LAMP Detection Of Genotype At Position 216 In Acetylcholinesterase In Apolygus Lucorum

Apolygus lucorum is one of the main pests that damage cotton in China.An alanine to serine mutation at position 216（A216S）of acetylcholinesterase-1（AChE-1）is the main resistance mechanism to chlorpyrifos in A.lucorum.The mutation frequency of A216S was significantly related to the resistance level to chlorpyrifos.On the basis of the resistance mechanism to chlorpyrifos in A.lucorum,a loop-mediated isothermal amplification（LAMP）method was developed for rapid genotype detecting at position216 in AChE-1 in A.lucorum,which can also use as a guide to precise pesticide selection.The LAMP technology for genotype detecting at position 216 in AChE-1 was established by specific primers designing and reaction condition optimization,the genomic DNA extracting time was shortened and the heating device was simplified,and the selection standard for chlorpyrifos applied in cotton fields based on LAMP detection results was established.The results will provide technical support for the resistance monitoring of A.lucorum in the field,and a criterion for the precise pesticide selection in the chemical control of A.lucorum,so as to implement the“Action to Achieve Zero Growth of Pesticide Use by 2020”and achieve the efficient prevention of A.lucorum.1.The developing of LAMP method for genotype detecting at position 216 in AChE-1 in A.lucorumBy designing and screening specific primers for an A216 allele,optimizing the reaction system and reaction conditions,and integrating the detection method for S216allele,the loop-mediated isothermal amplification（LAMP）technique for genotype detecting at position 216 in AChE-1 in A.lucorum was established.The reaction system was as follows:Bst DNA polymerase 0.16 U·μL^-1,d NTP 1.0 mmol·L^-1,Mg²⁺2.0-4.0mmol·L^-1,betaine 0.6-1.2 mmol·L^-1,a pair of internal primers FIP and BIP 1.6μmol·L^-1,a pair of external primers F3 and B3 0.4μmol·L^-1,2.5μL 10×Thermo Pol,2μL DNA template,2μL 1000×SYBR Green.The amplification protocol was 62°C for 45 min.Wild or mutant type internal primers and external primers were used for A216 or S216 allele detecting,respectively.The reaction solution in green was positive,while it in dark orange was negative.2.Simplification of LAMP technology for genotype detecting at position 216 in AChE-1 in A.lucorumLAMP technology for genotype detecting at position 216 in AChE-1 in A.lucorum was simplified from two aspects of time consuming and equipment.The results showed that the genomic that qualified DNA was extracted from samples within 5 minutes with Tiangen DNA rapid extraction kit,and no equipment were needed.The LAMP detecting was performed at a temperature range of 58°C to 66°C and a time range of 30 to75min.The temperature change of a daily insulation device（vacuum cup）was less than4°C within 1 hour.Therefore,the LAMP detection was simplified to perform in a vacuum cup within 50 minutes.3.Corresponding relationship between A216S mutation frequency and control efficacy of chlorpyrifos in fieldThe corresponding relationship between the frequency of A216S mutation in AChE-1 and chlorpyrifos control efficacy in field was determined by simulated field spraying.The results showed that the mutation frequency of A216S was significantly correlated with the control effect of chlorpyrifos at 25ppm(LC₉₉ of Chlorpyrifos against susceptible A.lucorum),150ppm(100 times the LC₅₀ of chlorpyrifos against susceptible A.lucorum)and 1000ppm（Field recommended dose of chlorpyrifos for controlling cotton field pests）,with a correlation coefficient of 0.92,0.93,0.88.When A216S mutation frequency was 0,the control efficacy of chlorpyrifos were 70%,96.7%and100%.With the increasing of mutation frequency of A216S,the control efficacy of chlorpyrifos decreased gradually.When the mutation frequency was 30%,the control efficacy of chlorpyrifos at different concentrations were 41.1%,78.9%and 86.7%,which could not meet the requirements of pest control.Therefore,this result could be used as a chlorpyrifos selection standard when the frequency of A216S mutation in AChE-1detected by LAMP was used to guide the control to A.lucorum.