Identification of pBC218/pBC210 Genes of Bacillus cereus G9241 in Five Florida Soils Using qPCR

Table 2

Oligonucleotide primers and probes used in qPCR assays.

ORF on pBC210	Protein or gene	Primer	Sequence (5′→3′)	Location^a	Amplicon length (bp)	Source
0047	Electron transport protein Rv1937	47F	GGTGATTCGATCAGGTTCATTA	54385–54406	140	This study
		47R	CGCCATCCCGGTATTAAAG	54506–54524
		47P^b	TTTGAATCGACAGCAGCCTGCTGA	54430–54453
0067^c	Transcriptional regulator	67F	CAATTGCCTTTAATACGTCACC	82127–82148	135	This study
		67R	CTCGTATGCGTTATGAAGACC	82241–82261
		67P^b	TGACGTTGCCGTATTTGACGACCGA	82204–82228
0072^d	Polysaccharide polymerase	72F	ACCCTAGTCCTTTCCCAAATA	87419–87439	111	This study
		72R	GCACAAACCAACAAGGAGA	87511–87529
		72P^b	TGCCAAGCTTCTTCCCTTCCAGAAAGA	87472–87498
	16S rRNA	16F	GCGGTGGAGCATGTGGTT	948–965	73	This study
		16R	AGGGTTTTCAGAGGATGTCAAGAC	997–1020
		16P^b	AATTCGAAGCAACGCGAAGAACCTTACCA	967–995

aThe location of oligonucleotide sequences was determined using the pBC210 plasmid sequence of B. cereus G9241 (GenBank accession number AAEK01000004.1) for ORF 0047, ORF 0067, and ORF 0072. For the internal amplification control, the B. cereus 16S rRNA sequence (GenBank accession number X55060) was used.
^bFor direct PCR amplicon detection during the reaction, all probe oligonucleotides were labeled with a 6-FAM (6-carboxy-flourescein) reporter at the 5′ end and a Black Hole Quencher-1 quencher at the 3′ end.
^cThe qPCR assay for ORF 0067 was used to confirm all positive tests determined by assays for ORF 0047 and ORF 0072 and was later performed on all of the bacterial and soil sample DNA.
^dORF 0072 is previously notated as pBC218-ORF 0073 (Hoffmaster et al., 2006) [11], while GenBank currently designates it as ORF 0072.