Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

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.

Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans

“…An exciting new DHPLC application is its use for separating and identifying bacterial, fungal, and microeukaryotic genes from mixed samples. For example, DHPLC has been used to identify different 16S rRNA genes present in mixed bacterial cultures (2,3,12) and in urine samples from human patients with polymicrobial genitourinary system infections (5,7,17). DHPLC has also been used for diagnosis of fungal infection by targeting the ribosomal internal transcribed spacer 2 sequence (11) and for typing of a wide variety of other microorganisms and target genes (1,18,38).…”

DEVELOPMENT AND CLINICAL VALIDATION OF A MULTIPLEX POLYMERASE CHAIN REACTION‐DENATURING HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE IDENTIFICATION OF FOODBORNE DIARRHEAGENIC ESCHERICHIA COLI

“…However, the PCR assay generally needs agarose gel electrophoresis stained with EB to analyze the PCR product, and the EB is very harmful to human health and pollutes the environment. DHPLC assay is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of foodborne pathogens (Franciosa et al . 2004.…”

“…Furthermore, the DHPLC technique was suggested to identify bacteria with rapid and sensitive feature (Hurtle et al . 2002, 2003; Franciosa et al . 2004; Edward et al .…”

A multiplex polymerase chain reaction coupled with high-performance liquid chromatography assay for simultaneous detection of six foodborne pathogens

“…Other investigations have used the DHPLC assay to identify bacteria (Edward, Barlaana, Sugimorib, Furukawab, & Takeuchib, 2005;Franciosa et al, 2004;Goldenberg et al, 2007). In our previous investigation, we have developed a new method for the detection of foodborne pathogens based on the DHPLC assay that multiplex PCR coupled with analysis of denaturing high-performance liquid chromatography apparatus under non-denaturing condition (PCReHPLC), which can efficiently identify different sizes of PCR products according to the differences of move rates in the stationary phase with the elution of the mobile phase (Xu et al, 2011).…”