Quantitative Duplex PCR of Clostridium botulinum Types A and B Neurotoxin Genes

Kasai, Yoshiaki; Kimura, Bon; Tajima, Yosuke; Fujii, Takeshi

doi:10.3358/shokueishi.48.19

Cited by 5 publications

(4 citation statements)

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“…showed a LOD of 60 copies of C. botulinum type A by SYBR green real-time PCR (Barker et al, 2016 ). The other researchers demonstrated an LOD between 16–200 copies for BoNT gene by q-PCR (Akbulut and Grant, 2004 ; Kasai et al, 2007 ; Fach et al, 2009 ; Takahashi et al, 2010 ; Malakar et al, 2013 ). A minimum of 100 copies BoNT Agene was detected in spiked rice (Sedlak et al, 2014 ).…”

Section: Discussionmentioning

confidence: 98%

“…The only currently admissive standard method for detection and identification of botulinum neurotoxin is the mouse bioassays (MBAs) which cause animal ethics issue and are time-consuming (Ferreira et al, 2004 ). At present, the detection methods for toxin-producing species mainly include isolation and culture (CfDCaP (CDC), 2016 ), PCR methods (Cordoba et al, 2001 ; Akbulut and Grant, 2004 ; Heffron and Poxton, 2007 ; Kasai et al, 2007 ; Dahlsten et al, 2008 ; Joshy et al, 2008 ; Fach et al, 2009 ; Hill et al, 2010 ; Kirchner et al, 2010 ; Lindberg et al, 2010 ; Peck et al, 2010 ; Satterfield et al, 2010 ; Anniballi et al, 2013 ; Fohler et al, 2016 ; Le Marechal et al, 2018 ; Masters and Palmer, 2021 ), sequencing (Gonzalez-Escalona et al, 2018 ; Gonzalez-Escalona and Sharma, 2020 ), and matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) based bacterial identification (Kalb et al, 2015 ; Bano et al, 2017 ; Xin et al, 2019 ; Drigo et al, 2020 ; Tevell Aberg et al, 2021 ). Most of these are time-consuming, labor-intensive, and not sensitive enough.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation and Optimization of Microdrop Digital PCR for Detection of Serotype A and B Clostridium botulinum

Gao

Zhang

et al. 2022

Front. Microbiol.

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Clostridium botulinum is the causative pathogen of botulism. Laboratory detection of C. botulinum is essential for clinical therapy treatment of botulism due to the difficulty in diagnosis, especially in infant botulism. The extreme toxicity of botulinum neurotoxin (BoNT) requires a sensitive detection method. Due to the detection limit of real-time quantitative PCR (q-PCR), a more sensitive detection method, micro-drop digital PCR (ddPCR) was applied in C. botulinum main serotypes A and B. The following performance criteria were evaluated by ddPCR: analytical sensitivity; repeatability; and diagnostic specificity. The limit of detection (LOD) was 0.84 and 0.88 copies/μl for BoNT A and B genes, respectively, by ddPCR with high specificity, compared to 5.04×102 and 6.91×102 copies/μl by q-PCR. It was increased 10 times compared with q-PCR in spiked stool samples. This improvement in sensitivity was especially important in clinical samples as more positive samples were detected by digital PCR compared with q-PCR. Meanwhile, enrichment time for low bacteria content samples was shortened by four hours both in serotypes A and B C. botulinum by ddPCR compared with q-PCR, which are important for laboratory diagnosis and epidemiology work.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Evaluation and Optimization of Microdrop Digital PCR for Detection of Serotype A and B Clostridium botulinum

Gao

Zhang

et al. 2022

Front. Microbiol.

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“…NIH B strain was used for C. botulinum type B spore preparation. Incubation was performed at 37°C anaerobically for 7 days, followed by incubation at room temperature aerobically until vegetative cells disappeared and the dominance of spores was observed (Kasai et al. 2007).…”

Section: Methodsmentioning

confidence: 99%

Rapid and simple detection ofClostridium botulinumtypes A and B by loop-mediated isothermal amplification

Sakuma

Kurosaki

Fujinami

et al. 2009

Journal of Applied Microbiology

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Aims: To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop‐mediated isothermal amplification (LAMP) method. Methods and results: The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT/A or BoNT/B, were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B. Conclusions: The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B. Significance and Impact of the Study: The LAMP assay would be useful for detection of C. botulinum in environmental samples.

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“…The detection of the target product is followed in real time as the reaction occurs, without the need for agarose gel electrophoresis. Fach et al (2009), Kimura et al (2001) and Yoon et al (2005) used real-time-based assays to target single botulinum toxin genes, whilst Akbulut et al (2004) and Kasai et al (2007) targeted multiple BoNT genes in a single real-time reaction. The single-target assays do not completely provide researchers or clinical laboratory technicians with the ability to identify which BoNT gene is present in an isolate.…”

Section: Introductionmentioning

confidence: 99%

A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F

Satterfield

Stewart

Lew

et al. 2010

Journal of Medical Microbiology

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Clostridium botulinum is the aetiological agent of botulism, a disease marked by flaccid paralysis that can progress to asphyxiation and death. This species is defined by the production of one of the botulinum neurotoxins (BoNTs), which are the most potent toxins known. Because of their potency, these toxins have the potential to be used as biological weapons, and therefore C. botulinum has been classified as a category A select agent. There are four related but antigenically distinct BoNT types that cause disease in humans, A, B, E and F. The mouse bioassay is the current gold standard by which BoNTs are confirmed. However, this method is expensive, slow and labour-intensive. Although PCR-based assays have been used extensively for the detection of BoNT-producing bacteria in food, animals and faecal samples, and recently to help diagnose disease in humans, no real-time quantitative PCR (qPCR) assay has yet been developed that can identify and differentiate all four BoNTs that cause disease in humans. This report describes the development of a qPCR single-tube assay that uniquely identifies these four BoNTs responsible for human disease. A total of 79 C. botulinum isolates with varying toxin types was evaluated in this study, as well as numerous near-neighbours and other bacterial species. The results showed that this quadruplex assay was capable of detecting any of the four toxin genes in a given sample at a sensitivity of about 130-840 fg genomic DNA and could detect the presence of up to all four BoNT genes simultaneously in a given sample. The assay was also functional in the presence of extraneous organic matter commonly found in various environmental samples. Abbreviations: BoNT, botulinum neurotoxin; qPCR, real-time quantitative PCR. Details of qPCR results for individual bacterial isolates are available as supplementary material with the online version of this paper.

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Quantitative Duplex PCR of Clostridium botulinum Types A and B Neurotoxin Genes

Cited by 5 publications

References 50 publications

Evaluation and Optimization of Microdrop Digital PCR for Detection of Serotype A and B Clostridium botulinum

Evaluation and Optimization of Microdrop Digital PCR for Detection of Serotype A and B Clostridium botulinum

Rapid and simple detection ofClostridium botulinumtypes A and B by loop-mediated isothermal amplification

A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F

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