A Novel and Highly Sensitive Real-Time nested RT-PCR Assay in a Single Closed Tube for Detection of Enterovirus

Publication date: Available online 2 December 2017
Source:Diagnostic Microbiology and Infectious Disease
Author(s): Xin-xin Shen, Fang-zhou Qiu, Huai-long Zhao, Meng-jie Yang, Liu Hong, Song-tao Xu, Shuai-feng Zhou, Gui-xia Li, Zhi-shan Feng, Xue-jun Ma
The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens.The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10⁻⁸ dilution with a corresponding Ct value of 38.20,36.45,36.75 and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2–10 fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzedin Silico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples.



from ! ORL Sfakianakis via paythelady.61 on Inoreader http://ift.tt/2AEg30H
via IFTTT