VP4基因鉴定非EV71非CoxA16手足口病病原体漏检的研究

Research on undetection of non-EV71 and non-CoxA16 related HFMD pathogens identified by using VP4 gene

目的探讨肠道病毒VP4基因通用引物PCR诊断非肠道病毒71型(EV71)非柯萨奇病毒A组16型(CoxA16)手足口病(HFMD)病毒方案的准确性。方法通过肠道病毒VP4基因通用引物PCR鉴定阜阳地区非EV71非CoxA16HFMD病毒时,显示两例病毒分离株为Sabin-1(命名为Fy-01和Fy-02株)。分别采用引物EVP4/Q8和VP1特异性引物对Fy-01和Fy-02分离株进行鉴定。结果采用VP4基因PCR扩增产物测序证实,两例病毒分离株为Sabin-1,而采用EVP4/Q8引物PCR扩增Fy-01分离株却存在异常的扩增条带,PCR扩增产物测序结果为柯萨奇病毒A组10型(CoxA10);将VP4基因PCR产物制备成克隆载体挑选多个单克隆测序结果显示,Fy-01分离株既存在Sabin-1 VP4基因,又存在CoxA10 VP4基因,证实为两种毒株混合感染。结论临床诊断为非EV71非CoxA16 HFMD的病例,如病原学诊断仅采用VP4基因PCR产物测序,特别是针对多种肠道病毒混合感染引起的HFMD,其结果并不完全可靠。

Objective To investigate the accuracy of PCR for detection of non-EV71 and non-CoxA16 by using universal primers with VP4 gene of HEV. Methods When identified by VP4 gene of EV71 and CoxA16 unrelated HFMD,two isolates of Sabin-1 were found(such as Fy-01 and Fy-02). They were identified by co-primers such as EVP4 /Q8 and specific primers of VP1 gene. Results Fy-01 isolates were appeared with an unusual frayment and identified as CoxA10 after being sequenced and the selected independent clones were sequenced and blasted in GenBank. It proved that both Sabin-1 and CoxA10 existed. Conclusion Clinical diagnosis of EV71 and CoxA16 unrelated HFMD,when identifying the type of HFMD pathogen only by using the amplification product of VP4 gene after being sequenced directly is not enough,especially HFMD with mixed infections caused by enterovirus.