Razu Mamudul Hasan, Ahmed Zabed Bin, Hossain Md Iqbal, Rabbi Mohammad Fazle Alam, Nayem Maksudur Rahman, Hassan Md Akibul, Paul Gobindo Kumar, Khan Md Robin, Moniruzzaman Md, Karmaker Pranab, Khan Mala
Bangladesh Reference Institute for Chemical Measurements, Dhaka 1205, Bangladesh.
DNA Solutions Ltd., Dhaka 1207, Bangladesh.
Diagnostics (Basel). 2022 Oct 28;12(11):2617. doi: 10.3390/diagnostics12112617.
In this study, we evaluated the performance of the in-house developed rRT-PCR assay for SARS-CoV-2 RNA targeting the envelope (E) and nucleocapsid (N) genes with internal control as human RNase P. A total of 50 positive samples and 50 negative samples of SARS-CoV-2 were tested by a reference kit at site 1 and a subset (30 positives and 16 negatives) of these samples are tested blindly at site 2. The limit of detection (LoD) was calculated by using a replication-deficient complete SARS-CoV-2 genome and known copy numbers, where Pseudo-virus samples were used to evaluate accuracy. On site 1, among the 50 SARS-CoV-2 positive samples 24, 18, and eight samples showed high (Ct < 26), moderate (26 < Ct ≤ 32), and low (32 < Ct ≤ 38) viral load, respectively, whereas in site 2, out of 30 SARS-CoV-2 positive samples, high, moderate, and low viral loads were found in each of the 10 samples. However, SARS-CoV-2 was not detected in the negative sample. So, in-house assays at both sites showed 100% sensitivity and specificity with no difference observed between RT PCR machines. The Ct values of the in-house kit had a very good correlation with the reference kits. LoD was determined as 100 copies/mL. It also displayed 100% accuracy in mutant and wild-type SARS-CoV-2 virus. This Bangasure™ RT-PCR kit shows excellent performance in detecting SARS-CoV-2 viral RNA compared to commercially imported CE-IVD marked FDA authorized kits.
在本研究中，我们评估了内部开发的针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）包膜（E）基因和核衣壳（N）基因的逆转录实时荧光定量聚合酶链反应（rRT-PCR）检测方法，并以人核糖核酸酶P作为内部对照。在第1检测点，使用参考试剂盒对50份SARS-CoV-2阳性样本和50份阴性样本进行检测，其中一部分样本（30份阳性和16份阴性）在第2检测点进行盲测。通过使用复制缺陷型完整SARS-CoV-2基因组和已知拷贝数来计算检测限（LoD），其中使用假病毒样本评估准确性。在第1检测点，50份SARS-CoV-2阳性样本中，分别有24份、18份和8份样本显示高（Ct<26）、中（26<Ct≤32）和低（32<Ct≤38）病毒载量，而在第2检测点，30份SARS-CoV-2阳性样本中，每份各有10份样本显示高、中、低病毒载量。然而，阴性样本中未检测到SARS-CoV-2。因此，两个检测点的内部检测方法均显示出100%的敏感性和特异性，逆转录聚合酶链反应（RT-PCR）仪器之间未观察到差异。内部试剂盒的Ct值与参考试剂盒具有非常好的相关性。检测限确定为100拷贝/毫升。它在突变型和野生型SARS-CoV-2病毒中也显示出100%的准确性。与商业进口的获得欧盟体外诊断医疗器械指令（CE-IVD）标志且经美国食品药品监督管理局（FDA）授权的试剂盒相比，这款Bangasure™ RT-PCR试剂盒在检测SARS-CoV-2病毒RNA方面表现出色。
