Establishment And Application Evaluation Of Rapid Detection System For Quinoline Resistant Gene Polymorphism Of Plasmodium Falciparum

Background: As one of the three global public health problems,malaria is a serious threat to human health and is mainly transmitted through the bite of female Anopheles mosquitoes,with Plasmodium falciparum causing the deadliest form of malaria.In 2020,there will be about 228 million new malaria cases and about 602,000 deaths worldwide.Although China has eliminated malaria,imported malaria cases still exist,seriously threatening the consolidation of the gains made after the elimination of malaria in China.Until a vaccine is available,drugs remain the primary means of malaria control.In recent years,the growing problem of P.falciparum drug resistance has posed a major barrier to global malaria elimination programs.Therefore,the monitoring of antimalarial drug resistance becomes crucial.Objective: This study intends to analyze the characteristics of drug resistance of imported malaria in Wuhan,Hubei Province.And then,establish two rapid detection systems for drug resistance based on POCT strategy,providing a feasible solution for malaria prevention and control.Methods: From 2017-2019,DNA was extracted from dried blood spot samples collected from African imported falciparum malaria patients in Wuhan,Hubei province,and polymorphic fragments in the pfcrt,pfmdr1 and pfk13 genes were amplified by nested PCR and sequenced for analysis.Subsequently,the wild type and mutant recombinant plasmids of pfcrt and pfmdr1 gene were constructed as templates.For pfcrt and pfmdr1 genes that different allele-specific primers were designed and screened;Optimization of AS-PCR reaction system;Establishment of AS-PCR combined with LFA detection system and evaluation of the applicability of the system by clinical samples,and compared with the sequencing results;RAA primers were designed and screened for pfcrt gene;The specific cr RNA of CRISPR-Cas12 a was designed for the pfcrt gene,and then genotyped;Preliminary establishment of RAA combined CRISPR-Cas12 a detection system.Results:1.The prevalence of Pf CRT 76 T,356,and Pf MDR1 86 Y,184F were 9.62%,6.41%,4.72% and 47.17%,respectively.For Pf CRT,including CVMNK（wild-type）,CVIET（mutant-type）,CVM /I N/E K/T（mixed-type）that were 87.50%,9.62% and2.88%,respectively.In Pf MDR1,NY（wild-type）,NF and YF（mutant-type）,N Y/F,Y Y/F and N/Y F（mixed-type）accounted for 34.91%,43.40%,3.77%,15.09%,0.94%and 1.89% of haplotypes,respectively.No mutation was found in pfk13 gene in this investigation.In addition,6 combinations of Pf CRT and Pf MDR1 gene haplotypes were detected,among which NY-CVMNK and NF-CVMNK were dominant,accounting for40.96% and 43.37%,respectively.2.Recombinant plasmids of pfcrt and pfmdr1 gene were successfully constructed and alleles specific primers of AS-PCR were screened.The CVMNK and CVIET of Pf CRT were genotypes by optimized AS-PCR combined with LFA.Clinical samples were used for verification,and the results were basically consistent with the sequencing results,with sensitivity and specificity reaching 95.83%（115/120）and 100%（120/120）respectively.The detection limit of plasmid DNA was about 1.5 pg/μl,and the clinical samples was as low as 100 parasites/μl.For the pfmdr1 gene,the sensitivity was 98.92%（274/277）and 100%（277/277）for A256 T and A551 T,respectively,and the specificity was 100% for both compared with the sequencing results.The detection limits were 1.5pg/μl and 150 fg/μl for plasmid DNA,respectively;the detection limits were 50plasmodium/μl for clinical samples.3.The RAA primer of pfcrt gene was successfully screened.In Cas12 a detection,wild-type and mutant-type cr RNA successfully distinguished wild-type and mutant-type plasmids.Conclusion:1.The pfcrt gene was found to be recovering its sensitivity to antimalarial drugs including CQ,AQ and MQ,while the frequency of mutation sites associated with piperaquine resistance remained relatively low.In addition,the elevation of N86 and184 F of Pf MDR1 gene suggests the risk of artemether-fluorenol resistance.Although no mutations were detected in pfk13,ongoing surveillance is necessary to prevent further development of resistance.2.The two rapid genotyping techniques established and optimized are not limited to antimalarial drug resistance genes,but can also be rapidly translated to SNPs of other drug resistance genes and other infectious diseases.It has realized the leap and transformation from scientific research theory to practical application.While actively responding to the policy of instant detection,it has also contributed to the global malaria eradication program.