SEARCH

SEARCH BY CITATION

Keywords:

  • Vibrio cholerae O1;
  • Vibrio cholerae O139;
  • Multiplex polymerase chain reaction

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences. The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V. cholerae O1 and O139, respectively. Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard. In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup. All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential of producing cholera toxin.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

The 1990s have witnessed a remarkable surge in the global incidence of cholera with the number of countries reporting cholera cases to WHO reaching an all-time high of 94 in 1994 [1]. Apart from the increasing global incidence of cholera, the 1990s have also witnessed the unprecedented emergence of a new serogroup associated with cholera. Cholera is, therefore, currently recognized to be caused by the O1 and O139 serogroups of Vibrio cholerae[2]. The O139 serogroup of V. cholerae was responsible for an explosive outbreak in Southern India [3]which subsequently spread to all cholera endemic areas in India [4]and neighboring countries [5, 6]. After the initial rapid spread between 1993 and 1994, the incidence of the O139 serogroup declined and it appeared as if the serogroup would become naturally extinct. However, in late 1996, V. cholerae O139 again reemerged in Calcutta and in other parts of India [7]indicating that the etiology of cholera in future is likely to swing between the O1 and O139 serogroups.

V. cholerae O139 differs from O1 El Tor in that it contains a distinct ‘O’ antigen which is not recognized by O1-specific antiserum. The genes responsible for O antigen biosynthesis and for generation of serotype specific determinants are located in the rfb region on the V. cholerae chromosome [8]. Subsequent to the genesis of the O139 serogroup, the rfb region has been analyzed in great detail to understand the origin of the O139 serogroup. Extensive studies have shown that V. cholerae O139 has most probably been derived from an El Tor O1 strain [8]or from a strain belonging to the non-O1 serogroup [9]by the acquisition of new polysaccharide genes and the subsequent or simultaneous deletion of the V. cholerae O1 rfb genes. Hybridization analysis using several DNA fragments derived from O139 rfb genes as probes has shown unique regions in the rfb gene which are specific for the O139 and O1 serogroups [10]. Primers for development of this multiplex PCR were selected from these regions of the O1 and O139 rfb genes.

Because of the devastating epidemic potential of O1 and O139 serogroups, the quick diagnosis and identification of the causative serogroup is important especially from a public health point of view. Realizing the need for quick diagnosis, a variety of rapid tests including co-agglutination tests [11–13], enzyme-linked immunosorbent assays [14, 15]and membrane based immunoassays [16]have been developed in the recent past. Recently, Albert et al. [17]have developed a polymerase chain reaction (PCR) assay using a primer pair corresponding to a chromosomal region of V. cholerae O139 for rapid detection of the serogroup from stool samples. However, these tests have limitations in that they are directed to detect either the O1 or the O139 serogroup and further these tests do not provide information on the toxigenic potential of the strains. To transcend these limitations, we embarked on a study to develop a multi-information multiplex PCR based test for concurrent detection of the O1 and/or the O139 serogroups directly from stool samples which could also provide information on the toxigenic potential of the strains.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

2.1Bacterial strains

V. cholerae O139 Bengal (MO45, ATCC 51394) and V. cholerae O1 El Tor biotype Inaba serotype (35A3) were used as the positive controls while V. cholerae O108 was used as the negative control for developing the PCR assay. To examine the specificity of the PCR developed in this study, additional strains were used which included reference strains of all the currently documented serogroups of V. cholerae (O2–O138 and O140–O193) [18]and other strains listed in Table 1. All the strains were cultured in LB broth or LB agar; the broth and the agar medium were supplemented with 1% NaCl for culturing V. parahaemolyticus strains.

Table 1.  Analysis of the specificity of the multiplex PCR using a variety of strains belonging to the families Vibrionaceae and Enterobacteriaceae
SpeciesNumber of strains testedNumber of strains positive for
  O1-rfbO139-rfbctxA
  1. aReference strains representing serogroups O2–O138 and O140–O193.

  2. bSerogroups O37, O105, O141 and O191.

Vibrio cholerae O11515011
Vibrio cholerae O1393603636
Vibrio choleraea191004b
Vibrio parahaemolyticus2000
Salmonella typhi1000
Salmonella paratyphi A1000
Shigella dysenteriae1000
Shigella sonnei1000
Enteroinvasive E. coli1000
Enterotoxigenic E. coli1000
Enterohemorrhagic E. coli 20000
Klebsiella oxytoca2000
Citrobacter freundii1000

2.2DNA preparation

The strains were grown in LB broth at 37°C. After overnight growth, the culture was diluted 10-fold in 10 mM Tris-HCl (pH 8.0) buffer containing 1 mM ethylenediaminetetraacetic acid disodium salt (EDTA), and boiled for 10 min. After centrifugation at 12 000×g for 10 min at 4°C, the supernatant was used as the template for PCR. For sensitivity determination, the bacterial cultures were grown to an optical density of 0.6 at 600 nm, and were serially diluted in sterile PBS. Quantification of V. cholerae O139 and O1 in the diluted sample was performed by determining the viable colony count on LB agar.

2.3PCR primers

The V. cholerae O139-rfb specific primers used were O139F2 [5′-AGCCTCTTTATTACGGGTGG-3′], sense strand and O139R2 [5′-GTCAAACCCGATCGTAAAGG-3′], antisense strand, while the V. cholerae O1-rfb specific primers were O1F2-1 [5′-GTTTCACTGAACAGATGGG-3′], sense strand and O1R2-2 [5′-GGTCATCTGTAAGTACAAC-3′], antisense strand. The cholera toxin gene (ctxA) primers were VCT1 [5′- ACAGAGTGAGTACTTTGACC-3′], sense strand and VCT2 [5′- ATACCATCCATATATTTGGGAG-3′], antisense strand. The O1 and O139 rfb primers were synthesized on an Oligo 1000M DNA synthesizer (Beckman, USA) and purified by reversed-phase HPLC while the ctxA primers were purchased from Shimadzu Co., Kyoto, Japan.

2.4PCR protocol

Amplification with the three primer pairs was performed simultaneously in 0.2-ml microcentrifuge tubes. Samples (3 μl) were added to the PCR mixture in a 30-μl volume of a final mixture containing 0.21 mM of each dNTP mixture, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 0.27 μM O139 primer pair, 0.5 μM O1 primer pair, 0.17 μM ctxA primer pair, and 0.75 units of Taq polymerase (Takara, Kyoto, Japan). The amplification condition used was 5 min at 94°C for initial denaturation of DNA and 35 cycles, each consisting of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, with a final round of extension for 7 min at 72°C in a DNA Robo Cycler Gradient Temperature Cycler (Stratagene, La Jolla, CA, USA). After amplification, 6 μl of each reaction mixture was separated by electrophoresis on a 3% agarose gel and the amplified gene products were visualized under UV light after staining with ethidium bromide.

2.5Evaluation of the multiplex PCR

A hospital based evaluation of the multiplex PCR study was conducted to determine the sensitivity and specificity of the test as compared to the conventional stool culture technique. The evaluation was conducted between March 19 and May 8, 1997 and constituted the period when V. cholerae O139 cases of cholera dominated over O1 cases in Calcutta. A total of 121 stool samples collected from patients suffering from acute secretory diarrhea admitted to the Infectious Diseases Hospital (IDH), Calcutta, constituted the source of samples. Stool samples were collected in sterile McCartney bottles using sterile catheters and were brought to the laboratory within 1 h of collection.

Thiosulfate-citrate bile salts-sucrose agar (TCBS; Eiken, Tokyo, Japan) was used as the selective medium for the isolation of V. cholerae. About 250 μl of the neat stool sample or the rectal swab was also introduced into 2 ml of alkaline peptone water (Bactopeptone 1%, NaCl 1%; pH 8.5) used as an enrichment medium and incubated at 37°C for 18 h. The inoculated plates (directly from stool samples or from enrichment medium) were incubated at 37°C for 18–24 h and subsequently examined for the growth of typical cholera-like colonies. A multitest medium was used for presumptive identification of V. cholerae[19]. All isolates were subsequently examined for the oxidase reaction and the identity of V. cholerae O1 was thereafter confirmed by serogrouping, using growth from the multitest medium, with polyvalent O1 and monospecific Inaba and Ogawa antisera. V. cholerae strains which did not agglutinate with the O1 antiserum were checked with monoclonal O139 antiserum prepared as previously reported [20]. V. cholerae strains which did not agglutinate with either O1 or O139 antisera were assumed to belong to the non-O1 non-O139 serogroups and these strains were further serogrouped by the somatic O antigen serogrouping scheme of V. cholerae[18].

An aliquot (250 μl) of each stool sample was taken in an Eppendorf tube and heated in a boiling water bath for 10 min. For PCR assay, the stool samples were then diluted using Tris-EDTA (pH 8.0) as diluent. Bovine serum albumin (BSA) was added at a concentration of 400 ng μl−1 to the stool samples to circumvent the problem of inhibitors of the PCR reaction usually present in stools [21]. Initially neat, 1:10, 1:20, 1:50 and 1:100 dilutions of the samples were used for PCR assay. The samples were then centrifuged at 2000 rpm for 2 min to remove the debris and the supernatant was transferred to a new Eppendorf tube and this was used as the template DNA. It was found that 1:10 dilution produced an intense band in all cases. However, in a few samples, the intensity of the band was stronger when the neat sample was used. The other dilutions exhibited bands of either the same intensity or lower intensity as compared to the 1:10 dilution. Therefore, neat and 1:10 dilution of stool samples were used for evaluation of the PCR assay. The PCR protocol was as described above except that an automated thermal cycler (Biometra, Göttingen, Germany) was used.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

3.1Sensitivity and specificity of the multiplex PCR assay

The V. cholerae O1 and O139 specific primers were designed from specific regions of the rfb cluster of the two serogroups [10]. The O139 specific primers yielded a 449-bp fragment while the O1 specific primers yielded a 192-bp fragment. The ctxA primers were designed from nucleotides 132–152 and 417–440 of the ctxA gene and yielded a 308-bp fragment. The upstream and downstream primers of the ctxA varied from the corresponding heat labile enterotoxin of enterotoxigenic Escherichia coli by 6 bp and 3 bp, respectively.

Using the three sets of primers listed above, a multiplex PCR format was developed to simultaneously identify toxigenic V. cholerae belonging to either the O1 or O139 serogroup. The specificity (amplification of a single specific sequence) of the multiplex PCR assay is shown in Fig. 1 with V. cholerae O139 strain MO45 yielding both the O139 rfb and ctxA specific amplicons while V. cholerae O1 strain 35A3 yielding both O1 rfb and ctxA specific amplicons.

image

Figure 1. Detection of V. cholerae O1 and O139 rfb-specific genes and of the ctxA gene by multiplex PCR. The PCR were performed as described in the text with O1 primers (lane 1), O139 primers (lane 2), ctxA primers (lane 3), mixed primers (lane 4), no primers, template only (lane 5) and no template, mixed primers only (lane C). The sizes of the amplified fragments were confirmed by the position of the bands relative to those of the molecular size markers (HaeIII digest of φX174 DNA, lane M).

Download figure to PowerPoint

Sensitivity (amount of amplified sequence) of the multiplex PCR assay was examined by inversely varying the amount of template DNA of both O1 and O139. The intensity of the amplified band, as shown in Fig. 2, varied with the amount of corresponding template DNA. The minimum detecting capability of the multiplex PCR was also established by examining serially diluted broth cultures of V. cholerae O1 and O139. The detection limits of V. cholerae O1 and O139 were 65 cfu and 200 cfu per assay, respectively.

image

Figure 2. Analysis of multiplex PCR products obtained by inversely varying the amount of O1 and O139 template DNA. The amounts of template present in each of the initial PCR mixtures were varied by diluting OD 0.6 at 600 nm by 10-fold in PBS. Numbers at the bottom indicate the amounts of template added to the initial PCR mixture with 10=3 μl, 9=2.7 μl, 8=2.4 μl, 7=2.1 μl, 6=1.8 μl, 5=1.5 μl, 4=1.2 μl, 3=0.9 μl, 2=0.6 μl, 1=0.3 μl, 0=0 μl. The sizes of the amplified fragments were confirmed by the position of the bands relative to those of the molecular size markers (HaeIII digest of φX174 DNA).

Download figure to PowerPoint

Further, the specificity of the multiplex PCR assay was determined by examining a large collection of strains belonging to the families Vibrionaceae and Enterobacteriaceae listed in Table 1. All the strains of V. cholerae O1 and O139 yielded the O1 or O139 specific bands and also the ctxA specific amplicons. Among the reference strains of V. cholerae representing serogroups O2–O138 and O140–O193, four strains belonging to serogroups O37, O105, O141, and O191 yielded the ctxA specific amplicon indicating that these non-O1 non-O139 strains had the genetic potential to produce cholera toxin.

3.2Application of the multiplex PCR assay to stool samples

Of the 121 stool samples examined, 38 were positive by the culture method as well as by PCR assay for V. cholerae belonging to either the O1 or O139 serogroup (Table 2). Of the 38 samples positive by the culture method and PCR assay, 34 were positive for serogroup O139 while four were positive for the O1 serogroup. An additional four stool samples which were negative by the culture method but positive by PCR assay belonged to the O139 serogroup. All 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential to produce cholera toxin. If the culture method is taken as the standard, the multiplex PCR was 100% (38/38) sensitive for a positive toxigenic V. cholerae O1/O139 culture and 95.2% (79/83) specific for a negative V. cholerae O1/O139 culture. Further, the multiplex PCR had an accuracy of 96.7% (117/121) with a positive predictive value (38/42) and negative predictive value (79/79) of 90.5% and 100%, respectively.

Table 2.  Results of the evaluation of the multiplex PCR for detection of toxigenic Vibrio cholerae O1 and O139
Culture methodMultiplex PCRbTotal
 PositiveNegative 
  1. aA positive culture test alludes to a positive V. cholerae O1 or O139 isolation by either direct or enrichment culture of stool sample.

  2. bAll multiplex PCR positive stool samples were positive for the ctxA amplicon also.

+a38038
47983
Total4279121

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

The unexpected and explosive entry of the O139 serogroup as another causative serogroup of cholera has complicated the diagnosis of this disease. Current rapid tests are all focused on the O1 serogroup. Against this background we initiated studies to develop a multiplex PCR capable of providing multiple information directly from stool specimens in approximately 5 h. The evaluation of the multiplex PCR as compared to routine stool culture performed in this study showed the multiplex PCR to be an efficient and sensitive method. The multiplex PCR also showed that all the O1 and O139 strains had the genetic potential to produce cholera toxin because of the presence of the ctxA amplicon. The multiplex PCR was further capable of detecting the O139 serogroup in four additional stool samples which were negative by both direct and enrichment culture on TCBS. It is unlikely that the four stool samples which were positive by multiplex PCR but negative by the culture method are false positive because all four stool samples apart from yielding the O139 rfb amplicon were also positive for the ctxA amplicon.

The multiplex PCR developed in this study is extremely versatile because apart from identifying toxigenic V. cholerae O1 and/or O139, this technique has the potential to identify non-toxigenic (non-cholera toxin producing) V. cholerae O1 or O139 and can also identify toxigenic V. cholerae belonging to a serogroup other than O1 or O139. Although the incidence of non-toxigenic V. cholerae O1/O139 or toxigenic V. cholerae non-O1 non-O139 is currently a rarity, the occurrence of cholera outbreaks caused by non-toxigenic V. cholerae O1 is on the increase as exemplified by the recent outbreak in South India [22]. Because of the unavailability of a rapid test which gives quick information on non-toxigenic V. cholerae O1 or O139 or on toxigenic V. cholerae non-O1 non-O139, the occurrence of infections caused by such atypical V. cholerae are invariably missed. However, as with other nucleic acid based assays the significance of detection of toxin genes in the absence of the organism or the gene product must be carefully evaluated [23]. The presence of the ctxA locus does not always suggest the production of active toxin as truncation, point mutation, and misreading of the ctxA gene can result in a ctxA positive, non-toxin producing strain.

Our previous studies on detection of the ctxA gene directly from stool samples of cholera patients have shown that rice-water stools are amenable to PCR analysis without pretreatment to remove substances that are inhibitory to the PCR assay [15, 24]. This study further confirms our previous observation. However, based on our previous and current experience with the PCR assay on stool samples of cholera patients, we recommend the concurrent use of neat and a 10-fold dilution of the stool sample from cholera patients while using the multiplex PCR.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References
  • 1
    World Health Organization (1996) Cholera in 1995. Weekly Epidemiol. Rec. 71, 157–164.
  • 2
    Nair, G.B., Albert, M.J., Shimada, T. and Takeda, Y. (1996) Vibrio cholerae O139 Bengal: the new serogroup causing cholera. Rev. Med. Microbiol. 7, 4351.
  • 3
    Ramamurthy, T., Garg, S., Sharma, R., Bhattacharya, S.K., Nair, G.B., Shimada, T., Takeda, T., Karasawa, T., Kurazono, H., Pal, A. and Takeda, Y. (1993) Emergence of novel strain of Vibrio cholerae with epidemic potential in Southern and Eastern India. Lancet 341, 703704.
  • 4
    Nair, G.B., Ramamurthy, T., Bhattacharya, S.K., Mukhopadhyay, A.K., Garg, S., Bhattacharya, M.K., Takeda, T., Shimada, T., Takeda, Y. and Deb, B.C. (1994) Spread of Vibrio cholerae O139 Bengal in India. J. Infect. Dis. 169, 10291034.
  • 5
    Albert, M.J., Siddique, A.K., Islam, M.S., Faruque, A.S.G., Ansaruzzaman, M., Faruque, S.M. and Sack, R.B. (1993) Large outbreak of clinical cholera due to V. cholerae non-O1 in Bangladesh. Lancet 341, 704.
  • 6
    Chongsa-nguan, M., Chaicumpa, W., Moolasart, P., Kandhasingha, P., Shimada, T., Kurazono, H. and Takeda, Y. (1993) Vibrio cholerae O139 Bengal from patients with cholera-like infection in Bangkok, Thailand. Lancet 342, 430431.
  • 7
    Mitra, R., Basu, A., Dutta, D., Nair, G.B. and Takeda, Y. (1996) Resurgence of Vibrio cholerae O139 Bengal with altered antibiogram in Calcutta, India. Lancet 348, 1181.
  • 8
    Manning, P.A., Stroeher, U.H. and Morona, R. (1994) Molecular basis for O-antigen biosynthesis in Vibrio cholerae O1: Ogawa-Inaba switching. In: Vibrio cholerae and Cholera: Molecular to Global Perspectives (Wachsmuth, K., Blake, P.A. and Olsvik, O., Eds.), pp. 77–94. ASM Press, Washington, DC.
  • 9
    Mooi, F.R. and Bik, E.M. (1997) The evolution of epidemic Vibrio cholerae strains. Trends Microbiol. 5, 161165.
  • 10
    Yamasaki, S., Hoshino, K., Shimizu, T., Garg, S., Shimada, T., Ho, S., Bhadra, R.K., Nair, G.B. and Takeda, Y. (1996) Comparative analysis of the gene responsible for lipopolysaccharide synthesis of Vibrio cholerae O1 and O139 and those of non-O1 non-O139 Vibrio cholerae. Abstr. 32nd Joint Conference U.S.-Japan Cooperative Medical Science Program, Cholera and Related Diarrhoeal Diseases Panel, p. 24.
  • 11
    Jesudasan, M.V., Thangavelu, C.P. and Lalitha, M.K. (1984) Rapid screening of fecal samples for Vibrio cholerae by a coagglutination technique. J. Clin. Microbiol. 19, 712713.
  • 12
    Rahman, M., Sack, D.A., Mahmod, S. and Hossain, A. (1987) Rapid diagnosis of cholera by a coagglutination test using four hour fecal enrichment cultures. J. Clin. Microbiol. 25, 22042206.
  • 13
    Galvez, S., Islam, M.S., Tamplin, M.L. and Bernstein, D. (1992) Development and evaluation of rapid, simple, sensitive, monoclonal antibody-based co-agglutination test for direct detection of Vibrio cholerae O1. FEMS Microbiol. Lett. 76, 215219.
  • 14
    Uesaka, Y., Otsuka, Y., Kashida, M., Oku, Y., Horigome, K., Nair, G.B., Pal, S.C., Yamasaki, S. and Takeda, Y. (1992) Detection of cholera toxin by a highly sensitive bead-enzyme linked immunosorbent assay. Microbiol. Immunol. 36, 4353.
  • 15
    Ramamurthy, T., Pal, A., Bag, P.K., Bhattacharya, S.K., Nair, G.B., Kurazono, H., Yamasaki, S., Takeda, T. and Takeda, Y. (1993) Detection of cholera toxin (CT) gene by the polymerase chain reaction in stool specimens: Comparison with the CT bead enzyme linked immunosorbent assay and with culture methods for laboratory diagnosis of cholera. J. Clin. Microbiol. 31, 30683070.
  • 16
    Hasan, J.A.K., Huq, A., Tamplin, M.L., Siebeling, R.J. and Colwell, R.R. (1994) A novel kit for rapid detection of Vibrio cholerae O1. J. Clin. Microbiol. 32, 249252.
  • 17
    Albert, M.J., Islam, D., Nahar, S., Qadri, F., Falklind, S. and Weintraub, A. (1997) Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR. J. Clin. Microbiol. 35, 16631635.
  • 18
    Shimada, T., Arakawa, E., Itoh, K., Okitsu, T., Matsushima, A., Asai, Y., Yamai, S., Nakazato, T., Nair, G.B., Albert, M.J. and Takeda, Y. (1994) Extended serotyping scheme for Vibrio cholerae. Curr. Microbiol. 28, 175178.
  • 19
    Nair, G.B., Misra, S., Bhadra, R.K. and Pal, S.C. (1987) Evaluation of the multitest medium for rapid presumptive identification of Vibrio cholerae from environmental sources. Appl. Environ. Microbiol. 53, 12031205.
  • 20
    Garg, S., Ramamurthy, T., Mukhopadhyay, A.K., Deb, B.C., Nair, G.B., Shimada, T., Takeda, T., Huq, A., Colwell, R.R. and Takeda, Y. (1994) Production and cross reactivity patterns of a panel of high affinity monoclonal antibodies to Vibrio cholerae O139 Bengal. FEMS Immunol. Med. Microbiol. 8, 293298.
  • 21
    Kreader, C.A. (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 11021106.
  • 22
    Saha, P.K., Koley, H., Mukhopadhyay, A.K., Bhattacharya, S.K., Nair, G.B., Ramakrishna, B.S., Krishnan, S., Takeda, T. and Takeda, Y. (1996) Nontoxigenic Vibrio cholerae O1 serotype Inaba biotype El Tor associated with a cluster of cases of cholera in Southern India. J. Clin. Microbiol. 34, 11141117.
  • 23
    Popovic, T., Fields, P.I. and Olsvik, O. (1994) Detection of cholera toxin genes. In: Vibrio cholerae and Cholera: Molecular to Global Perspectives (Wachsmuth, K., Blake, P.A. and Olsvik, O., Eds.), pp. 44–52. ASM Press, Washington, DC.
  • 24
    Shirai, H., Nishibuchi, M., Ramamurthy, T., Bhattacharya, S.K., Pal, S.C. and Takeda, Y. (1991) Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae. J. Clin. Microbiol. 29, 25172521.