Selection of primers for specific detection of Clostridium botulinum types B and E neurotoxin genes using PCR method

Alsallami, Ali Ahmed; Kotłowski, Roman

doi:10.1016/s0168-1605(01)00499-8

Cited by 11 publications

(5 citation statements)

References 25 publications

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“…PCR-based assays have been reported for BoNT gene fragments using conventional block based assays with gel electrophoresis for detection of amplified products (Campbell et al, 1993;Szabo et al, 1993;Ferreira et al, 1994;Franciosa et al, 1994Franciosa et al, , 1998Fach et al, 1995;Takeshi et al, 1996;Aranda et al, 1997;Hyytia et al, 1998;Alsallami and Kotlowski, 2001;Dahlenborg et al, 2001;Lindström et al, 2001;Nevas et al, 2002;Craven et al, 2002). These assays either detected a single toxin type or suffered from a lack of specificity due to low melting temperatures of the primers, some requiring subsequent use of hybridisation probes to confirm the specific toxin type.…”

Section: Discussionmentioning

confidence: 99%

Development and Application of Real-Time PCR Assays to Detect Fragments of theClostridium botulinumTypes A, B, and E Neurotoxin Genes for Investigation of Human Foodborne and Infant Botulism

Akbulut¹,

Grant²,

McLauchlin³

2004

Foodborne Pathogens and Disease

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Real-time PCR assays for detection of Clostridium botulinum neurotoxin (BoNT) gene fragments specific to BoNTA, B, and E were developed as alternatives to the mouse bioassay. The expected specificities of the PCR assays were demonstrated by in silico analysis as well as empirical testing of target DNA extracted from 83 pure cultures of C. botulinum, and 44 bacteria from other species. The sensitivities of the assays were found to be equivalent to 16, 10, and 141 genomes for BoNT A, B, and E, respectively. The assays were shown to be applicable to both purified DNA, as well as crude DNA extracted from cultures and enrichment broths. The assays were evaluated using DNA extracted directly from clinical and food specimens as well as from inoculated broths using material collected from seven confirmed and one suspected case of botulism. The appropriate BoNT genes were detected in material from seven of the eight cases of botulism and provided a supportive diagnosis faster than the conventional bioassay. These assays have already proven useful for pubic health microbiological investigation of suspected cases of human botulism by substantially improving the diagnostic process.

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Section: Discussionmentioning

confidence: 99%

Development and Application of Real-Time PCR Assays to Detect Fragments of theClostridium botulinumTypes A, B, and E Neurotoxin Genes for Investigation of Human Foodborne and Infant Botulism

Akbulut¹,

Grant²,

McLauchlin³

2004

Foodborne Pathogens and Disease

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show abstract

“…Several qPCR detection and enumeration methods for nonproteolytic C. botulinum have been developed in the past (Alsallami and Kotłowski, 2001;De Medici et al, 2009;ISO, 2017;Kimura et al, 2001). Those methods were designed to quantify C. botulinum by amplifying sequences from botulinum neurotoxin genes.…”

Section: Qpcr Enumeration Of Cb-mixmentioning

confidence: 99%

Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum – Evaluation and validation with MAP and smoked foods

2022

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“…Since human and other eukaryotic genomes are larger than those of prokaryotes, the probability of nonspecific amplification in the PCR method increases in the presence of genomic DNA isolated from eukaryotic cells. Non-specific bands can be removed and sensitivity increased (Alsallami & Kotlowski, 2001) by performing amplification reactions at higher annealing temperatures.…”

Section: Optimization Of Cell Lysis and Dna Extractionmentioning

confidence: 99%

One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish

Kotłowski

Martin

Ablordey

et al. 2004

Journal of Medical Microbiology

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The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.

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Selection of primers for specific detection of Clostridium botulinum types B and E neurotoxin genes using PCR method

Cited by 11 publications

References 25 publications

Development and Application of Real-Time PCR Assays to Detect Fragments of theClostridium botulinumTypes A, B, and E Neurotoxin Genes for Investigation of Human Foodborne and Infant Botulism

Development and Application of Real-Time PCR Assays to Detect Fragments of theClostridium botulinumTypes A, B, and E Neurotoxin Genes for Investigation of Human Foodborne and Infant Botulism

Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum – Evaluation and validation with MAP and smoked foods

One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish

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