Table 1.
Primers and probes used in quantitative RT-PCR for unspliced and spliced viral RNA.
| A. MLV and hybrid viruses | |||
|---|---|---|---|
| Target transcript | Primer or probe | Sequencea | Coordinates in MLV genomeb |
| Unspliced | Forward primer | 5′-AACAAGCGGGTGGAAGACATC-3′ | 2952-2972 |
| Probe | 5′-FAM-CCCACCGTGCCCAACCCTTACAACC-TAMRA-3′ | 2976-3000 | |
| Reverse primer | 5′-CAAAGGCGAAGAGAGGCTGAC-3′ | 3088-3108 | |
| Spliced | Forward primer | 5′-CTCCTCTGAGTGATTGACTACCC-3′ | 103-125 |
| Probe | 5′-VIC-TGCCGCCAGAGGTCTCCAGACTTCG-TAMRA-3′ | 5522-5546 | |
| Reverse primer | 5′-CGGTCCAGTTGTTCTTGGTAGG-3′ | 5548-5569 | |
| B. GALV viruses | |||
|---|---|---|---|
| Target transcript | Primer or probe | Sequencea | Coordinates in GALV genomec |
| Unspliced | Forward primer | 5′-TGGTATACAGACGGTAGCAGTTTC-3′ | 4102-4125 |
| Probe | 5′-FAM-CCCTGCTCTCCGTTTACCTTCCGTGA-TAMRA-3′ | 4127-4152 | |
| Reverse primer | 5′-CCGTCCGCTTGCCATCTAC-3′ | 4162-4180 | |
| Spliced | Forward primer | 5′-ACCACCGACCCACCAACG-3′ | 182-199 |
| Probe | 5′-VIC-TTCCACCGAGGCTCAAGGCTGCTG-TAMRA-3′ | 5481-5504 | |
| Reverse primer | 5′-GGTAGTCAGCAACACCAGGTATG-3′ | 5510-5532 | |
a
Abbreviations: FAM, 5-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine. VIC is a proprietary fluorescent label of Applied Biosystems.
b
Numbered in accordance with the MLV RNA genome sequence of GenBank accession no. J02255. The MLV splice donor and acceptor sites are located at nucleotides 206 and 5490, respectively.
c
Numbered in accordance with the GALV RNA genome sequence of GenBank accession no. M26927. The GALV splice donor and acceptor sites are located at nucleotides 204 and 5445, respectively.