马梨形虫病双重PCR检测方法的建立

Establishment of duplex PCR method for detection of equine piroplasma

为了建立马梨形虫病双重PCR检测方法,试验根据Gen Bank发表的驽巴贝斯虫和马泰勒虫18S rRNA保守基因序列分别合成2对特异性引物,以核酸混合物为模板,优化反应条件建立马梨形虫病双重PCR检测方法,并检验该方法的特异性和敏感性。同时用该双重PCR检测方法对采自昭苏种马场的马疑似病例全血进行检测,并与镜检法和单重PCR法进行比较。结果表明：该双重PCR检测方法能对驽巴贝斯虫和马泰勒虫核酸扩增出大小为529 bp和789 bp特异性目的片段,而对双芽巴贝斯虫、环形泰勒虫、羊泰勒虫、牛巴贝斯虫核酸的扩增均为阴性;驽巴贝斯虫和马泰勒虫阳性DNA被稀释1×108倍时均能检出其相应目的片段;对采集的46份马疑似病例血样进行PCR诊断,其中驽巴贝斯虫感染率为30.4%（14/46）,马泰勒虫感染率为41.3%（19/46）;双重PCR检测方法与单重PCR方法的符合率为100%。说明建立的双重PCR检测方法具有一定的特异性和敏感性,可用于驽巴贝斯虫和马泰勒虫临床感染隐性病例的联合检测与鉴别诊断。

The aim of the present study was toestablish a duplex PCR method for detection of equine piroplasma.Aceording to the 18S rRNA con- served gene sequences of Babesia caballi and Theileria equi published by GenBank,two pairs of specific primers were synthesized.Then, using nucleic acid mixture as template, the reaction conditions were optimized to establish a duplex PCR assay method for detection of equine piro- plasma, and the specificity and sensitivity of the method were tested.At the same time, the horse blood samples of suspected cases collected from Zhaosu equestrian farm were detected by the duplex PCR method, and compared with the microscopic examination and routine PCR method.The results showed that the specific fragments of 529 bp and 789 bp were amplified by using the duplex PCR from DNA samples of B.caballi and T. equi, respectively. But the nucleic acid amplification results of B. bigemina, T. annulata, T. sergenti and B. bovis were negative. When the posi- tive DNA concentrations of both B.cabaUi and T. equi were diluted 1 x l0s fold, the corresponding target fragments could still be detected.46 blood samples collected from suspected cases of horses were diagnosed by PCR. The infection rate of B.caballi was 30. 4% （14/46）. The infec- tion rate of T.equi was 41.3%（19/46）. The coincidence rate between the results of duplex PCR and the routine PCR was 100%.The results in- dicate that the duplex PCR detection method has certain specificity and sensitivity, and can be used for the detection and differential diagnosis of recessive cases of clinical infection of B.cabaUi and T.equi.