Table 1.
Primers used to detect and to sequence arbovirus from mosquito pools in Kenya
| Target | Primer name | Nucleotide sequence (5′ to 3′) | Polarity | Product (bp) | Cycle condition | Reference |
|---|---|---|---|---|---|---|
| Universal primers for flavivirus | MAMD | AACATGATGGGRAARAGRGARAA | Forward | 252 | 94°C, 2 min, 1 cycle; 94°C, 1 min, 53°C, 1 min, 72°C, 1 min, 35 cycles; 72°C, 5 min, 1 cycle | Scaramozzino et al. (2001) [48] |
| cFD2 | GTGTCCCAGCCGGCGGTGTCATCAGC | Reverse | ||||
| Universal primers for flavivirus | FLAVI-1 | AATGTACGCTGATGACACAGCTGGCTGGGACAC | Forward | 854–863 | 94°C, 5 min, 1 cycle; 94°C, 1 min, 58°C, 1 min, 72°C, 90 s, 45 cycles; 72°C, 10 min, 1 cycle | Ayers et al.(2006) [49] |
| FLAVI-2 | TCCAGACCTTCAGCATGTCTTCTGTTGTCATCCA | Reverse | ||||
| Universal primers for flavivirus (mainly YF) | YF-1 | GGTCTCCTCTAACCTCTAG | Forward | 675 | 94°C, 2 min, 1 cycle; 94°C, 30 s, 53°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 5 min, 1 cycle | Tanaka et al. (1993) [50] |
| YF-3 | GAGTGGATGACCACGGAAGACATGC | Reverse | ||||
| Universal primers for alpha viruses (mainly chikungunya and o’nyong’nyong viruses) | nsP1-S | TAGAGCAGGAAATTGATCC | Forward | 354 | 94°C, 2 min, 1 cycle; 94°C, 30 s, 53°C, 30 s, 72°C, 45 s, 35 cycles; 72°C, 5 min, 1 cycle | Hasebe et al. (2002) [51] |
| nsP1-C | CTTTAATCGCCTGGTGGTA | Reverse | ||||
| Universal primers for alpha viruses (mainly chikungunya and o’nyong’nyong viruses) | E1-S | TACCCATTCATGTGGGG | Forward | 294 | 94°C, 2 min, 1 cycle; 94°C, 30 s, 53°C, 30 s, 72°C, 45 s, 35 cycles; 72°C, 5 min, 1 cycle | Hasebe et al. (2002) [51] |
| E1-C | GCCTTTGTACACCACGAT | Reverse | ||||
| Rift Valley virus | RVF009 | CCAAATGACTACCAGTCAGC | Forward | 400–500 | 94°C, 2 min, 1 cycle; 94°C, 30 s, 50°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 5 min, 1 cycle | Jupp et al. (2000) [52] (modified) |
| RVF007 | GACAAATGAGTCTGGTAGCA | Reverse | ||||
| Mosquito RNA marker | Act-2F | ATGGTCGGYATGGGNCAGAAGGACTC | Forward | 683 | 94°C, 2 min, 1 cycle; 94°C, 30 s, 54°C, 30 s, 72°C, 45 s, 35 cycles; 72°C, 5 min, 1 cycle | Staley et al. (2010) [53] |
| Act-8R | GATTCCATACCCAGGAAGGADGG | Reverse | ||||
| Culex quinquefaciatus | ACEpip | GGAAACAACGACGTATGTACT | Forward | 610 | 94°C, 5 min, 1 cycle; 94°C, 30 s, 54°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 5 min, 1 cycle | Kasai et al. (2008) [39] |
| ACEquin | CCTTCTTGAATGGCTGTGGCA | Forward | 274 | |||
| B1246s | TGGAGCCTCCTCTTCACGG | Reverse | ||||
| Aedes aegypti | 18SFHIN | GTAAGCTTCCTTTGTACACACCGCCCGT | Forward | 550 | 97°C, 4 min, 1 cycle; 96°C, 30 s, 48°C, 30 s, 72°C, 2 min, 30 cycles; 72°C, 4 min, 1 cycle | Higa et al. (2010) [54] |
| aeg.r1 | TAACGGACACCGTTCTAGGCCCT | Reverse | ||||
| Anopheles funestus, Anopheles rivulorum | UV | TGTGAACTGCAGGACACAT | Forward | 94°C, 2 min, 1 cycle; 94°C, 30 s, 45°C, 30 s, 72°C, 40 s, 30 cycles; 72°C, 5 min, 1 cycle | Koekemoer et al. (2002) [55] | |
| FUN | GCATCGATGGGTTAATCATG | Reverse | 505 | |||
| RIV | CAAGCCGTTCGACCCTGATT | Reverse | 411 |
Note: Each 25 μl reaction mixture contained. (Accupower TM PCR PreMix kit with 2 μl template, 15.2 μl sterile water, and 1.4 μl of 100 pmol/μl each of primers)