PDF(Pubmed)
Abstract：
BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology.
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.
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.
摘要：
背景：聚合酶链反应（PCR）已广泛用于许多病原体检测。然而,PCR技术仍然存在检测时间长、灵敏度不足的问题。重组酶辅助扩增（RAA）是一种功能强大的核酸检测工具，具有较高的灵敏度和扩增效率，但其复杂的探针和无法多重检测阻碍了该技术的进一步应用。
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。
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。
