环形泰勒虫与牛无浆体双重PCR方法的建立

A duplex PCR method for detection of Theileria annulata and Anaplasma bovis

旨在建立快速检测环形泰勒虫和牛无浆体的双重PCR方法,调查吐鲁番地区这2种病原感染情况。根据NCBI库中上传的环形泰勒虫Spm2、Tams1基因和牛无浆体16S rRNA基因保守序列设计、合成3对特异性引物,使用PCR扩增并测序。根据Spm2和16S rRNA基因设计双重PCR并对反应体系及条件进行优化,对该方法进行特异性、灵敏性和重复性检测,建立一种双重PCR方法。结果显示,双重PCR方法特异性扩增出环形泰勒虫和牛无浆体相应目的片段,片段大小分别为853,351 bp,而驽巴贝斯虫、马泰勒虫、绵羊无浆体和伊氏锥虫均未扩增出条带。对环形泰勒虫Spm2、Tams1和牛无浆体16S rRNA基因序列进行系统发育分析,环形泰勒虫Spm2基因序列与印度株同源关系最近(MH844677)、Tams1基因序列与突尼斯株同源关系近(AF214899),牛无浆体16S rRNA基因序列与突尼斯株同源关系近(KY655808)。此方法能检测环形泰勒虫与牛无浆体最低浓度分别为2.9×10^(-16),1.8×10^(-19)g/μL。用该方法对临床60份牛血DNA进行双重PCR扩增,其中环形泰勒虫阳性率为66.7%(40/60),牛无浆体阳性率为53.3%(32/60),混合感染阳性率为43.3%(26/60)。双重PCR方法与已建立的环形泰勒虫和牛无浆体单一PCR方法检测结果的符合率为93.3%。成功建立了一种能同时检测2种病原的双重PCR方法。

The aim of this study was to establish a duplex PCR method for rapid detection of Theileria annulate(T.annulata)and Anaplasma bovis(A.bovis),and to investigate the infection of these two pathogens in Turpan.Three pairs of specific primers were designed and synthesized according to the conserved sequences of the T.annulata Spm2,Tams1 gene and A.bovis 16S rRNA gene uploaded in the NCBI library,the genes were amplified by PCR and sequenced.Duplex PCR was designed according to Spm2 and 16S rRNA genes,and the reaction system and conditions were optimized.The method was tested for specificity,sensitivity and reproducibility,a duplex PCR method was established.The results showed the duplex PCR method specifically amplified the corresponding target fragments of T.annulata and A.bovis,and the fragment sizes were 853 bp and 351 bp,respectively.However,no bands were amplified in Babesia caballi,T.equi,A.ovis and Trypanosoma evansi.Phylogenetic analysis of the Spm2 and Tams1 gene sequences of T.annulata and the 16S rRNA gene sequences of A.bovis was carried out,it was found that the Spm2 gene sequence of the T.annulata was the closest to the Indian strain(accession number:MH844677),the sequence of Tams1 gene was closely related to the Tunisian strain(accession number:AF214899),the 16SrRNA gene sequence of A.bovis had a close homology relationship with the Tunisian strain(accession number:KY655808).The minimum concentration of T.annulataand A.bovis detected by this method were 2.9×10^(-16)and 1.8×10^(-19)g/μL,respectively.This method was used for duplex PCR amplification of 60clinical samples of bovine blood DNA,among them,the positive rate of T.annulata was 66.7%(40/60),the positive rate of A.bovis was 53.3%(32/60),and the positive rate of mixed infection was 43.3%(26/60).The coincidence rate between the duplex PCR method and the established single PCR method for T.annulata and A.bovis was 93.3%.A duplex PCR method for simultaneous detection of two pathogens was successfully established.