Singh Ajay Vir, Chauhan Devendra Singh, Singh Abhinendra, Singh Pravin Kumar, Sohal Jagdip Singh, Singh Shoor Vir
Canadian Food Inspection Agency, QC, Canada.
Indian J Med Res. 2015 Jan;141(1):55-61. doi: 10.4103/0971-5916.154497.
BACKGROUND & OBJECTIVES: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), 'Bison type' is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn's disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between 'Indian Bison type' and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions.
A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods.
All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to 'Bison type'. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as 'Bison type', 'Cattle type' and 'Sheep type', respectively. IS1311 L2 PCR-REA method showed different restriction profiles of 'Bison type' genotype as compared to non-Indian DNA samples.
INTERPRETATION & CONCLUSIONS: IS1311 L2 PCR-REA method successfully discriminated 'Indian Bison type' from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates.
在副结核分枝杆菌（MAP）的三种主要基因型中，“野牛型”是该国家畜物种中最常见的基因型，并且也从克罗恩病患者体内分离得到。最近，一种基于IS1311基因座2聚合酶链反应-限制性内切酶分析（REA）的新检测方法被设计用于区分“印度野牛型”和非印度基因型。本研究在筛选一组来自不同基因型和不同地理区域的MAP分离株时，研究了这种新检测方法的鉴别潜力。
本研究共纳入53株分枝杆菌分离株（41株MAP和12株非MAP分枝杆菌）、3份MAP基因组DNA以及36份来自不同家畜物种（牛、水牛、山羊、绵羊和野牛）和不同地理区域（印度、加拿大、美国、西班牙和葡萄牙）的MAP阳性粪便DNA样本。使用聚合酶链反应（PCR）分析提取的DNA样本（n = 92）中MAP特异性序列（IS900、ISMav 2和HspX）的存在情况。DNA样本进一步使用IS1311 PCR-REA和IS1311 L2 PCR-REA方法进行基因型区分。
所有DNA样本（非MAP分枝杆菌分离株的DNA除外）基于PCR的三种MAP特异性序列检测均为阳性。IS1311 PCR-REA显示，印度来源的MAP DNA样本属于“野牛型”。而在总共19份非印度MAP DNA样本中，分别有2份、15份和2份被基因分型为“野牛型”、“牛型”和“羊型”。与非印度DNA样本相比，IS1311 L2 PCR-REA方法显示“野牛型”基因型具有不同的限制性图谱。
IS1311 L2 PCR-REA方法成功地将“印度野牛型”与其他非印度基因型区分开来，并显示出有潜力成为未来的流行病学工具以及用于MAP分离株的基因分型。
