Real-time molecular beacon NASBA reveals hblC expression from Bacillus spp. in milk.
Nucleic acid sequence-based amplification (NASBA) was applied in combination with a fluorescein-conjugated molecular beacon specific for a sequence flanked by transcript-specific primers in order to monitor hblC enterotoxin gene expression in real-time from milk separately contaminated with Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus circulans. Maximal enterotoxin expression was noted following 16, 15, and 16 h, respectively, when grown in artificially contaminated nonfat dried milk incubated aerobically at 32 degrees C, corresponding to 1.6 x 10(5), 5 x 10(7), and 9.8 x 10(4)cfu/ml, for B. amyloliquefaciens, B. cereus, and B. circulans, respectively. This RNA amplification assay allows for simultaneous detection and confirmation of target transcripts in a closed tube format and may be performed in a high DNA background. The development of a rapid, sensitive, real-time method to quantitate the expression of virulence genes in pathogenic spore-formers is useful in shelf life determination of foods and other quality assurance measures.
Gore
Hope M
HM
Louisiana State University School of Dentistry, New Orleans, LA, USA.
Wakeman
Catherine A
CA
Hull
Rhiannon M
RM
McKillip
John L
JL
D000818:Animals; D001407:Bacillus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D005504:Food Analysis / Q000379:methods; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D020869:Gene Expression Profiling / Q000379:methods*; D006460:Hemolysin Proteins; D008892:Milk / Q000382:microbiology*; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D013045:Species Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; C083961:hemolysin BL protein, Bacillus