Abstract
Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.
| Original language | English |
|---|---|
| Pages (from-to) | 4170-4176 |
| Number of pages | 7 |
| Journal | Applied and Environmental Microbiology |
| Volume | 70 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - Jul 2004 |
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ASJC Scopus subject areas
- Environmental Science(all)
- Biotechnology
- Microbiology
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Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography. / Franciosa, Giovanna; Pourshaban, Manoocheher; De Luca, Alessandro; Buccino, Anna; Dallapiccola, Bruno; Aureli, Paolo.
In: Applied and Environmental Microbiology, Vol. 70, No. 7, 07.2004, p. 4170-4176.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography
AU - Franciosa, Giovanna
AU - Pourshaban, Manoocheher
AU - De Luca, Alessandro
AU - Buccino, Anna
AU - Dallapiccola, Bruno
AU - Aureli, Paolo
PY - 2004/7
Y1 - 2004/7
N2 - Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.
AB - Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.
UR - http://www.scopus.com/inward/record.url?scp=3242742301&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3242742301&partnerID=8YFLogxK
U2 - 10.1128/AEM.70.7.4170-4176.2004
DO - 10.1128/AEM.70.7.4170-4176.2004
M3 - Article
C2 - 15240298
AN - SCOPUS:3242742301
VL - 70
SP - 4170
EP - 4176
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 7
ER -