猪弓形虫特异性PCR诊断方法的建立及应用

Establishment and Application of a PCR Assay for Detection of Toxoplasma gondii in Pigs

以猪弓形虫核糖体DNA第一内转录间隔区(ITS1)序列为模板自行设计引物,建立了猪弓形虫病特异PCR诊断方法,并从弓形虫国际标准强毒株RH速殖子和疑似T.gondii感染猪全血及肺脏组织样品基因组DNA中扩增出了预期长度273bp的目的DNA片段。敏感性和特异性试验结果显示,该PCR方法能检测到的最低DNA量为0.001ng,且与相关的9种对照寄生虫、细菌和病毒无交叉反应。用建立的PCR诊断方法对临床30份猪弓形虫疑似病料和60份健康猪抗凝全血样品进行检测,结果30份病料中有24份呈现阳性;60份健康猪血中有5份为阳性;随机取两个临床样品的阳性PCR扩增片段进行克隆测序表明,二者序列与Gen-Bank中已登录的猪弓形虫ITS1基因相应部分序列完全相同。以上表明所建立的PCR方法具有高度的敏感性和特异性;本研究为猪弓形虫病的快速诊断提供了一种新方法。

A highly sensitive and specific PCR method yeas developed using the primers designed based on the first internal transcribed spacer （ITS1） of nuclear ribosomal DNA genome sequences of Toxoplasma gondii （T. gondii） to detect the suspectedly infected pigs. An expected PCR product about 273bp in length was amplified from T. gondii RH strain tachyzoite and blood samples and pulmonary tissues from the pigs with clinical signs. The sensitivity and specificity assays indicated that the PCR method could detect at least 0.001ng DNA of T. gondii RH strain tachyzoite, and no products were amplified from the genomic DNA of the other 9 species of pathogenic microorganism. The PCR was used to amplify the DNA of 30 samples from suspiciously infected pigs and 60 samples from healthy pigs.And 24 from 30 suspicious samples as well as 5 from 60 healthy samples showed positive reactions. Two PCR products amplified from suspicious samples were randomly picked out and sequenced, and the homology ratios of nucleotide and amino acids sequences of the two PCR products with the corresponding region of published T. gondii genome in GenBank were both 100%. respectively, suggesting that the established PCR method was highly specific and sensitive,and was suitable to clinic rapid T. gondii diagnosis.