猪圆环病毒2型交叉引物恒温扩增检测方法的建立

Establishment of Cross Priming Isothermal Amplification for Porcine Circovirus 2 Detection

拟建立一种简便、快速、准确的现场快速分子检测方法,为猪圆环病毒2型（PCV2）疾病的生物安全防控提供技术支撑。采用交叉引物恒温扩增技术原理,根据PCV2 Cap基因设计一组交叉扩增引物,用构建的Cap基因重组质粒作为标准DNA模板,对反应体系的引物浓度、Betaine、MgSO4、dNTP、Bst酶、反应温度和时间进行优化,并结合核酸试纸条技术,建立可视化检测PCV2的交叉引物恒温扩增检测方法（PCV2-CPA）。用建立的CPA检测方法、常规PCR和ELISA方法检测经IFA方法标定的127份临床样品,比较这些方法的敏感性、特异性和符合率。结果显示,最佳反应体系为引物F3、B3各0.5μmol·L-1,CPR 1μmol·L-1,DF5F0.7μmol·L-1、DF5B0.9μmol·L-1,Betaine 0.075mol·L-1,MgSO43mol·L-1,dNTPs 0.4mmol·L-1,Bst DNA聚合酶9.6U。建立的检测方法58℃扩增50min,检测限可达7.6拷贝·μL-1,灵敏度是常规PCR的10倍;与PCV1、PRV、CSFV、PRRSV、PEDV、TGEV和RV无交叉反应。三种检测方法的敏感性分别为78.08%～94.52%,特异性为87.03%～92.59%,符合率为84.25%～91.34%,ROC曲线图显示三种检测方法中,CPA检测方法效能最优。建立的猪圆环病毒2型交叉引物恒温扩增检测方法不需要PCR仪,仅一台水浴锅或金属浴即可完成病毒DNA的扩增,密闭的核酸检测试纸条使结果判定直观、客观,且可有效避免气溶胶污染,可应用于猪圆环病毒2型的现场快速检测。

This experiment was conducted to establish a simple, sensitive and rapid detection method for the biosecurity control of the porcine circovirus 2 （PCV2） diseases. The Cap gene of PCV2 was used as the signature sequence for cross amplification primers design and the construc- ted Cap gene plasmid DNA was used as the standard template. Amplification temperature, time and constituents of the reaction, including concentration of primers, MgSO4 , dNTP, Betaine and Bst DNA polymerase were systematically optimized. Used the nucleic acid test strip detect the am- plified products, compared the sensitivity, specificity and consistency of CPA,PCR and ELISA by 127 standard clinical samples. The detection results showed that the cross amplification could be realized at 58 ℃ within 50 min,could specifically detect PCV2 with a sensitivity that was about 10 times higher than conventional PCR method. The detection limit of recombinant plasmid was 7.6 copies · gL 1 and the assay gave no amplification from PCV1, PRV, CSFV, PRRSV, PEDV, TGEV and RV. The final optimal CPA condition established for rapid detection of PCV2 was as follows：0.5 μmol · L-1 each of F3 and B3,1.0 μmol· L-1 of CPR,0.7 μmol· L-1 of DF5F,0.9μmol · L-1 of DF5B,0. 075 mol · L-1 Betaine,3 mmol · L-1 MgSO4 , 0. 4 mmol · L -1 dNTPs,9.6 U Bst DNA polymerase, 2μL 10 ×ThermoPol Reaction Buffer, 4 μL templates and sterilized double-distilled water in 20 μL volumes. The sensitivity,specificity and consistency were 78.08% to 94.52% 87.03% to 92.59%and 84.25% to 91.34% among the three methods,respectively. Receiver operating characteristic curve showed that CPA was the best detection method. PCV2 cross priming amplification method does not require expensive equipment and skilled technicians, the visual detection could provide an intuitive and objective decision for people,and could also ef fectively avoid the aerosol pollution,which could be used in the on-site diagnosis for effective pre- vention and control of porcine circovirus 2 diseases.