Barry Rhiannon, Nissly Ruth H, Feria Willard, Thirumalapura Nagaraja, Tewari Deepanker, Jayarao Bhushan M, Kuchipudi Suresh V
Penn State Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, Wiley Lane, University Park, PA, 16802, USA.
Pennsylvania Veterinary Laboratory, Pennsylvania Department of Agriculture, Bureau of Animal Health and Diagnostic Services, 2305 N Cameron St, Harrisburg, PA, 17110, USA.
Vet Parasitol. 2019 May;269:2-6. doi: 10.1016/j.vetpar.2019.04.002. Epub 2019 Apr 4.
Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.
犬新孢子虫是一种顶复门原生动物寄生虫，是牛流产的主要原因。通过聚合酶链反应（PCR）检测寄生虫特异性DNA是一种在多种组织中鉴定犬新孢子虫存在的高度灵敏方法。我们开发并验证了一种基于探针的实时PCR检测方法，该方法靶向犬新孢子虫的保守Nc5基因。使用犬新孢子虫Nc-1株基因组DNA和合成基因片段作为扩增标准品，我们确定PCR扩增效率和检测限分别为95.60%和3个拷贝。对五种经常与牛流产相关的病原体进行了检测，即I型和II型牛病毒性腹泻病毒、牛α疱疹病毒-1、衣原体和钩端螺旋体，以确保分析特异性。同时使用标准常规PCR和新的基于探针的实时PCR检测方法对103份来自流产胎儿的临床样本进行了检测。所有检测样本在这两种检测方法之间显示出100%的一致性。总之，基于探针的实时PCR检测方法有助于准确、快速地从牛流产样本中检测犬新孢子虫。
