High-throughput dual barcoding method for major Anopheles species and their insecticide resistance genes in China
Objective To establish a high-throughput method that can simultaneously,quickly,and accurately detect main malaria-transmitting Anopheles species and their resistance genes in China,providing a high-throughput monitoring tool for monitoring the main malaria vectors in China after malaria elimination.Methods In different sampling locations,including Tengchong City,Yunnan Province;Wenchang City,Hainan Province;and Donggang City,Liaoning Province,adult specimens of mosquitoes,including Anopheles sinensis,Anopheles minimus,Anopheles dirus,and Anopheles anthropophagus,were collected.Polymerase chain reaction(PCR)technology and Sanger sequencing were employed to detect the ITS2,kdr(L1014),rdl(A296),and ace-1(G119)genes in individual mosquitoes.For the analysis of mixed samples,an optimized multiplex PCR reaction system,custom-designed dual barcode primers,and next-generation sequencing(NGS)technology were utilized to detect the aforementioned genes.The consistency was assessed using Kappa consistency tests and Chi-square tests for multiple rates.Sensitivity,specificity,and the Youden index were calculated using a four-grid table calculation method.The costs associated with each step of the normal operational process for each method were statistically summarized,and the optimal quantity of mixed samples for detection was determined by a comprehensive approach.Results Conventional PCR amplification of gDNA from 300 mosquitoes resulted in 144 individuals of Anopheles sinensis,53 individuals of Anopheles dirus,62 individuals of Anopheles anthropophagus,and 41 individuals of Anopheles minimus,as identified by Sanger sequencing.The mutation frequencies of resistance genes kdr(L1014),rdl(A296),and ace-1(G119)were found in 73,27,and 41 specimens,respectively.Using a newly established multiplex PCR reaction system based on custom dual barcode and NGS sequencing technology,samples corresponding to Sanger sequencing were detected under different sample sizes.The two methods showed high consistency in the results(all Kappa＞0.900).Multiple comparison tests showed significant differences in the consistencies of the two methods across different sample sizes N(40,80,160),N(120,200,240,280),and N(300)(x2=26.547,P＜0.001).The new method demonstrated high sensitivity and specificity across various sample sizes,with the Youden index ranging from highest to lowest as follows:1(40,80,160)＞0.994(120)＞0.990(280)＞0.988(200)＞0.987(240)＞0.985(300).With an increase in sample size from 40 to 300,the cost per sequencing site for the new method decreased from 20.0 yuan to 8.3 yuan,while the cost per sequencing site for the conventional method decreased from 16.7 yuan to 15.4 yuan.The optimal mixed sample size for the new method was determined to be 280.Conclusion The newly developed multiplex PCR and barcode NGS detection method enables simultaneous screening of four major malaria vector mosquito species and the presence of mutations in the ace-1,kdr,and rdl resistance genes,exhibiting excellent stability,high sensitivity,and specificity.It allows for the efficient analysis of large sample sizes in a single run,offering a cost-effective alternative compared to other types of detection methods.
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