摘要
为建立一种不提取病毒RNA,直接采用Taq Man荧光定量RT-PCR检测样本上清液中犬瘟热病毒的方法。通过扩增CDV的NP基因部分片段构建重组质粒,建立Taq Man荧光定量PCR方法,对所建立的方法进行特异性、敏感性、重复性检测;比较了提取核酸后进行荧光定量RT-PCR与不提取核酸对原液进行荧光定量RT-PCR检测的结果,最后对疑似CDV病料进行检测。结果显示,建立的CDV质粒Taq Man荧光定量PCR检测灵敏度比普通PCR灵敏度高1 000倍。将核酸提取液和病毒原液稀释后用该研究建立的方法进行检测,最小检测稀释倍数分别为10~7和10~5,表明提取核酸后样本中的有效c DNA浓度比直接使用病毒原液检测高100倍。特异性和重复性检测结果表明,该方法特异性良好且重复性较高。对97份临床病料进行检测,建立的方法共检出60份阳性,阳性检出率高于普通RT-PCR和胶体金快速检测试纸板,提取核酸法与原液法的一致率为100%。标准曲线分析表明,当病毒含量高于10~2拷贝·μL^(-1)时,与普通RT-PCR以及胶体金快速检测试纸板相比,CDV原液Taq Man荧光定量RT-PCR检测技术阳性检出率更高、准确性更好,值得临床推广应用。
To establish a direct Taq Man fluorescent quantitative RT-PCR（ direct-qRT-PCR） method without extracting nucleic acid for detecting canine distemper virus（ CDV）. Specificity,sensitivity and reproducibility of the method were tested by using constructed recombinant plasmid. Fluorescence quantitative RT-PCR was used to compare the detection limitation in both the nucleic acid extraction and the viral stock. Clinical samples were also collected and applied for verification the established method. Our results showed that the sensitivity of this method was 1 000 timeshigher than ordinary PCR. Nucleic acid extraction and viral stock was detected by the established method,the minimum detectable dilution times were 10~7 and 10~5,respectively,indicated that the effective c DNA concentration in the sample after nucleic acid extraction was 100 times higher than that using viral stock to test. Specificity and reproducibility results showed that the method had good specificity and reproducibility. 97 clinical samples were detected by this method,60 samples showed positive. The detectable rate of this method was higher than those of ordinary RTPCR and colloidal gold rapid detection plate. The positive detectable rate using nucleic acid extraction was perfectly matched with that using viral stock. Compared with ordinary RT-PCR and colloidal gold rapid detection plate,standard curve showed direct-qRT-PCR had high accuracy and positive detectable rate while the virus concentration was higher than 10~2 copies·μL^（-1）,and it was worthy of clinical application.
出处
《浙江农业学报》
CSCD
北大核心
2017年第11期1819-1826,共8页
Acta Agriculturae Zhejiangensis
基金
国家重点研发计划(2016YFD0501009)
国家自然科学基金项目(31000548)
成都大熊猫繁育研究基金会项目(CPF2015-4)
