Amplification of pVGC region for Listeria monocytogenes strains
Region 1: prfA-hly
prf1046R AAATGCTTCTGTAAGTTC
hly1541R GTCATCAATTACCGTTCTC
Amplifications were performed in 25µl reactions with 1X PCR buffer, 2mM MgSO4, 0.2mM dNTP’s, 0.6µM each primer, 1 U of Taq polymerase, and 75ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 40 cycles of 94ºC (30 sec), 52ºC (30 sec), and 68ºC (4 min).
Region 2: mplA-actA
mplA-F GCTCATTTCACATCGTCCATC
actA-R CTTCTGTTGGGATTGGTG
Amplifications were performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 56ºC (30 sec), and 68ºC (4 min).
Region 3: actA-orfB
actA-F CGGATAGTGAGCTTGAAAGCC
orfB-R GCGCAAATGCCGTTATCGGTG
Amplifications were performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 56ºC (30 sec), and 68ºC (4 min).
Amplification of pVGC region for Listeria seeligeri, strain 33019
Region 1: prfA-hly
s-prfA-3 AAGGCGCGACAAGAAATCCC
s-hly-1 TAGAATGTCTTGGGTAAAGCG
Amplification was performed in 25µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.6µM each primer, 1.5 U of Taq polymerase, and 75ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 40 cycles of 94ºC (30 sec), 55ºC (30 sec), and 68ºC (5 min).
Region 2: hly-actA
s-actA-R CCTCACTGACAACTGGCAAAG
s-hly-A GATCATTCTGGTGGTTATGTAG
Amplification was performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 56ºC (30 sec), and 68ºC (6 min).
Region 3: actA-orfH
s-actA-F1 ARTTGCTAAAAGTGCWGARSRCG
s-orfH-R CTGTATAGCTTGAAGCATGGACC
Amplification was performed in 25µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.6µM each primer, 1.5 U of Taq polymerase, and 75ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 40 cycles of 94ºC (30 sec), 54ºC (30 sec), and 68ºC (2 min).
Amplification of pVGC region for Listeria ivanovii, strain 33017
Region 1: prfA-hly
prf1046R AAATGCTTCTGTAAGTTC
hly1541R GTCATCAATTACCGTTCTC
Amplification was performed in 25µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.6µM each primer, 1.5 U of Taq polymerase, and 75ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 40 cycles of 94ºC (30 sec), 50ºC (30 sec), and 68ºC (4 min).
Region 2: mplA-actA
impl-A CTCTAGTGTTGCATATGGTCGTG
iact-AR CGTGGGCGAATCAAGGCAAC
Amplification was performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 54ºC (30 sec), and 68ºC (5 min).
Region 3: actA-orfB
iact-A GCTTGCTTCCGYCTAGGG
siorf-BR GCTATTAAAGARATGGAACARCGCGC
Amplification was performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 56ºC (30 sec), and 68ºC (4 min).
Amplification of pVGC region for Listeria ivanovii, strain 33021
Region 1: prfA-hly
prf1046R AAATGCTTCTGTAAGTTC
hly-2iv GCAGCCTTCACTCTGGTGC
Amplification was performed in 25µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.6µM each primer, 1.5 U of Taq polymerase, and 75ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 40 cycles of 94ºC (30 sec), 52ºC (30 sec), and 68ºC (4 min).
Region 2: hly-mplA
hly-Aiv CACTATACGCTGGATAAAGC
i21hly-4R CATGGTGTCATGCGTTCC
Amplification was performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 1.0µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 54ºC (30 sec), and 68ºC (90 sec).
Region 3: mplA-actA
impl-A CTCTAGTGTTGCATATGGTCGTG
iorf-act1R CTGCTCGAGTTGGCGGTGATGGG
Amplification was performed in 20µl reactions with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM each primer, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 58ºC (30 sec), and 68ºC (4 min).
Region 4: actA-orfB
iact-A GCTTGCTTCCGYCTAGGG
siorf-BR GCTATTAAAGARATGGAACARCGCGC
Amplification was performed in 20µl with 1X PCR buffer, 1.5mM MgSO4, 0.2mM dNTP’s, 0.75µM iact-A and siorf-BR primers, 1.5 U of Taq polymerase, and 100ng of genomic DNA. PCR consisted of an initial denaturation at 94°C (90 sec), followed by 30 cycles of 94ºC (30 sec), 56ºC (30 sec), and 68ºC (4 min).