基于 SYBR Green I 的实时逆转录聚合酶链反应检测猫星状病毒的建立。
Development of SYBR Green I-based real-time reverse transcription polymerase chain reaction for the detection of feline astrovirus.
机构信息
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, PR China.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, PR China.
出版信息
J Virol Methods. 2021 Feb;288:114012. doi: 10.1016/j.jviromet.2020.114012. Epub 2020 Nov 4.
In this study, a SYBR Green I-based real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for the clinical diagnosis of feline astroviruses (FeAstVs). Specific primers were designed based on the conserved region of the FeAstV ORF1b gene. Experiments for specificity, sensitivity, and repeatability of the assay were carried out. In addition, the assay was evaluated using clinical samples. Specificity analysis indicated that the assay showed negative results with samples of Feline Parvovirus, Feline Herpesvirus, Feline Calicivirus, Feline Bocavirus, and Feline Coronavirus, indicating good specificity of the assay. Sensitivity analysis showed that the SYBR Green I-based real-time RT-PCR method could detect as low as 3.72 × 10 copies/μL of template, which is 100-fold more sensitive compared to the conventional RT-PCR. Both intra-assay and inter-assay variability were lower than 1 %, indicating good reproducibility. Furthermore, an analysis of 150 fecal samples showed that the positive detection rate of SYBR Green I-based real-time RT-PCR was higher than that of the conventional RT-PCR, indicating the high reliability of the method. The assay is cheap and effective. Therefore, it could provide support for the detection of FeAstV in large-scale clinical testing and epidemiological investigation.
在这项研究中，开发了一种基于 SYBR Green I 的实时逆转录-聚合酶链反应（RT-PCR），用于猫星状病毒（FeAstV）的临床诊断。根据 FeAstV ORF1b 基因的保守区设计了特异性引物。进行了特异性、敏感性和重复性实验。此外，还使用临床样本评估了该检测方法。特异性分析表明，该检测方法对猫细小病毒、猫疱疹病毒、猫杯状病毒、猫博卡病毒和猫冠状病毒的样本呈阴性结果，表明该检测方法具有良好的特异性。敏感性分析表明，基于 SYBR Green I 的实时 RT-PCR 方法可检测低至 3.72×10 拷贝/μL 的模板，比常规 RT-PCR 灵敏 100 倍。内和间试验变异性均低于 1%，表明具有良好的重现性。此外，对 150 份粪便样本的分析表明，基于 SYBR Green I 的实时 RT-PCR 的阳性检出率高于常规 RT-PCR，表明该方法具有较高的可靠性。该检测方法廉价且有效。因此，它可以为大规模临床检测和流行病学调查中 FeAstV 的检测提供支持。
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