Development and preliminary application of a novel multiplex reverse transcriptase-polymerase chain reaction assay for simultaneous detection for enterovirus 71 and coxsackievirus A16
WANG Ting-Ting1; ZHU Ru-Nan1,2; QIAN Yuan1,2; DENG Jie2; ZHAO Lin-Qing2; WANG Fang2; DENG Li3
1. Capital Institute of Pediatrics, Peking University, Beijing 100020, China ; 2. Laboratory of Virology, Capital Institute of Pediatrics, Beijing 100020, China; 3. The Children’s Hospital Affiliated to Capital Institute of Pediatrics, Beijing 100020, China
Abstract：The present paper aims to develop a rapid, accurate and efficient technique to detect enterovirus 71 (EV71) and coxsackievirus A16 (CA16) simultaneously for the etiological investigation of hand, foot and mouth disease (HFMD) in children during epidemic seasons in Beijing. Primers including one pair of universal enterovirus primers based on highly conserved region of 5’UTR of enteroviruses and two pairs of type-specific primers based on highly conserved VP1 region of EV71 and CA16 were designed and used at different concentrations in one single reaction tube to develop a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) which was carried out with two annealing temperatures. After the sensitivity and specificity of the multiplex RT-PCR were evaluated, the technique was applied to use for detecting EV71 and CA16 from clinical specimens collected from pediatric patients with HFMD who visited the Children’s Hospital Affiliated to Capital Institute of Pediatrics during the period of March to October in 2010. All the specimens were also inoculated into the Vero cells for virus isolation, which was used as a golden standard for this study. The minimum detectable concentration for CA16 and EV71 were 5.32 pg/ml and 0.64 pg/ml, respectively. The specificity of multiplex RT-PCR was 100%. The application of multiplex RT-PCR in 381 clinical samples collected from 371 HFMD cases showed that the total positive rate was 78.4%, of which the detection positive rates of CA16 and EV71 were 32.6% and 35.8% (the tested positive ratio of CV16:EV71 was 1:1.1). Comparative analysis with virus isolation demonstrated that the detection accuracy of this method was up to 95.2% for CA16 and 98.6% for EV71. The results indicate that this novel multiplex RT-PCR offers a rapid, sensitive and time-saving method to detect EV71 and CA16 from clinical specimens and can be used for the etiological surveillance of HFMD. Both CA16 and EV71 were still the major pathogens of HFMD in Beijing in 2010.
王婷婷1; 朱汝南1,2; 钱渊1,2; 邓洁2; 赵林清2; 王芳2; 邓莉3. 肠道病毒71型和柯萨奇病毒A16型多重反转录聚合酶链反应的建立及初步应用[J]. 微生物与感染
, 2011, 6(1): 11-17.
WANG Ting-Ting1; ZHU Ru-Nan1,2; QIAN Yuan1,2; DENG Jie2; ZHAO Lin-Qing2; WANG Fang2; DENG Li3. Development and preliminary application of a novel multiplex reverse transcriptase-polymerase chain reaction assay for simultaneous detection for enterovirus 71 and coxsackievirus A16. Journal of Microbes and Infections, 2011, 6(1): 11-17.
2011, Vol. 6 
