Table 2.
PCR conditions for the detection and genotyping of avian respiratory viruses in samples from Pakistan
| Virus | Primers | Sequences (5–3) | Target | Amplicon size (bp) | PCR conditions | Reference | |
|---|---|---|---|---|---|---|---|
| Detection PCR | AAVV-1 | FIP-1 | 5′ TACTTTGCTCACCCCCCTT 3’ | Fusion gene (F) | 280 | 94 C for 2 min; 40 cycles of 94 C for 30 s, 58 C for 30 s, 72 C for 1 min; final extension at 72 C for 5 min | [48] |
| FIP-2 | 5’ CATCTTCCCAACTGCCACT 3’ | ||||||
| IBV | N791 | 5’ GTGATGACAAGATGAATGAGGA 3’ | Nucleo-protein gene (N) | 380 | 94 C for 2 min; 40 cycles of 94 C for 30 s, 54 C for 30 s, 72 C for 1 min; final extension at 72 C for 5 min | [49] | |
| N1129 | 5’ CAGCTGAGGTCAATGCTTTATC 3’ | ||||||
| ILTV |
gEU gEL |
5’ GCTGGGTTCTGGGCTACACAAC 3′ 5′ TGCGCGTGACTCGGAGAG 3’ |
Glyco-protein E gene (gE) | 626 | 94 C for 2 min; 40 cycles of 94 C for 30 s, 61 C for 30 s, 72 C for 1 min; final extension at 72 C for 5 min | [50] | |
| aMPV | G1a | 5′ GGGACAAGTATCYMKAT 3’ | Attachment glycoprotein gene (G) | 441 | 94 C for 2 min; 40 cycles of 94 C for 30 s, 50 C for 30 s, 72 C for 1 min; final extension at 72 C for 5 min | [51] | |
| G6a | 5′ CTGACAAATTGGTCCTGATT 3’ | ||||||
| AIV | M52C | 5′ CTTCTAACCGAGGTCGAAAG 3’ | Matrix gene (M) | 280 | 95 C for 30 s; 40 cycles of 95 C for 30 s, 55 C for 30 s, 72 C for 30s; final extension at 72 C for 1 min | [52] | |
| M253R | 5’AGGGCATTTTGGACAAAKCGTCTA 3’ | ||||||
| Genotyping PCR | AIV | HA-1134F | 5′ GGAATGATHGAYGGNTGGTATG 3′ | hemma-gglutinin gene (HA) | 600 | 95 C for 30 s; 40 cycles of 95 C for 30 s, 55 C for 30 s, 72 C for 30s; final extension at 72 C for 1 min | [53] |
| NS-890 R | 5′ ATATCGTCTCGTATTAGTAGAAAC AAGG 3′ | ||||||
| IBV | S15 | 5′ TGAAAACTGAACAAAAGACA 3’ | Spike gene (S) | 700 | 95 °C for 2 min; 40 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 30 s; final extension of 72 °C for 12 min | [54] | |
| CK2 | 5′ CTCGAATTCCNGTRTTRTAYTGRCA 3′ | ||||||
bp base pairs
athis primer pair does not detect serotype C viruses