METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process.
RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR.
CONCLUSIONS: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.
方法:在本研究中,我们开发并验证了人腺病毒3(HADV3)的多重逆转录重组酶辅助PCR(多重RT-RAP)检测方法,人腺病毒7(HADV7),和人呼吸道合胞病毒(HRSV)在1小时内以人RNaseP蛋白为参考基因监测整个过程。
结果:使用重组质粒,多重RT-RAP检测HADV3、HADV7和HRSV的灵敏度为每个反应18、3和18个拷贝,分别。多重RT-RAP显示与其他呼吸道病毒没有交叉反应,证明了其良好的特异性。通过多重RT-RAP测试了总共252个临床样本,发现结果与相应的RT-qPCR测定的结果一致。在对选定的阳性标本进行系列稀释测试后,多重RT-RAP的检测灵敏度比相应的RT-qPCR高2至8倍。
结论:我们得出结论,多重RT-RAP是一种稳健的,快速,高度敏感,和特异性测定,有可能用于筛选低病毒载量的临床样品。