Pillai S R, Jayarao B M
Department of Veterinary Science, The Pennsylvania State University, University Park 16802, USA.
J Dairy Sci. 2002 May;85(5):1052-7. doi: 10.3168/jds.S0022-0302(02)74165-9.
A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 10(8) cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosis by DNA sequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.
开发了一种基于聚合酶链反应（PCR）的检测方法，用于检测生牛奶中副结核分枝杆菌插入序列900（IS900）。这种IS900 PCR检测方法包括使用市售试剂盒进行DNA提取和PCR检测。对DNA提取和PCR检测进行了优化，以便直接从生牛奶中检测IS900序列。通过向生鲜大罐牛奶和Middlebrook's 7H9肉汤中接种0至10⁸ cfu/ml的四株美国典型培养物保藏中心副结核分枝杆菌菌株，对IS900 PCR检测方法进行了评估。在实验条件下，Herrold蛋黄培养基斜面上的牛奶培养和IS900 PCR均可检测到10至100 cfu/ml的副结核分枝杆菌。当存在约100 cfu/ml时，通过IS900 PCR检测副结核分枝杆菌的结果是一致的（24次PCR检测中有24次），而在低至10 cfu/ml的浓度下，检测结果则存在差异（24次PCR检测中有12次）。基于实验研究结果，使用来自五个有约翰氏病史牛群的211头奶牛的混合四分奶样本对IS900 PCR进行了进一步评估。在检测的211只动物中，通过牛奶培养和牛奶中的IS900 PCR检测，分别有9只（4%）和69只（33%）副结核分枝杆菌呈阳性。还对总共20个大罐牛奶样本等分试样（每个牛群一个样本，四个等分试样）进行了检测，其中10个（50%）通过IS900 PCR检测副结核分枝杆菌呈阳性。相比之下，20个大罐牛奶样本等分试样中只有1个（5%）通过培养呈阳性。通过DNA序列分析证实，对四分奶和大罐牛奶样本检测时获得的229 bp的IS900 PCR扩增产物为副结核分枝杆菌的IS900。本研究结果表明，通过IS900 PCR可直接从四分奶和大罐牛奶中检测到副结核分枝杆菌。
