卵形巴贝斯虫与中华泰勒虫双重PCR检测方法的建立

Establishment and application of multiplex PCR method for detection of Babesia ovata and Theileria sinensis

为建立一种能快速对卵形巴贝斯虫(Babesia ovata)和中华泰勒虫(Theileria sinensis)同时进行检测的双重PCR方法。根据GenBank已报道的卵形巴贝斯虫AMA1基因和中华泰勒虫MPSP基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。结果显示:双重PCR可特异扩增出卵形巴贝斯虫和中华泰勒虫目的条带,片段大小分别为500 bp和986 bp。该方法具有较好的特异性,对卵形巴贝斯虫和中华泰勒虫的最低检出浓度为16 fg/μL。对采集的90份牛血液样本进行双重PCR检测,卵形巴贝斯虫阳性率为30%(27/90),中华泰勒虫阳性率为16.67%(15/90),混合感染率为10%(9/90)。结果表明,双重PCR方法可用于卵形巴贝斯虫和中华泰勒虫的快速诊断和流行病学调查。

In order to establish a fast duplex PCR method to simultaneously detect Babesia ovata and Theileria sinensis.The primers which could specificall amplify the sequences of the AMA1of Babesia ovata,and the 16S rRNA of Theileria sinensis.A multiplex PCR was developed to amplify specific DNA fragment of Babesia ovata and Theileria sinensis with the target Sequence of 986 bp and 500 bp.This method was specific in detecting Babesia ovata and Theileria sinensis.The detection limit was 16 fg/μL for Babesia ovata and Theileria sinensis,respectively.Clinical samples from Shulan in jilin province were detected by the multiplex PCR and the detection rate was 30%for Babesia ovata;16.67%for Theileria sinensis and 10%for mixed infection.It showed that the established multiplex PCR is suitable for quickly clinical detection and molecular epidemiological investigation.