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Table 1 PCR primers, probes and reaction conditions

From: Investigating molecular mechanisms of insecticide resistance in the Eastern Democratic Republic of the Congo

 

Primers and probes (concentrations)

5′ modifications

Sequences

Reaction conditions

References

An. gambiae s.l. mutation

 L1014F-kdr

IPCF (2.5 pmol/µL)

AltRev (2.5 pmol/µL)

WT (25 pmol/µL)

West (8 pmol/µL)

 

GATAATGTGGATAGATTCCCCGACCATG

TGCCGTTGGTGCAGACAAGGATG

GGTCCATGTTAATTTGCATTACTTACGAATA

CTTGGCCACTGTAGTGATAGGAAATGTT

1 cycle: 5 min at 95 °C

35 cycles: 30 s at 95 °C, 30 s at 59 °C, 30 s at 72 °C

1 cycle: 5 min at 72 °C

[25]

 L1014S-kdr

IPCF (2.5 pmol/µL)

AltRev (2.5 pmol/µL)

WT (5 pmol/µL)

East (2.5 pmol/µL)

 

GATAATGTGGATAGATTCCCCGACCATG

TGCCGTTGGTGCAGACAAGGATG

GGTCCATGTTAATTTGCATTACTTACGAATA

CTTGGCCACTGTAGTGATAGGAAAATC

1 cycle: 5 min at 95 °C

35 cycles: 30 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C

1 cycle: 5 min at 72 °C

[25]

 N1575Y

Forward (1 µM)

Reverse (1 µM)

N (0.5 µM)

Y (0.5 µM)

HEX

FAM

TGGATCGCTAGAAATGTTCATGACA

CGAGGAATTGCCTTTAGAGGTTTCT

ATTTTTTTCATTGCATTATAGTAC

TTTTTCATTGCATAATAGTAC

1 cycle: 15 min at 95 °C

40 cycles: 15 s at 94 °C, 1 min at 60 °C

[24]

 G119S-ace1

Forward (0.8 µM)

Reverse (0.8 µM)

G (0.2 µM)

S (0.2 µM)

HEX

FAM

GGCCGTCATGCTGTGGAT

GCGGTGCCGGAGTAGA

TTCGGCGGCGGCT

TTCGGCGGCAGCT

1 cycle: 10 min at 95 °C

40 cycles: 10 s at 95 °C, 35 s at 60 °C

[25]

 L43F-CYP4J5

Forward (1 µM)

Reverse (1 µM)

Probe 1 (0.5 µM)

Probe 2 (0.5 µM)

FAM

HEX

AGCCTGCGCGTGTGATA

CTTCTTCTCCTGTGGTTCGTTTG

TTGCCGGAAGGCAGT

TTGCCGGAGGGCAGT

1 cycle: 10 min at 95 °C

40 cycles: 15 s at 92 °C, 1 min at 60 °C

[26]

 CYP6P4-I236M

CYP6P4_I236M_Forward (0.35 µM)

CYP6P4_I236M_Reverse (0.35 µM)

CYP6P4_I-Wild (0.2 µM)

CYP6P4_M-Mutant (0.2 µM)

FAM

HEX

AGTTTATGTTTGCGACCACGTT

TCCACCGTCTCGCGCACAAC

TTC+ATGC+C+G+ATGC

TTC+ATGC+C+C+ATGC

1 cycle: 3 min at 95 °C

20 cycles: 15 s at 95 °C, 30 s at 66 °C

23 cycles: 10 s at 95 °C, 20 s at 58 °C, 11 s at 72 °C

[27]

 Zanzibar-like TE

ZZB_Flank_A (0.35 µM)

ZZB_Flank_B (0.35 µM)

ZZB_Int_A (0.35 µM)

ZZB_Mutant (0.2 µM)

ZZB_WT (0.2 µM)

FAM

HEX

CAAAATCAATGKCACRGAGC

CGCTACAATGAAKGGAAAGTC

CATTACATGGCGACCGTACCT

A+C+ATTA+CA+CTTTGT+CA+GTA

GATG+TT+CTTTK+T+CA+G+TATT

1 cycle: 3 min at 95 °C

40 cycles: 10 s at 95 °C, 20 s at 58 °C, 11 s at 72 °C

[27]

 CYP6AA1 duplication

AA1_Dup1_ins1 (0.35 µM)

AA1_Dup1_outs (0.35 µM)

AA1_Dup1_ins2 (0.35 µM)

AA1_Dup1_outs (0.2 µM)

AA1_Dup1_ins (0.2 µM)

AA1_Dup1_junct (0.2 µM)

Cy5

HEX

FAM

CAGTGCGGTACGCTCGTTAA

GGATCGGTTTACAGCGGACG

CATCACCTGTGCTCGCAARTT

CCAT+CA+C+CGAA+CG

AGAA+CCTGCA+C+CAA

A+CA+AT+TAATT+G+CAT+CGG

1 cycle: 3 min at 95 °C

40 cycles: 10 s at 95 °C, 20 s at 57 °C, 15 s at 72 °C

[27]

 2La inversion

23A2 (25 pmol/µL)

27A2 (25 pmol/µL)

DPCross 52 L+ (25 pmol/µL)

 

CTCGAAGGGACAGCGAATTA

ACACATGCTCCTTGTGAACG

GGTATTTCTGGTCACTCTGTTGG

1 cycle: 2 min at 94 °C

35 cycles: 30 s at 94 °C, 30 s at 60 °C, 45 s at 72 °C

1 cycle: 10 min at 72 °C

[25]

An. funestus s.l. mutation

 L119F-GSTe2

Forward (1 µM)

Reverse (1 µM)

L119 (0.5 µM)

119 F (0.5 µM)

HEX

FAM

AACAATTTTTCATTTCTTATTCTCATTTAC

CGACTCGATCTTCGGGAATGTC

AGGAGCGTATTCTTTTCTAC

AGGAGCGTATTTTTTTCTA

1 cycle: 10 min at 95 °C

40 cycles: 15 s at 92 °C and 1 min at 60 °C

[28]

 CYP6P9a

Forward (1 µM)

Reverse (1 µM)

 

TCCCGAAATACAGCCTTTCAG

ATTGGTGCCATCGCTAGAAG

1 cycle: 95 °C for 3 min

40 cycles: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min

1 cycle: 72 °C for 10 min

[18]

 CYP6P9b

Forward (1 µM)

Reverse (1 µM)

 

CCCCCACAGGTGGTAACTATCTGAA

TTATCCGTAACTCAATAGCGATG

1 cycle: 95 °C for 3 min

40 cycles: 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min

1 cycle: 72 °C for 10 min

[19]