Table 1 Primers probes and protocols used for the assay

A. funestus Species ID

FUNUNIV_F: CCGATGCACACATTCTTGAGTGCCTA

FUN _R: CTCGGGCATCGATGGGTTAATCATG

VAN _R: AACTCTGTCGACTTGGTAGCCGAAC

RIV_R: AATCAGGGTCGAACGGCTTGCCG

PAR_R: GCCCTGCGGTCCCAAGCTAGATT

RIVLIKE_R: CTCCCGTGGAGTGGGGGATC

LEES_R: GACGGCATCATGGCGAGCAGC

KapaTaq [500 nM primers]

2.0 mM MgCl2

94 °C/4 min × 1 cycle

(94 °C/30 s 58 °C/30 s, 72 °C/45 s) × 30 cycles; 72 °C/7 min × 1 cycle

496 bp An. vaneedeni, 424 bp An. funestus, 346 bp, An. rivulorum, 241 bp An. rivulorum-like (West Africa), 176 bp An. parensis and 93 bp An. leesoni [run 10 μl sample]

Kdr detection

F: GGMGAATGGATYGAATCMATGTGGGA

R: GATGAACCRAAATTKGACAAAAGCAA 3'

KapaTaq [500 nM primers]

94 °C/ 5 min, 94 °C/ 1 min, 50 °C/ 2 min, 72 °C/ 2 min (35 cycles), 72 °C/ 2 min

200 bp

CYP6P9a

F: 5′-TCCCGAAATACAGCCTTTCAG-3′,

R: 5′-ATTGGTGCCATCGCTAGAAG

PCR: KapaTaq; 30 uL reaction [500 nM primers]

The 30 μl solution underwent a denaturing step at 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min and 30 s, followed by a final extension step of 72 °C for 10 min

450 bp [run 10 uL, keep the rest for digestion]

Digestion: 20 uL final volume [10.0 uL PCR product; 1 uL from 10U TaqI; 2 uL TaqI buffer 10x; BSA up to 100 ug/mL; water up to 20 uL]

Digestion: Incubate at 65 °C for 2 h

Restriction digest was separated on 2.0% agarose gel. The Taq I enzyme cut the 450-bp fragment from the putative pyrethroid resistance haplotype into two fragments of 350 and100 bp

N485I acetylcholinesterase1 detection

F: CATGCGATACTGGTCAAACTTTGC

R: GCCATTCGGGAAATTCGCTACTA

P (wt) HEX-CAAACCCCAACACGGC-MGB

P (mut): FAM-CAAACCCCATCACGGC-MGB

The TaqMan reactions were performed in a 10 uL final volume containing 1 × TaqManunivesalmastermix (Applied Biosystems), 800 nM of each primer and 200 nM of each probe,

Cycling conditions were: 10 min at 95 °C, 40 cycles of 15 s at 92 °C and 1 min at 60 °C

N/A

Plasmodium detection

F: GCTTAGTTACGATTAATAGGAGTAGCTTG

R: GAAAATCTAAGAATTTCACCTCTGACA

P: Falcip + (FAM-TCTGAATACGAATGTC-MGB)

P: OVM + (HEX-CTGAATACAAATGCC-MGB')

The TaqMan reactions were performed in a 10 uL final volume containing 1 × TaqManunivesalmastermix (Applied Biosystems), 800 nM of each primer and 300 nM of each probe

Cycling conditions were: 10 min at 95 °C, 45 cycles of 15 s at 92 °C and 1 min at 60 °C

N/A