An. gambiae s.l. target site mutation screening
Mosquito individuals morphologically identified as An. gambiae s.l. (n=192) were screened for eight mutations: L1014S-kdr, L1014F-kdr, G119S-ace1, N1575Y, L43F-CYP4J5, CYP6P4 (I236M), Zanzibar-like transposable element (TE) and CYP6AA1 duplication.
For L1014F-kdr and L1014S-kdr detection, the following primers were used: IPCF (5’-GATAATGTGGATAGATTCCCCGACCATG-3’), AltRev (5’-TGCCGTTGGTGCAGACAAGGATG-3’), WT (5’-GGTCCATGTTAATTTGCATTACTTACGAATA-3’), West (5’-CTTGGCCACTGTAGTGATAGGAAATGTT-3’) and East (5’- CTTGGCCACTGTAGTGATAGGAAAATC-3’) [29]. For L1014F-kdr, amplifications were performed in 25 µL reactions containing 2 µL template DNA, 1 µL IPCF (2.5 pmol/µL), 1 µL AltRev (2.5 pmol/µL), 1 µL WT (25.0 pmol/µL), 3 µL West (8.0 pmol/µL), 4.5 µL H2O, and 12.5 µL 2X Hot Start Taq PCR Master Mix (New England Biolabs). For L1014S-kdr, amplifications were performed in 25 µL reactions containing 2 µL template DNA, 2 µL IPCF (2.5 pmol/µL), 2 µL AltRev (2.5 pmol/µL), 2 µL WT (5 pmol/µL), 2 µL East (2.5 pmol/µL), 2.5 µL H2O, and 12.5 µL 2X Hot Start Taq PCR Master Mix (New England Biolabs). Samples were run on a Bio-Rad T100TM Thermal Cycler, according to the following conditions: for 5 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 59°C (L1014F) or 57oC (L1014S), and 30 seconds at 72°C, and a final extension step of 5 minutes at 72°C. PCR products were separated in 2% E-GelTM agarose gels with SYBR Safe (Invitrogen, UK). A control band at 314 base pairs (bp) indicated a successful reaction, a band at 214 bp indicated the susceptible wild type allele, and a band at 156 bp indicated the resistant allele. No template controls (NTCs) were run in parallel for all assays.
For N1575Y, PCR reactions were prepared with 10 µL of 2X QuantiTectTM Probe PCR master mix (Qiagen, UK), a final concentration of 1 µM of forward (5’-TGGATCGCTAGAAATGTTCATGACA-3’) and reverse (5’-CGAGGAATTGCCTTTAGAGGTTTCT-3’) primers, 0.5 µM of N probe (5’-HEX-ATTTTTTTCATTGCATTATAGTAC-3’) and Y probe (5’-6-FAM-TTTTTCATTGCATAATAGTAC-3’), and 2 µL template DNA for a final reaction volume of 20 µL [30]. Reaction plates were run on the Agilent Technologies Stratagene Mx3005P qPCR system, according to the following conditions: 15 minutes at 95oC followed by 40 cycles of 15 seconds at 94oC and 1 minute at 60oC. Positive controls from gDNA extraction from known An. gambiae s.s. with and without the N1575Y mutation were included in each run, alongside NTCs.
For G119S-ace1, PCR reactions were prepared with 5 µL 2X PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies, UK), a final concentration of 0.8 µM of forward (5’-GGCCGTCATGCTGTGGAT-3’) and reverse (5’- GCGGTGCCGGAGTAGA-3’) primers, 0.2 µM of G probe (5’-HEX-TTCGGCGGCGGCT-3’) and S probe (5’-6-FAM-TTCGGCGGCAGCT-3’), and 2 µL template DNA for a final reaction volume of 10 µL [29]. Reaction plates were run on the Agilent Technologies Stratagene Mx3005P qPCR system, according to the following conditions: 10 minutes at 95oC followed by 40 cycles of 10 seconds at 95oC and 35 seconds at 60oC. Positive controls from gDNA extraction from known An. gambiae s.s. with and without the G119S mutation were included in each run, alongside NTCs.
For L43F-CYP4J5, PCR reactions were prepared with 10 µL of 2X QuantiTectTM Probe PCR master mix (Qiagen, UK), a final concentration of 1 µM of forward (5’-AGCCTGCGCGTGTGATA-3’) and reverse (5’-CTTCTTCTCCTGTGGTTCGTTTG-3’) primers, 0.5 µM of SNP-specific probes (5’-6-FAM-TTGCCGGAAGGCAGT -3’) and (5’-HEX-TTGCCGGAGGGCAGT-3’), and 2 µL template DNA for a final reaction volume of 20 µL [31]. Reaction plates were run on the Agilent Technologies Stratagene Mx3005P qPCR system, according to the following conditions: 10 minutes at 95oC followed by 40 cycles of 15 seconds at 92oC and 1 minute at 60oC.
Three recently designed, independent, locked-nucleic acid (LNA) probe-based PCR assays were used to genotype the point mutation CYP6P4-I236M, the insertion of the partial Zanzibar-like TE and the duplication of CYP6AA1 [23]. For CYP6P4-I236M, PCR reactions were prepared with 5 µL 2X PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies, UK), 0.35 µM of each primer, CYP6P4_I236M_Forward (5’- AGTTTATGTTTGCGACCACGTT-3’) and CYP6P4_I236M_Reverse (5’-TCCACCGTCTCGCGCACAAC-3’), 0.2 µM of each probe, CYP6P4_I-Wild (5’-6-FAM-TTC+ATGC+C+G+ATGC-3’) and CYP6P4_M-Mutant (5’-HEX-TTC+ATGC+C+C+ATGC-3’), and 2 µL template DNA for a final reaction volume of 10 µL. Reaction plates were run on the Agilent Technologies Stratagene Mx3005P qPCR system, according to the following conditions: 3 minutes at 95oC followed by 20 cycles of 15 seconds at 95oC and 30 seconds at 66oC, followed by 23 cycles of 10 seconds at 95oC, 20 seconds at 58oC and 11 seconds at 72oC. For the Zanzibar-like TE, PCR reactions were prepared with 5 µL 2X PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies, UK), 0.35 µM of each primer, ZZB_Flank_A (5’-CAAAATCAATGKCACRGAGC-3’), ZZB_Flank_B (5’- CGCTACAATGAAKGGAAAGTC-3’), ZZB_Int_A (5’-CATTACATGGCGACCGTACCT-3’), 0.2 µM of each probe, ZZB_Mutant (5’-6-FAM- A+C+ATTA+CA+CTTTGT+CA+GTA-3’) and ZZB_WT (5’-HEX-GATG+TT+CTTTK+T+CA+G+TATT -3’), and 2 µL template DNA for a final reaction volume of 10 µL. Reaction conditions were 3 minutes at 95oC followed by 40 cycles of 10 seconds at 95oC, 20 seconds at 58oC and 11 seconds at 72oC. For the CYP6AA1 duplication, PCR reactions were prepared with 5 µL 2X PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies, UK), 0.35 µM of each primer, AA1_Dup1_ins1 (5’-CAGTGCGGTACGCTCGTTAA-3’), AA1_Dup1_outs (5’-GGATCGGTTTACAGCGGACG-3’), AA1_Dup1_ins2 (5’-CATCACCTGTGCTCGCAARTT-3’), 0.2 µM of each probe, AA1_Dup1_outs (5’-Cy5-CCAT+CA+C+CGAA+CG-3’), AA1_Dup1_ins (5’-HEX-AGAA+CCTGCA+C+CAA-3’) and AA1_Dup1_junct (5’-6-FAM-A+CA+AT+TAATT+G+CAT+CGG-3’), and 2 µL template DNA for a final reaction volume of 10 µL. Reaction conditions were 3 minutes at 95oC followed by 40 cycles of 10 seconds at 95oC, 20 seconds at 57oC and 15 seconds at 72oC.
An. funestus s.l. target site mutation screening
Mosquito individuals morphologically identified as An. funestus s.l. (n=160) were screened for three target site mutations: L119F-GSTe2, CYP6P9a and CYP6P9b.
For L119F-GSTe2, PCR reactions were prepared with 10 µL of 2X QuantiTectTM Probe PCR master mix (Qiagen, UK), a final concentration of 1 µM of forward (5’-AACAATTTTTCATTTCTTATTCTCATTTAC-3’) and reverse (5’-CGACTCGATCTTCGGGAATGTC -3’) primers, 0.5 µM of probes L119 (5’-5-HEX-AGGAGCGTATTCTTTTCTAC-3’) and 119F (5’-6-FAM-AGGAGCGTATTTTTTTCTA-3’), and 2 µL template DNA for a final reaction volume of 20 µL [32]. Reaction plates were run on the Agilent Technologies Stratagene Mx3005P qPCR system, according to the following conditions: 10 minutes at 95oC followed by 40 cycles of 15 seconds at 92oC and 1 minute at 60oC.
For CYP6P9a, PCR reactions were performed in a final volume of 25 µL, containing 2X Hot Start Taq PCR Master Mix (New England Biolabs), 1 µM of forward (5’-TCCCGAAATACAGCCTTTCAG -3’) and reverse (5’-ATTGGTGCCATCGCTAGAAG -3’) primers, and 2 µL template DNA [18]. Samples were run on a Bio-Rad T100TM Thermal Cycler, according to the following conditions: 95oC for 3 minutes, 40 cycles of 94oC for 30 seconds, 55oC for 30 seconds and 72oC for 1 minute; followed by 72oC for 10 minutes. Ten microlitres of each PCR product was digested by adding 1 µL 10X buffer TaqI, 0.2 µL TaqI restriction enzyme (Thermo Scientific, UK) and 3.8 µL of H2O. Digests were incubated at 65oC for 4 hours. Digested products were separated in 2% E-GelTM agarose gels with SYBR Safe (Invitrogen, UK). A band at 450 bp indicated the susceptible wild type allele, a band at 350 bp and 100 bp indicated the resistant allele.
For CYP6P9b, PCR reactions were performed in a final volume of 25 µL, containing 2X Hot Start Taq PCR Master Mix (New England Biolabs), 1 µM of forward (5’-CCCCCACAGGTGGTAACTATCTGAA -3’) and reverse (5’-TTATCCGTAACTCAATAGCGATG -3’) primers, and 2µl template DNA [19]. Samples were run on a Bio-Rad T100TM Thermal Cycler, according to the following conditions: 95oC for 3 minutes, 40 cycles of 94oC for 30 seconds, 58oC for 30 seconds and 72oC for 1 minute; followed by 72oC for 10 minutes. Ten microlitres of each PCR product was digested by adding 1 µL CutSmart buffer, 0.2 µL Tsp45I restriction enzyme (New England Biolabs, UK) and 3.8 µL of H2O. Digests were incubated at 65oC for 4 hours. Digested products were separate in 2% E-GelTM agarose gels with SYBR Safe (Invitrogen, UK). A band at 550bp indicated the resistant allele and two bands at 400bp and 150bp indicated the susceptible wild type allele.
For both CYP6P9a (n=8) and CYP6P9b (n=8), PCR-RFLP results for a subset of resistant and susceptible individuals were confirmed by sequencing as previously described [33]. In brief, PCR products used in the enzyme digests were submitted to Source BioScience (Source BioScience Plc, Nottingham, UK) for PCR reaction clean-up, followed by chain termination sequencing. Sequencing analysis was conducted in Geneious Prime®2021.1.1.