Target gene |
Primer Sequenza 5’ – 3’ |
Amplicon size (bp) |
Concentration (µM) |
Reference |
A16S F |
GGG AGT GCC TTC GGG AAT CAG A |
356 bp |
0.24 |
Wang et al, 2003 |
A16S R |
TCA CCG CAA CAT TCT GAT TTG |
|
0.24 |
|
A-hyd F |
AGT CTG CCG CCA GTG GC |
144 bp |
0.48 |
S.Persson et al,2015 |
A-hyd R |
CRC CCA TCG CCT GTT CG |
|
0.48 |
|
astF |
ATG CAC GCA CGT ACC GCC AT |
260 bp |
0.12 |
Kingombe et al,2010 |
astR |
ATC CGG TCG TCG CTC TTG GT |
|
0.12 |
|
lipF |
ATC TTC TCC GAC TGG TTC GG |
382 bp |
0.8 |
K.SenandM.Rodgers, 2004 |
lipR |
CCG TGC CAG GAC TGG GTC TT |
|
0.8 |
|
elaF |
ACA CGG TCA AGG AGA TCA AC |
513 bp |
0.2 |
K.SenandM.Rodgers, 2004 |
elaR |
CGC TGG TGT TGG CCA GCA GG |
|
0.2 |
|
|
GAG AAG GTG ACC ACC AAG AACA |
232 bp |
0.8 |
Kingombe et al,2010 |
actF |
|
|
||
actR |
AAC TGA CAT CGG CCT TGA ACT C |
|
0.8 |
|
altF |
GCA CGG CGT GAC TTC GGT GA |
576 bp |
0.8 |
Kingombe et al,2010 |
altR |
ACC GCG GTC TTG CAG TTG GG |
|
0.8 |
|
aerF |
AAC CGA ACT CTC CAT |
301 bp |
1.2 |
Pabbs et al, 2009 |
aerR |
CGC CTT GTC CTT GTA |
|
1.2 |
|
|
GGC CGG TGG CCC GAA GAT ACGGG |
597 bp |
0.12 |
Wong et al, 1998 |
hlyAF |
|
|
|
|
hlyAR |
GGC GGC GCC GGA CGA GAC GGG |
|
0.12 |
|
Table 1: PCR Primers for Aeromonas genus-specific and virulence associated
Multiplex 1 |
|
Multiplex 2 |
|
Multiplex 3 |
|
dNTPs |
0,5 µl |
dNTPs |
0,5 µl |
dNTPs |
0,5 µl |
A16S-F |
0,5 µl |
alt-F |
2 µl |
hlyA-F |
0,3 µl |
A16S-R |
0,5 µl |
alt-R |
2 µl |
hlyA- R |
0,3 µl |
A-hyd -F |
0,5 µl |
lip-F |
2 µl |
ela-F |
0,5 µl |
A-hyd -R |
0,5 µl |
lip-R |
2 µl |
ela-R |
0,5 µl |
|
|
ast-F |
0,3 µl |
aer-F |
3 µl |
|
|
Ast-R |
0,3 µl |
aer-R |
3 µl |
|
|
|
|
act-F |
2 µl |
|
|
|
|
act-R |
2 µl |
H2O PCR |
14,2 µl |
|
9,2 µl |
|
6,2 µl |
DNA template |
2 µl |
|
2 µl |
|
2 µl |
Taq Polymerase |
0,2 µl |
|
0,2 µl |
|
0,2 µl |
MgCl2 |
2 µl |
|
2 µl |
|
2 µl |
Buffer |
2,5 µl |
|
2,5 µl |
|
2,5 µl |
Volume totale |
25 µl |
|
25 µl |
|
25 µl |
Table 2: Mix Reaction Multiplex PCR 1,2,3
Step |
Temperature |
Time (Min) |
Cycle |
Initial denaturisation |
95°C |
0.208333333 |
1 |
Denaturisation |
95°C |
0.020833333 |
30 |
Coupling |
60°C |
0.020833333 |
|
Extension |
72°C |
0.041666667 |
|
Final Extension |
72°C |
0.291666667 |
1 |
Table 3: Multiplex 1,2,3 PCR: thermic profiles
|
|
Alt |
Lip |
Ast |
Hly |
Ela |
Aer |
Act |
1C |
A. h |
+ |
+ |
- |
+ |
+ |
- |
- |
2C |
A. spp |
- |
- |
- |
- |
- |
- |
+ |
3C |
A. spp |
- |
+ |
- |
+ |
- |
- |
+ |
4C |
A. h |
+ |
+ |
+ |
+ |
+ |
- |
- |
5C |
A. h |
+ |
+ |
- |
- |
+ |
- |
+ |
6C |
A. h |
+ |
+ |
- |
+ |
+ |
- |
- |
7C |
A. spp |
- |
+ |
- |
- |
+ |
- |
- |
8C |
A. h |
+ |
+ |
- |
+ |
+ |
- |
- |
9C |
A.spp |
- |
+ |
- |
+ |
- |
- |
+ |
10C2 |
A. spp |
- |
- |
- |
- |
- |
- |
+ |
10C1 |
A. h |
+ |
+ |
+ |
- |
+ |
- |
- |
13C |
A. spp |
- |
+ |
- |
- |
+ |
- |
- |
11C |
A. h |
+ |
+ |
- |
+ |
+ |
- |
- |
12C |
A. h |
+ |
+ |
+ |
+ |
+ |
- |
- |
14C |
A. spp |
- |
+ |
- |
- |
+ |
- |
- |
17C |
A. spp |
- |
+ |
- |
+ |
- |
- |
+ |
Table 4: Detection enterotoxin gene in isolates of Aeromonas hidrophila and spp in the meat samples
Tables at a glance