Amonsin Alongkorn, Li Ling Ling, Zhang Qing, Bannantine John P, Motiwala Alifiya S, Sreevatsan Srinand, Kapur Vivek
Department of Microbiology and Biomedical Genomics Center, University of Minnesota, St. Paul, Minnesota 55108, USA.
J Clin Microbiol. 2004 Apr;42(4):1694-702. doi: 10.1128/JCM.42.4.1694-1702.2004.
We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.
我们描述了一种用于鸟分枝杆菌副结核亚种（副结核分枝杆菌）菌株基因分型的多位点短序列重复（MLSSR）测序方法。初步分析确定了185个单核苷酸、二核苷酸和三核苷酸重复序列，它们分散在副结核分枝杆菌基因组中，其中78个是完美重复序列。对来自不同宿主物种和地理位置的6株副结核分枝杆菌分离株的78个位点进行比较核苷酸测序，确定了11个多态性短序列重复（SSR）的一个子集，每个位点平均有3.2个等位基因。对这11个位点进行比较测序，用于对代表不同IS900位点多重PCR（MPIL）或扩增片段长度多态性（AFLP）类型的33株副结核分枝杆菌分离株进行基因分型。分析将33株副结核分枝杆菌分离株分为20种不同的MLSSR类型，与地理和流行病学相关性一致，鉴别指数为0.96。MLSSR分析还能够清楚地区分副结核分枝杆菌的绵羊和牛分离株，并且能够轻松且可重复地区分代表先前描述的副结核分枝杆菌主要MPIL基因型（基因型A18）和AFLP基因型（基因型Z1和Z2）的菌株。综上所述，我们的研究结果表明，MLSSR测序能够为分子流行病学和群体遗传学分析提供便捷且可重复的副结核分枝杆菌分离株高分辨率亚型分型。
