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Table 2 Common molecular approaches used in Pseudomonas fluorescens detection

From: Pseudomonas fluorescens: a potential food spoiler and challenges and advances in its detection

Strains used

Target site

Primers

Probe

Amplification conditions

References

Laboratory isolates

16S rRNA

DNA 16S–23S intergenic spacer (ITS1)

16S rRNA gene partial region

16SF 5′ AGAGTTTGATCCTGGCTCAG-3′

16SR 5′-CTACGGCTACCTTGTTACGA-3′)

(16F945 5′-GGGCCCGCACAAGCGGTGG-3′; 23R458 5′-CTTTCCCTCACGGTAC-3′)

(16SPSEfluF 5′-TGCATTCAAAACTGACTG-3′; 16SPSER 5′AATCACACCGTGGTAACCG-3′)

NA

2 min at 94 °C; 5 cycles consisting of 94 °C for 45 s, 55 °C for 1 min, 72 °C for 2 min; 35 cycles consisting of 92 °C for 45 s, 60 °C for 45 s, 72 °C for 2 min; final extension of 72 °C for 2 min and final cooling at 4 °C

5 min at 94 °C; 30 cycles consisting of 94 °C for 1 min, 72 °C for 2 min; final extension of 72 °C for 2 min

Franzetti and Scarpellini (2007)

Laboratory isolates

16S rDNA

forward 5′-CTACGGGAGGCAGCAGTGG-3′ and reverse 5′-TCGGTAACGTCAAAACAGCAAAGT-3′

NA

NS

Márta (2012)

Laboratory isolates

16S rDNA

metalloprotease gene (aprX)

F:5′-TGCATTCAAAACTGACTG-3

R:5′-AATCACACCGTGGTAACCG-3′

SM2F (5′-AAA-TCG-ATA-GCT-TCA-GCC-AT-3′),

SM3R (5′-TTG-AGG-TTG-ATC-TTC-TGG-TT-3′)

NA

2 min at 94 °C for 1 cycles, 35 cycles of 45 s at 94 °C, 45 s at 59 °C, 2 min at 68 °C with 1 final extension of 5 min at 72 °C and cooling at 4 °C

Al-Rodhan and Nasear (2016)

Laboratory isolates

16S DNA

16S rRNA gene partial region

alkaline protease gene AprX

PA-GS-F (GACGGGTGAGTAATGCCTA) and PA-GS-R (CACTGGTGTTCCTTCCTATA)

16SPS EfluF (TG-CATTCAAAACTGACTG) and 16SPSER (AATCA-CACCGTGGTAACCG)

FP apr I (TAYGGBTTCAAYTCCAAYAC) and RF apr II (VGCGATSGAMACRTTRCC)

NA

NS

Hammad (2015)

Referred strains

alkaline protease gene aprX

F/5′-ACCGAGAACACCAGCTTGTC-3′

R/5′-CTCACGGTCAATGGCAAAC-3′

5′-CTCACGGTCAATGGCAAACTTTTTTTTTTTTTTTTTTTTT-3′

Denaturation at 94 °C for 5 min followed by 35 cycles of amplification at 94 °C for 20 s, annealing at 58 °C for 20 s, extension at 72 °C for 20 s final extension at 72 °C for 7 min

Chiang et al. (2012)

Laboratory isolates

partial sequence rpoB gene

aprX

PSF (5′-AGTTCA-TGG-ACC-AGA-ACA-ACC-3′) as forward/rpoB-PTR

(5′-CCT-TGA-CGG-TGA-ACT-CGT-TTC-3′) as reverse

SM2F (5′-AAA-TCG-ATA-GCT-TCA-GCC-AT-

3′)/SM3R (5′-TTG-AGG-TTG-ATC-TTC-TGG-TT-3′

NA

Denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and elongation at 72 °C for 1 min, final extension at 72 °C for 10 min

Initial denaturation at 95 °C fpr 5 min followed by 30 cycles with denaturation for 30 s at 95 °C, annealing for 30 s at 60 °C, extension for 1 min at 72 °C and final elongation at 72 °C for 8 min

Decimo et al. (2014)

Laboratory isolates

16S rRNA

PA-GS-F (5′-GACGGGTGAGTAATGCCTA-3′) and PA-GS-R (5′-CACTGGTGTTCCTTCCTATA-3′)

NA

95 °C denaturation for 5 min, 10 cycles at 94 °C for 15 s, annealing at 53 °C for 30 s and elongation at 72 °C for 45 s 25 cycles repeated increasing elongation step at 72 °C by 5 s every cycle and final extension at 72 °C for 10 min

Ardura et al. (2013)

Referred strains

16S rRNA

DNA 16S–23S intergenic spacer (ITS1)

16S rRNA gene partial region

16SF 5′ AGAGTTTGATCCTGGCTCAG-3′

16SR 5′-CTACGGCTACCTTGTTACGA-3′)

(16F945 5′-GGGCCCGCACAAGCGGTGG-3′; 23R458 5′-CTTTCCCTCACGGTAC-3′)

(16SPSEfluF 5′-TGCATTCAAAACTGACTG-3′; 16SPSER 5′AATCACACCGTGGTAACCG-3′)

NA

2 min at 94 °C; 5 cycles consisting of 94 °C for 45 s, 55 °C for 1 min, 72 °C for 2 min; 35 cycles consisting of 92 °C for 45 s, 60 °C for 45 s, 72 °C for 12 min; final extension of 72 °C for 2 min and final cooling at 4 °C

5 min at 94 °C; 30 cycles consisting of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min; final extension of 72 °C for 2 min

Scarpellini et al. (2004)

Laboratory isolates

odc (ornithine decarboxylase)

PUT2-F

ATHWGNTWYGGNAAYACNATHAARAA

PUT2-R

GCNARNCCNCCRAAYTTNCCDATRTC

NA

10 min enzyme activation at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 53 °C and 2 min at 72 °C and final extension 20 min at 72 °C

De las Rivas et al. (2006)

Laboratory isolates

alkaline protease gene AprX

FP apr I (TAYGGBTTCAAYTCCAAYAC) and RF apr II (VGCGATSGAMACRTTRCC)

NA

1 min denaturation at 94 °C, 30 s annealing at 55 °C and a 30 s extension at 72 °C

Martins et al. (2005)

Laboratory isolates

16S rRNA gene partial region

16SPS EfluF (TG-CATTCAAAACTGACTG) and 16SPSER (AATCA-CACCGTGGTAACCG)

NA

NS

Caldera and Franzetti (2014)

Laboratory isolates

16S rRNA gene partial region

16SPS EfluF (TG-CATTCAAAACTGACTG) and 16SPSER (AATCA-CACCGTGGTAACCG)

NA

1 min at 94 °C; 30 cycles consisting of 56 °C for30 s, 72 °C for 1 min; a final extension of 72 °C for 7 min; final cooling at 4 °C

Morales et al. (2016)

  1. NA not applicable, NS not specified