帕利亚姆病毒实时荧光定量RT-PCR检测方法的建立与应用
Development and Application of Serotyping Real-time Quantitative RT-PCR Methods for Palyam Virus
杨恒 1李占鸿 2宋子昂 3高林 2李卓然 2廖德芳 2肖雷 2李华春2
作者信息
- 1. 昆明学院农学与生命科学学院,昆明 650214;云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,昆明 650224
- 2. 云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,昆明 650224
- 3. 南京农业大学动物医学院,南京 210095
- 折叠
摘要
本研究拟建立帕利亚姆病毒(Palyam virus,PALV)血清型特异性实时荧光定量RT-PCR(qRT-PCR)方法用于临床样本或媒介中PALV血清型鉴定.根据我国流行PALV毒株的基因节段2序列,设计扩增引物和Taq-Man探针,建立PALV血清型特异型qRT-PCR方法,对方法的特异性、灵敏性与重复性进行评估;以我国分离的28株PALV和90份核酸阳性血液样本评估检测方法的可靠性;利用建立的方法对采集库蠓样本中携带的PALV进行血清型鉴定.结果显示,建立的PALV血清型qRT-PCR检测方法具有良好的特异性与灵敏性,可检出核酸拷贝数下限在22至28 copies·μL.对28株PALV的qRT-PCR检测结果与病毒测序鉴定结果一致;对PALV不同感染阶段哨兵动物血液(90份)中的qRT-PCR鉴定结果与分离病毒的血清型鉴定结果一致;建立的方法可准确鉴定库蠓中携带PALV的血清型.本研究建立的PALV血清型qRT-PCR定型方法具有良好的特异强、敏感性与重复性,可用于PALV感染动物与媒介中PALV血清型的鉴定,具有良好的应用价值.
Abstract
The aim of present study was to develop serotyping real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)methods for serotype identification of Palyam virus(PALV)from clinical samples or insect vector.According to segment 2(Seg-2)sequences of Chinese epidemic PALV strains,PALV serotype-specific qRT-PCR methods were established and their specificity,sensitivity and repeatability were evaluated.The reliability of the established qRT-PCR methods was assessed by 28 Chinese PALV strains and 90 PALV-positive blood samples,then the established methods were applied to identify the serotypes of PALV from the collected Culicoides samples.The established PALV serotyping qRT-PCR methods showed highly specificity and sensitivity,with the minimum copy number of detectable viral nucleic acid ranging from 22 to 28 copies·μL-1.The qRT-PCR identification results of 28 PALV strains are consistent with the results of virus sequencing.Furthermore,serotype qRT-PCR identification results of 90 PALV-positive blood samples from infected sentinel animals were consistent with serotyping results of the isolated PALVs.The established method can accurately identify the serotype of PALV collected from Culicoides samples.With features of strong specificity,high sensitivity and positive repeatability,the PALV serotype-specific qRT-PCR methods established in this study could be used for rapid and accurate diagnosis of the serotypes of PALV in vectors and animals.
关键词
帕利亚姆病毒/血清型鉴定/实时荧光定量RT-PCR/检测方法Key words
Palyam virus/serotype identification/real-time quantitative reverse transcription polymerase chain reaction/detection method引用本文复制引用
基金项目
昆明学院引进人才科研项目(YJL2210)
国家公益性行业(农业)科研专项(201303035)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据