LAMP-LFD技术在检测猪圆环病毒3型中的应用研究

Development and Application of Loop-Mediated Isothermal Amplification in Combination with Lateral Flow Dipstick for Detection of Porcine Circovirus Type 3

为快速了解猪群中猪圆环病毒Ⅲ型(PCV3)的感染状况,本研究将环介导等温扩增技术(LAMP)与侧向流动试纸条(LFD)相结合,建立了快速检测PCV3 LAMP-LFD方法,并对该方法的特异性、灵敏性以及临床初步应用进行了验证分析。试验结果显示:常规PCR能检测到0.2 pg/μL的病毒,而本研究建立的LAMP-LFD能检测到0.2 fg/μL的病毒;引物、杂交探针只与PCV3发生反应,与其他病毒模板不发生反应;相较于常规PCR方法,LAMP-LFD方法时间更短(约1 h),且对仪器要求更低;在临床样品的检测中,常规PCR能检出的阳性样品,同时采用LAMP-LFD方法均能检出,且LAMP-LFD方法的敏感性更高于常规PCR。以上结果表明,本研究建立的PCV3 LAMP-LFD检测方法具有极高的灵敏性和特异性,操作简单反应快捷,且检测结果的可视化效果优于PCR和单一LAMP试验,在实际生产应用中具有更大的应用前景。

In order to quickly obtain the infection status of porcine circovirus type 3(PCV3)in pigs,it is an important to develop a fast and efficient detection method for use in the breeding industry.In this experiment,a rapid and convenient detection method(LAMP-LFD)was developed by combining loop-mediated isothermal amplification(LAMP)with lateral flow dipstick(LFD).The LAMP-LFD method utilized specially designed primers and probes to make its sensitivity higher than conventional PCR method.The results showed that a clear detection line was visible on the test strip when the nucleic acid concentration was 0.2 fg/μL while the bands of conventional PCR were blurred at the same concentration.Specificity testing showed that the primers and hybridization probes only reacted with PCV3.Moreover,the LAMP-LFD method took less time(about 1 h)and required less equipment as compared with PCR method.These results indicated that the LAMP-LFD method developed here had advantages of high sensitivity and specificity,simple and quick operation and easier visualization than that of PCR and single LAMP test,which brought great convenience for practical production and application.