Universal and specific quantitative detection of botulinum neurotoxin genes

BMC Microbiology

Methodology Article

Daniel C Douek¹  Brenna J Hill¹  Stephen S Arnon²  Theresa J Smith³  Janet C Skerry³ 

[1] Human Immunology Section, Vaccine Research Center, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, 20892, Bethesda, MD, USA;Infant Botulism Treatment and Prevention Program, California Department of Public Health, 94804, Richmond, CA, USA;Integrated Toxicology Division, USAMRIID, Fort Detrick, 21702, MD, USA;

关键词: Botulism;    Botulinum Neurotoxin;    Mouse Bioassay;    Crude Toxin;    Wound Botulism;   

DOI  :  10.1186/1471-2180-10-267

 received in 2010-04-20, accepted in 2010-10-20,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundClostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin.ResultsWe assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies.ConclusionsWhile other studies have reported conventional or quantitative PCR-based assays for the detection of C. botulinum genes, our procedure's high-throughput capability and its portability allows most laboratories to quickly assess the possible presence of BoNTs either in food processing samples or in suspected cases of botulism. Thus, this assay provides rapid and specific detection of BoNT and toxin complex genes and would enable the targeting of appropriate therapeutics to infected individuals in a timely manner.

【 授权许可】

Unknown   
© Hill et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.