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  1. Laboratory Methods (Food)

BAM Chapter 28: Detection of Enterotoxigenic Vibrio cholerae

Bacteriological Analytical Manual (BAM) Main Page

 January 2001

Authors: Walter H. Koch (ret.), William L. Payne (ret.), and Thomas A. Cebula (ret.)

For additional information, contact Jessica Jones

Revision History: Contact information updated.

Recent epidemics of cholera in various parts of the world have emphasized the urgent need for rapid and reliable detection methods for Vibrio cholerae, especially in food and water. Classical microbiological methods are sensitive and specific; however, they require several days to complete (see Chapter 9) and may result in considerable loss of perishable foods. Since cholera toxin production (encoded by the ctxAB genes) is the major factor in the pathogenesis of cholera, a polymerase chain reaction (PCR) method that selectively amplifies a DNA fragment within the ctxAB operon of V. cholerae has been developed and applied to various foods (19).

The PCR was first described by Mullis et al. in 1985, and since then has revolutionized most of the biological sciences (25). In this technique, double-stranded target DNA is denatured to provide single-stranded templates to which specific oligonucleotide primers are hybridized, followed by primer extension with a thermostable DNA polymerase (26). Primer pairs complementary to opposite strands of a DNA region are chosen so that 5' to 3' directional extensions are toward one another. Thus repetitive denaturation, annealing, and primer extension cycles exponentially amplify a unique DNA fragment bordered by the primers. The process is extremely rapid (as little as 30 min for 25 cycles with certain thermocyclers) and sensitive (amplification of gene sequences from a single cell is possible). Further, the process can be designed to be specific for genus, species, or allele.

Use of the PCR as a detection method for microbial pathogens in foods has been documented in approximately two dozen PCR-based, detection procedures published by early 1994 (Table 1). PCR-based methods have been developed to detect a wide variety of foodborne pathogens, including Listeria monocytogenes, enterotoxigenic Escherichia coli (see Chapter 4), V. vulnificus, V. cholerae, Shigella flexneri, Yersinia enterocolitica, various Salmonella and Campylobacter species, and the Hepatitis A (15) (see Chapter 26) and Norwalk viruses.

Table 1. PCR methods developed for the detection of pathogenic microbes in foods
Organism Target gene/gene product Reference
Campylobacter spp. 16S rRNA 14
C. jejuni flaA-flaB intergenic sequences 32
C. coli flaA-flaB intergenic sequences 32
Escherichia coli malB, LT1, ST1 5
LT 34
SLTI, SLTII 13
VT1, VT2 and VTE 23
plasmid invasion gene (ial) 1,20
Listeria monocytogenes listeriolysin O (hly) 3,4,9,10,12,21,22,24,29,34
16S rRNA 30
Listeria cell surface protein 31
Salmonella spp. oriC, chromosomal origin of replication 11
Shigella flexneri plasmid invasion gene 20
Vibrio cholerae ctxAB 19
V. vulnificus cytotoxin-hemolysin 16
Yersinia enterocolitica virulence plasmid virF gene 35
yadA 17
Hepatitis A virus(a) polymerase gene 2,15
Norwalk virus(a) polymerase gene 2

a Reverse transcription PCR (RT-PCR).

Many of these PCR methods have relied on extraction of DNA from contaminated foods, an additional step which adds several hours to the procedure and often requires modification of each diverse food matrix tested. An advantage of PCR is that the amplification reaction often proceeds well with crude lysates of cells, in some cases requiring only brief boiling of a bacterial suspension.

Although the PCR method, in principle, can detect a single bacterial cell with extended cycle regimens (50-60), the detection limit of direct PCR is effectively confined to about 104 bacteria per gram of food. This limitation is due to reaction volume constraints (25-100 µl), the increased propensity to amplify nonspecific products at high cycle numbers, and the inhibitory effect of many food components on Taq polymerase. Thus, the coupling of enrichment procedures with the PCR has effectively served two purposes: 1) It increases the sensitivity of detection to as few as 0.1 organism per gram of food; 2) it demonstrates by comparison of pre- and post-enrichment inocula that the food contains viable organisms. The added sensitivity and information regarding cell viability warrants the additional 4-24 hours (depending on the growth characteristics of the organism) required for such a procedure, and overcomes an early criticism that PCR would give false-positive results because it amplifies any DNA, including that of dead or nonviable organisms.

Results of amplification reactions are usually obtained by the resolution of products based on size via agarose gel electrophoresis and visualization by UV-induced fluorescence after staining with ethidium bromide. The complete PCR amplification and agarose gel analysis generally requires only about 2-4 hours after enrichment. In terms of sensitivity, specificity, and analysis time, selective enrichment followed by PCR is clearly a powerful, rapid, and robust methodology for detecting foodborne bacterial pathogens.

V. cholerae of the Inaba and Ogawa serotypes which lack the cholera toxin genes have been isolated; however, such strains are generally nonpathogenic. Since the presence of the cholera toxin operon is a prerequisite for pathogenicity, various PCR methods for the detection of pathogenic V. cholerae have all used the ctxAB genes as a target for amplification (8,18,28); these and the PCR method described here, will not detect nontoxigenic V. cholerae. As a practical matter, this PCR detection method allows one to define food samples as negative for the presence of toxigenic V. cholerae much more quickly than by following the complete microbiological identification scheme. However, it is recommended that alkaline peptone water (APW) enrichment broths used for PCR analysis also be plated onto selective thiosulfate-citrate-bile salts-sucrose (TCBS) agar (see Chapter 9) for isolation and direct confirmation of the presence of V. cholerae in samples that give positive PCR results.

  1. Equipment and materials
    1. For APW enrichment of V. cholerae (see Chapter 9).
    2. Programmable automatic thermocyler
    3. Horizontal gel electrophoresis apparatus
    4. Electrophoresis constant-voltage power supply
    5. Heating plate
    6. Microcentrifuge tubes, 1.5 and 0.6 ml
    7. Variable digital micropipettors (e.g., 0.5-20 µl, 20-200 µl)
    8. Aerosol-resistant pipet tips
    9. Microcentrifuge
    10. UV transilluminator
    11. Polaroid camera
    12. Polaroid film
  2. Media and Reagents
    1. Alkaline peptone water (APW) (see Chapter 9)
    2. Cholera toxin PCR primers, 10 pmol/µl stock solutions (5'-TGAAATAAAGCAGTCAGGTG-3', 5'-GGTATTCTGCACACAAATCAG-3'; see ref. 19)
    3. Taq DNA polymerase (native available from various vendors) or Amplitaq® (Perkin-Elmer)
    4. 2'-Deoxynucleoside-5'-triphosphates (dATP, dCTP, dGTP, dTTP); stock solution 1.25 mM of each dNTP
    5. 10× PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2)
    6. Light mineral oil
    7. Sterile deionized water
    8. 10× TBE (0.9 M Tris-borate, 0.02 M EDTA, pH 8.3)
    9. Agarose (nucleic acid electrophoresis grade)
    10. Ethidium bromide solution, 10 mg/ml
    11. 6× sample loading buffer (see ref. 27)
    12. DNA molecular weight markers (e.g., 123 bp ladder, Bethesda Research Laboratories, Gaithersburg, MD)

  3. Procedure for amplification of cholera toxin gene sequences from V. cholerae using APW enrichment broth

    Food sample preparation and APW enrichment (see Chapter 9).

    APW enrichment lysate preparation. Prepare APW washes or blends (see Chapter 9). Sample and freeze immediately (about 1 ml). After enrichment (6-24 h), prepare crude APW lysates for PCR by boiling 1 ml samples in 1.5 ml microcentrifuge tubes for approximately 5 min. Lysates may be used for PCR immediately or stored in a -20°C freezer until use. NOTE: Due to the enormous amplification possible with the PCR, minute levels of contamination can result in false positives. It is recommended that sample preparation, PCR reaction set-up, and PCR product analysis be physically separated from one another to minimize contamination. For an excellent discussion of considerations in setting up a PCR laboratory, see PCR Methods and Applications 3(2):S1-S14, (1993) A Manual Supplement, Section 1: Establishing PCR in the Laboratory (6,7). Minimally, use of aseptic technique in handling all PCR reagents and solutions is absolutely necessary. Use aerosol-resistant pipet tips for preparing samples and reagents for PCR reactions, and, if possible, a separate set of pipettors for analysis of PCR reaction products.

    PCR reaction preparation. To minimize cross contamination of PCR reagents, it is recommended that master mix solutions be prepared, aliquoted, and stored frozen. Master mixes contain all necessary reagents except Taq polymerase and the lysates being amplified. The final reaction contains 10 mM Tris-HCl, pH 8.3; 50 mM KCl, 1.5 mM MgCl2, 200 µM each dATP, dCTP, dGTP, and dTTP; 2 to 5% (v/v) APW lysate; 0.5 µM of each primer and 2.5 U Taq polymerase per 100 µl; reaction volumes of 25-100 µl may be used. Add Taq polymerase to the master mix and add APW lysate upon distribution to 0.6 ml microcentrifuge tube reaction vessels. Cover with approximately 50-70 µl of mineral oil.

    Temperature cycling. While there is some variability in the heating and cooling dynamics of thermocylers from different manufacturers, use of the following temperature cycling regimen should yield efficient amplification of the ctx gene fragment: Denaturation for 1 min at 94°C, primer annealing for 1 min at 55°C, and primer extension 72°C for 1 min, repeated for no more than 35 cycles. Increasing the cycle number beyond 35 cycles often leads to the formation of nonspecific amplification products, including primer dimers.

    Agarose gel analysis of PCR products. Mix 10-20 µl portions of PCR reactions with 6× gel loading buffer (choose one of four common buffers from Molecular Cloning: A Laboratory Manual by Sambrook et al. (27) and load into sample wells of 1.5-1.8% agarose gel submerged in 1× TBE containing 1 µg/ml ethidium bromide. After appropriate migration with a constant voltage of 5-10 V/cm, illuminate the agarose gel with a UV transilluminator and visualize bands relative to molecular weight marker migration. The primers listed above give rise to a 777 bp fragment (19). Take Polaroid photographs of gels to document results. Further details regarding gel electrophoresis analyses may be found in the above-mentioned Molecular Cloning Laboratory Manual (27).

    Proper controls. The need for a number of control reactions to ensure accurate interpretation of PCR results cannot be overemphasized. Minimally, for PCR analysis of food types previously optimized for this method (e.g., vegetable washes, oyster, crab and shrimp blends; see ref. 19), include a master mix contamination control containing no lysate and a toxigenic V. cholerae APW positive control in every analysis. For every new food blend to be analyzed by this PCR method, determine the potential inhibitory effects of that food. Minimally, this entails spiking 1 ml of a 1:10 and 1:100 APW food blend post-enrichment with about 5 × 106 organisms per ml (or an equivalent amount of positive control lysate). A direct comparison of these spiked samples with the APW (no food) lysate containing identical numbers of ctx+ cells, allows one to determine if any inhibition occurs at either of the two food concentrations and prevents the occurrence of false negatives. It is unlikely that food washes (e.g., fruits and vegetables) will inhibit the PCR reaction unless the fruits are bruised and washing releases excessive acidity to the APW wash.

References

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Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 28.

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