Study On The Genotype Of Plasmodium Falciparum From China-Myanmar Border

Objective:To acknowledge the genotype distribution of Plasmodium falciparum from China-Myanmar border area in Yunnan province, together with the polymorphism of each allele in MSP1 that we use in vitro culture and molecular biology methods. And on the basis of which we find out the growth and development tendency of short-time culture strains and advantageous ones.Methods:We extract genome DNA from plasmodium falciparum blood samples of 271 filter paper or in vitro culture, then amplify the fragment with specificity in MSP1(merozoite surface protein 1), MSP2 (merozoite surface protein 2) and GLURP (glutamate rich protein) of plasmodium falciparum using nested-PCR, and finally study on the genotyping. With the above as base, we sequence each allele in 36 plasmodium falciparum strains which have been identified as MSP1 genes and further study the polymorphism of MSP 1 genes in the samples from China-Myanmar border area in Yunnan province. Through the short-time culture of 19 samples strains, we respectively extract the genome DNA that 8 days,14 days before and after culture.And then assay the relative expression using RT-PCR separately. By contrast of the DNA contents variation as shown above to judge the development tendency of plasmodium strains and advantageous ones under the laboratory experiment condition that with mixed infection.Result:1. Genotyping (1) The genotyping of MSP1:Of the case of simplex infection,87,78 and 25 were respectively amplified single gene segment as MAD20, K1 and RO33 from 271 blood samples; (2) The genotyping of MSP2:Of these simplex infection, there are 100 were amplified gene segments as IC/3D7 while 100 as FC27;(3) The genotyping of GLURP:Of these referred 271 cases,262 were identified simplex infection whose gene segments are between 500-1000. So isolates between 750-850bp from these cases occurred most frequently that they belong to the advantageous strains.That 40 samples were identified mixed infection with both MAD20 and Kl (The genotyping of MSP1 genes) that occurred highest in freqency,9 cases were identified as mixed infection with K1, MAD20 and PO33; While the mixed infection with MSP2 gene were 57 cases. So from the integrated estimation of these three alleles, we conclude that the single infetion strains were 162 and the mixed ones were 109. Yet there were just 21 samples which simultaneously infected with both MSP1 and MSP2 genes,and only 1 sample was mixed infected with MSP1,MSP 2 and GLURP genes. In summary,the above data indicate that the cross infection rate between different genotypes is quite low. But from 2007 to 2009 there is a rising tendency of chances of mixed infection in the China-Myanmar border area, Yunnan province.2.On the basis of genotyping, we sequenced 36 blood samples which had been indentified to be MSP1 gene. And 3 strains were found mixedly infected,2 of which were mixed with Kl and RO33,1 with MAD20 and RO33.Then after the analysis and processing with software DNASTAR, we finally found Kl occurred in 10 strains of these cases. This genotype has three different size of gene sequence segments (2 werel59bp,6 were 168bp,2 were 195bp).And 20 of these samples were infected with MAD20, which had four different size of sequence segments(2 were 159bp,2 were 168bp,6 were 186bp and 10 were 195bp).Of which the 186bp sequences showed four variance (2 strains with 186-1,2 with 186-2,1 with 186-3 and 1 with 186-4), while the 195bp sequences showed two discrepancies(9 strains with 195-1,1 with 195-2). That 9 strains were found to be RO33, of which there was 1 sample with 159bp sequence,in which two differences arised. Then by further sequence contrast and analysis, we finally found out a new genotype of MAD20-PF-M6.3. In the course of dynamic study on the mixed strains of MSP1 genes, we used 19 blood samples,18 of which were infected with both Kl and MAD20, the DNA content were found increased in 13 strains of MAD20 and 5 strains of Kl in the short-time culture; There was only one sample infected with K1,MAD20 and RO33, yet the DNA content increased merely in RO33 in the meantime of culture.Conclusion:Of MSP1 gene MAD20 in Plasmodium falciparum from China-Myanmar border area in Yunnan province occurred most frequently.So this type is advantageous strain, while Kl is secondary to it and RO33 is the least;The allele IC/3D7 and FC27 in MSP2 gene respectively accounts for 36.9% of the whole population; Glurp gene from Isolates between 750-850bp as an advantageous strain occurred highest in frequency. Of the mixed infection of MSP1 genes, K1 mixed with MAD20 were the main type, yet the cross infection rate among MSP1, MSP2,GLURP was quite low. By statistical analysis, there is no distinguish discrepancy between different years from 2007 to 2010 (P=0.077>0.05), while the mixed infection rate showed a rising tendency year by year. And there was a regional variance by contrast of the infection of plasmodium falciparum in Lasa and wabang(P=0.019<0.05). The seqencing of MSP1 indicated that there was a highly complex genetic diversity in the Plasmodium falciparum from China-Myanmar border area, while MAD20 take predominant advantages in the overall distribution of each allele.The contrast and analysis of sequences confirmed allele K1,MAD20 and RO33 of Plasmodium falciparum from China-Myanmar border area were in accordance with the corresponding genotype sequences in Thailand, Vietnamese and Tanzania. So on the basis of that a new genotype---PF-M6 was found. After in vitro short-time culture, the allele MAD20 prior to others in the state of growth and development. While K1 is secondary to it and RO33 is the least of all, which accord with the above genotyping result and further confirmes that the strain type MAD20 is the most advantageous one.