Möbius Petra, Hotzel Helmut, Rassbach Astrid, Köhler Heike
Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Naumburger Str. 96a, Germany.
Vet Microbiol. 2008 Jan 25;126(4):324-33. doi: 10.1016/j.vetmic.2007.07.016. Epub 2007 Jul 25.
For molecular biological detection of Mycobacterium avium subsp. paratuberculosis (MAP), PCR methods with primers targeting different regions specific for MAP are used worldwide. However, some uncertainties exist concerning the specificity of certain target regions and the sensitivity. To identify the methods which are best suited for diagnostics, 8 single-round and 5 nested PCR systems including 12 different primer pairs based on IS900 (9 x), ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared regarding their analytical sensitivity and specificity under similar PCR conditions. Reference strains and field isolates of 17 Mycobacterium species and subspecies, 16 different non-mycobacterial bovine pathogens and commensals were included in this study. Single-round PCR resulted in a detection limit of 100 fg to 1 pg, and nested PCR in 10 fg or below. Depending on the specific primer sequences targeting IS900, false positive results occurred with one of the five single-round and two of the four nested PCR systems. This also applied to the single-round PCR based on ISMav2 and the nested PCR based on f57. A high number of non-specific products were primarily detected for the single-round PCR assay based on ISMav2, but also for a single-round PCR targeting the IS900 and the locus 255. In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a favourite target sequence for amplification of MAP specific loci. The studied PCR systems based on f57, and locus 255 can also be recommended. Revision of ISMav2 primers is necessary. Single-round PCR systems are very reliable. Nested PCR assays were occasionally disturbed by contaminations, thus bearing a risk for routine diagnostics.
对于副结核分枝杆菌（MAP）的分子生物学检测，全球范围内都在使用针对MAP不同特异性区域的引物进行PCR方法。然而，某些靶区域的特异性和灵敏度存在一些不确定性。为了确定最适合诊断的方法，在相似的PCR条件下，对8种单轮和5种巢式PCR系统（包括基于IS900（9种）、ISMav2（1种）、f57（1种）和基因座255（1种）序列的12种不同引物对）的分析灵敏度和特异性进行了比较。本研究纳入了17种分枝杆菌属物种和亚种的参考菌株及现场分离株、16种不同的非结核分枝杆菌牛病原体和共生菌。单轮PCR的检测限为100 fg至1 pg，巢式PCR的检测限为10 fg或更低。根据靶向IS900的特定引物序列，5种单轮PCR系统中的1种以及4种巢式PCR系统中的2种出现了假阳性结果。这也适用于基于ISMav2的单轮PCR和基于f57的巢式PCR。基于ISMav2的单轮PCR检测中主要检测到大量非特异性产物，但针对靶向IS900和基因座255的单轮PCR也有此类情况。总之，严格选择IS900特异性引物可确保IS900仍然是扩增MAP特异性位点的首选靶序列。基于f57和基因座255的研究PCR系统也值得推荐。有必要对ISMav2引物进行修订。单轮PCR系统非常可靠。巢式PCR检测偶尔会受到污染干扰，因此存在常规诊断风险。
