Table 3.
Candidate primer pairs for species-specific identification of the target DNAs
| Forward primer name | Sequence 5′ to 3′ | Tm | Reverse primer name | Sequence 5′ to 3′ | Tm | Amplicon length (bp) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Hand |
F |
1 |
2 |
ACATCGTCGCCCCAAACC |
67.0 |
Hand |
R |
1 |
2 |
CCATTATCCGCCCCATCCTC |
66.5 |
65 |
| Hand |
F |
1 |
3 |
AAATGTGATACTTGGTGTGAATTGC |
64.7 |
Hand |
R |
1 |
3 |
CGAGGTGTTGGGTTTGGG |
64.8 |
135 |
|
Hand |
F |
1 |
4 |
CACATCGTCGCCCCAAAC |
65.6 |
Hand |
R |
1 |
4 |
ACCATTATCCGCCCCATCC |
65.9 |
67 |
| Hand |
F |
1 |
4 |
CACATCGTCGCCCCAAAC |
65.6 |
Hand |
R |
1 |
5 |
TTATCCGCCCCATCCTCTTC |
65.4 |
63 |
| Hand |
F |
1 |
5 |
CGGCTGTCCTCCTGTTCATAAC |
67.1 |
Hand |
R |
1 |
6 |
TCACACCAAGTATCACATTTCGCTAC |
67.3 |
98 |
| Hand |
F |
2 |
1 |
CGAAATGTGATACTTGGTGTGAATTG |
65.1 |
Hand |
R |
1 |
3 |
CGAGGTGTTGGGTTTGGG |
64.8 |
137 |
| Hasc |
F |
1 |
2 |
TTCCTTCGGTTCATAACTAAAAC |
60.9 |
Hasc |
R |
1 |
2 |
ACCCAATGAACTCGAAAGAG |
61.7 |
225 |
|
Hasc |
F |
1 |
4 |
GTGGCTTTCCTTCGGTTC |
62.5 |
Hasc |
R |
1 |
2 |
ACCCAATGAACTCGAAAGAG |
61.7 |
231 |
| Hasc |
F |
1 |
3 |
TTTCCTTCGGTTCATAACTAAAAC |
61.5 |
Hasc |
R |
1 |
1 |
GAACTCGAAAGAGGCATTG |
60.4 |
219 |
| Hasc |
F |
1 |
5 |
TCCTTCGGTTCATAACTAAAAC |
60.2 |
Hasc |
R |
1 |
1 |
GAACTCGAAAGAGGCATTG |
60.4 |
217 |
| Hath |
F |
1 |
1 |
CCCCGAAATTCCGATATCTC |
61.8 |
Hath |
R |
1 |
1 |
CTTACAACCACCGCTAGTC |
61.7 |
137 |
| Hath |
F |
1 |
1 |
CCCCGAAATTCCGATATCTC |
61.8 |
Hath |
R |
1 |
3 |
CAACCACCGCTAGTCGTG |
64.6 |
133 |
| Hath |
F |
1 |
1 |
CCCCGAAATTCCGATATCTC |
61.8 |
Hath |
R |
1 |
4 |
CCGCTAGTCGTGGCTTTG |
64.9 |
127 |
|
Hath |
F |
1 |
3 |
GTGTCACACATCGTTGCC |
63.2 |
Hath |
R |
1 |
3 |
CAACCACCGCTAGTCGTG |
64.6 |
151 |
| Hper |
F |
1 |
1 |
TGTAACGCTCCCGGCTGTG |
69.0 |
Hper |
R |
1 |
1 |
CCGATTGTCTCTTGCGAGATATC |
65.0 |
273 |
|
Hper |
F |
4 |
1 |
GGGGCTTCCTTCTGTTCATAAC |
65.1 |
Hper |
R |
4 |
1 |
TCTTGCGAGATATCGGGATTTTG |
64.9 |
222 |
| Hper |
F |
1 |
2 |
ATAAGAAGTGTAACGCTCCCGGCTGTG |
72.5 |
Hper |
R |
1 |
1 |
CCGATTGTCTCTTGCGAGATATC |
65.0 |
281 |
| Hper |
F |
1 |
3 |
GAAGTGTAACGCTCCCGGCTGTG |
71.9 |
Hper |
R |
1 |
1 |
CCGATTGTCTCTTGCGAGATATC |
65.0 |
277 |
| Hper | F | 1 | 4 | AGTGTAACGCTCCCGGCTGTG | 71.2 | Hper | R | 1 | 1 | CCGATTGTCTCTTGCGAGATATC | 65.0 | 275 |
The selected pairs are indicated in bold. Candidate selection was based on specificity, and further analysis of theoretical interactions in the multiplex PCR and discrimination of amplicons by capillary electrophoresis were used to make the final selection.