Xiao S M, Li G Q, Zhou R Q, Li W H, Yang J W
Department of Veterinary Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
Vet Parasitol. 2007 Nov 10;149(3-4):185-90. doi: 10.1016/j.vetpar.2007.08.010. Epub 2007 Sep 11.
A rapid and sensitive assay for the detection of Cyclospora species in dairy cattle faecal specimens has been developed. The method utilizes a nested PCR to amplify a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. and an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) to detect the amplified product. In this study, the OLA technique was compared with conventional gel electrophoresis for the detection of amplified product. In evaluating the PCR-OLA for Cyclospora sp. and non-Cyclospora parasites, A(405) reading value for Cyclospora species was significantly higher than those for non-Cyclospora control. At known concentrations of purified amplicons from cattle-derived Cyclospora sp., the OLA was able to detect more than 0.5 ng of the amplified DNA. Of 168 clinical specimens collected from four dairy cattle farms, 6 were positive by both PCR-gel electrophoresis and the PCR-OLA procedure, and 2 were positive only by PCR-OLA, indicating the PCR-OLA procedure was more sensitive than the common way with gel electrophoresis. The results indicated that the PCR-OLA is simple, rapid and suitable in clinical detection of cattle-derived Cyclospora species.
已开发出一种用于检测奶牛粪便样本中环孢子虫属的快速灵敏检测方法。该方法利用巢式PCR扩增牛源环孢子虫属18S rRNA基因的168 bp DNA片段，并采用基于酶联免疫吸附测定（ELISA）的寡核苷酸连接测定（OLA）来检测扩增产物。在本研究中，将OLA技术与用于检测扩增产物的传统凝胶电泳进行了比较。在评估针对环孢子虫属和非环孢子虫寄生虫的PCR-OLA时，环孢子虫属的A(405)读数显著高于非环孢子虫对照。在已知浓度的牛源环孢子虫属纯化扩增子中，OLA能够检测到超过0.5 ng的扩增DNA。在从四个奶牛场收集的168份临床样本中，6份通过PCR-凝胶电泳和PCR-OLA程序均呈阳性，2份仅通过PCR-OLA呈阳性，这表明PCR-OLA程序比普通的凝胶电泳方法更灵敏。结果表明，PCR-OLA简单、快速，适用于牛源环孢子虫属的临床检测。
