A Mismatch-tolerant Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Method And Its Application On Detection Of Insect-borne Disease

Dengue is one of the most rapidly transmitted insect-borne diseases and widely distributed in the world,which has caused a great burden on public health.However,more than half of patients with dengue fever show symptoms or no symptoms of common fever.Therefore,it is difficult to determine whether dengue virus infection is caused by clinical symptoms alone.Therefore,it has become very important to establish a highly sensitive and specific detection method.Because of constant reaction temperature,short reaction time,high sensitivity and strong specificity,loop-mediated isothermal amplification(LAMP)has been widely used in various assays for the detection of infectious diseases.However,mismatches between primers(especially in the 3'-end)and templates significantly reduced the amplification efficiency of LAMP,and limited its application to genetically diverse viruses.To solve this problem,conventional LAMP system was optimized in this study,and high-fidelity enzymes with 3'-5' exonuclease activity were firstly used in RT-LAMP to detect dengue virus.The amount of high-fidelity DNA polymerase was optimized at 0.15 U per 25 ?l LAMP reaction mixture,at which the mismatched bases at the 3'-end of primers could be efficiently removed by the 3'-5' exonuclease activity of high-fidelity DNA polymerase,thereby resulting in excellent tolerance for various mismatches.When novel RT-LAMP was used to detect wild and mutant DENV-4 templates with concentration of 30000 copies/reaction,the time threshold of the novel method was about 5.6-22.6 minutes faster than the conventional LAMP method regardless of the presence or absence of mismatches between primers and templates.The detection time threshold of the novel method is significantly different from that of the traditional method(P<0.01).Using the novel method,we improved a previously established pan-serotype assay for DENV,and demonstrated greater sensitivity for detection of four DENV serotypes than the previous one.The limit of detection(LOD)of the novel assay was 74,252,78 and 35 virus copies/reaction for DENV-1,DENV-2,DENV-3 and DENV-4,respectively.10 viruses with similar clinical symptoms to DENV were detected by modified DENV mismatch-tolerant RT-LAMP,and all the detection results were negative,indicating the high specificity of the novel method.Among 153 clinical samples from patients with suspected DENV infection,the improved assay detected 94.8% samples being DENV positive,higher than that detected by the commercial NS1 antigen assay(92.2%),laboratory-based RT-PCR method(78.4%),and the conventional RT-LAMP assay(86.9%).Furthermore,the novel RT-LAMP assay has been developed into a visual determination method by adding HNB and cresol red dyes.The mismatch-tolerant RT-LAMP method established in this study is not only limited to detection of DENV,but also may be widely applied to the detection of Zika,Japanese encephalitis,Chikungunya and other insect-borne diseases in the future to improve the detection sensitivity and shorten the detection time,which is especially suited for application in resource-limited settings.