SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A

Abstract

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 ؋ 10 1 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

“…BOTULINUM GROUP I GENOTYPING BY 15-LOCUS VNTR 4253 (2,10,14,23,25,26, and 35 RU) were observed for cbms04; four new alleles (7,8,13, and 15 RU) were observed for cbms05; three new alleles (3, 3.5, and 5 RU) were observed for cbms07; one new allele (2 RU) was observed for cbms11; and four new alleles (3, 10, 11, and 12 RU) were observed for cbms14.…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…By far, the most commonly employed methods are PCR-based techniques (Mullis et al 1986;Saiki et al 1988), many of which aim at detecting bont genes by conventional or quantitative amplification reactions (Szabo et al 1992(Szabo et al , 1993Franciosa et al 1994Franciosa et al , 1996Fach et al 1995Fach et al , 2009Takeshi et al 1996;Aranda et al 1997;Braconnier et al 2001;Kimura et al 2001;Craven et al 2002;Popoff and Walker 2003;Akbulut et al 2004;Takeda et al 2005;Yoon et al 2005;Lindström and Korkeala 2006;Artin et al 2007;Fenicia et al 2007;Heffron and Poxton 2007;Prévot et al 2007;Sánchez-Hernández et al 2008;Sakuma et al 2009;Hill et al 2010;Lindberg et al 2010;Takahashi et al 2010). Since conventional PCR is difficult to quantify and requires a post-PCR step to visualize and to verify the PCR product, many modern approaches use quantitative PCR (qPCR) formats.…”

Bodas de plata de la reacción en cadena de la polimerasa (PCR)

“…Siete tipos de toxinas antigénicas han sido identificadas (A-G) BoNT (neurotoxina botulínica) tipo A, B E y raramente F son las responsables de botulismo humano (53). Esta bacteria puede contaminar alimentos mal enlatados o almacenados en recipientes abiertos o inapropiados.…”

“…Los métodos microbiológicos estándar toman sólo en consideración la detección de C. botulinum; el bioensayo en ratones es el método universal, altamente sensible, pero costoso y con problemas éticos ya que requiere gran cantidad de animales. La detección de genes de BoNT por PCRtr es más sensible y una alternativa mas rápida que los bioensayos (53,54).…”