Molecular Method For The Dection Of Target Mutation Related To Insecticide Resistance To Chlorpyrifos And Fipronil In Nilaparvata Lugens St?l

Nilaparvata lugens Stil(Brown planthopper,BPH)is one of the most important agricultural pest insects on rice in Asia.BPH migrates with the monsoon and it is easy to break out.At present,the prevention and control of BPH mainly relies on the use of chemical insecticides.However,the long-term,vast and unreasonable application of chemical insecticides has caused multiple levels of resistance to various commonly used insecticides.The effect of insecticide is reducing,and the BPH population is prone to rampant again.Currently,insecticide resistance of BPH to organophosphates,carbamates and phenylpyrazole insecticides of BPH in China is at middle levels,and that to most neonicotinoid insecticides such as imidacloprid,thiamethoxam is at high levels.Target resistance is one of the most important mechanisms of insecticide resistance.Gene mutations result in the variation of insecticide target,which is the main mechanism for insect to develop target resistance.Once the target resistance is formed,it can be stable in the insect population that is adverse to insect control.Therefore,efficient and precise detections of insect target mutation can assess the variation of the insecticide target,which is of great significance to insect control.Acetylcholinesterase(AChE)is the target of organophosphates and carbamates such as chlorpyrifos,phoxim,leafhopper;and the gamma-aminobutyric acid receptor(GABAR)is the target of phenylpyrazole insecticide such as fipronil,buprofezin,butenaphthosone.It is found that mutations in AChE and GABAR in BPH result in resistance to insecticides such as organophosphates,carbamates and phenylpyrazole.In the present study,Bidirectional PCR Amplification of Specific Alleles(Bi-PASA)and Loop-Mediated Isothermal Amplication(LAMP)were developed for the rapid detection of target mutations in AChE and GABAR(RDL)related to insecticide resistance in BPH.1.Detection of G119S mutation in AChE by Bi-PASA methodAt present,it has been found that there is a mutation G119S in insect AChE.Position 119 is in the oxyanion hole at the base of the active site gorge of the enzyme.The substitution of glycine(Gly)by serine(Ser)is believed to cause a steric shift and hence to enhance the turnover of organophosphates and carbamates molecules or to block the initial reaction of the enzyme with insecticides.A previous study found that mutant G119S consists in AChE of BPH,resulting in decreased susceptibility to chlorpyrifos.Based on Bi-PASA technology,two pairs of specific primers were designed to establish a method for detecting G119S mutation in BPH and identifying the resistance genotype.PCR was performed using the DNA of individual BPH as a template,while the amplified products were tested by electrophoresis.The bands in the electrophoresis gel are employed to interpret the result with the following rules:three bands indicate resistant heterozygotes;two large bands indicate resistant homozygote;and two bands of the largest and the smallest size indicate sensitive homozygotes.This method displays similar accuracyas the sequencing method.2.Detection of G119S Mutation in AChE by LAMP MethodLAMP technology is simpler,faster,more efficient and with less cost when compared with Bi-PASA technology.LAMP reaction can be performed under constant temperature conditions without the requirement of a thermal cycle instrument.The result can be easily observed by adding colorant,instead of electrophoresis test.Based on LAMP technique,the method of rapid detection of G119S mutation of AChE in BPH was established.Through the screening of multiple sets of primers,a set of specific primers was obtained.The reaction system and conditions were optimized.The LAMP detection system established in this chapter was:2.5 ?L 10 × Isothermal Amplification Buffer,8 U Bst 2.0 DNA polymerase,I ?L genomic DNA of individual BPH,final concentration of 0.25 mmol/L dNTPs,1 mmol/L MgSO4,0.2 ?mol/L Primers,0.8 ?mol/L primers,0.4 mol/L betaine,200 mmol/L hydroxynaphthol blue(HNB)in a total reaction volume of 25 ?L.Hydroxylnaphthol blue(HNB)was applied as an indicator and the reaction was performed at 64 ? for 60-70 min.The results were obtained by observing the change of color before(purple)and after the reaction(purple to sky blue).After the reaction,if the reaction solution is still purple,the individual is sensitive individuals.If the reaction solution turns from purple to sky blue,the individual is resistant individuals.3.Detection of A302S Mutation in RDL by LAMP MethodGamma-aminobutyric acid receptors(GABARs)of insects are important targets for phenylpyrazole insecticides in which the RDL subunits can form functional GABA-gated anion channels.Position 302 is in the second transmembrane region of the RDL subunit.The substitution of Ala by Ser leads to insecticide resistance,such as the resistance to fipronil in BPH.Based on LAMP technique,a set of primers was successfully designed and employed to detect the A302S mutation of GABA receptor in BPH.The LAMP detection system established in this chapter was:2.5 ?L 10 × Isothermal Amplification Buffer,6 U Bst 2.0 DNA polymerase,1 ?L genomic DNA of BPH,the final concentration of 0.25 mmol/L dNTPs,1 mmol/L MgSO4,0.2 ?mol/L Primers,1.2 ?mol/L primers,0.6 mol/L betaine,200 mmol/L hydroxynaphthol blue(HNB)in a total reaction volume of 25 ?L.The reaction was performed at 64? for 70 min.The detection method is easy to operate with only about one hour for one reaction and the results are straightforward in the end of the reaction.