Determination of neurotoxin gene expression in Clostridium botulinum type A by quantitative RT-PCR.
Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and 4 mg ml-1, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk.
Shin
Na-Ri
NR
Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health, Seoul 122-701, Korea.
Shin
Ji-Hun
JH
Chun
Jeong Hoon
JH
Yoon
So-Yeon
SY
Kim
Bong Su
BS
Oh
Hee-Bok
HB
Rhie
Gi-Eun
GE
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000235:genetics* / Q000633:toxicity; D001906:Botulism / Q000401:mortality*; D047013:Clostridium botulinum type A / Q000235:genetics* / Q000254:growth & development; D004275:DNA, Ribosomal / Q000235:genetics; D005520:Food Preservatives / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D051379:Mice; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012977:Sodium Nitrite / Q000494:pharmacology; D013011:Sorbic Acid / Q000494:pharmacology; D015996:Survival Rate
D004275:DNA, Ribosomal; D005520:Food Preservatives; D012336:RNA, Ribosomal, 16S; D019274:Botulinum Toxins, Type A; D012977:Sodium Nitrite; D013011:Sorbic Acid