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Research Article

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

Table 1

Results of the qPCR assay targeting ORF 0047 and ORF 0072 unique to pBC210 using bacterial and soil DNA extracts as template.
DNA source ( ) PCR assay
ORF 0047 ORF 0072 ORF 0067
Bacteria (244)   
 B. cereus G9241  ( ) +++
Other Bacillus species (151)   
 B. cereus   ( ) −−−
 B. anthracis  ( ) −−−
 Bacillus badius  ( )−−−
 B. marisflavi  ( ) −−−
 B. megaterium  ( )−−−
 B. mycoides  ( )−−−
 B. pseudomycoides  ( ) −−−
 B. pumilus  ( ) −−−
 B. subtilis   ( ) −−−
 B. thuringiensis  ( )−−−
Other bacteria (71)   
Gram-positive (16)   
 Enterococcus faecalis  ( )−−−
 Lactobacillus rhamnosus   ( )−−−
 Staphylococcus aureus  ( )−−−
 Streptococcus pneumoniae  ( )−−−
Gram-negative (55)   
 Achromobacter xylosoxidans  ( )−−−
 Acinetobacter baumannii  ( )−−−
 Acinetobacter calcoaceticus  ( )−−−
 Burkholderia cepacia  ( )−−−
 Burkholderia mallei   ( ) −−−
 Burkholderia pseudomallei   ( )−−−
 Burkholderia species  ( ) −−−
 Cedecea neteri  ( )−−−
 Citrobacter freundii  ( )−−−
 Enterobacter cloacae   ( )−−−
 Escherichia coli  ( ) −−−
 Klebsiella pneumoniae  ( )−−−
 Pseudomonas aeruginosa   ( )−−−
 Salmonella enterica   ( )−−−
 Serratia marcescens  ( )−−−
 Shigella flexneri  ( )−−−
 Stenotrophomonas maltophilia  ( )−−−
 Vibrio parahaemolyticus  ( )−−−
Soils from United States (256)   
 Florida  ( ) 5+, 64−5+, 64−5+, 64−
 Texas  ( ) −−−
 Elsewhere  ( ) −−−
a“+” denotes a positive amplicon that was produced by the assay with a cycle threshold (CT) value of ≤39.99. For ORF 0047, the average CT for the two controls was 16.66 with a range of 15.92–18.73 using plasmid DNA and 29.21 with a range of 20.93–38.25 using genomic DNA. For ORF 0072, the average CT for the two controls was 16.10 with a range of 15.24–18.03 for plasmid DNA and 27.09 with a range of 21.29–33.60 using genomic DNA. All samples positive for the two ORF sequences were also positive for ORF 0067. For ORF 0067, the average CT for the two controls was 15.24 (range of 12.25–15.91) using plasmid DNA and 30.72 (range of 28.59–33.99) using genomic DNA. For the 16S assay, the average CT for the two positive controls was 20.40 with a range of 13.68 to 27.20 for genomic DNA. For all DNA extractions of both bacterial and soil samples, the average CT value was 21.24 and the range was from 12.89 to 39.97. The two B. cereus carrying pBC210 (CBD 1056 and CBD 1057) were tested a minimum of 250 times.
b“−” denotes a negative result as an amplicon was not detected in the qPCR assay. All samples including the five Florida soils that produced positive results for the target sequences (ORF 0047, ORF 0067, and ORF 0072) were tested in duplicate in multiple runs by two different personnel.