土壤中弓形虫半套式PCR检测方法的建立

Establishment of the hemi-nested PCR for detecting Toxoplamsa goodii in soil

根据NCBI上公布的弓形虫RH株的529bp基因序列(登录号:AF146527)用Primer Premier 5.0设计3条特异性引物TX1、TX2和TX3,建立土壤中弓形虫的半套式PCR检测方法,并将引物TX1与TX3扩增产物克隆到pMD18-T载体上进行测序.结果表明:该方法能扩增出约350bp的片段,TX1与TX3扩增片段约520bp,克隆到pMD18-T载体上的521bp片段与所选取的弓形虫RH株的目的基因序列相似性100%.该检测方法特异性强,以犬新孢子虫、牛微小隐孢子虫、牛巴贝斯虫、环形泰勒虫、牛链球菌、猪链球菌、蒙氏肠球菌基因组为模板均未扩增出条带;灵敏性高,检测极限为20fg弓形虫DNA;在22份土壤样品中检出3份阳性样品.

Three primers, TX1, TX2 and TX3 were designed using Primer Premier 5. 0 according to 529 bp sequence from RH strain of T. gondii for the establishment of the hemi-nested PCR. Combining the product of PCR with the primers TX1 and TX3 to PMD18-T vector for sequencing. The product of the hemi-nested PCR was about 350 bp long and the product of primer TX1 and TX3 was about 520 bp long,The 521 bp fragment ligated to the pMD18-T vector was 100% identical to the published 529 sequence （AF146527）. The special test showed that no fragment was amplified using DNA of Neospora caninum, Cryptosporidium boris, Babesia boris, Theileria, annulata, Streptococcus boris, Streptococcus suis , Entero- coccus mundtii. Result of sensitivity showed the detection limit of the method was 20fg DNA. Three soil samples were detected positive among 22 soil samples using the detection method.