Junlong Liu, Li Youquan, Liu Aihong, Guan Guiquan, Xie Junren, Yin Hong, Luo Jianxun
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu, 730046, People's of Republic China,
Parasitol Res. 2015 Jul;114(7):2715-21. doi: 10.1007/s00436-015-4478-z. Epub 2015 Apr 18.
Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.
为构建一种简单高效的环形泰勒虫和瑟氏泰勒虫诊断检测方法，本研究开发了一种多重聚合酶链反应（PCR）方法。在对相关序列进行比对后，设计了两组引物，分别特异性靶向环形泰勒虫细胞色素b（COB）基因和瑟氏泰勒虫内部转录间隔区（ITS）序列。结果发现，所设计的引物可在一个PCR体系中反应，分别扩增出瑟氏泰勒虫的818碱基对和环形泰勒虫的393碱基对。对两种泰勒虫的标准基因组DNA进行10倍系列稀释以检测灵敏度，特异性试验证实两组引物均与其他泰勒虫和巴贝斯虫物种无交叉反应。此外，使用378份野外样本评估多重PCR检测方法对病原体感染的检测效用。将检测结果与另外两种分别靶向环形泰勒虫COB基因和瑟氏泰勒虫主要梨形虫表面蛋白（MPSP）基因的已发表PCR方法进行比较。所开发的多重PCR检测方法与COB和MPSP PCR具有相似的高效检测效果，这表明该多重PCR可能是用于环形泰勒虫和瑟氏泰勒虫流行病学研究的一种有价值的检测方法。
