Development And Application Of A DNA Microarray For Analysis Of The Drug Resistance Molecular Markers In Plasmodium Falciparum

Objective（1） To develop a microarray technique for detection of the drug resistance molecular markers（21 SNPs） in Plasmodium falciparum,including polymorphisms of pfcrt 391 T/A,392 G/C,399 G/T,400 A/G,402 T/A and 404 A/C（involving the 72nd-76th amino acids,associated with resistance to chloroquine and amodiaquine）, polymorphisms of pfmdr1 256 A/T and 257 A/T（involving the 86th amino acid, associated with resistance to mefloquine and lumefantrine）,polymorphisms of pfdhps 1482 T/G,1483 C/T/G,1486 G/C,1794 A/G,1918 C/G and 2013 G/T/A（involving the 436th,437th,540th,581st and 613rd amino acid,associated with resistance to sulfadoxine）,polymorphisms of pfdhfr 148 T/C,152 A/T,153 T/C,175 T/C,323 G/A/C and 490 A/T（involving the 50th,51st,59th,108th and 164th amino acid, associated with resistance to pyrimethamine）,polymorphisms of pfATPase6 2306 G/A (involving the 769th amino acid,associated with increased IC₅₀ of artemisinin).（2） To evaluate the accuracy,sensitivity,specificity,reliability and sample applicability of the DNA microarray;to see whether the microarray has the ability to analyze the mixed infection and all possible genotypes;and to estimate the cost and time consumed of the microarray method.（3） To analyze the distribution of drug resistance molecular markers in Plasmodium falciparum in China’s Yunnan Province,Hainan Province and Myanmar’s Shan State by the microarray method,thereby to understand the antimalarial drug resistance situation of China and Sino-Myanmar border area.Methods（1） Specific oligonucleotide probes and control probes were designed according to the reference sequences from NCBI（http://www.ncbi.nlm.nih.gov） by using Primer Premier 5.0,Oligo 6.0 and Array Designer 2.0 software.The probes were spotted on silylated slide to fabricate a microarray.A multiplex PCR was used for amplification of the five genes in P.falciparum followed by DNase I fragmentation and Cy3 labeling.The fluorescently labeled products were hybridized to the probes on the microarray.After hybridization and washing,slides were scanned using a microarray scanner,and the fluorescent signals obtained were combined to deduce the genotype of genes.Based on the results of repeating analysis of the five laboratory isolates,sequences of probes and hybridization condition were optimized.（2） Analyzing the genomic DNA of P.falciparum FCC1/HN isolate at different concentration to evaluate the sensitivity of the microarray;comparing microarray results with sequencing results of the 30 field samples to evaluate the accuracy of the microarray;analyzing the genomic DNA of P.vivax,P.berghei,P.cynomolgi, Leishmania donovani and Cryptosporidium to evaluate the specificity of the microarray;comparing the results of three parallel tests within a slide and comparing the results of two independent tests of the same samples to evaluate the reliability of the microarray;analyzing the mixture of genomic DNA of P.falciparum 3D7 with P. falciparum Dd2,P.falciparum isolate FCC1/HN and P.falciparum isolate CMH/YN, respectively,to examine the ability of the microarray for analysis of mixed infection; analyzing 20 combinations of 37 synthesized oligos harboring all known genotypes to see whether the microarray has the ability to detect all possible genotypes;analyzing filter paper samples preserved for five years and genomic DNA samples preserved for three years to evaluate the sample applicability of the microarray;calculating the cost and time consumed of the microarray method.（3） P.falciparum samples on the filter paper were collected from China’s Yunnan Province,Hainan Province and Myanmar’s Shan State,and were analyzed by the microarray to detect the 21 SNPs associated with drug resistance in P.falciparum.Whole genome amplification technique was applied to the samples that failed to be amplified by multiplex PCR,and then these samples were retested by the microarray.Results（1） Through repeating adjustment,45 specific probes of 11 groups and 5 control probes were determined.The optimal hybridization condition was:probes spotting at 25μmol/L,hybridization at 52℃for 2 hours.After optimization of the probes and hybridization condition,the analysis results of the five laboratory isolates by microarray were in accordance with that of the nested-PCR sequencing assay.（2） Out of 30 field samples 2 results could not be obtained due to failure in DNA extraction or mPCR amplification.Among 308 genotyped positions of the 28 successful hybridizations,301（97.7%） yielded usable data,6 had inconsistent genotyping results between sequencing assay and microarray assay.The detection limit of the microarray method was 60 pg DNA.Intra-assay and inter-assay reproducibility were 97.3%and 95.7%,respectively.No positive signal was detected in any specific probes for five other parasites.The testing results of the three simulative mixed infections were in accordance with the reality.The microarray could successfully analyze the filter paper samples preserved for five years and genomic DNA samples preserved for three years.It took about 8 hours to accomplish a whole microarray assay and the cost was about 2.9 RMB per SNP.（3） Out of 92 field samples,14 failed to be amplified by multiplex PCR at the first time.After whole genome amplification,among them 6 could be successfully analyzed by the microarray.Totally 84 samples obtained analysis results of the microarray,there were several samples with one or two genotyped positions that could not be detected.The main genotype at pfcrt 72-76 in the three fields was CVIET;the rates of mutation at pfcrt 76 were 52.6%,92.1%and 100.0%in China’s Yunnan Province,Hainan Province and Myanmar’s Shan State,respectively,and there was a significant difference of the mutation rate among the three fields（x²=21.57,P＜0.01）.The rates of mutation at pfmdr1 86 were 10.5%,12.5%and 8.3%in China’s Yunnan Province, Hainan Province and Myanmar’s Shan State,respectively,and there was no significant difference of the mutation rate among the three fields（x²=0.27,P=0.87）. Yunnan Province had more mutations at pfdhfr 50,51,59,108 and 164 than that of Myanmar’s Shan State and Hainan Province（P＜0.01）.Yunnan Province also had more mutations at pfdhps 436,437,540,581 and 613 than that of Hainan Province （P=0.03）.No pfATPase6 769 mutation was found in any samples collected from the three fields.The mean mutation number in the five drug resistance related genes were 5.21,8.32 and 7.00 in China’s Yunnan Province,Hainan Province and Myanmar’s Shah State,respectively,and there was a significant difference of the mean mutation number among the three fields（F=11.41,P＜0.01）.Yunnan Province and Myanmar’s Shah State had more mutations in the five genes than that of Hainan Province（P＜0.01 and P=0.04）.Conclusions（1） The present study has established a microarray method for simultaneous analysis of 21 SNPs associated with drug resistance in Plasmodium falciparum.（2） The microarray method is sensitive and specific,and the analysis results of the microarray show high accuracy and reliability.It is more convenient as compared with traditional methods and significantly improves the efficiency of analysis of the drug resistance molecular markers in P.falciparum.As a limitation,the microarray could only be used for analysis of the known SNPs in the resistance related genes,while could not be used for detection of the copy number of pfmdr1, which has been associated with resistance against chloroqnine,mefloquine and probably artemisinins.（3） By using the microarray method developed in this study, we accomplished the analysis of the drug resistance molecular markers in Plasmodium falciparum in China and Sino-Myanmar border area.The situation of antimalarial drug resistance in Yunnan Province and Myanmar’s Shan State is more serious than that in Hainan Province.The resistance to chloroquine,sulfadoxine and pyrimethamine still exist in the three fields although the use of these drugs has been terminated for a quite long period of time.The molecular marker related to decreased sensitivity of artemisinin can not be found at present in samples from the three fields.