Detection Of Enterovirus 71,Coxsackievirus A16 And RpoB Gene Mutations In The Mycobacterium Tuberculosis Based On Recombinase Aided Amplification

Recombinase aided amplification(RAA)is an emerging nucleic acid isothermal amplification technology,with China-owned intellectual property rights.Because of the advantages of rapidity,high sensitivity,specificity,high efficiency,low equipment requirements,low test cost and simple operation,it is very suitable for rapid screening in remote areas and on-site investigation.Since the invention of RAA technology,it has been applied to the detection of a variety of pathogenic microorganisms,but many reports only detected the specific sequence of pathogenic targets,lacking of internal amplification control(IAC).The probe directed recombinase amplification(PDRA)is an improvement of RAA technique.Single probe and single primer are used to identify SNP sites.The SNP site of MTHFR A1298C was detected by this assay,proving that the technique has the potential to detect SNP sites and point mutations.Clustered regularly interspaced short palindromic repeats(CRISPR)and its related protein 13a(Cas13a)system is a new RNA targeting system.Cas13a has RNase activity.When crRNA and Cas13a protein form a complex,it recognizes the target RNA,specifically cleavages target RNA,activates the non-specific cleavage activity of Cas13a,the CRISPR-Cas13a system is developed as a nucleic acid detection tool.Single copy nucleic acid and single base mutation can be detected using CRISPR-Cas13a coupled with RPA technology.Hand-foot-mouth disease(HFMD),caused by various enterovirus,is an acute infectious disease in children,which has become an important public health problem in China.Enterovirus 71(EV71)and coxsackievirus A16(CVA16)are the two main pathogens causing HFMD.Therefore,rapid detection of pathogens,early diagnosis and early treatment are of great significance for the prevention and control of the epidemic situation.Tuberculosis,especially drug-resistant tuberculosis,has caused a great disease burden in China,and rifampicin is very important in the treatment of tuberculosis.If rifampicin resistance occurs in Mycobacterium tuberculosis(MTB),it will increase the difficulty and cost of treatment,and increase the risk of tuberculosis transmission.There are many studies on the mechanism of rifampicin resistance in Mycobacterium tuberculosis,the most important aspect is the mutation of rpoB gene,which leads to drug resistance.Therefore,it is necessary to establish a method for rapid detection of rpoB gene mutation.In this study,based on RAA,PDRA and CRISPR-Cas13a techniques,RT-RAA methods for detection of EV71 and CVA16 were established and evaluated,PDRA and RAA-Cas13a methods for detecting rifampin-resistant rpoB gene mutations of Mycobacterium tuberculosis were established and evaluated.Part Ⅰ.Application of reverse-transcription recombinase aided amplification assays for detection of Enterovirus 71 and Coxsackievirus A16Objective:We aimed to establish two sensitive and specific assays for rapid detection of enterovirus 71(EV71)and coxsackievirus A16(CVA16)based on RAA.The first is the RT-RAA assay based on the constant temperature fluorescence detection system(duplex real-time RT-RAA assay),the second is the RT-RAA assay combined with lateral flow strip(LFS RT-RAA).Methods:(1)The whole gene sequences of EV71 and CVA16 were downloaded from GenBank database,and the conserved regions of VP1 gene were selected as the design region of RAA primers and probes after sequence alignment,several pairs of primers were designed according to the design principles,and the best primer pairs were selected by real-time fluorescence method.(2)Internal amplification control(IAC)probe and IAC plasmid were included to establish a duplex real-time fluorescent RT-RAA assay.(3)Reverse transcription and reaction temperature were optimized.(4)According to sequences of the best primers and probes selected by real-time RT-RAA,LFS RT-RAA assays were established.(5)The sensitivity of the two assays was analyzed respectively by EV71 and CVA16 recombinant plasmid standards,and other enteroviruses were used to analyze specificity.(6)The established real-time RT-RAA assays,LFS RT-RAA assays and literature reported RT-qPCR were used to detect 445 clinical samples of suspected HFMD,and the detection efficiency of these RT-RAA assays were evaluated.Results:Duplex real-time RT-RAA assays for detection of EV71 and CVA16 were established,they were performed at 42℃ within 30 min using a portable real-time fluorescence detector.The limit of detection(LoD)of the real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction,respectively.There was no cross reactivity with other enteroviruses.Compared with RT-qPCR,the diagnostic sensitivity of real-time RT-RAA for EV71 and CVA16 was 92.3%and 99.0%,respectively,and the diagnostic specificity was 99.7%and 100%,respectively.Visible RT-RAA assays combined with lateral flow strip(LFS)for detection of EV71 and CA16 were developed respectively.LFS RT-RAA assays were performed at 42℃ within 30 min in an incubator and the product was detected by lateral flow strip.The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction.There was no cross reactivity with other enteroviruses.Compared with RT-qPCR,the diagnostic sensitivity of LFS RT-RAA for EV71 and CVA16 was 90.1%and 94.9%,respectively,and the diagnostic specificity was 99.7%and 100%,respectively.Conclusion:The developed EV71 and CVA16 duplex real-time RT-RAA assays and lateral flow strip RT-RAA assays have the potential to be applied in resource limited areas with backward medical conditions or in the field.Part Ⅱ.Application of probe directed recombinase amplification to detect MTB rpoB gene mutationsObjective:We aimed to develop assays based on probe directed recombinase amplification(PDRA)for rapid detection of rpoB gene mutation in Mycobacterium tuberculosis.Methods:(1)The wild-type and mutant-type exo probes and common reverse primers were designed for the three(516,526,531)common mutant sites within the rpoB gene of Mycobacterium tuberculosis(MTB).Then the detection system was constructed and was performed at 39℃ for 40 minutes.(2)The specificity and sensitivity of the detection system were evaluated by wild-type and mutant-type recombinant plasmids.The specificity of the detection system was assessed by detecting the different type of recombinant plasmid with certain probes,while the sensitivity of the detection system was assessed by detecting the recombinant plasmid standards with the corresponding probe.(3)The established PDRA assay was used to detect 6 rifampicin-resistant MTB strains and 35 MTB culture-positive sputum samples,and compared with the results of Sanger sequencing.Results:(1)The PDRA assay of 516 site was preliminarily established,which included four detection systems(TB-516-W,TB-516-GGC,TB-516-GTC and TB-516-TAC).The four detection systems detected the samples in parallel,and the base type of the site was determined according to the positive threshold time.The specificity of the four detection systems was good,when the probe and the target sequence completely matched,the positive threshold time was short,otherwise,the positive threshold time was long and there was a stable difference in positive threshold time.The sensitivity of the detection systems was 103copies/μl.The PDRA detection results of 6 rifampicin-resistant MTB strains and 35 MTB culture-positive sputum samples were consistent with the sequencing results.(2)Although specific exo probes were designed for 526 site and 531 site,PDRA assay could not distinguish wild type from mutant type.Conclusion:This study has preliminarily established a PDRA assay which can be used to detect the 516 mutation in rpoB gene of Mycobacterium tuberculosis.It has certain application value in laboratory.Part Ⅲ.Application of RAA combined with CRISPR-Cas13a in the detection of MTB rpoB gene mutationsObjective:The aim of this study was to establish a rapid method for detection of rifampin-resistant rpoB gene mutation of Mycobacterium tuberculosis by combining recombinase aided amplification(RAA)technique with CRISPR-Cas13a system.Methods:(1)RAA primers were designed for amplification of rpoB gene,and T7 promoter sequence was added to the 5’end of one of the primers,which was used for subsequent in vitro transcription of RNA.(2)Specific wild-type and mutant-type crRNA for sites 516,526 and 531 were designed and synthesized,and CRISPR-Cas13a detection systemss were constructed respectively.(3)Wild-type and mutant-type recombinant plasmids were used to evaluate its specificity and sensitivity.(4)Rifampin resistant strains of Mycobacterium tuberculosis and clinical samples were selected to evaluate the method,and compared with the results of Sanger sequencing.Results:The sensitivity of two-step and one-step RAA-Cas13a methods for detecting recombinant plasmids was 106 cupies/μ 1 and 107copies/μ 1,respectively.The detection methods of sites 516,531 were preliminarily evaluated by rifampicin-resistant MTB strains,and the results were consistent with those of Sanger sequencing.Conclusion:RAA-Cas13a method can identify MTB rpoB gene mutations,but the method needs to be optimized.