RT-LAMP可视化检测猪瘟病毒及特异性鉴别猪瘟兔化弱毒疫苗

Visual detection of classical swine fever virus and identification of hog cholera lapinized virus by reverse transcription loop-mediated isothermal amplification assay

  • 摘要: 【目的】建立可视化检测猪瘟病毒(CSFV)及特异性鉴别猪瘟兔化弱毒疫苗株(HCLV)的逆转录环介导等温扩增技术(RT-LAMP),为临床检测CSFV野毒株及特异性鉴别HCLV毒株提供技术支持。【方法】根据GenBank中CSFV非结构蛋白NS5B基因的保守序列,设计两套针对CSFV和HCLV的特异性引物,并优化RT-LAMP反应条件,同时用LA-320C浊度仪实时监测其浊度变化。【结果】优化的RT-LAMP仅需在63℃水浴锅中反应50 min即可完成检测,且能通过肉眼观察反应液的颜色变化直接判定结果;所有CSFV样本的检测结果均为阳性(翠绿色),其他非CSFV样本的检测结果均为阴性(桔红色),且能特异性鉴别HCLV毒株,与实时浊度监测结果一致。该方法对CSFV和HCLV的最小检测限分别是100和101 copies,敏感度分别是常规RT-PCR的100倍和10倍。【结论】建立的RT-LAMP特异性强、灵敏度高、方法简便,尤其适合基层进行CSFV感染的快速检测及特异性鉴别HCLV,对有效防控CSF具有重要意义。

     

    Abstract: ObjectiveA rapid method for visual detection of classical swine fever virus (CSFV) and identification of hog cholera lapinized virus ( HCLV ) vaccine strain by reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was established to provide technical support for clinical test of classical swine fever virus (CSFV) and identification of hog cholera lapinized virus (HCLV) vaccine strain. MethodTwo sets of specific primers were de-signed to target CSFV and HCLV based on the conserved sequences of non-structural protein NS5B gene of CSFV from GenBank. The reaction conditions LAMP assay were optimized as well as real-time measurement of turbidity by the turbidity meter LA320. ResultThe LAMP assay reaction was conducted in a 63°C water bath condition for 50 min. This method could be directly judged by the naked eyes through calcein color change results. Detection of CSFV was positive (emerald green). Detection of other common pathogens of pig was negative (orange). Specific discriminating HCLV was performed, in which the results were consistent with real-time measurement of turbidity. The detection limits of the LAMP assay to CSFV and HCLV were 100 and 101 copies, respectively. This indicated that the sensitivities of the LAMP assay to CSFV and HCLV were 100-fold and 10-fold higher than that of conventional PCR, respectively. Con-clusionRT-LAMP method is simple , sensitive , specific and can be used for rapid detection of CSFV and specific iden-tification of HCLV. It is of great significance for effective control of CSF.

     

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