Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
Parasitol Res. 2013 May;112(5):2087-93. doi: 10.1007/s00436-012-3267-1. Epub 2013 Jan 19.
Different molecular detection methods require diverse molecular platforms, but there is no uniform standard for people to reference in the detection of Trichinella. In this study, real-time PCR, loop-mediated isothermal amplification (LAMP), and conventional PCR were developed for the detection of Trichinella by targeting mitochondrial large subunit ribosomal DNA (mt-lsrDNA). We compared the performance of the three newly developed assays. The results revealed that the detection limits of the real-time PCR, LAMP, and conventional PCR assays were 10 and 100 fg/μL and 1 pg/μL of Trichinella spiralis genomic DNA, respectively. The assays were used in the detection of Trichinella in the field. A total of 192 samples were obtained from pigs: 75 samples from free range farming and 117 from intensive feeding factory. The infection rate was 8/192 (4.2 %), 7/192 (3.6 %), and 1/192 (1.0 %) through the real-time PCR, LAMP, and conventional PCR assays, respectively. These data indicate that Taqman real-time PCR was a rapid, specific, and sensitive tool as a preferred option for investigation of valuable samples, but that LAMP assay was closed tube, highly sensitive, cost-effective, rapid, easy-to-perform, and was the optimal choice for detection of Trichinella in the field. The results of a model of experimental infection in mice indicated that spleen can be used as sampling site for the detection of early T. spiralis infection. However, the diaphragm and myocardium were the most suitable sampling sites for the detection of T. spiralis.
不同的分子检测方法需要不同的分子平台，但在旋毛虫检测方面，人们没有统一的参考标准。在本研究中，我们针对线粒体大亚基核糖体 DNA（mt-lsrDNA），开发了实时 PCR、环介导等温扩增（LAMP）和常规 PCR 三种检测旋毛虫的新方法，并对这三种新方法的性能进行了比较。结果表明，实时 PCR、LAMP 和常规 PCR 检测方法的检测限分别为 10 和 100 fg/μL 以及 1 pg/μL 的旋毛虫基因组 DNA。我们将这些方法应用于野外旋毛虫的检测。从猪身上共获得 192 个样本：75 个来自自由放养农场，117 个来自集约化养殖场。通过实时 PCR、LAMP 和常规 PCR 检测，感染率分别为 8/192（4.2%）、7/192（3.6%）和 1/192（1.0%）。这些数据表明 Taqman 实时 PCR 是一种快速、特异、敏感的工具，是有价值样本调查的首选方法，但 LAMP 检测是闭管检测，具有高度敏感、经济高效、快速、易于操作等特点，是野外旋毛虫检测的最佳选择。在小鼠实验感染模型中，结果表明脾脏可作为检测早期旋毛虫感染的采样部位，但膈肌和心肌是检测旋毛虫的最适宜采样部位。
