应用手足口病病原体验证改良的DNase-SISPA方法检测未知病毒感染的特异性

Apply hand-foot-mouth disease(HFMD) pathogens to verify the specificity of the improved DNase-SISPA method for the detection of an unknown viral infection

目的以引起手足口病的肠道病毒EV71为研究对象,在DNaseI-非序列依赖的单引物扩增技术的基础上建立感染性疾病RNA病毒性病原体的检测方法。方法根据卫生部发布的《手足口病预防控制指南》(2008年版)附件手足口病实验室检测方案(试行),收集临床诊断手足口病的患儿粪便,经实验室RT-PCR检测阳性后,进行过滤、DNase消化处理。反转录、合成病毒RNA的双链cDNA,Taq I酶切双链cDNA,加接头分子,并以接头分子为引物非特异扩增病原基因,测序并进行序列分析。结果非序列依赖的单引物扩增粪便标本中外源核酸得到多个DNA片段;将所有PCR产物克隆测序,有的序列与已知病原EV71基因序列同源性达99%;有的序列与单核细胞增生李斯特菌(Listeria monocytogenes) strain CDC 54007同源性达81%。结论应用DNase处理及非序列依赖的单引物扩增方法可以检测粪便标本中的RNA病毒性病原,并应用该改良技术检测到单核细胞增生李斯特菌。

Objective Apply the enterovirus EV71 that caused the hand-foot-mouth disease to set up a methods to detect RNA viral pathogen of the infectious disease based on DNase-sequence-independent single primer amplification.Methods According to hand-foot-mouth disease laboratory detect program（for trial implementation）,that is,the appendix of ＂the Guide of Hand-Foot-Mouth Disease Prevention and Control＂（2008 edition） that Ministry of Public Health issued.To collect the faeces of infants that had been diagnosed hand-foot-mouth disease in the clinical and were positive by reverse transcription-polymerase chain reaction（RTPCR） in the laboratory,the specimens were filtered,digested by DNase,then reverse transcripted and synthesized double-stranded cDNA of the RNA virus,double-stranded cDNA were digested by Taq Ⅰand added to adapter molecule,pathogenic gene were amplified non-specificly with the adapter molecule as primers,then sequenced and analysed.Results we got a number of exogenous DNA fragments from faeces specimens of infants using DNase-sequence-independent single primer amplification.All PCR products were sequenced and analysed,we find that some sequences are 99% homologous to the known pathogens EV71 gene sequence,while some are 81% homologous to the Listeria monocytogenes strain CDC54007.Conclusion we can detect viral RNA pathogen from faeces specimens,and detect Listeria monocytogenes bacteria using DNase-sequence-independent single primer amplification.