Journal
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
Volume 90, Issue 3, Pages 181-185Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.diagmicrobio.2017.11.015
Keywords
Human enteroviruses; Detection; RTN RT-PCR
Categories
Funding
- National key research and development plan [2016TFC1202700, 2016YFC1200900, 2017YFC1200503]
- Beijing Municipal Science&Technology Commission project [D151100002115003]
- Guangzhou Municipal Science & Technology Commission project [201582150820]
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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. (C) 2017 Elsevier Inc. All rights reserved.
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