猪附红细胞体半巢式PCR诊断方法的建立

目的为能够更加准确、敏感的检测出猪附红细胞体(Mycoplasma suis,M.suis),本试验建立一种新的诊断方法—半巢式聚合酶链反应(hemi-nested Polymerase Chain Reaction,hemi-nested PCR)。方法利用Oligo 6.0和Primer 5.0软件设计筛选出合适的PCR引物,经酶切分析和半巢式PCR进一步鉴定后进行序列测定。同时与普通PCR诊断方法进行比较。结果测得的猪附红细胞体基因序列与GenBank中发表的猪附红细胞体基因序列(AJ504999)同源性为100%。特异性试验结果表明本实验设计的PCR引物不能从弓形虫、链球菌、大肠杆菌、猪肺炎支原体和猪伪狂犬病病毒的基因组DNA中扩增出条带。通过敏感性试验,半巢式PCR诊断方法最低能够检测出0.12fg的标准模板DNA。通过与普通PCR比较,证明本试验建立的半巢式PCR更具敏感性和实用性。结论本试验建立的半巢式PCR诊断方法具有特异、敏感、实用等特点,为猪附红细胞体的检测提供了一种可靠的诊断方法。

A diagnostic method to detect Mycoplasma suis using the semi-nested PCR

A novel diagnostic method was established to detect the presence of Mycoplasma suis by using the semi-nested PCR,in which the proper primers for PCR were designed by Oligo 6.0 and Primer 5.0 softwares and the cloned genes as demonstrated by analysis with enzyme digestion and the semi-nested PCR were proved to be the specific genes for M suis.Meanwhile,the sequence of clone M.suis was compared with that of M.suis published in GenBank(AJ504999).It was found that the sequence homology the clone M.suis with AJ504999 was 100%.As demonstrated by the specificity testing,the PCR primers designed in the present study could not be amplified to form bands from the genomic DNA of Toxoplasma gondii,Steptococcus species,E.coli,Mycoplasma hyopneumoniae of swine and pseudorabies virus.The lowest value of sensitivity of the semi-nested PCR as proved by the sensitivity testing was 0.12 fg DNA of the standard template.Compared with the conventional PCR,semi-nested PCR appeared to be more sensitive and more practicable,thus providing a reliable method to detect M.suis.