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 |