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Multiplex PCR for the detection of Clostridium botulinum & C. perfringens toxin genes.

Sir,

Food-borne diseases constitute a major public health problem. Due to increased morbidity and mortality leading to time loss in the work place and reduced productivity, food-borne diseases across the world cost billions of dollars annually (1). The Clostridium group of bacteria is commonly found in soil everywhere in the world, and some of its species live harmlessly in our intestines. Clostridium produces a chemical that is toxic to human body. One of the major illnesses caused by the Clostridium group of bacteria is food poisoning which includes perfringens food poisoning by C. perfringens and food-borne botulism by C. botulinum. The enterotoxin produced by C.perfringens and neurotoxin produced by C. botulinum are responsible for the illness (2,3).

One of the inherent difficulties in the detection of food pathogens is that they are generally present in very low numbers (< 100 cfu/g) in the midst of up to a million or more other bacteria and these microbes may be lost among a background of indigenous microflora in the samples. The isolation of these organisms in the samples is frequently complicated by the presence of nontoxigenic strains that phenotypically and genetically resemble and exhibit a high relatedness with their toxigenic counterparts (4,5). Diagnosis of these food pathogens is confirmed by clinical observation together with the detection of toxin in a patient or suspected food sample (6). Presently, the only method of sufficient sensitivity for detection of botulinum neurotoxin is the mouse lethality test or bioassay, which is not only time-consuming and expensive but also raises ethical concern (7). Alternative in vitro immunological methods for toxin detection, which include electroimmuno diffusion, reversed passive latex haemagglutination, radioimmunoassay, and enzyme-linked immunosorbent assays (ELISA), have been described previously (7). Only the ELISA procedure has demonstrated sensitivity approaching that of the bioassay, but for C.botulinum neurotoxin none has been accepted for commercial production (7).

For C. botulinum and C. perfringens several PCR-based detection methods have been reported during the last decade (8-11). Compared to conventional methods, these protocols provide rapid and sensitive detection of these organisms. However, none of the reported PCR was able to detect the enterotoxin gene and neurotoxin gene simultaneously. The multiplex PCR method may provide a more sophisticated approach, enabling a simultaneous and specific detection of C. perfringens and more than one serotype of C. botulinum (9).

We therefore attempted to standardize a multiplex PCR using a pair of C.perfringens enterotoxin primer (melting temperature, 55[degrees]C) and two pairs of C. botulinum neurotoxin -specific primers with almost equal melting temperatures (60[degrees]C) for the simultaneous detection of C. perfringens and C. botulinum toxin genes using reference strain DNA in the microbiology department of All India Institute of Medical Sciences (AIIMS), New Delhi. The sensitivity and specificity of the assay was also analyzed. A few suspected cases of food poisoning were analyzed to see the applicability of this assay.

The DNA of enterotoxigenic strain C. perfringens type A--CN 3418 cpe +, C.botulinum type A (proteolytic C. botulinum types A, ATCC 25763) and B (nonproteolytic types B, Eklund 2B) were used for the standardization of multiplex PCR. Other Clostridium species including C. sporogenes, C. bifermentans, C. hastiforme as well as other bacteria viz., Staphylococcus aureus, Salmonella typhi, S. typhimurium, Escherichia coli, and Pseudomonas aeruginosa were used to confirm the specificity of PCR.

A total of 8 suspected cases of botulinum food poisoning (age group of 4 to 20 yr) received in the Anaerobic Laboratory, Microbiology Department, All India Institute of Medical Sciences, during the period of l year (January 2005-December 2005) were analyzed using this assay. Total genomic DNA of C.perfringens reference strain were extracted by the boiling method (11). All the diarrhoeal stool samples (0.1 g) were enriched overnight in Robertson's Cooked Meat broth (RCM), and DNA was extracted by Instagene Matrix (BioRad, USA).

The primers for the enterotoxin gene (cpe) of C.perfringens was chosen from the sequences that had been developed and validated by Fach and Popoff (10) to yield a 426 bp fragment for the cpe gene and for C. botulinum, the primers were selected from the nonhomologous regions of the BoNT types A, and B gene (9).

The PCR analysis was carried out in 25 [micro]l volume. The mixture contained 10 mM Tris HCl, 50 mM KCl, 0.0001 per cent gelatin, 3 mM Mg[Cl.sub.2], 100 [micro]M of dNTPS (MBI, Fermentas, USA), 50 pmols of primers of C. botulinum A and B and 20 pmols of primers of C.perfringens enterotoxin (BioBasic Inc, Canada), 1.5 U of Taq polymerase (MBI, Fermentas, USA) and 1 [micro]l of DNA. PCR analysis was performed by initial denaturation step at 95[degrees]C for 30 sec, followed by 25 cycles of amplification (denaturation at 95[degrees]C for 30 sec, primer annealing at 60[degrees]C for 25 sec, primer extension at 72[degrees]C for 1 min 25 sec) and 15 cycles of amplification (denaturation at 94[degrees]C for 30 sec, primer annealing at 55[degrees]C for 30 sec, primer extension at 72[degrees]C for 30 sec) ending with a final extension at 72[degrees]C for 10 min using a thermocycler (Gene Amp PCR system 9700; AB Applied Biosystems, USA).

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Specificity of the PCR assay was evaluated using several isolates belonging to the genus Clostridium and other isolates belonging to other genera. Sensitivity of PCR was confirmed using DNA of the reference strains. A 10-fold dilution of purified DNA was used from type A and type B C. botulinum and enterotoxigenic C.pefringens prepared in distilled water. The highest dilution yielding an amplicon with 3 bands (426 bp of C.perfringens and 782 and 205 bp of C.botulinum) in multiplex PCR from a 10 [micro]l sample volume was taken as the end point.

C. botulinum types A, B and enterotoxigenic C.perfringens yielded the expected amplification products: type A, 782 bp; type B, 205 bp; enterotoxigenic C.perfringens type A 426 bp. The mixed DNA suspensions yielded the corresponding DNA fragments (Fig.). The PCR products were clearly visualized in agarose gels; a 150 to 200 bp difference in the size of each amplification product enabled an easy distinction between the fragments without the use of high-resolution agarose. No detectable amplification occurred with DNA from other bacterial species analyzed. The sensitivity of multiplex PCR was 20-25 pg/10 [micro]l respectively. All expected amplification products were easily differentiated in low-resolution agarose gels.

Of the eight diarrhoeal cases analyzed, none showed the presence of neurotoxin or enterotoxin genes. All these samples were further analyzed for the presence of C. perfringens phospholipase C gene by PCR described previously (12) and enterotoxin by reverse passive latex agglutination (RPLA) and ELISA (12). Two of the analyzed samples were positive for phospholipase C gene (283 bp) which shows the presence of C. perfringens in the samples. The possibility of C. perfringens as the causative agent of food poisoning in these cases was ruled out as all of them were negative for enterotoxin by PCR, RPLA and ELISA.

This multiplex PCR might be useful in the diagnosis of food-borne clostridial pathogens- C. botulinum and C. perfringens, since the previously described methods require more than one PCR for simultaneous detection and identification of C. botulinum types and enterotoxigenic C. perfringens. The assay can thus markedly improve the PCR diagnostics of food-borne clostridial infections. Further, this may be used to overcome the cumbersome and time consuming animal tests used to identify toxigenic Clostridium. Its application for the direct detection of pathogenic Clostridium in food samples needs to be evaluated further.

Acknowledgment

Authors thank Dr E. Augustynowicz, Faculty, Department of Sera and Vaccine Evaluation, National Institute of Hygiene, Warsaw, Poland, for providing us the reference strains of C.perfringens and Dr Riikka Keto, Department of Food and Environmental Hygiene, University of Helsinki, Finland, for providing the DNA of C. botulinum. This work was supported by the Indian Council of Medical Research, New Delhi, India.

References

(1.) Todd ECD. Preliminary estimates of costs of foodborne disease in the United States. J Food Prot 1989; 52 : 595-601.

(2.) Buchanan RE, Gibbons NE, editors. Bergey's manual of determinative bacteriology, 8th ed. Baltimore, MD: Williams and Wilkins Company; 1974.

(3.) Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev 1990; 3 : 66-98.

(4.) Hatheway CL. Clostridium botulinum and other Clostridia that produce botulinum neurotoxin. In: Hauschild AHD, Dodds KL, editors. Clostridium Botulinum. ecology and control in foods'. New York: Marcel Dekker; 1992. p. 3-20.

(5.) Broda DM, Boerema JA, Bell RG. A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes. Lett Appl Microbiol 1998; 27 : 219-23.

(6.) Nordic Committee on Food Analysis. Botulinum toxin. Detection in foods, blood and other test materials. Method no. 79, 2nd ed. Espoo, Finland: Nordic Committee on Food Analysis; 1991.

(7.) Szabo EA, Pemberton JM, Desmarchelier PM. Specific detection of Clostridium botulinum type B by using the polymerase chain reaction. Appl Environ Microbiol 1992; 58 : 418-20.

(8.) Braconnier A, Broussolle V, Perelle S, Fach P, Nguyen-The C, Carlin F. Screening for Clostridium botulinum type A, B, and E in cooked chilled foods containing vegetables and raw material using polymerase chain reaction and molecular probes. J Food Prot 2001; 64 : 201-7.

(9.) Lindstrom M, Keto R, Markkula A, Nevas M, Hielm S, Korkeala H. Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material. Appl Environ Microbiol 2001; 67: 5694-9.

(10.) Fach P, Popoff MR. Detection of enterotoxigenic Clostridium perffringens in food and fecal samples with a duplex PCR and the slide latex agglutination test. Appl Environ Microbiol 1997; 63 : 4232-6.

(11.) Tansuphasiri U. Development to duplex PCR assay for rapid detection of enterotoxigenic isolates of Clostridium perfringens. Southeast Asian J Trop Med Public Health 2001; 32 : 105-13.

(12.) Joshy L, Chaudhry R, Dhawan B, Kumar L, Das BK. Incidence and characterization of Clostridium perfringens isolated from antibiotic-associated diarrhoeal patients: a prospective study in an Indian hospital. J Hosp Infect 2006; 63 : 323-9.

Lovely Joshy, Rama Chaudhry * & D.S. Chandel

Department of Microbiology

All India Institute of Medical Sciences

New Delhi 110 029, India

* For correspondence: drramach@rediffmail.com, ramach003@yahoo.com
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Title Annotation:polymerase chain reaction
Author:Joshy, Lovely; Chaudhry, Rama; Chandel, D.S.
Publication:Indian Journal of Medical Research
Article Type:Letter to the editor
Geographic Code:9INDI
Date:Aug 1, 2008
Words:1707
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