中国农业科学院哈尔滨兽医研究所/ 兽医生物技术国家重点实验室/ 黑龙江省实验动物与比较医学重点实验室/ 国家禽类实验动物资源库,哈尔滨 150069
R-33
SU Shibo
Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin 150069, ChinaPU Lusha
Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin 150069, ChinaCHEN Xiaohan
Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin 150069, ChinaZHAO Lili
Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin 150069, ChinaCHEN Hongyan
Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin 150069, ChinaHarbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin 150069, China
目的 建立一种可快速诊断鹅源星状病毒的荧光定量 PCR 方法。 方法 分析比对 NCBI 下载的鹅源星状病毒全基因组序列,在其 ORF1a 保守区设计一对特异性引物及 TaqMan 探针,优化反应条件,建立标准曲 线,并验证其特异性,敏感性和稳定性。 结果 该方法最低检测线限为 1. 50×102 copies/ μL,批内和批间检测变异系数分别小于 0. 5%和 4%,且对其他常见水禽病毒无特异性扩增。 临床应用结果显示检出率明显高于常规 RT-PCR 方法。 结论 建立了一种鹅源星状病毒的 TaqMan 荧光定量 PCR 方法,该方法特异性强,灵敏度高且重复性良好, 适用于鹅源星状病毒的早期检测和定量分析。
Objective In this study, we established a fluorescence quantitative PCR method for rapidly diagnosing a new type of goose-origin astrovirus (GoAstV). Methods After analyzing and comparing the whole-genome sequences of GoAstV downloaded from NCBI, a pair of specific primers and TaqMan probes were designed in its open reading frame-1a conservative region. By optimizing the reaction conditions, a standard curve was established, and the specificity, sensitivity and stability were verified. Results The minimum sample size was 1. 5×102 copies/ μL. The coefficients of variation for the intra-assay and inter-assay detection were < 0. 5% and 4%, respectively, with no specific amplification of other common waterfowl viruses. The clinical application result showed that the detection rate was significantly higher than that of conventional real-time PCR. Conclusions This fluorescence quantitative PCR method exhibited stronger specificity, higher sensitivity and better reproducibility for early detection and quantitative analysis of GoAstV.
苏世博,蒲路莎,陈肖韩,赵丽丽,陈洪岩.鹅源星状病毒 TaqMan 荧光定量 PCR 检测方法的建立及初步应用[J].中国比较医学杂志,2021,31(3):43~48.
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