摘要
参考GenBank中牛霉形体和无乳霉形体P81基因的序列差异,设计了针对牛霉形体P81基因的PCR引物,建立了牛霉形体PCR检测方法,并对该方法进行了敏感性和特异性试验。该方法可以从100ccu/mL(ccu:color change unit,颜色变化单位)的牛霉形体培养物中检出目的片段;经对丝状霉形体丝状亚种SC型(MmmSC)PG1株、丝状霉形体丝状亚种LC型(MmmLC)Y-goat株、山羊霉形体山羊亚种(Mccp)87001株、无乳霉形体(M.agalactiae)PG2株、绵羊肺炎霉形体Y-98株等亲缘关系相近的霉形体纯培养物检测,结果证实该方法具有良好的特异性。利用建立的PCR检测方法可以从人工感染牛的鼻拭子和临床发病牛肺中扩增出目的片段,与分离培养结果完全符合,本方法为国内首次建立的敏感、特异、快速的牛霉形体PCR检测方法。
With a pair of primers,which was designed for amplification of P81 gene of Mycoplasrna boris based on difference of P81 gene sequence between Mycoplasma boris and Mycoplasma agalactiae, a PCR assay was established for detection of M. boris and the sensitivity and specificity of the PCR assay were evaluated. The test of sensitivity showed that a target gene could be amplified by the PCR assay from 100 ccu/mL cultures with M. boris. The specificity of the PCR assay was confirmed by the amplifications of P81 genes from the other organisms including PGl（Mycoplasma mycoides subsp, mycoides small colony type, MmmSC）, Y-goat（Mycoplasma mycoides subsp, mycoides Large colony type, MmmSC）, 87001 （Mycoplasma capricolum subsp, capripneumoniae （Mccp） , PG2 （ Mycoplasrna agalactiae ）, Y98 （ Mycoplasma ovipneumoniae）, which were phylogenetically related to M. boris. The target P81 genes were amplified from nose swabs of the infected calves and lung samples of the diseased calves by the PCR assay,which were the same as the result of isolation of M. bowls from the nose swabs and the lung samples. The results showed that a rapid,sensitive,specific and reliable PCR assay was developed successfully for detection of M.bovis for the first time in China.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第5期416-420,共5页
Chinese Veterinary Science
基金
兽医生物技术国家基本科研项目(NKLVBP200809)
