Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.3 Insecticide Resistance Allele Assay by PCR5.3.4 ACE-1 Resistance in An. gambiaePage 1 of 25.3.4 ACE-1 Resistance in An. gambiaeMR4 StaffIntroductionInsensitive acetylcholinesterase (AChE) is a resistance mechanism associated with tolerance tocarbamate and organophosphate insecticides. Mutations within AChE genes in Dipterans are widespreadand have varying effects on the tolerance levels to insecticides. This is of great importance due to theincreased interest in utilizing bendiocarb as a potential treatment for bed-nets and increased resistancelevels to common insecticides across Africa. (Weill et al. 2004) isolated a unique mutation found in bothold and new world vectors in the ace-1 allele. From this a PCR-RFLP was designed based on the G119Smutation isolated from An. gambiae. An RT-PCR based assay can be found in Chapter 8.5.1.4.Anopheles spp. (Weill et al. 2004)Prepare PCR Master Mix for 96, 48 or 1 25μl PCR reactions. Add reagents in the order presented.96 48 1 Reagent1635 μl 817.5 μl 16.35 μl sterile H 2 O500 μl 250 μl 5.0 μl 5X GoTaq PCR Buffer100 μl 50 μl 1.0 μl dNTP (2.5 mM mix)125 μl 62.5 μl 1.25 μl MOUSTDIR1 (25 pmol/µl) [CCGGGNGCSACYATGTGGAA]125 μl 62.5 μl 1.25 μl MOUSTREV1 (25 pmol/µl) [ACGATMACGTTCTCYTCCGA]15 μl 7.5 μl 0.15 μl Taq DNA polymerase (5 U/μl)2.5 ml 1.25 ml 25 μl TotalTable 5.3.4.1. Use 1 μl DNA template.PCR Cycle conditions93 o C/5min x 1 cycle(93 o C/1min -0- 53 o C/1min -0- 72 o C/1.5min) x 35 cycles72 o C/10min x 1 cycle4 o C holdRestriction enzyme digestAdd 1μl AluI restriction enzyme, 2 µl of H20, and 2µl of buffer to 15 μl PCR product from above reaction.Allow to incubate at 37°C for 8-24 hr. For shorter times, incomplete digests could be a problem.Visualize on a 2% agarose ethidium bromide gel.Anopheles spp.Primers create a 194 bp amplicon, after restriction enzyme digest homozygous resistant individuals willhave 120bp and 74bp fragments (Figure 5.3.4.1).