Table 3.
Common methods used for the species delineation of the Culex pipiens complex.
| Method | Target | Primer Sequences | Identification Output | References |
|---|---|---|---|---|
| Gel electrophoresis | Electrophoreticpolymorphisms in various genetic targets, often enzymes | n/a | Provides estimates of genetic differentiation between populations in target genes | [40,92] |
| Multiplex end-point PCR | ace-2 |
FOR ACEtorr 5′-TGCCTGTGCTACCAGTGATGTT-3′ FOR ACEpip (5′-GGAAACAACGACGTATGTACT-3′) REV B1246s (5′-TGGAGCCTCCTCTTCACGG-3′) |
Cx. pipiens complex: Cx. pipiens, Cx. quinquefasciatus, Cx. p. pallens, Cx. australicus, Cx. torrentium, Cx. pervigilans, Cx pipiens/Cx. quinquefasciatus hybrids | [98] |
| CQ11 |
FOR CQ11F (5′-GATCCTAGCAAGCGAGAAC-3′) REV pipCQ11R (5′-CATGTTGAGCTTCGGTGAA-3′ REV molCQ11R (5′-CCCTCCAGTAAGGTATCAAC-3′ |
Cx. pipiens form pipiens and form molestus | [97] | |
| PCR-DNA sequencing | COI |
FOR LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) REV HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) |
Enables universal identification to species level with comparison to sequence database | [104] |
| COI |
FOR TY-J-1460 (5′-TACAATCTATCGCCTAAACTTCAGCC-3′) REV UEA10 (5′ -TCCAATGCACTAATCTGCCATATTA-3′) |
[105,106,107] | ||
| PCR-RFLP | COI |
FOR COIF (5′-TTGAGCTGGA-ATAGTTGGAACTT-3′) REV COIR (5′-CCTCCAATTGGATCAAAGAATGA-3′) |
Cx. pipiens form pipiens and form molestus, Cx. torrentium | [91] |
| ace-2 |
FOR F1457 (5′-GAGGAGATGTGGAATC CCAA-3′) REV B1246 (5′-TGGAGCCTCCTCTTCACGG C-3′) |
Cx. pipiens, Cx. quinquefasciatus and their hybrids | [108] | |
| Wolbachia pipientis markers, ank2, pk1 |
ank2 FOR (5′-CTTCTTCTGTGAGTGTACGT-3′) ank2 REV (5′-TCCATATCGATCTACTGCGT-3′) pk1 FOR (5′-CCACTACATTGCGCTATAGA-3′) pk1 REV (5′-ACAGTAGAACTACACTCCTCCA-3′) |
Five groups of W. pipientis: wPip-I to wPip-V | [102,109] | |
| Real-time PCR | CQ11 |
FOR
Culex pipiens (5′-GCGGCCAAATATTGAGACTT-3′) REV Culex pipiens (5′-CGTCCTCAAACATCCA-GACA-3′) Probes Cx. pipiens all (59-Cy55-GGAACATGTTGAGCTTCGGK-BBQ-1-39 Cx. pipiens pipiens form pipiens (5′-JOE-GCTTCGGTGAAGGT TTGTGT-BHQ1-3′) Cx. pipiens pipiens form molestus (5′-Rox-TGAACCCTCC AGTAAGGTATCAACTAC-BHQ2-3′) |
Collectively enables separation Cx. pipiens and its ecoforms and hybrids, plus Cx. torrentium | [110] |
| ace-2 |
FOR
Cx. torrentium (5′ -GACACAGGACGACAGAAA-3′) REV Cx. torrentium (5′-GCCTACGCAACTACTAAA-3′) Probe Cx. torrentium (5′-FAM-CGAT-GATGCCTGTGCTACCA-3BHQ1-3′) |
|||
| CQ11 |
FOR Cx_pip_F (5′-GCGGCCAAATATTGAGACTTTC-3′) REV Cx_pip_R (5′- ACTCGTCCTCAAACATCCAGACATA-3′) Probes Cpp_mol_P (5′-FAM-TGAACCCTCCAGTAAGGTA-MGB-3′) Cpp_pip_P1 (5′-VIC-CACA CAAAYCTTCACCGAA-MGB-3′) Cpp_pip_P2 (5′-VIC-ACACAAACCTTCATCGAA-MGB-3′) |
Collectively enables separation Cx. pipiens and its ecoforms and hybrids, plus Cx. torrentium | [100] (modified from Rudolf et al. [110]) | |
| ace-2 |
FOR Cx_tor_F (5′-CTTATTAGTATGACACAGGACGACAG AAA-3′) Cx_tor_R (5′-GCATAAACGCCTACGCAACTACTAA-3′) Probe Cx_tor_P (5′-FAM-ATGATGCCTGTG CTACCA-MGB-3′) |