Table 2 List of primers and PCR conditions used for amplification of virulence factors in the T. pyogenes bacteria isolated from samples of mastitis and metritis [19, 20]
Target gene | Primer sequence (5′-3′) | PCR product (bp) | PCR programs | PCR volume (50 μL) |
|---|---|---|---|---|
nanH | F: CGCTAGTGCTGTAGCGTTGTTAAGT R: CCGAGGAGTTTTGACTGACTTTGT | 781 | 1 cycle: 94 0C ------------ 3 min. 35 cycle: 94 0C ------------ 60 s 60 0C ------------ 60 s 72 0C ------------ 3 min 1 cycle: 72 0C ------------ 7 min | 5 μL PCR buffer 10X 1.5 mM Mgcl2 200 μM dNTP (Fermentas) 0.5 μM of each primers F & R 1.25 U Taq DNA polymerase (Fermentas) 2.5 μL DNA template |
nanP | F: TTGAGCGTACGCAGCTCTTC R: CCACGAAATCGGCCTTATTG | 150 | ||
cbpA | F: GCAGGGTTGGTGAAAGAGTTTACT R: GCTTGATATAACCTTCAGAATTTGCA | 124 | ||
fimC | F: TGTCGAAGGTGACGTTCTTCG R: CAAGGTCACCGAGACTGCTGG | 843 | ||
plo | F: GGCCCGAATGTCACCGC R: AACTCCGCCTCTAGCGC | 270 | 1 cycle: 94 0C ------------ 2 min 35 cycle: 94 0C ------------ 60 s 55 0C ------------ 60 s 72 0C ------------ 60 s 1 cycle: 72 0C ------------ 5 min | 5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 μL DNA template |
fimA | F: CACTACGCTCACCATTCACAAG R: GCTGTAATCCGCTTTGTCTGTG | 605 | 1 cycle: 94 0C ------------ 3 min. 35 cycle: 94 0C ------------ 60 s 57 0C ------------ 60 s 72 0C ------------ 3 min 1 cycle: 72 0C ------------ 7 min | 5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 μL DNA template |
fimG | F: ACG CTT CAG AAG GTC ACC AGG R: ATC TTG ATC TGC CCC CAT GCG | 929 | ||
fimE | F: GCCCAGGACCGAGAGCGAGGGC R: GCCTTCACAAATAACAGCAACC | 775 | 1 cycle: 94 0C ------------ 3 min. 35 cycle: 94 0C ------------ 60 s 55 0C ------------ 60 s 72 0C ------------ 3 min 1 cycle: 72 0C ------------ 7 min | 5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 μL DNA template |