Prevalence of β-lactam (blaTEM) and Metronidazole (nim) Resistance Genes in the Oral Cavity of Greek Subjects

Table 1: Polymerase chain reaction (PCR) conditions for 16S, bla_TEM and nim.

PCR conditions	16S (625 bp)	bla_TEM (618 bp)	nim (458 bp)
Initial denaturation	95°C for 5 min	94°C for 5 min	94°C for 10 min
Denaturation	94°C for 1 min	94°C for 1 min	94°C for 30 s
Annealing	55°C for 1 min	62°C for 1 min	62°C for 1 min
Extension	72°C for 1 min	72°C for 1 min	72°C for 1 min
Step 2 to 4	34 cycles	35 cycles	32 cycles
Final extension	72°C for 5 min	72°C for 10 min	72°C for 10 min
DNA template	5 µl	5 µl	5 µl
dNTPs mix, 10 mM	1 µl (200 µM)	2 µl (400 µM)	1 µl (200 µM)
Reaction Buffer 5x	10 µl	10 µl	10 µl
MgCl₂ Solution, 25 mM	3 µl (1.5 mM)	4 µl (2 mM)	3 µl (1.5 mM)
Upstream Primer, 10 µM	2.5 µl (0.5 µM)	5 µl (1 µM)	5 µl (1 µM)
Downstream Primer, 10 µM	2.5 µl (0.5 µM)	5 µl (1 µM)	5 µl (1 µM)
Taq polymerase, 5 U/µl	0.25 µl (1.25 U)	0.25 µl (1.25 U)	0.25 µl (1.25 U)
Water for injection	25.75 µl	18.75 µl	20.75 µl
Values in parenthesis are final concentration

16S primers 5'-CAG GAT TAG ATA CCC TGG TAG TCC ACG C-3' and 5'- GAC GGG CGG TGT GTA CAA GGC CCG GGA ACG-3' [¹²] bla_TEM primers 5'-AGA TCA GTT GGG TGC ACG AG -3' and 5'-CAG TGC TGC AAT GAT ACC GC -3' [¹⁵] nim primers 5'-ATG TTC AGA GAA ATG CGG CGT AAG CG-3' and 5'-GCT TCC TTG CCT GTC ATG TGC TC-3' [¹⁴]