A rapid qualitative assay for detection of Clostridium perfringens in canned food products
Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A–E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other experimental strains like C. tetani, C. botulinum, C. acetobutyricum, Bacillus subtilis, and Escherichia coli. This assay is extramly rapid and provides reliable and reproducible results within one hour of incubation at 37°C.
Berry PR, Rodhouse JC, HughesS, Bartholomew BA, Gilbert RJ (1988) Evaluation of ELISA, RPLA, and Vero cell assays for detecting Clostridium perfringens enterotoxin in faecal specimens. J Clin Pathol 41: 458–461. PMCID: PMC1141476
Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, Cai J, Hippe H, Farrow JA (1994) The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44: 812–826. DOI: 10.1099/00207713-44-4-812
Flick JS, Thorner J (1993). Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae. Mol Cell Biol 13: 5861–5876
Hatheway CL (1990). Toxigenic clostridia. Clin Microbiol Rev 3: 66
Itabe H, Takeshima E, Iwasaki H, Kimura J, Yoshida Y, Imanaka T, & Takano T (1994). A monoclonal antibody against oxidized lipoprotein recognizes foam cells in atherosclerotic lesions. Complex formation of oxidized phosphatidylcholines and polypeptides. J Biol Chem 269: 15274–15279
Kirk DG, Dahlsten E, Zhang Z, Korkeala H, Lindström M (2012) Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol 78: 4590–4596. doi:10.1128/AEM.00304-12
Jihong L, Adams V, Bannam TL, Miyamoto K, Garcia JP, Uzal FA, Rood JI, McClane BA (2013) Toxin plasmids of Clostridium perfringens. Microbiol Mol Biol Rev 77: 208–233. doi: 10.1128/ MMBR.00062-12
Read TD, Peterson SN, Tourasse N, Baillie LW, Paulsen IT, Nelson KE, Hanna PC (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423: 81–86: doi: 10.1038/nature01590
Kanakaraj R, Harris DL, Songer JG, Bosworth B (1998). Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed. Vet Microbiol 63: 29–38. https://doi.org/10.1016/S0378-1135(98)00229-6
Kokai-Kun JF, Songer JG, Czeczulin JR, Chen F, McClane BA (1994) Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol 32: 2533–2539
Rodrigues AS, Aguiar APD, de Aguiar MR, Santa Maria LCD (2007) Quaternization reaction of 2-vinylpyridine and 4-vinylpyridine network copolymers for 4-nitrophenol adsorption. J Brazil Chem Soc 18: 431–436. http://dx.doi.org/10.1590/S0103-50532007000200028
Sheikhnejad RG, Srivastava PN (1986) Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma. J Biol Chem 261: 7544–7549
Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppée JY, Dupuy B (2013) Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. PLoS Genet 9: e1003493. https:// doi.org/10.1371/journal.pgen.1003493
Thorpe TC, Miller RD (1981). Extracellular enzymes of Legionella pneumophila. Infection and Immunity 33: 632–635
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