Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay

Abstract

Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID(50)) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.

Fig 1

Specificity of the multiplex RT-PCR assay. Cy5-labeled PCR products were separated via GeXP capillary electrophoresis and detected by fluorescence spectrophotometry, given as dye signals in arbitrary units on the y axis. Each peak was identified by comparing the expected to the actual PCR product size on the x axis. EV71, CVA16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3, and CVB5 were assayed by using enterovirus RNAs extracted from various cell-cultured strains. Nuclease-free water was used as the negative control (NTC) (J).

Fig 2

Sensitivity of GeXP detection of 10 premixed RNA templates with multiplex primers. All 10 target genes could be detected at 10³ copies/μl (A) and 10² copies/μl levels (B); only CVA16 and CVA9 could be detected at 10 copies/μl levels (C). Nuclease-free water was used as the negative control (D).

Fig 3

Diagram of the GeXP amplification workflow using a temperature switch PCR strategy.