Table 1.
Molecular assays used in the study: primers and PCR conditions
| Species | Assay | Primers (5′ → 3′) | PCR condition | Reference | |
|---|---|---|---|---|---|
| Insecticide resistance assays | Culex pipiens | VGSC (1014F)/kdr - allele specific PCR |
Cgd1: GTGGAACTTCACCGACTTC Cgd2: GCAAGGCTAAGAAAAGGTTAAG Cgd3: CCACCGTAGTGATAGGAAATTTA Cgd4: CCACCGTAGTGATAGGAAATTTT |
95 °C 5 min, 40 cycles × (94 °C 30 s, 48 °C 30 s, 72 °C 1 min), 72 °C 10 min | [26] |
| Cx. pipiens | CHS (1043 L) - allele specific PCR |
External_F: GCAGTCCTTCGGCGATCTT External_R: GAACAGTCCGGCGATGGATA ATC_ R: AACAGCAAGTACATAGACGGGAT CTC_ F: GGCTTGATCTACCTGCTGTCTC |
95 °C 5 min, 28 cycles × (95 °C 30 s, 68 °C 30 s, 72 °C 1 min), 72 °C 5 min | [17] | |
| Cx. pipiens | CHS (1043 M) - PCR and RFLP |
Diagnostic I1043M_R: CCCAGGAGACGACGTTCAG Diagnostic I1043M_F: GCCTGTCTCCATCCGCAAG |
95 °C 5 min, 30 cycles × (95 °C 30 s, 60 °C 30 s, 72 °C 30 s),72 °C 5 min | [17] | |
| Aedes albopictus | VGSC (1016G)/kdr - PCR and sequencing |
F1016: TTCACCGACTTCATGCACTC R1016: CGCAATCTGGCTTGTTAACTT |
95° 3 min, 40 cycles × (94 °C 30 s, 55 °C 30 s, 72 °C 1 min), 72 °C 5 min | – | |
| Ae. albopictus | VGSC (1532 T,1534C)/kdr - PCR and sequencing |
For: GAGAACTCGCCGATGAACTT Rev.: GACGACGAAATCGAACAGGT Rev. for sequencing: AGCTTTCAGCGGCTTCTTC |
95 °C 3 min, 40 cycles × (94 °C 30 s, 57 °C 30 s, 72 °C 1 min), 72 °C 5 min | – | |
| Ae. albopictus | CHS (1043 L/M) - PCR and sequencing |
kkv F3: TCGGAAGTCCTTCGGCTTAT TC kkv R3: TGGATACTTCAATGGAACCTTCC |
95 °C 5 min, 40 cycles × (94 °C 30 s, 55 °C 30 s, 72 °C 1 min), 72 °C 10 min | – | |
| Anopheles (An. sacharovi, An. hyrcanus, An. maculipennis, An. claviger) | VGSC (1014F/C/S) / kdr - PCR and sequencing |
KDRF: GGMGAATGGATYGAATCMATGTGGGA KDR R2: GATGAACCRAAATTKGACAAAAGCAA |
94 °C 5 min, 35 cycles × (94 °C 1 min, 50 °C 2 min, 72 °C 2 min), 72 °C 2 min | – | |
| Sand flies (Phlebotomus perfiliewi, P. tobbi, P. simici, P. neglectus) | VGSC (1011, 1014, 1016, and 1020) / PCR and sequencing |
Vssc8F: AATGTGGGATTGCATGCTGG Vssc1bR: CGTATCATTGTCTGCAGTTGGT |
95 °C 5 min, 36 cycles × (94 °C 45 s, 51 °C 50 s, 72 °C 50 s),72 °C 7 min | [27] | |
| Species id assays | Cx. pipiens complex | AChE2- PCR diagnostic assay |
ACEquin: CCTTCTTGAATGGCTGTGGCA ACEpall: ATGGTGGAGACGCATGACG ACEpip: GGAAACAACGACGTATGTACT ACEtorr: TGCCTGTGCTACCAGTGATGTT B1246s: TGGAGCCTCCTCTTCACGG |
94 °C 5 min, 35 cycles × (94 °C 30 s, 55 °C 30 s, 72 °C 1 min), 72 °C 5 min | [25] |
| Cx. pipiens biotype | CQ11 - allele specific PCR |
pipRev: CATGTTGAGCTTCGGTGAA molRev: CCCTCCAGTAAGGTATCAAC biotype-forward: GATCCTAGCAAGCGAGAAC |
95 °C 5 min, 40 cycles × (94 °C 30 s, 54 °C 30 s, 72 °C 40 s), 72 °C 5 min | [28] | |
| Anopheles | ITS2 - PCR and sequencing |
5.8 s primer: TGTGAACTGCAGGACACATG 28 s primer: ATGCTTAAATTTAGGGGGTA |
94 °C 2 min, 40 cycles × (94 °C 30 s, 53 °C 30 s, 72 °C 50 s), 72 °C 10 min | [29] | |
| Anopheles | COI - PCR and sequencing |
Cl -N-2191: CCCGGTAAAATTAAAATATAAACTTC Cl-J-1718: GCAGGATTTGGAAATTGATTAGTTCC |
94 °C 2 min, 40 cycles × (94 °C 30 s, 50 °C 30 s, 72 °C 50 s), 72 °C 10 min | [29] | |
| Ae. albopictus | ITS2 - PCR and sequencing |
5.8 s primer: TGTGAACTGCAGGACACATG 28 s primer: ATGCTTAAATTTAGGGGGTA |
94 °C 10 min, 40 cycles × (94 °C 1 min, 50 °C 1 min, 72 °C 1 min), 72 °C 10 min | [30] | |
| Sand flies | Direct multiplex PCR |
Forward: TGCGGTTAAAACGTTCGTAG perf: TAAACCCACAAATCAGAAT sim: CTTTCATTAAATTTAGCCTGCC papat: TAACATAAGGGGCGTATTAATATGCTTT tob: GCGTRTTAACAATAGAGTCCATTAAA |
95 °C 5 min, 32 cycles × (94 °C 40 s, 54 °C 30 s, 72 °C 1 min), 72 °C 10 min | [24] | |
| Pathogen detection assays | Plasmodium (P. vivax, P. falciparum) | PCR 1 (Nested PCR) |
rPLU5: CCTGTTGTTGCCTTAAACTTC rPLU6: TTAAAATTGTTGCAGTTAAAACG |
95 °C 5 min, 24 cycles × (94 °C 1 min, 57 °C 2 min,72 °C 2 min), 72 °C 5 min | [31] |
| PCR 2 (Nested PCR) |
VIV-1: CGCTTCTAGCTTAATCCACATAACTGATAC VIV-2: ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA FAL-1: TTAAACTGGTTTGGAAAACCAAATATATT FAL-2: ACACAATGAACTCAATCATGACTACCCGTC |
95 °C 5 min, 30 cycles × (94 °C 1 min, 57 °C 2 min, 72 °C 2 min), 72 °C 5 min | [31] | ||
| Flavivirus detection | Conventional one-step reverse transcriptase-PCR (RT-PCR) |
Forward: TACAACATGATGGGAAAGAGAGAGAA Reverse: GTGTCCCAGCCGGCGGTGTCATCAGC |
50 °C 30 min, 95 °C 3 min, 40 cycles × (95 °C 15 s, 55 °C 20 s, 72 °C 60 s), 72 °C 10 min | [32] | |
| West Nile virus detection | Multiplex Real Time one step RT-PCR TaqMan assay |
Forward: GTGATCCATGTAAGCCCTCAGAA Reverse: GTCTGACATTGGGCTTTGAAGTTA FAM probe (MGB): AGGACCCCACATGTT HEX probe (MGB): AGGACCCCACGTGCT |
50 °C 15 min, 95 °C 3 min, 45 cycles × (95 °C 3 s and 60 °C 30 s) | [33, 34] |
VGSC Voltage gated sodium channel; CHS Chitin synthase; kdr Knock down resistance; AChE2 Acetylcholinesterase-2; CQ11 Microsatellite locus; ITS2 Internal transcribed spacer two; COI Cytochrome oxidase subunit 1; [REF] References