A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus

作者：Shen, Xin-xin; Qiu, Fang-zhou; Zhao, Huai-long; Yang, Meng-jie; Hong, Liu; Xu, Song-tao; Zhou, Shuai-feng; Li, Gui-xia; Feng, Zhi-shan; Ma, Xue-jun^*

来源：Diagnostic Microbiology and Infectious Disease, 2018, 90(3): 181-185.

DOI：10.1016/j.diagmicrobio.2017.11.015

• Human enteroviruses
• Detection
• RTN RT-PCR

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 RTPCR (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 3820, 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 RN RT-PCR assay was extensively analyzed in 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.

• 出版日期2018-3
• 单位中国疾病预防控制中心; 河北医科大学; 河北省儿童医院; 山东理工大学; 中国疾病预防控制中心病毒病预防控制所