Analysis Of Polymorphisms In Drug Resistance Related Genes In Imported Plasmodium Falciparumin Wuhan And Establishment Of Its Detection Methods

Objective:Malaria is one of the three global public health problems that seriously endanger human health.Among them,falciparum malaria caused by Plasmodium falciparum is the most dangerous and deadly.At present,the prevention and treatment of malaria mainly rely on antimalarial drugs,while P.falciparum has generally developed resistance to traditional antimalarial drugs.Therefore,malaria resistance monitoring and related molecular research are very important in the global malaria eradication process.We investigated the polymorphisms of pfnhe1,pfcrt,pfmdr1 and pfk13 in imported P.falciparum in Wuhan,Hubei Province in order to identify molecular markers associated with drug resistance.The methods of detecting SNP of P.falciparum resistance genes by q PCR,AS-PCR and AS-LAMP were preliminarily established,and to use the established methods for preliminary clinical application.Methods:A total of 211 dry filter blood spots were collected from returnees diagnosed with P.falciparum in areas of Africa and Southeast Asia during 2011to 2016 in Wuhan,Hubei Province.Genomic DNA was extracted,and the polymorphisms in pfnhe1,pfcrt,pfmdr1 and pfk13 genes and the haplotype patterns of pfnhe1,pfcrt,pfmdr1 were analyzed by nested PCR with DNA sequencing.Wild type and mutant type recombinant plasmids were constructed and verified by sequencing according to the mutations of drug-resistant genes pfcrt coding 72 to 76 and pfmdr1 coding 86 and 184.Probes and primers were designed,q PCR,AS-PCR and AS-LAMP reaction systems and conditions were optimized,and clinical samples were tested.Results:1.(1)pfnhe1 gene:a total of 28 different ms4760 microsatellite profiles were observed.The three main prevalent ms4760 profiles were ms4760-1,ms4760-3 and ms4760-7.Furthermore,five ms4760 profiles were found that had not been previously reported.(2)pfcrt gene:coding 72 to 76 showed four haplotypes,50.57%were CVMNK(wild type),1.14%were SVMNT(mutant type),25%were CVIET(mutant type),23.30%were CV M/I N/E K/T(mixed type).(3)pfmdr1 gene:coding 86 and 184 were the main prevalent mutations,with 22.28%and 60.01%prevalence,respectively.(4)pfk13 gene:four mutations at position S550S、R561H、R575R/K、V589I,with 1.09%,0.54%,0.54%and0.54%prevalence,respectively.2.The genotyping of pfmdr1 coding 86 by q PCR showed that a total of 183samples were consistent with the results of previous nested PCR with sequencing,and the limit of detection was 10~5copies/L.The whole detection only took 1 hour.AS-PCR primers were designed to detect coding 86 and 184 in pfmdr1 gene,and AS-LAMP primers to detect SVMNT(mutant type)in pfcrt gene.The detection results of clinical samples were consistent with the results of nested PCR with sequencing.Conclusion:1.Pfnhe1 ms4760 microsatellite profiles has moderately diverse in isolates from imported P.falciparum in Wuhan,Hubei province.2.Isolates from imported P.falciparum in Wuhan,Hubei province,polymorphism mutations of pfcrt and pfmdr1 were associated with resistance to chloroquine and amodiaquine,while the limited mutations in pfk13 might be associated with artemisinin resistance.3.Pfcrt,pfmdr1 and pfk13 can continue to be used as molecular markers to monitor chloroquine,amodiaquine and artemisinin resistance.4.The q PCR technique established in this study is suitable for rapid genotyping of pfmdr1 coden 86 and AS-PCR was used for genotyping of pfmdr1coden 86 and 184.