.001）；Ct≤25，对于第2组为87.2（95%置信区间：80.2，94.0，p<0.001）；Ct≥26 - 30，具有优异的准确性（0.9<AUC<1.0），且特异性为100%。
我们的研究结果表明该策略是可行的，并且可能有助于开发一种快速、省力且经济实惠的rtRT-PCR检测方法，以应对大流行地区的SARS-CoV-2疾病。
