利用套式PCR检测牛羊乏质体

Detection of Anaplasma marginale,Anaplasma centrale,and Anaplasma ovis by Nested Polymerase Chain Reaction Assay

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较。结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段。套式PCR检测乏质体DNA量为0.2pg(相当于6个感染红细胞)。检测1 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份。首次在分子生物学水平证明中央乏质体存在于中国。同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体。上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5%(835/848)。检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定。

To detect and differentiate A.marginale,A.centrale,and A.ovis,an msp4 nested PCR assay was developed by two universal and 3 specific pairs of primers.Sensitivity test revealed that this method was able to detect as little as 0.2 pg of DNA（approximately 6 infected erythrocytes）.No cross-reaction with Babesia bovis,B.bigemina,Mycoplasma wenyonii,and DNA from other organisms was observed in the specificity test.1 119 clinical blood samples of cows,beef,buffalos and sheep from six different areas were tested by this method and 106 of them were positive for Anaplasma spp.Of the 106 samples,46 were positive for A.marginale,15 for A.centrale,and 35 for A.ovis,and 4 showed mixed infections with A.centrale and A.ovis,3 with A.marginale and A.centrale,and 3 with A.marginale and A.ovis.The results had 98.5%（835/848） coincidence with the OIE recommended msp5 nested PCR method.The results also provided molecular evidence for the existence of A.centrale in China.The study reveals that cattle can be simultaneously infected by more than one Anaplasma spp.This study indicated that the msp4 nested PCR assay is specific and sensitive for the detection and differentiation of A.marginale,A.centrale,and A.ovis.