JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1997, p. 714–718 0095-1137/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 35, No. 3
An Enzyme-Linked Immunosorbent Assay To Detect PCR Products of the rfbS Gene from Serogroup D Salmonellae: a Rapid Screening Prototype JOHN M. LUK,1* URIRAT KONGMUANG,1† RAYMOND S. W. TSANG,2
AND
ALF A. LINDBERG1‡
Department of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital F82, S-141 86 Huddinge, Sweden,1 and National Laboratory for Enteric Pathogens, Bureau of Microbiology, LCDC, Ottawa, Ontario, Canada2 Received 15 July 1996/Returned for modification 12 November 1996/Accepted 16 December 1996
We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody, MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 82 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 6 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 6 0.08; n 5 63) and non-Salmonella isolates (A405, 0.16 6 0.04; n 5 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible. ing of the related rfb genes from major Salmonella serogroups have revealed substantial sequence variations among the Oantigen genes (14, 28). On the basis of these distinctive molecular fingerprints, a panel of specific primers was designed to differentiate major serogroups of Salmonella (serogroups A, B, C2, and D) in a PCR (16). The rfb primers were shown to be highly specific for these major serogroups, and even in the presence of fecal materials, the PCR specificity remained after appropriate pretreatment of the samples (12). However, the current PCR procedures followed by gel electrophoresis and Southern hybridization are limited in the number of samples that can be conveniently analyzed during one electrophoresis run. To facilitate the detection process for large-scale screening, we developed and describe here a simple and rapid prototype method for the semiquantitative analysis of PCR products that can be carried out in a microtiter plate. This method is developed according to the format of an enzyme-linked immunosorbent assay (ELISA), having great potential for future automation. Biotinylated primers were used together with digoxigenin (DIG)-119-dUTP, which can be incorporated into the amplified PCR product. The amplified DNA (amplicon) was captured onto the solid phase via the avidin-biotin interaction by using a streptavidin-coated microtiter plate (8, 29) and was then detected with an enzyme-conjugated anti-digoxigenin antibody (Fig. 1). Performance of the PCR and DIGbased ELISA (PCR DIG-ELISA) was evaluated with clinical specimens and was compared with the culture technique for the detection of serogroup D salmonellae in stool specimens.
Lipopolysaccharide (LPS) or endotoxin is a pyrogenic glycolipid component of the gram-negative bacterial cell wall and plays an essential role in the pathogenesis of septic shock and systemic inflammatory reaction syndrome (20, 24). Structurally, the Salmonella LPS is composed of the O-antigenic polysaccharide chain, the core oligosaccharide domain, and lipid A. While lipid A is highly conserved in many genera of gramnegative bacteria, the O-polysaccharide chains are antigenically extremely variable. More than 2,000 serotypes have been identified for the Salmonella species according to the Kauffmann-White serologic typing scheme (10, 23). Although Salmonella remains an important group of enteric bacterial pathogens causing gastroenteritis (food poisoning) and enteric fever in humans, the laboratory procedures for the detection and/or identification of Salmonella by conventional culture methods are still laborious and time-consuming (normally taking 3 to 4 days to obtain a definitive result) (10, 21). PCR has proven to be an indispensable tool for the detection of infectious agents in the clinical microbiology laboratory (2, 3, 7, 9, 13, 22, 30). In attempts to develop alternative diagnostic methods for salmonellae, we recently described a PCR assay for the selective amplification of the LPS rfb genes of the major serogroups of Salmonella (16). The rfb gene clusters, which are responsible for biosynthesis of the O antigens of Salmonella LPS, have been targeted as molecular markers for these organisms. Cloning and sequenc* Corresponding author. Mailing address: Department of Surgery, University of Hong Kong, Queen Mary Hospital, Hong Kong. Fax: 852-2872-8425. E-mail:
[email protected]. † Present address: Department of Pathology, Faculty of Medicine, Prince of Songkla University, Hat-yai 90112, Thailand. ‡ Present address: Pasteur Me´rieux Connaught, 69280 Marcy L’Etoile, France.
MATERIALS AND METHODS Bacterial strains and samples. The salmonellae and other members of the family Enterobacteriaceae examined in this study were clinical isolates obtained from the culture collection at the Department of Clinical Bacteriology, Huddinge University Hospital, Stockholm, Sweden. Isolation and biochemical identification of the salmonellae were performed by standard procedures as described by
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FIG. 1. Schematic diagram of the PCR DIG-ELISA on a microtiter plate. dNTP, deoxynucleoside triphosphate.
Ewing (10) and Murray et al. (21). Serological typing of the salmonellae was based on the Kauffmann-White scheme (23) with anti-H and anti-O sera. In order to evaluate the reliability of the DIG-ELISA PCR method, the three types of samples described below were examined. (i) Pure culture suspension. Pure cultures of an S. enteritidis (serogroup D) and an S. typhimurium (serogroup B) strain isolated from the stools of patients with diarrhea were used to determine the sensitivity and specificity of the method. Serial dilutions of pure broth cultures of these two Salmonella strains were prepared in sterile phosphate-buffered saline (PBS) for colony counting by the standard spread-plate technique and for heat extraction at 958C for 10 min to provide DNA templates for the PCRs. (ii) Artificial Salmonella-spiked stool samples. Known numbers (predetermined by the standard viable plate count technique) of S. enteritidis isolates in pure culture (same strain described above) were added to 0.2 g of normal stool samples (pretested to be free of Salmonella by both conventional culture and PCR assays). The Salmonella-spiked samples were then processed by the immunomagnetic bead separation (IMS) technique by our previously described method (17) to study the effect of interfering material. Purified monoclonal antibodies specific for the Salmonella O9 antigen (monoclonal antibody MATyO9) (19) were immobilized on Dynabeads (M-280; Dynal AS, Oslo, Norway) precoated with sheep anti-mouse immunoglobulin G antibodies. Thus, the MATy-O9-coated Dynabeads were used to capture serogroup D salmonellae and separate them from the other microorganisms in the stool specimens. After two washes with PBS, the captured Salmonella bacteria on the beads were subjected to DNA extraction by a brief heat treatment (958C, 10 min). (iii) Diarrheal stool samples. In order to evaluate the usefulness of the PCR DIG-ELISA method for routine application in a diagnostic bacteriology laboratory, stool specimens from patients with diarrhea (submitted for Salmonella testing by physicians) were inoculated into enrichment Rappaport broth (Oxford Ltd., Hampshire, United Kingdom) and were incubated overnight at 378C. After low-speed centrifugation to remove stool particles, the bacteria were pelleted by high-speed centrifugation and were washed twice with PBS before heat extraction (958C, 10 min) to provide the DNA templates. PCR primers and conditions. A pair of 18-mer nucleotide primers (59-TCA CGA CTT ACA TCC TAC-39 and 59-CTG CTA TAT CAG CAC AAC-39) targeted to the rfbS gene, which is responsible for the biosynthesis of the ADPparatose in serogroup A and D salmonellae, was synthesized, and the primers were biotinylated at their 59 ends (Scandinavian Gene Synthesis AB, Ko ¨ping, Sweden). The specificities and physical properties of these primers were described previously (16). The PCR conditions were essentially as described before (12, 16), with minor modifications. Ten microliters of sample DNA template was diluted with an equal volume of Milli Q water and was then added to 80 ml of a PCR mixture (0.2 mg of each of the biotinylated primers, 200 mM [each] dATP, dCTP, and dGTP plus 150 mM dTTP and 50 mM DIG–119-dUTP [Boehringer Mannheim, Indianapolis, Ind.], 2 U of Taq DNA polymerase [Perkin-Elmer, Norwalk, Conn.],
0.002% [wt/vol] gelatin, 50 mM KCl, and 1.5 mM MgCl2 in 20 mM Tris-HCl buffer [pH 8.0]). The samples were then overlaid with 100 ml of sterile mineral oil (molecular grade; Perkin-Elmer). The PCR was taken through 30 cycles in a DNA thermal cycler (Perkin-Elmer); each cycle consisted of 948C for 1 min (denaturation), 458C for 1 min (annealing), and 728C for 2 min (extension). A buffer control to which no template DNA sample was added was included in each PCR assay as surveillance for interior contamination. Detection of PCR products. (i) DIG-ELISA. Enzyme immunoassay microplates (96-well plates; Corning Costar, Cambridge, Mass.) were coated with 100 ml of streptavidin (various concentrations from 1 to 10 mg/ml) per well in 0.01 M PBS (pH 7.4) at 48C overnight. Unsaturated binding sites were blocked with 1% (wt/vol) bovine serum albumin in PBS, and then the plates were washed with PBS. Samples (10 ml of PCR product diluted in 100 ml of PBS) were added in duplicate to the streptavidin-coated wells, and the plates were incubated at 378C for 1 h. After three washes with PBS, 100 ml of a 1:2,000 dilution of anti-DIG Fab–alkaline phosphatase conjugate (Boehringer Mannheim) was added to each well for incubation at 378C for 1 h. After four more washes with PBS containing 0.05% Tween 20, substrate solution (1 mg of p-nitrophenyl phosphate per ml in 0.05 M diethanolamine-HCl [pH 9.8]) was added and the color was allowed to develop at 378C for 30 minutes. ELISA absorbance was measured at 405 nm with a Dynatech microplate reader. (ii) Agarose gel electrophoresis. Ten microliters of each PCR-amplified sample was loaded onto each lane on a 1.5% agarose gel. Electrophoresis of the amplified DNA fragment was carried out in TBE (Tris-borate-EDTA) buffer at a constant voltage of 120 V for 1 h. HaeIII-digested fX174 DNA was used as a marker for determining the sizes of the amplified DNA bands. Afterward, the gel was stained with ethidium bromide and was visualized by UV illumination by standard techniques (26).
RESULTS AND DISCUSSION In attempts to improve the sensitivity of the PCR assay for the detection of salmonellae, we developed a DIG-ELISA to further enhance the detection level of amplified PCR products. While most of the PCR conditions were already established, several optimization procedures for the DIG-ELISA were studied: (i) various coating concentrations of streptavidin were used, (ii) various amounts of PCR products were added to the streptavidin-coated microplate, and (iii) excess free primers and nucleotides were removed from the PCR products by a chromatography method (Wizard DNA Clean-up system; Promega, Madison, Wis.) before addition to the streptavidin plate.
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FIG. 2. Detection sensitivity of PCR, followed by agarose gel electrophoresis (A) and DIG-ELISA (B), for pure cultures of S. enteritidis. The negative control was 107 CFU of S. typhimurium (serogroup B).
The optimal amount of streptavidin coating was found to be 5 mg/ml (0.5 mg per well). Increasing the coating concentration from 1 to 10 mg/ml showed a dose-dependent response, with 5 mg of streptavidin per ml appearing to be optimal for capture of the biotinylated PCR product. Further increases in the coating streptavidin concentration did not markedly increase the signal in the DIG-ELISA. We also found that 10 ml of the PCR mixture products (diluted in 100 ml of PBS) was sufficient for the DIG-ELISA and that overloading the samples onto the plate actually lowered the sensitivity of the assay. Conceivably, there is competition between the excess primers and biotinylated PCR products for binding to the streptavidin solid phase. To this end, we used the resin chromatography method to remove unwanted free primers and nucleotides from the amplified DNA. However, the procedure was relatively cumbersome and did not result in any measurable enhancement of the detection sensitivity in the DIG-ELISA. It is thus considered unsuitable for screening purposes. To evaluate the reliability and applicability of the DIGELISA method for large-scale PCR screening of serogroup D salmonellae (a major group of Salmonella pathogens in humans), we compared the DIG-ELISA technique with the standard agarose gel electrophoresis procedure for testing samples from pure cultures of Salmonella, normal stool samples artificially spiked with salmonellae, and stool samples from patients with diarrhea. With a pure culture of S. enteritidis (serogroup D), both DIG-ELISA and gel electrophoresis were able to detect the amplified gene products from #10 bacteria (Fig. 2). The PCR assay was negative with 107 CFU of S. typhimurium (serogroup B), showing the assay specificity for serogroup D salmonellae. An obligatory feature of any assay for the detection of infectious agents is satisfactory performance with clinical specimens
J. CLIN. MICROBIOL.
(in this case, stool specimens). Much higher rates of inhibition and lower sensitivity would be expected if the method were applied directly to stool specimens. Due to the presence of endogenous interfering materials in feces (e.g., bile salts and bilirubin), a 102- to 103-fold reduction in the sensitivity of the PCR assay was demonstrated in earlier studies (12, 15). The IMS technique and enrichment cultivation of target bacteria in samples have proven to be effective approaches to circumventing these inhibition and/or interference problems. Thus, we tested these stool-processing methods and examined the sensitivity and specificity of the PCR-ELISA with normal stool specimens artificially spiked with known numbers of S. enteritidis and S. typhimurium. For IMS, Dynabeads coated with an O9-antigen-specific monoclonal antibody (MATy-O9) were used to capture the serogroup D salmonellae directly in stool samples before the PCR. About 10 to 102 CFU of S. enteritidis (O:9,12) in the samples generated a positive signal, as analyzed by DIGELISA (Fig. 3B), whereas a minimum of 103 CFU was required before a demonstrable amplified DNA band could be visualized in the ethidium bromide-stained agarose gel (Fig. 3A). Several stool samples spiked with 10 CFU of S. enteritidis, followed by IMS and PCR, were negative when analyzed by gel electrophoresis, but were weakly positive by DIG-ELISA (data not shown). Although the IMS technique has its advantages in the removal of fecal substances which inhibit PCR, it is apparently not a cost-effective approach for detecting enteric pathogens like salmonellae that are easily and rapidly grown in culture (15). Since enrichment broth cultivation of fecal samples from patients with diarrhea is a routine practice, we accommodated
FIG. 3. Results for artificially spiked stool samples of S. enteritidis. Salmonella bacteria were first isolated with monoclonal antibody MATy-O9 (serogroup D specific)-coated Dynabeads by the IMS technique, followed by PCR. The amplified DNA was determined by agarose gel electrophoresis (A) and DIGELISA (B). The negative control was 107 CFU of S. typhimurium (serogroup B).
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TABLE 1. Comparison of three different methods for detection of salmonellae in diarrheal stool samples No. of positive samples Bacterium identifieda
PCR followed by:
Culture technique
DIG-ELISA
Gel electrophoresis
Salmonella isolates Serogroup B Serogroup C1 Serogroup C2 Serogroup D Serogroup E
31 15 12 82 5
0 0 0 82 0
0 0 0 82 0
Non-Salmonella isolates
58
0
0
a
A total of 203 clinical isolates (145 salmonellae and 58 other species) were identified from the stool samples. After overnight enrichment in Rappaport broth, 1 ml of the broth culture was subjected to PCR, followed by DIG-ELISA and agarose gel electrophoresis.
the PCR ELISA following the enrichment cultivation procedure (in lieu of IMS), as would occur in a clinical microbiology laboratory. After 2 to 4 h of cultivation, about 102 to 103 bacteria in the initial inoculum were detectable, and as few as 10 salmonellae could be detected with prolonged (12 to 16 h) incubation. With an overnight enrichment step, it is likely that all or almost all samples will yield enough salmonellae to allow detection by the PCR DIG-ELISA. Presumably, the overnight enrichment in broth could have led to a relative overrepresentation of the bacterial targets, thus resulting in a relatively clean study with few problems with inhibition. In order to determine the performance of the assay with clinical specimens, 203 stool samples from patients with diarrhea were tested by the PCR ELISA in parallel with the conventional culture method for the identification of serogroup D salmonellae. After overnight enrichment cultivation in Rappaport broth, a portion (ca. 1 ml) of the broth culture was taken for DNA extraction, followed by PCR with the rfbS primers as described in the Materials and Methods. On the same day, the PCR products were analyzed by both gel electrophoresis and DIG-ELISA. The results are summarized in Table 1. Eightytwo samples that yielded serogroup D salmonellae by the conventional culture technique also gave a positive result on the agarose gel and in the DIG-ELISA after PCR amplification. A clear-cut positive colorimetric signal (ELISA optical density of $1.0; A405, 2.54 6 0.74) was obtained with the PCR products from these positive samples. On the contrary, 63 samples identified by culture as non-group D Salmonella did not give positive PCR results, regardless of which detection method was used, agarose gel electrophoresis or DIG-ELISA (A405, 0.26 6 0.08). Likewise, the 58 samples which did not yield salmonellae in culture were also negative by PCR (DIG-ELISA A405, 0.16 6 0.04). Thus, the results demonstrated a strong correlation between the standard culture technique and PCR, as analyzed by either electrophoresis or the ELISA method. In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), in addition to brief enrichment cultivation of samples (ranging from 4 to 16 h). One shortcoming of the DIG-ELISA for analysis of PCR products is the inability of this method to confirm the identity of the amplified DNA. In this regard, oligonucleotide capture formats for the detection of PCR products on microplates are in common clinical use (4–6, 11, 25) and, indeed, may have the advantage of achieving higher-stringency specificity. This approach usually requires a hybridization step with a biotinylated
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oligonucleotide probe immobilized onto microplate wells with streptavidin. Similarly, detection of the PCR products involves the use of anti-DIG antibody and, in some cases, a specific antibody against the antigen-hybridized DNA. In this study, the accuracy of the assay heavily relied on the exquisite specificity of the rfbS primers designed for the study. These primers have been shown extensively to be specific for Salmonella serogroups A and D (12, 16; unpublished data). Although the rfbS gene is expressed in Salmonella serogroups A and D, there are only four serotypes of A salmonellae (23). Among them, S. paratyphi A, which causes paratyphoid fever, is the most commonly encountered serotype (1). However, it is rarely isolated in Sweden. The immunomagnetic separation technique with O2-specific (serogroup A) and O9-specific (serogroup D) monoclonal antibodies (18, 19) accomplishes the desired differentiation between these two serogroups. The advantage of the DIG-ELISA is the simplicity of the method, together with the solid-phase 96-well (or even 384well) microplate format, which allows many samples to be analyzed at one time. Automation of the PCR and ELISA procedures with robotic equipment also makes large-scale screening of samples possible. Examples of potential applications are rapid identification of infectious agents in areas experiencing outbreaks and epidemics, as well as detection of food-borne pathogens in food processing and assembly plants according to the hazard analysis and critical control point programs (27). It is conceivable that hundreds of samples could be screened and proven to be free of salmonellae within 48 h, and in the meantime, for those positive samples that are found, another day or two would be needed for serotype identification. While the PCR ELISA offers a rapid screening test, it is important that any samples showing positive results be cultured to yield the putative Salmonella organism for further studies, including identity check, serotyping, and antibiotic susceptibility testing. ACKNOWLEDGMENTS This work was supported by grant 16X-656 from the Swedish Medical Research Council (to A.A.L.). We thank Peter R. Reeves of Sydney University for designing the rfbS primer sequences and Jon A. Jonasson of Huddinge Hospital and W. C. Yam of Hong Kong University for helpful discussions. REFERENCES 1. Bannister, B. A. 1983. Infectious diseases, p. 84–90. Bailliere Tindall, London, United Kingdom. 2. Bobo, L., F. Coutlee, R. H. Yolken, T. Quinn, and R. Viscidi. 1990. Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay. J. Clin. Microbiol. 28:1968–1973. 3. Butcher, A., and J. Spadoro. 1992. Using PCR for detection of HIV-1 infection. Clin. Immunol. Newsl. 12:73–76. 4. Chevrier, D., M. Y. Popoff, M. P. Dion, D. Hermant, and J. L. Guesdon. 1995. Rapid detection of Salmonella subspecies I by PCR combined with nonradioactive hybridization using covalently immobilized oligonucleotide on a microplate. FEMS Immunol. Med. Microbiol. 10:245–252. 5. Coutlee, F., O. Saint Antoine, C. Olivier, H. Voyer, A. Kessous-Elbaz, F. Berrada, P. Begin, L. Giroux, and R. Viscidi. 1991. Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA. J. Clin. Microbiol. 29:2461–2467. 6. De Beenhouwer, H., Z. Liang, P. De Rijk, C. van Eekeren, and F. Protaels. 1995. Detection and identification of mycobacteria by DNA amplification and oligonucleotide-specific capture plate hybridization. J. Clin. Microbiol. 33:2994–2998. 7. De Wit, D., L. Steyn, S. Shoemaker, and M. Sogin. 1990. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J. Clin. Microbiol. 28:2437–2441. 8. Diamandis, E. P., and T. K. Christopoulos. 1991. The biotin-(strept) avidin system: principles and applications in biotechnology. Clin. Chem. 37:625– 636. 9. Donofrio, J. C., J. D. Coonrod, J. N. Davidson, and R. F. Betts. 1992.
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