Rapid detection of hand, foot and mouth disease enterovirus genotypes by multiplex PCR
Rapid detection of hand, foot and mouth disease enterovirus genotypes by multiplex PCR
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DOI：
10.1016/j.jviromet.2018.05.005
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发表时间：
2018-08-01
影响因子：
3.1
通讯作者：
Shi, Weifeng
中科院分区：
文献类型：
--
作者：
Wang, Min;Ren, Qian;Shi, Weifeng
手足口病（HFMD）是一种与肠道病毒（EV）基因型感染相关的儿科疾病。主要的手足口病EV病原体是肠道病毒A71（EVA 71）和柯萨奇病毒A16（CVA 16）;然而，最近也出现了柯萨奇病毒A6（CVA 6）和柯萨奇病毒A10（CVA 10）。EV基因型不能在临床上区分，迫切需要一种新的方法来快速检测四种主要的手足口病EV基因型。本研究建立了同时检测CVA 6、CVA 10、CVA 16和EVA 71的多重荧光定量PCR方法。在临床样本的验证组上确定测定的特异性和灵敏度，所述临床样本包括脑脊液（n = 51）、血液（n = 39）、粪便（n = 58）和咽拭子（n = 29）。结果表明，该方法特异性高，与其他EV基因型无交叉反应，对CVA 6、CVA 10、CVA 16和EVA 71的检测下限分别为4 × 10（3）、4 × 10（2）、5 × 10（2）和3 × 10（3）copies/pL，与临床标本的单重检测方法具有相当的灵敏度。本研究建立的多重实时PCR方法可用于快速检测四种最流行的手足口病相关EV，用于流行病学监测EV基因型，评估治疗反应和疫苗研究。
Hand, foot and mouth disease (HFMD) is a pediatric disease associated with infection by enterovirus (EV) genotypes. The major HFMD EV pathogens are enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16); however, recently, coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have also emerged. EV genotypes cannot be distinguished on clinical grounds and a new methodology for the rapid detection of the four major HFMD EV genotypes is urgently required. In the present study, a multiplex real-time PCR assay was established for the simultaneous detection of CVA6, CVA10, CVA16 and EVA71. The specificity and sensitivity of the assay was determined on a validation panel of clinical samples, comprising cerebrospinal fluid (n = 51), blood (n = 39), feces (n = 58) and throat swabs (n = 29). The results showed that the multiplex real-time PCR exhibited high specificity, no cross-reactivity with other EV genotypes, lower limits of detection for CVA6, CVA10, CVA16 and EVA71 were 4 x 10(3), 4 x 10(2), 5 x 10(2), and 3 x 10(3) copies/pL, respectively and had comparable sensitivity to singleplex assays testing clinical samples. The multiplex real-time PCR methodology established in this study can be employed for the rapid detection of the four most prevalent HFMD-associated EVs, for epidemiologic surveillance of circulating EV genotypes and for assessing treatment responses and vaccine studies.