应用16S rDNA克隆文库筛查腹泻病暴发病原体及特异毒力基因验证

目的以16S rDNA克隆文库筛查某地腹泻病暴发的病原体,并验证筛查结果的可靠性。方法收集12份暴发期内腹泻患者的粪便标本,抽提粪便中总细菌基因组,随机抽取3例患者,应用中国传染病预防控制国家重点实验室建立的16S rDNA克隆文库方法筛查可能的致腹泻病原菌,根据筛查结果,设计致腹泻病原菌特异性毒力基因的引物进行PCR验证,同时对其它9份粪便标本进行该致病菌的PCR检测,根据致病菌的检出比例,确定可能导致本次腹泻病暴发的病原体。结果用16S rDNA克隆文库在3例患者标本中筛查到2例可能存在志贺菌或大肠杆菌,用针对志贺菌的特异性毒力基因ipaH对该2例患者标本进行PCR检测为阳性。在其它9例腹泻患者粪便标本中6例检测到志贺菌,志贺菌在腹泻病例中的总检出率为66.7%(8/12)。16S rDNA克隆文库筛查结果与志贺菌ipaH基因的验证结果均显示,3号病例的志贺细菌基因组在粪便总细菌基因组中所占的比例比2号病例高,两种检测结果具有一致性。结论志贺菌可能是导致某地本次腹泻病暴发的主要病原体。16S rDNA基因克隆文库法能快速筛查到可能的病原菌,其筛查结果比较可靠,因此,该方法对于不明原因疾病暴发流行的病原体筛查具有指导作用。

Pathogen screening in diarrheal disease outbreak with 16S rDNA clone library and specific virulence gene verification

In order to investigate the pathogen causing an outbreak of diarrhea and test the reliability of 16S rDNA library in screening suspicious pathogen,the feces from 12 diarrheal patients in the period of outbreak were collected and all the bacterial genome in stool was extracted.Samples from three patients were randomly selected to analyze bacterial species based on 16S rDNA sequence analysis method established by State Key Laboratory for Infectious Disease Prevention and Control（China） and to screen for the pathogen causing diarrhea.Then,the specific gene of diarrheal pathogen screened by 16S rDNA library was further verified by PCR.This specific gene was also tested in samples from other 9 diarrheal patients.Eventually,the diarrheal pathogen which led to the outbreak of diarrhea this time was confirmed according to the detection rate of diarrheal pathogen in 12 stool samples.Results showed that Shigella or E.coli was tested to be positive in 2 out of 3 diarrheal patients by 16S rDNA sequence analysis.Shigella was proved to exist in stool of those two patients after the PCR test of Shigella specific virulence gene ipaH.Meanwhile,Shigella was also detected positively in 6 out of other 9 diarrheal patients with PCR.The total detection rate of Shigella was 66.7%（8/12）.The result of 16S rDNA sequence analysis was consistent with that of PCR test because both results showed that the proportion of Shigella genome to bacteria genome in stool of patient Ⅲ was higher than that in stool of patient Ⅱ.Consequently,Shigella might be the primary pathogen causing the outbreak of diarrhea.In conclusion,the method of 16S rDNA library could find out the suspicious pathogen in a short time.Therefore,it is a novel method to conduct the screening for unknown disease pathogen in epidemics and outbreaks.