套式PCR应用于恙虫病鼠类标本的检测研究

DETECTION OF ORIENTIA TSUTSUGAMUSHI DNA FROM THE SPECIMENS OF BLOOD CLOT AND SPLEEN COLLECTED BOTH FROM EXPERIMENTAL INFECTED MICE AND FIELD RODENTS BY NESTED POLYMERASE CHAIN REACTION

目的 评价基因扩增法应用于检测鼠类标本调查恙虫病疫源地的作用。方法 用一定量恙虫病东方体人工感染昆明种小鼠 ,攻击后第 3、6、9天处死小鼠取血块及脾脏用特异性套式 PCR技术进行恙虫病东方体 DNA的检测 ,再用该技术测定现场采集的鼠类标本 ,以可从标本中扩增出一段长 88bp的 DNA片段作为实验或现场捕获鼠感染了恙虫病东方体的指标 ,继而估计该地区是否存在恙虫病疫源地。结果 实验感染恙虫病东方体 3天时在两类标本中均不能检出该病原 DNA,而第6天测定时均可发现含有恙虫病东方体 ;从福建闽西北地区采集的 111份鼠类脾脏中检出 1份阳性标本 ,另从江西送检的 2 9份野鼠血块中也检出 1份阳性标本 ,说明采集标本区域已成为恙虫病疫源地。结论 套式 PCR扩增技术具有很好的特异性和敏感性 ,可用于现场鼠类标本的恙虫病病原学的调查。

Aim\ To evaluate the efficiency of gene amplification technique used in detecting the specimens colleted from rodents to identify natural epidemic foci of scrub typlus.Method\ Mice of Kunining strain were experimentally infected by a certain amount of Oriential tsutsugamushi.The specimens of blood clot and spleen from the infected animals were detected by nested polymerase chain reaction(nPCR)specific to O.T sutsugamush at the day 3,6 and 9 of post-infection.Then the technique was used for detection of samples collected from field.As an infected index,the specimen was considered to be positive only if a 88-bp DNA fragment from Sta 58kDa gene of O.Tsutsugamushi could be produced.According to the study ,it was estimated whether or not that the sampling area is a natural epidemic focus of the disease.Results\ The specimens of both blood clot and spleen from the mica at day 3 of post-infecction showed negative to the specific PCR product,but positive when detected at day 6 and hereafter.Of 111 spleen samples from the field collections in the northwest of Fujian province,one was positive ,and another positive sample was in the 29 blood clots from Jiangxi province.It is demonstrated that these areas have been the natural epidemic foci.Conclusion \ The nPCR method is of highly sensitive and specific to be used in the etiologic study on specimens from field rats.