TABLE 2.
Primers selected for the quantification of the negative strand of the BTV-4 segments
| Segment | Direction | Sequence | 5′ position | Amplicon size (bp) | Efficiency (%) | Mismatcha | Specificity threshold (CT) |
Final concn (μM) | |
|---|---|---|---|---|---|---|---|---|---|
| Biting midges | Sheep blood | ||||||||
| 1 | Forward | AACATGGCTATTGGGACC | 2213 | 125 | 91.9 | >5 | >39.5 | >38.5 | 0.5 |
| Reverse | TCTGTAGTGTGTAGCTTTGTGTAA | 2337 | |||||||
| 2 | Forward | TCTAGCTTCTCTATGTTTAGGGC | 2541 | 70 | 93.0 | >5 | >39.8 | >40 | 0.4 |
| Reverse | TTTCTTTGGATGCGCGAC | 2610 | |||||||
| 3 | Forward | ATTCCGAGCGGCTTTAAGA | 2287 | 75 | 94.5 | >5 | >40 | >40 | 0.5 |
| Reverse | CGTACAGTGCGTAATACACC | 2361 | |||||||
| 4 | Forward | TATGCGTAAGGGATTTGGTG | 343 | 88 | 94.2 | >5 | >35 | >35.3 | 0.5 |
| Reverse | GTTCAACGTCTCCGCTTC | 430 | |||||||
| 5 | Forward | TGATCGCGGCAACTGAC | 189 | 100 | 92.4 | 5 | >40 | >40 | 0.8 |
| Reverse | CAGACTGTTTCCCGATCATAC | 279 | |||||||
| 6 | Forward | GTGAATCTTATGGAGAGTCGG | 212 | 70 | 91.8 | >5 | >37.4 | >40 | 0.5 |
| Reverse | GGTAATTCTTCACCTGTACCCAA | 281 | |||||||
| 7 | Forward | ATGGCTACGATTGGTGTACTA | 278 | 74 | 95.2 | >5 | >38.4 | >38.7 | 0.4 |
| Reverse | ACGCGAGCAATCTCATTC | 351 | |||||||
| 8 | Forward | AAGAGATGATTCCGGGAACTA | 608 | 78 | 91.8 | >5 | >34.2 | >34.8 | 0.4 |
| Reverse | CCAGCTTCCACCTCCTTA | 686 | |||||||
| 9 | Forward | GGAACCCAAAGAGGAAGACA | 133 | 110 | 95.1 | >5 | >37.5 | >36.4 | 0.6 |
| Reverse | CCACATCTGCATCTTTAGC | 242 | |||||||
| 10 | Forward | AAGGCTGCATTCGCATCGTA | 242 | 115 | 93.5 | >5 | >40 | >40 | 0.5 |
| Reverse | AGCCTCCTAGGTCGCTTTTC | 356 | |||||||
a
Number of positions differing from sequence of the segment positive strand.