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

Abstract

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.

Report of two unlinked cases of infant botulism in the UK in October 2007

“…We reported previously on the evaluation of these assays for the diagnosis of botulism, which allows online monitoring of amplified gene fragments at each cycle of PCR, thus permitting simultaneous amplification and detection at high sensitivities of pathogen-specific nucleic acids within 1-2 h (Akbulut et al, 2004(Akbulut et al, , 2005. Additionally here, we included the use of a fourth real-time PCR to detect a fragment of the bontF gene.…”

“…Real-time PCR assays for bontA, bontB and bontE gene fragments were performed as described previously (Akbulut et al, 2004) except that PCR primers (F1: 59-CCTGC-AATTTCACTAGCTCATGA-39; F2: 59-GCCTTATGGGTTTTTC-GGCTAT-39) and a 59 Yakima Yellow-labelled fluorescent probe (59-TTGATACATGCACTGCATGGATTATACGGG-39) were also included for the specific detection of a fragment of the bontF gene. PCR analysis was performed as two duplex reactions: one for the detection of bontA and bontB gene fragments, and the other for the detection of bontE and bontF gene fragments.…”

“…DNA was extracted from pure cultures of C. botulinum growing on Columbia blood agar plates after 48 h at 30 uC in an anaerobic cabinet using a previously described automated method (Akbulut et al, 2004). Restriction digestion of DNA and ligation of the adaptors were performed in a single step using a modification of the method of Valsangiacomo et al (1995).…”

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.…”

“…From a diagnostic point of view, assays including an internal amplification control allow for a more accurate evaluation of results, which is mandatory under certain quality control schemes, and procedures have thus been implemented accordingly (Braconnier et al 2001;Akbulut et al 2004;Messelhäusser et al 2007;De Medici et al 2009;Kirchner et al 2010;Fach et al 2011;Fenicia et al 2011;Anniballi et al 2012). …”