扇棘单睾吸虫Real-time PCR与常规PCR检测方法的建立

Establishment of Real-time PCR and Conventional PCR for Identifing Haplorchis taichui

根据扇棘单睾吸虫核糖体DNA第一内转录间隔区(ITS1)序列,应用Primer premier 5.0软件自行设计一对可同时应用于real-time PCR和常规PCR的特异引物,建立real-time PCR与常规PCR鉴定扇棘单睾吸虫的方法,评估其特异性及灵敏性。应用建立的方法鉴定犬猫内脏中收集的吸虫,并用测序进行验证,检验方法的准确性和实用性。结果表明,建立的两种方法均只能特异性扩增扇棘单睾吸虫目的片段,不与横川后殖吸虫、钩棘单睾吸虫、华支睾吸虫、瓦氏瓦特松吸虫、棘口属吸虫、背孔属吸虫、心形咽口吸虫、野牛平腹盘吸虫、东方次睾吸虫、卫氏并殖吸虫、异尖属线虫、宫脂属线虫发生交叉扩增;敏感性试验表明,real-time PCR和常规PCR检测扇棘单睾吸虫质粒的最低检测限分别为43拷贝和86拷贝。建立的realtime PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(R2=0.998)。鉴定来自犬猫的吸虫17条,结果显示两种方法均能准确鉴定出扇棘单睾吸虫。

To develope SYBR Green-Ⅰreal-time PCR and conventional PCR for identification of Haplorchis taichui,the specific primers used in real-time PCR and conventional PCR were designed by Premier 5.0based on ITS1 sequences,through optimizing reaction conditions,the real-time PCR and conventional PCR for H.taichuiidentification were established and the assay sensitivity and specificity were detected.Those methods were employed to identify H.taichui collected from cats and dogs to verify the practicality.The results showed that those two methods can amplified fragments of H.taichui,and no cross reaction with H.pumilio,Metagonimus yokogawai,Clonorchis sinensis,Watsonius watsoni,Echinostomasp.,Notocotylus sp.,Pharyngostomum cordatum,Homalogaster paloniae,Metorchisorientalis tanabe,Paragonimus westermani,Anisakis sp.and Hysterothylaciumsp.genome DNA.The real-time PCR and conventional PCR could detect H.taichui DNA as limit as 43 copies and 86 copies,respectively.The cycle threshold of real-time PCR showed a good linear relationship with the number of template copies.Those two methods were all efficient to identify H.taichui from cats and dogs and no cross amplification with other 17 isolated trematodes.