Kirvar E, Ilhan T, Katzer F, Hooshmand-Rad P, Zweygarth E, Gerstenberg C, Phipps P, Brown C G
Centre for Tropical Veterinary Medicine, University of Edinburgh, Midlothian, UK.
Parasitology. 2000 Mar;120 ( Pt 3):245-54. doi: 10.1017/s0031182099005466.
A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 microl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at -20 degrees C.
本文描述了一种用于检测环形泰勒虫的聚合酶链反应（PCR）和Southern印迹杂交方法。该PCR使用引物扩增环形泰勒虫基因的一段785个碱基对的片段，该基因编码30 kDa主要裂殖子表面抗原Tams1。PCR在牛血液中的灵敏度为每微升血液中1个梨形虫。未检测到水牛泰勒虫、小泰勒虫、双芽巴贝斯虫、牛巴贝斯虫和分歧巴贝斯虫。PCR检测到在静止和部分进食的成年小亚璃眼蜱中低至每只蜱1个受感染腺泡，对由该蜱传播但对牛无感染性的莱氏泰勒虫和马泰勒虫呈阴性。使用30株环形泰勒虫菌株检查PCR的特异性，所有菌株均被检测到。使用3株莱氏泰勒虫、4株马泰勒虫以及各1株水牛泰勒虫、小泰勒虫、双芽巴贝斯虫、牛巴贝斯虫和分歧巴贝斯虫来确定是否存在交叉反应。在-20℃长期保存的经皂素提取的DNA样本中，使用分别用于第一次反应的引物和用于第二次反应的相同引物进行巢式PCR，检测环形泰勒虫的灵敏度和特异性相同。
