基于PCR蜱体内巴贝虫检测方法的评价  

Evaluation of PCR-based diagnostic methods for identifying Babesia in ticks

作　　者：危芙蓉[1] 兰勤娴[1] 朱丹[1] 施文琦[1] 周正斌[1] 张仪[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾,血吸虫病和丝虫病合作中心

出　　处：《国际医学寄生虫病杂志》2015年第2期89-93,共5页International JOurnal of Medical Parasitic Diseases

基　　金：卫生行业科研专项(201202019);国家科技重大专项(2012ZX10004-220,2008ZX10004-011)~~

摘　　要：目的 分别对4种牛体表蜱体内巴贝虫和两种犬体表蜱体内巴贝虫基于PCR的检测方法进行评价. 方法 在广西壮族自治区6个市的62个村,现场采集犬和牛体表蜱.巢式PCR特异性扩增巴贝虫18s rDNA,纯化阳性样本的PCR产物、测序、比对,以确定感染的病原体.应用双重PCR、双重巢式PCR、双重环介导等温扩增 （loop-mediated isothermal amplification,LAMP）检测牛体表蜱内病原体双芽巴贝虫（Babesia bigemina）和牛巴贝虫（B.bovis）的感染情况.应用多重PCR检测犬体表蜱的犬巴贝虫（B.canis canis）、佛氏巴贝虫（B.canis vogeli）和罗氏巴贝虫（＆canis rossi）的感染情况.将各方法的检测结果进行比较,对检测方法进行评价. 结果 采集牛体表蜱102只.巢式PCR、双重PCR、双重巢式PCR检测牛体表蜱内双芽巴贝虫和牛巴贝虫均为阴性,双重LAMP的浊度仪检测结果为5例阳性,颜色观测结果阳性样本为29例,该29例包含浊度仪检测的5例阳性.采集犬体表蜱184只.巢式PCR检测出犬体表蜱佛氏巴贝虫阳性样本9例.其中4例被多重PCR检测出佛氏巴贝虫阳性.同时多重PCR检测检出罗氏巴贝虫阳性样本4例,经测序均为罗氏巴贝虫阴性. 结论 双重PCR、双重巢式PCR检测牛体表蛑体内巴贝虫未出现假阳性情况,巢式PCR检测微小巴贝虫,双重PCR、双重巢式PCR未发生交叉反应,说明其具有一定特异性,但因无阳性样本,敏感度需要进一步评价.双重LAMP颜色观测结果阳性率15.76％,浊度仪测出阳性率2.72％,其中双重LAMP颜色观测法2例阳性样本为巢式PCR微小泰勒虫阳性（Theileria microti.双重LAMP法直观快捷,但对其检测的阳性样本需进行进一步确认,同时由于该方法的高度敏感性,需注重预防实验污染.犬体表蜱多重PCR可一次鉴别3种犬巴贝虫,鉴于在本研究中的假阳性和假阴性情况,多重PCR不适用,建议将病原体独立检测.Objective To evaluate 4 PCR-based diagnostic methods to detect Babesia in ticks from dogs and cattle,respectively.Methods Specimens of ticks were collected from 62 counties of 6 municipals in Guangxi Zhuang Autonomous Region.The 18s rDNA genes of Babesia were specifically amplified by nested PCR.The PCR products of positive samples were purified,sequenced and blasted in GenBank.Duplex PCR and duplex nested PCR,duplex Loop-mediated isothermal amplification （LAMP） were evaluated for detection of Babesia bigemina and B.bovis infections in cattle.Multiplex PCR was evaluated for B.canis canis,B.canis vogeli and B.canis rossi in dogs.The comparison of the diagnostic methods results was taken to evaluate the performance of each method.Results A total of 102 tick samples was collected from cattle.No positive sample was found by nested PCR,duplex PCR and duplex nested PCR,while 5 and 29 positive samples were detected by duplex LAMP of which results determined by turbidity reader and visual color change,respectively.The 5 positive samples by turbidity reader are also positive by visual color change.184 tick samples were collected from dogs.9 B.canis vogeli,positive samples were detected by nested PCR.4 of the 9 samples were B.canis vogeli,positive by multiplex PCR.5 B.canis rossi were determined by multiplex PCR,while sequencing results were totally negative.Conclusion Methods of duplex PCR and duplex nested PCR for bovine babesiosis,showed high specificity,with no false positive claimed.The sensitivity should be further evaluated for the next step due to the lack of positive specimens.Positive rates of 15.76% and 2.72% were determined by duplex LAMP with turbidity reader and visual color change,respectively.While 2 of the 29 visual color change were B.microti positive by nested PCR.Despite of easy and quick operation,positive results should be confirmed.And contamination should be prevented efficiently due to the high sensitivity of LAMP.Though the diagnosis of B.canis,B.vogeli,B.rossi infections in dogs could be made

关 键 词：巴贝虫 蜱 PCR 评价 

分 类 号：R378.21[医药卫生—病原生物学]