Masri S A, Nguyen P T, Gale S P, Howard C J, Jung S C
Animal Diseases Research Institute, Agriculture and Agri-Food Canada, Lethbridge, Alberta.
Can J Vet Res. 1997 Jan;61(1):15-20.
A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.
本文描述了一种使用聚合酶链反应（PCR）检测牛精液中致病性钩端螺旋体属的快速且特异的方法。所用引物源自一个EcoR1/BamH1片段，该片段与hardjobovis血清型的染色体DNA强烈杂交。本研究评估了三种不同的提取方法：酚 - 氯仿提取法、在1%十二烷基硫酸钠（SDS）中使用蛋白酶K（PK），然后进行酚 - 氯仿提取，以及酚 - 氯仿提取后再用1%十六烷基三甲基溴化铵（CTAB）。当使用纯钩端螺旋体培养物的DNA作为PCR模板时，无论采用何种DNA提取方法，均获得了长度约为500个碱基对（bp）的PCR产物。该产物与基因序列预测的结果一致。然而，在体外接种精液以及感染公牛的精液中，只有当使用CTAB方法从标本中提取钩端螺旋体DNA时，才能获得PCR产物。相比之下，用于DNA提取的其他方法未能为PCR程序生成合适的模板。这是首个开发用于检测牛精液中钩端螺旋体的PCR方案。该PCR方案直接且明确地证明了在感染动物的精液中可检测到钩端螺旋体。CTAB方法也成功用于检测感染动物尿液中的钩端螺旋体。结果表明，PCR程序在检测尿液中的该微生物方面比荧光抗体试验（FAT）或培养法更敏感。
