基于PCR检测和基因测序分析的鹿源副结核分枝杆菌鉴定及亚型分型

Genotype identification of Mycobacterium avium subsp. paraenberculosis from Red Deer based on PCR and sequence analysis

目的快捷、准确地鉴定马鹿副结核分枝杆菌基因型和亚型,为马鹿副结核病防控工作提供理论依据。方法从疑似感染副结核病马鹿的病料样品中提取其DNA,参照副结核分枝杆菌插入序列IS900鉴定引物、亚型分型引物,PCR扩增、测序及GenBank中BLAST比对。结果所测2份病例与副结核分枝杆菌核苷酸同源性均在99%以上,属于副结核分枝杆菌,采用亚型分型鉴定,属于Ⅱ型(牛型)副结核分枝杆菌。结论结合发病马鹿的临床症状、病理变化、抗酸性染色,证实该马鹿感染牛型副结核病。

The aim of this study is to identify quickly and accurately genotype of Mycobacterium avium subsp.paraenberculosis and provide reference to the prevention and control of paratuberculosis,DNA was extracted from two suspected samples of red deer paratuberculosis.Primers were designed based on IS900 of M.avium subsp.paraenberculosis according to reference.The amplified fragments were sequenced.Analysis of the nucleotide sequences and construction of phylogenetic tree were based on IS900 gene of Mycobacterium avium subsp.paraenberculosis with computer programs.The sequencing results showed that two suspected samples had 99% similarity to reference strain from GenBank.Two suspected samples were tested to infect M.avium subsp.paraenberculosis.According to DMC primers,the pathogen was genotype Ⅱ Mycobacterium avium subsp.paraenberculosis.It was confirmed that two red deer infected genotype Ⅱ paratuberculosis according to symptoms,pathological change,acid-fast stain and PCR.