Visual Deteetion Of EV71,CVA16,HPV6and16by Loop-mediated Isothermal Amplification

Objective:To establish a simple, rapid, sensitive and colorimetric loop-mediated isothermal amplification method to detect human enterovirus71subgenotype C4, coxsackievirus A16and human papillomavirus genotypes6and16.Methods:A colorimetric RT-LAMP method was developed to detect human enterovirus71subgenotype C4and coxsackievirus A16. Two sets of six primers were designed according to the conserved sequences of human enterovirus71subgenotype C4and coxsackievirus A16by Primer Explore4.0., respectively. To develop and evaluate RT-LAMP method with different control enteroviruses and stool samples.A colorimetric LAMP method was developed to detect human papillomavirus genotype6and16. Two sets of four pairs of primers were designed according to the conserved sequences of human papillomavirus genotypes6and16by Primer Explore4.0.respectively. To develop and evaluate LAMP method with different control papillomaviruses and swab samples.Results:The detection limits of the RT-LAMP assay for EV71subgenotype C4and CVA16were0.33and1.58of a50%tissue culture infective dose (TCID50) per reaction based on10-fold dilutions of a titrated EV71or CVA16strain, respectively. No cross-reaction was observed with cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14, and24), coxsackievirus B (CVB) viruses (CVB1,2,3,4, and5) or ECHO viruses (ECHO3,6,11, and19). The assay was further evaluated with47clinical stool specimens and compared with virus isolation assay. Both assays were in100%agreement.The detection limits of both HPV6and HPV16type-specific LAMP were1000copies per reaction.Thirteen cervical swab samples having single infection with13different HPV genotypes were examined to evaluate the specificity. The results showed that no cross-reaction with other HPV genotypes was observed. The assay was further evaluated with62clinical specimens and100%consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. Conclusion:We have successfully established colorimetric RT-LAMP and LAMP methods to detect EV71subgenotype C4, CVA16, and HPV6, HPV16, respectively, which improves the monitoring ability for surveillance network to detect HEV71subgenotype C4, CVA16infections and the quality for the screening of HPV6and HPV16infections in China.