Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producingClostridium botulinum,Clostridium baratiiandClostridium butyricum

Abstract

Aims: To develop real‐time PCR assays for tracking and tracing clostridia responsible for human botulism. Methods and Results: Real‐time PCR assays based on the detection of the genes ntnh encoding the nontoxin‐nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real‐time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type‐specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type‐specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 103 to 104 cells ml−1. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of ‘foie gras’ suspected in a C. botulinum outbreak. Conclusion: These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism. Significance and Impact of the Study: Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…Inclusion of a fluorogenic probe in qPCR assays was shown to increase specificity and to allow simultaneous detection of a number of genes via differently labeled probes. Using qPCR, 10-100 genome equivalents (GE) can be readily detected in about 2 h. Including DNA purification and dilution steps, this has led to detection limits of 10 3 -10 5 GE/mL (Fach et al 2009;Kirchner et al 2010).…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…Inclusion of a fluorogenic probe in qPCR assays was shown to increase specificity and to allow simultaneous detection of a number of genes via differently labeled probes. Using qPCR, 10-100 genome equivalents (GE) can be readily detected in about 2 h. Including DNA purification and dilution steps, this has led to detection limits of 10 3 -10 5 GE/mL (Fach et al 2009;Kirchner et al 2010). Since in botulism diagnosis more than one serotype is of concern, multiplex reactions covering several serotypes simultaneously have been reported in recent years.…”

“…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.…”

Comparison of Direct Culture, Immunomagnetic Separation/culture, and Multiplex PCR Methods for Detection of <i>Salmonella</i> in Food

“…PCR targeting Salmonella virulence genes, such as fimA (Moreira et al, 2008), invA (Rahn et al, 1992;Upadhyay et al, 2010), and hilA (Guo et al, 2000), has been developed. Recently, multiplex PCR (mPCR) targeting two or more genes has been widely used in Salmonella detection to increase the specificity (Fach et al, 2009;Kim et al, 2006;Woods et al, 2008).…”