Table 1.
Target genes, oligonucleotide primers and PCR conditions used for detection of antibiotic resistance genes in the MRSA strains isolated from hospital cockroaches (16–22)
| Target gene | Primer sequence (5′-3′) | PCR product (bp) | PCR programs | PCR volume (50 μL) |
|---|---|---|---|---|
| AacA-D | F: TAATCCAAGAGCAATAAGGGC R: GCCACACTATCATAACCACTA |
227 | 1 cycle: 94 0C ------------ 5 min. 25 cycle: 94 0C ------------ 60 s 55 0C ------------ 70 s 72 0C ------------ 60 s 1 cycle: 72 0C ------------ 10 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 |
| ermA | F: AAGCGGTAAACCCCTCTGA R: TTCGCAAATCCCTTCTCAAC |
190 | ||
| tetK | F: GTAGCGACAATAGGTAATAGT R: GTAGTGACAATAAACCTCCTA |
360 | ||
| tetM | F: AGTGGAGCGATTACAGAA R: CATATGTCCTGGCGTGTCTA |
158 | 1 cycle: 94 0C ------------ 6 min. 34 cycle: 95 0C ------------ 50 s 55 0C ------------ 70 s 72 0C ------------ 60 s 1 cycle: 72 0C ------------ 8 min |
5 μL PCR buffer 10X 2 mM Mgcl2 200 μM dNTP (Fermentas) 0.5 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 5 μL DNA template |
| vatA | F: TGGTCCCGGAACAACATTTAT R: TCCACCGACAATAGAATAGGG |
268 | ||
| msrA | F: GGCACAATAAGAGTGTTTAAAGG R: AAGTTATATCATGAATAGATTGTCCTGTT |
940 | 1 cycle: 94 0C ------------ 6 min. 34 cycle: 95 0C ------------ 60 s 50 0C ------------ 70 s 72 0C ------------ 70 s 1 cycle: 72 0C ------------ 8 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 |
| vatB | F: GCTGCGAATTCAGTTGTTACA R: CTGACCAATCCCACCATTTTA |
136 | 1 cycle: 94 0C ------------ 6 min. 35 cycle: 95 0C ------------ 50 s 55 0C ------------ 70 s 72 0C ------------ 80 s 1 cycle: 72 0C ------------ 10 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 |
| linA | F: GGTGGCTGGGGGGTAGATGTATTAACTGG R: GCTTCTTTTGAAATACATGGTATTTTTCGA |
323 | 1 cycle: 94 0C ------------ 6 min. 30 cycle: 95 0C ------------ 60 s 57 0C ------------ 60 s 72 0C ------------ 60 s 1 cycle: 72 0C ------------ 10 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 |
| blaZ | F: ACTTCAACACCTGCTGCTTTC R: TGACCACTTTTATCA CAACC |
490 | 1 cycle: 94 0C ------------ 5 min. 30 cycle: 94 0C ------------ 20 s 60 0C ------------ 30 s 72 0C ------------ 90 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 |
| cat1 | F: AGTTGCTCAATGTACCTATAACC R: TTGTAATTCATTAAGCATTCTGCC |
547 | 1 cycle: 94 0C ------------ 8 min. 32 cycle: 95 0C ------------ 60 s 55 0C ------------ 70 s 72 0C ------------ 2 min 1 cycle: 72 0C ------------ 8 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 |
| gyrA | F: AATGAACAAGGTATGACACC R: TACGCGCTTCAGTATAACGC |
223 | 1 cycle: 94 0C ------------ 10 min. 25 cycle: 94 0C ------------ 20 s 52 0C ------------ 20 s 72 0C ------------ 50 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 |
| grlA | F: ACTTGAAGATGTTTTAGGTGAT R: TTAGG AAATCTTGATGGCAA |
459 | ||
| dfrA |
F: CTCACGATAAACAAAGAGTCA
R: CAATCATTGCTTCGTATAACG |
201 | 1 cycle: 94 0C ------------ 2 min. 30 cycle: 94 0C ------------ 60 s 50 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 |
| rpoB | F: ACCGTCGTTTACGTTCTGTA R: TCAGTGATAGCATGTGTATC |
460 | 1 cycle: 94 0C ------------ 5 min. 32 cycle: 94 0C ------------ 60 s 56 0C ------------ 45 s 72 0C ------------ 60 s 1 cycle: 72 0C ------------ 10 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 |