旋毛虫实时荧光PCR检测方法的建立与应用研究

根据旋毛虫隔离种线粒体Ls-rRNA基因序列,设计一对特异性引物及TaqMan MGB探针。以Ls-rRNA阳性重组质粒为模板,建立了旋毛虫实时荧光PCR检测方法。该检测方法对各旋毛虫隔离种具有良好的特异性,而对小鼠、狗和猪正常肌肉组织、猪鞭虫、猪蛔虫、猪钩虫、犬蛔虫等无交叉反应。最小检出量为1.32×10^2拷贝（10^-5条旋毛虫）,建立的标准曲线拟合良好,扩增方程Y=-3.43X＋19.70,相关系数R^2=0.9984,扩增效率为95%。平行检测组内变异系数为1.11%（n=20）,同一组不同浓度梯度样本（n=9）间隔30d重复检测,结果无显著性差异。人工感染不同剂量猪旋毛虫的小鼠,在攻虫14d后检出率100%。对经压片镜检、人工胃液消化、胶体金试纸条检测结果为阴性的63份送检狗肉样本中敏感地检出8个阳性。

Establishment and Preliminary Application of the Real-Time PCR Assay for Trichinella

According to the Ls - rRNA gene sequences of different Trichinella isolates, a pair of specific primers and TaqMan MGB probe were designed using Primer Express 3.0. The real - time PCR method for Trichinella was established successfully using the recombinant plasmid contained Ls- rRNA gene as template. The results showed that the method was specific for Trichinella isolates and no cross - reaction between different Trichinella isolates and normal mouse, dog, pig muscular tissue, Trichuris suis, Ascaris suis, swine ancylostome, and Ascaris canis. The detection limit was 1.32 × 10^2 copies （ 10^ -5 single nematode）. Valid points of standard curve were fitted well with plot, amplification equations was Y= - 3.43X ＋ 19.70, correlation coefficient was 0. 9984 and amplification efficiency was 95%. The coefficient of variation （CV） for the parallel same positive quality control （QC） was 1.11% （ n = 20）. The positive QC of different concentration gradient （ n = 9） were detected with 1 month interval, the results showed that there were no significant difference by T test. The detection ratio of positive specimens that mouse artificial infected with different dose of Trichinella spiralis past 14 days, was 100% using established real -time PCR method. Eight samples were positive detected by real -time PCR method from sixty -three negative samples detected by Microscope Test, Artificial Digestion and Indicator paper, which indicated that the Real -Time PCR was more sensitive.