罗氏沼虾野田村病毒和十足目虹彩病毒1二重荧光定量PCR检测方法的建立及应用

Development and application of duplex fluorescent quantitative PCR method for detecting Macrobrachium rosenbergii nodavirus and decapod iridescent virus 1

  • 摘要: 【目的】建立能同时检测罗氏沼虾野田村病毒(MrNV)和十足目虹彩病毒1(DIV1)的二重荧光定量PCR,为罗氏沼虾白尾病(WTD)及虹彩病毒病(DIV1D)的临床诊断和疫情监测提供更简便和高效的检测方法。【方法】分别根据MrNV-CP基因和DIV1-MCP基因的保守序列设计特异性引物和TaqMan-MGB探针,将目的基因片段分别克隆至pGM-T载体上制备重组质粒(pGM-T-CPMrNV和pGM-T-MCPDIV1),其中,pGM-T-CPMrNVSal I酶切后经体外转录获得MrNV标准品RNA,pGM-T-MCPDIV1则直接作为DIV1标准品DNA;以标准品为模板进行反应条件优化及标准曲线制定,建立可同时检测MrNV和DIV1的二重荧光定量PCR,并对该方法进行特异性、敏感性、重复性及临床应用试验。【结果】制备的MrNV标准品RNA和DIV1标准品DNA均具有很好的稳定性,可作为标准品和阳性对照使用;标准品起始模板范围为1.0×101~1.0×108 Copies/反应时,建立的标准曲线具有良好线性关系。优化后的二重荧光定量PCR灵敏度高,对标准品的检测灵敏度可达10 Copies/反应,对罗氏沼虾组织样品的检测灵敏度约10 Copies/mg;与其他虾类常见病原无交叉反应,且整个检测过程仅需1 h左右。二重荧光定量PCR检测MrNV的组内试验和组间试验变异系数分别为0.31%~0.93%和0.96%~1.33%,检测DIV1的组内试验和组间试验变异系数分别为0.35%~0.81%和0.78%~1.04%。应用建立的二重荧光定量PCR和国内现行有效标准的套式PCR分别对117份临床样品进行MrNV和DIV1检测,结果显示对MrNV和DIV1检测的符合率分别为99.15%和97.44%,且二重荧光定量PCR检测目标病毒拷贝数较低样品时较套式PCR具有更高的灵敏度。2020年广西罗氏沼虾样品MrNV和DIV1的阳性检出率分别为6.0%和11.0%。【结论】结合TaqMan-MGB探针技术建立的二重荧光定量PCR能同时检测MrNV和DIV1,且具有灵敏度高、特异性强、重复性好、简便快速的特点,可为WTD和DIV1D的临床诊断及实时监测提供技术保障。

     

    Abstract: 【Objective】To develop duplex fluorescent quantitative PCR detection method for Macrobrachium rosenbergii nodavirus(MrNV)and decapod iridescent virus(DIV1),so as to provide a simpler and more effective detection technology for clinical diagnosis and epidemic monitoring of M. rosenbergii white tail disease(WTD)and decapod iridescent virus 1 disease(DIV1D).【Method】Specific primers and TaqMan-Minor Groove Binder(TaqMan-MGB)probes were designed based on the conserved sequence of MrNV-CP gene and DIV1-MCP gene respectively,and the target gene fragments were cloned into the pGM-T carrier to construct recombinant plasmids,pGM-T-CPMrNV and pGM-T-MCPDIV1. Among them,pGM-T-CPMrNV was digested by SalⅠand transcribed in vitro to obtain MrNV standard RNA,while DNA of pGMT-MCPDIV1 were served as DIV1 standard DNA. The reaction conditions were optimized with standard being taken as templates. And the standard curves were established. Then the duplex fluorescent quantitative PCR detection method for MrNV and DIV1 was developed,and the specificity,sensitivity,reproducibility tests and detection of clinical samples were carried out to evaluate the applicability of this method.【Result】The prepared RNA transcribed and DNA had good stability and could meet the requirements of the standards,and the standard curves showed a good linear relationship with quantitative range from 1×101 to 1×108 copies/reaction. The optimized duplex fluorescent quantitative PCR method had a high sensitivity with the detection limit as low as to 10 copies/reaction for the standards and approximately 10 copies/mg for the M. rosenbergii tissue samples. It had no cross reaction with common shrimp pathogens,and the entire detection could be completed within 1 h for a single sample;the variation coefficients of intra-group and inter-group were 0.31%-0.93% and 0.96%-1.33% for MrNV detection,which were 0.35%-0.81% and 0.78%-1.04% for DIV1 detection. The duplex fluorescent quantitative PCR method and nested PCR method recommended by current standards were adopted to detect 117 clinical samples,the results showed that most of the detection result were the same with coincidence rate of 99.15% for MrNV detection and 97.44% for detecting DIV1. But the duplex fluorescent quantitative PCR method had better sensitivity in detecting samples with low copies target virus. The MrNV and DIV1 positive rates of Guangxi were 6.0% and 11.0% in 2020 respectively.【Conclusion】The duplex fluorescent quantitative PCR method developed based on TaqMan-MGB probe technology can detect MrNV and DIV1 virus at the same time and it is sensitive,specific,reproducible,simple and rapid,so it can provide technological support for real-time diagnosis and monitoring of WTD and DIV1D.

     

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