Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing

Table 1 Oligonucleotide primer pairs and PCR running conditions used in this study

Primer	Sequence 5' to 3'	Targeted site	Amplicon size (bp)	Reference
SG-F	GGGTTGTCTGCGAAAGCGAA^a	ITS-G	282	Liu et al., [44]
SG-R	GTCTTCGTGCTGCGAGTTTG	ITS-G & ITS-IA
SI-F	CAGGAGTTGAAGAGGTTTAACT^b	ITS-IA	251	Liu et al., [44]
SI-R	GTGCTGCGAGTTTGAGAGACTC	ITS-G & ITS-IA
Saka 1a	ACAGGGAGCAGCTTGCTGC^c	V1^g	952	Hassan et al., [45]
Saka 2b	TCCCGCATCTCTGCAGGA	V3^h
Zpx F	GAAAGCGTATAAGCGCGATTC^d	zpx	94	Kothary et al., [13]
Zpx R	GTTCCAGAAGGCGTTCTGGT
BAM122	AWATCTATGACGCGCAGAACCG^e	zpx	350	Kothary et al., [13]
BAM123	AAAATAGATAAGCCCGGCTTCG
EsgluAf	TGAAAGCAATCGACAAGAAG^f	gluA	1680	Lehner et al., [3]
EsgluAr	ACTCATTACCCCTCCTGATG
EsgluBf	TGAGTGAAGCACCGACGCAG^f	gluB	1720	Lehner et al., [47]
EsgluBr	GTTACGTCACAGGTTTTGAT
ESSF	GGATTTAACCGTGAACTTTTCCⁱ	ompA	469	Nair and Venkitanarayanan [46]
ESSR	CGCCAGCGATGTTAGAAGA

^a&^b Running conditions; 94°C for 10 min; 30 cycles of 94°C for 30 sec each; 57°C for 1 min; 72°C for 1 min; a final extension period of 5 min at 72°C.
^c Running conditions; 95°C for 4 min; 30 cycles of 95°C for 60 sec each; 50°C for 1 min; 72°C for 90 sec; final extension period of 4 min at 72°C.
^d&^e Running conditions; The hot start polymerase was activated by incubation for 15 min at 95°C; followed by 35 cycles of 1 min at 95°C; 62°C for zpx primers (50.5°C was used for 8 isolates) or 50°C for BAM primers for 1 min; 72°C for 1 min; final extension of 10 min at 72°C for zpx and 7 min for BAM primers.
^f Running conditions were as described by Lehner et al. [3, 47]; The hot start polymerase was activated by incubation for 15 min at 95°C; followed by 30 cycles of 30 s at 94°C; 56°C (gluA) or 58°C (gluB) for 1 min; 72°C for 1.5 min; final extension period of 5 min at 72°C.
^g&^h: Variable regions of the 16S rRNA gene.
ⁱ Running conditions: 94°C for 2 min; 30 cycles 94°C for 15 sec each; 60°C for 15 sec; 72°C for 30 sec; final extension period of 5 min at 72°C.

ISSN: 1471-2180