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Clinical Chemistry 50: 2037-2044, 2004. First published September 13, 2004; 10.1373/clinchem.2004.036814
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(Clinical Chemistry. 2004;50:2037-2044.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

Nicole J. Morin1, Zhilong Gong1 and Xing-Fang Lia,1

1 Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

aAddress correspondence to this author at: Department of Public Health Sciences, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta T6G 2G3, Canada. Fax 780-492-7800; e-mail xingfang.li{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens.

Methods: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates.

Results: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis.

Conclusion: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi can cause serious gastrointestinal illness. These bacteria are commonly found in untreated and ineffectively treated sewage and water and pose a threat to public health. Food crops can also become contaminated through the use of untreated water for irrigation(1).

E. coli O157:H7 is an important pathogen causing serious illness in humans(2). It is the leading cause of morbidity and mortality among children in developing countries(3). Although infection is usually self-limiting, the bacterium can cause life-threatening complications, including hemorrhagic colitis and hemolytic uremic syndrome in children and the immunocompromised(4). Salmonella Typhi is responsible for typhoid fever. It is estimated that 16 million new cases of typhoid fever occur each year around the world, mostly in developing countries(5). Infection is characterized by a variety of clinical manifestations ranging from high-grade fever to complications including "encephalopathy, peritonitis, perforation and hemorrhage"(6). V. cholerae O1 is a waterborne pathogen that was responsible for seven recent pandemics(1). Infection is characterized by vomiting and rice-like diarrhea(7).

The ubiquitous and virulent nature of these bacterial pathogens creates a need for specific, sensitive, and rapid detection techniques. Traditional detection techniques involve culturing of the bacteria or use of immunologic assays(8). Culture techniques distinguish bacteria by their ability to grow on differential media. However, positive identification cannot be confirmed by growth on a selective medium alone because many enteric bacteria possess similar biochemical characteristics. A second culture medium is often used for confirmation, and as a result, the analysis time is increased. Because pathogenic bacteria are often present in very low numbers, several plates may be needed to streak the entire sample(3). Moreover, the simultaneous detection and identification of several pathogens by use of one differential medium plate is not possible. As a result, culture methods are time-consuming and tedious(9).

Immunologic methods of detection are usually specific, but cross-reactions can occur(10). In the case of E. coli O157:H7, a monoclonal antibody that is specific for the O157 lipopolysaccharide (LPS)1 is used. Cross-reactivity with other bacteria results from the presence of 4-amino-1,6-dideoxy-D-mannopyranosyl, a constituent sugar of LPS(11). The O157 antibody also binds the LPS of V. cholerae O1 and Salmonella Typhi(11). The potential for false positives dictates use of additional analyses to achieve identification of E. coli O157:H7.

Molecular techniques such as PCR have been invaluable tools for the detection of pathogens(12). When multiple target genes need to be amplified, multiplex PCR (MPCR) can be performed and may provide a simple and sensitive tool for the simultaneous detection of multiple pathogenic bacteria(13). PCR, however, does not provide information related to cell viability because it cannot distinguish the DNA molecules from live and dead cells(14)(15). To improve discrimination power, a preenrichment step is typically performed to allow viable cells to grow and multiply(12). However, the preenrichment process takes 6–48 h to complete, which becomes the bottleneck for the subsequent PCR(16). mRNA can be used as a marker of viability because it is present only in replicating cells and degrades quickly after cell death(17). As a result, mRNA can more accurately reflect the viability of the cells in a sample than can DNA(3). Several studies have investigated mRNA as a viability marker for bacteria(18)(19)(20).

We report here the simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi. MPCR methods for detecting several genes of these bacteria have been reported previously for E. coli O157:H7(9)(10)(13)(16)(21)(22)(23)(24), Salmonella spp(13)(21)(25)(26)(27), and V. cholerae(21)(28)(29). However, none of the reported MPCR methods simultaneously detected all three viable pathogens present in the same sample.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
bacteria and growth conditions
E. coli O157:H7, V. cholerae O1, and Salmonella Typhi were obtained from Dr. Glen Armstrong at the Enterobacteria Culture Collection, Department of Medical Microbiology and Immunology, University of Alberta (Alberta, Canada). All bacteria were previously identified by use of differential media plates. Frozen E. coli O157:H7, Salmonella Typhi, and V. cholerae O1 were streaked on tryptic soy agar plates, tryptic soy agar plates containing 50 mL/L sheep blood, and nutrient agar plates, respectively, and incubated at 37 °C overnight. A negative control consisting of a nonstreaked plate was included. Single colonies were inoculated in 5 mL of broth [tryptic soy broth for E. coli O157:H7, brain heart yeast extract broth for Salmonella Typhi, and nutrient broth for V. cholerae O1] and were incubated along with negative controls (broth alone) at 37 °C with shaking at 125 rpm overnight. The next day, 5 mL of each culture was separately transferred into 50 mL of broth and incubated at 37 °C with shaking at 125 rpm until the culture reached log phase. The cells were harvested in late log phase (A615 = 0.7). To confirm that only viable replicating cells contained transcripts of target RNA, we included a dead cell control from 1 mL of E. coli O157:H7 cells (~107 cells), which was boiled for 10 min at 100 °C. All cell suspensions were pelleted in a Micromax RF centrifuge (Thermo IEC).

cultures of clinical isolates
Nonpathogenic E. coli (ATCC 25922) was used as culture control as well as in the subsequent tests. E. coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica, and Salmonella Typhi were previously obtained from patient stool samples and serotyped by the Provincial Laboratory of Public Health (Microbiology) at the University of Alberta. Each bacterium (1 mL) was grown in LB broth (Fisher Scientific) in a 37 °C incubator overnight. The following day, each bacterial culture (~107 cells) was provided to us for testing. Two groups of bacteria were prepared in sterile phosphate-buffered saline (130 mmol/L NaCl, 10 mmol/L sodium phosphate buffer, pH 7.4). Group 1 contained five bacteria: nonpathogenic E. coli, E. coli O157:H7, L. monocytogenes, Y. enterocolitica, and Salmonella Typhi. No clinical sample of V. cholerae O1 was available for inclusion in this experiment. Group 2 contained only nonpathogenic E. coli, L. monocytogenes, and Y. enterocolitica. This group was included to demonstrate the specificity of the primers for the rfbE and fliC genes of E. coli O157:H7 and the tyv gene of Salmonella Typhi. For each group, five serial dilutions were prepared to obtain samples containing various numbers of bacterial cells. Total RNA was separately extracted from these samples and analyzed by the reverse transcription-multiplex PCR (RT-MPCR) assay.

total rna extraction
Trizol (Invitrogen Life Technologies) was used to extract total RNA from the cell pellets. The concentration and purity of the extracted RNA were measured by use of a Bio-Rad SmartSpec spectrophotometer. According to the recommended Trizol protocol, an A260/A280 ratio >1.6 indicated that the potential protein contamination in the extracted RNA sample was minimal. All RNA extracts were found to have a ratio >1.6. The RNA sample was immediately frozen at –70 °C until use.

primer design
Forward and reverse primer pairs were designed for the E. coli O157:H7 rfbE and fliC genes, the V. cholerae O1 rfbE gene, and the Salmonella Typhi tyv gene, as shown in Table 1 . The primers were analyzed for melting temperature (Tm), hairpin structures, and dimers by use of software available from Integrated DNA Technologies (www.idtdna.com). In addition, the primers were designed to include an EcoRI restriction site at their 5' ends to facilitate cloning. A 4-base GCGC cap was added to the 5' end of the EcoRI restriction site to improve the efficiency of EcoRI restriction digestion. The specificity of each primer was analyzed by use of BLAST, available at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). All primers (desalted) were purchased from Integrated DNA Technologies.


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  		Table 1. Sequences and Tms of primer pairs for E. coli O157:H7, V. cholerae O1, and SalmonellaTyphi.

reverse transcription and pcr
Reverse transcription was performed to convert mRNA to cDNA according to the procedures for the first-strand cDNA (Invitrogen Life Technologies). All reverse transcription and DNase reagents were purchased from Invitrogen Life Technologies. Total RNA was treated with DNase to remove any contaminating DNA and to ensure that only RNA was being amplified. Each 200-µL reaction tube contained 1 µg of total RNA, 250 ng of random primers [oligodeoxyribonucleotide hexamers in the Random Primers DNA Labeling Kit from Invitrogen Life Technologies (cat. no. 18187-013/part no. Y01101)], 1 µL of 10 mM deoxynucleotide triphosphate mixture, 4 µL of 5x first-strand buffer, 2 µL of 0.1 M dithiothreitol, 1 µL of RNaseOUT, 1 µL of SuperScript RNase H– reverse transcriptase (250 U/µL), and sterile water up to a final volume of 20 µL. Reverse transcription was performed in a PTC-1000 programmable thermal controller (MJ Research Inc.). At the initial assay development stage, reverse transcription was carried out for individual target genes in separate tubes. Reverse transcription reaction tubes were prepared for each bacteria gene: E. coli O157:H7 rfbE, E. coli O157:H7 fliC, V. cholerae O1 rfbE, and Salmonella Typhi tyv. Controls were also prepared: a negative control containing all reagents except RNA template and a DNase control for each bacteria containing RNA template and all reagents except SuperScript RNase H– reverse transcriptase. For the blinded tests of mixed RNA samples (Fig. 4 ) and the clinical isolates (Fig. 5 ), reverse transcription was carried out for mixed RNA samples.



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  		Figure 4. Agarose gel electrophoresis analysis of RT-MPCR products from seven unknown RNA samples containing various combinations of the three bacterial RNA.

Lane 1, 100-bp ladder; lane 2, MPCR negative control containing only the MPCR reagents; lane 3, reverse transcription negative control containing only the reverse transcription reagents; lane 4, unknown sample 1 (V. cholerae O1 rfbE gene); lane 5, unknown sample 2 (E. coli O157:H7 rfbE and fliC genes); lane 6, unknown sample 3 (Salmonella Typhi tyv gene); lane 7, unknown sample 4 (V. cholerae O1 rfbE and E. coli O157:H7 rfbE and fliC genes); lane 8, unknown sample 5 (V. cholerae O1 rfbE, Salmonella Typhi tyv, and E. coli O157:H7 rfbE and fliC genes); lane 9, unknown sample 6 (V. cholerae O1 rfbE and Salmonella Typhi tyv genes); lane 10, unknown sample 7 (Salmonella Typhi tyv gene and E. coli O157:H7 rfbE and fliC genes); lane 11, 100-bp ladder



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  		Figure 5. Agarose gel electrophoresis analysis of RT-MPCR products of clinical isolates containing nonpathogenic E. coli, E. coli O157:H7, L. monocytogenes, Y. enterocolitica, and Salmonella Typhi.

Lane 1, 100-bp DNA ladder; lane 2, HindIII marker; lane 3, cells diluted 1:75 000; lane 4, cells diluted 2:7500; lane 5, cells diluted 3:1500; lane 6, cells diluted 4:150; lane 7, cells diluted 5:30.

PCR was performed to test the specificity of all primer pairs before they were used in a MPCR assay. PCR reagents were purchased from Invitrogen Life Technologies. Each 200-µL PCR tube contained 32 µL of sterile water, 2 µL of 10 mM deoxynucleotide triphosphate mixture, 1 µL of 50 mM MgSO4, 5 µL of 10x amplification buffer, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, 1 µL of (2.5 U/µL) Platinum Pfx DNA polymerase, and sterile water up to a final volume of 50 µL. Four PCR reaction tubes were prepared: two for E. coli O157:H7, each containing 5 µL of O157:H7 reverse transcription product as the cDNA template; one for V. cholerae O1, containing 5 µL of V. cholerae reverse transcription product as the cDNA template, and one for Salmonella Typhi, containing 5 µL of reverse transcription product as the Salmonella Typhi cDNA template. Six controls were included: a reverse transcription negative control, containing all PCR reagents including 5 µL of the negative-control reverse transcription product as template; a PCR negative control, containing PCR reagents and no template; and a DNase control for each gene, which contained all of the PCR reagents and 5 µL of the reverse transcription DNase control as template. PCR was performed in an Applied Biosystems GeneAmp PCR System 2700 with an initial denaturation of 3 min at 94 °C; amplification for 45 cycles with denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min. The resulting PCR products were individually cloned into the EcoRI site of a pBK-CMV vector. DNA sequencing was performed at the DNA Services Laboratory, Department of Biochemistry, University of Alberta.

mpcr
MPCR master mixture containing all of the reagents except primers was purchased from Qiagen. MPCR procedures were adapted from the accompanying Qiagen multiplex PCR handbook. Primers were diluted to 2 µM for E. coli O157:H7 rfbE, 2 µM for V. cholerae O1 rfbE, 2 µM for Salmonella Typhi tyv, and 4 µM for E. coli O157:H7 fliC genes in a 10x Tris-EDTA mixture (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). A MPCR reaction mixture was prepared containing 25 µL of Qiagen MPCR master mixture, 5 µL of 10x Tris-EDTA primer mixture, 5 µL from the E. coli O157:H7 reverse transcription reaction, 5 µL from the V. cholerae O1 reverse transcription reaction, and 5 µL from the Salmonella Typhi reverse transcription reaction, with sterile water added to a final volume of 50 µL. Three controls were included to demonstrate that the assay was free of contamination. The first control contained PCR reagents and 5 µL from the reverse transcription negative control as template. The second control contained only the PCR reagents. The third control contained all of the PCR reagents and 5 µL of each reverse transcription DNase control as template. MPCR was performed in an Applied Biosystems GeneAmp PCR system. PCR conditions consisted of initial denaturation at 94 °C for 15 min; amplification for 45 cycles with denaturation at 94 °C for 30 s, annealing at 60 °C for 90 s, and extension at 72 °C for 90 s; and a final extension at 72 °C for 10 min. The MPCR protocol took 3.5 h to complete.

agarose gel electrophoresis
Amplification products (5 µL) were separated and characterized on a 1.5% agarose gel containing 0.5 mg/L ethidium bromide. Ultra PureTM Agarose was purchased from Invitrogen Life Technologies. We prepared 1x Tris–acetic acid–EDTA electrophoresis buffer a 50x stock (242 g of Tris Base, 57 mL of acetic acid, and 100 mL of 0.5 mol/L EDTA, pH 8.0; Fisher Scientific). DNA size markers were included in each gel to determine the molecular sizes of the PCR products. All products were visualized on a Syngene UV illuminator.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
design of primers
In designing primers, we evaluated the sequence similarities of potential primers used in this assay against all known sequences found in GenBank. To achieve specificity, we designed PCR primers that were unique to a single region of each target gene. The sequences and Tms of the forward and reverse primers are summarized in Table 1Up . These primers enabled the generation of different-sized PCR products, allowing easy differentiation of these products by subsequent agarose gel electrophoresis.

Initial attempts to design each primer pair with a similar Tm were not successful because of the potential formation of primer-dimers and hairpin interactions. Consequently, primer pairs with different Tms were used (Table 1Up ). Under optimized conditions, we achieved a similar efficiency for the subsequent MPCR assay using each primer pair.

rt-pcr for evaluation of primers
Each primer pair was initially evaluated separately for specificity and functionality by traditional RT-PCR. As shown in Fig. 1 , all four target genes were successfully amplified. They include a 419-bp region of the V. cholerae O1 rfbE gene, a 319-bp region of the Salmonella Typhi tyv gene, a 239-bp region of the E. coli O157:H7 rfbE gene, and a 179-bp region of the E. coli O157:H7 fliC gene.



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  		Figure 1. Agarose gel electrophoresis of RT-PCR products of the rfbE gene of V. cholerae O1, the tyv gene of Salmonella Typhi, and the rfbE and fliC genes of E. coli O157:H7.

Lane 1, 100-bp DNA ladder; lane 2, negative control containing only PCR reagents with no template; lane 3, negative control containing reverse transcription and PCR reagents with no template; lane 4, 419-bp product of the rfbE gene of V. cholerae O1; lane 5, 329-bp product of the tyv gene of Salmonella Typhi; lane 6, 239-bp product of the rfbE gene of E. coli O157:H7; lane 7, 179-bp product of the fliC gene of E. coli O157:H7.

To confirm the identity of the PCR products, they were sequenced after cloning into a pBK-CMV vector. Sequencing results confirmed that the primers specifically amplified the intended gene sequences (data not shown).

rt-mpcr
To develop a MPCR assay, we further confirmed the specificity and functionality of all primer pairs, using this format. In addition, we optimized the MPCR conditions, including annealing temperature, concentration of the starting material (RNA), and primers. We found that an annealing temperature of 60 °C and 1 µg of template (1 µg of total RNA from each bacterium) were optimum. The optimum primer concentrations were 4 µM for fliC of E. coli O157:H7 and 2 µM each for rfbE of E. coli O157:H7, rfbE of V. cholerae O1, and tyv of Salmonella Typhi.

The results from the agarose gel electrophoresis analysis of the MPCR products obtained with the optimized conditions are shown in Fig. 2 . Four bands corresponding to the targets were clearly detected. These results demonstrate that the four sets of primers maintain their specificity in the MPCR format and that they can be applied to the simultaneous detection of E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.



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  		Figure 2. MPCR of the rfbE gene of V. cholerae O1, the tyv gene of Salmonella Typhi, and the rfbE and fliC genes of E. coli O157:H7

Lane 1, 100-bp DNA ladder; lane 2, Lambda DNA/HindIII fragments; lane 3, negative control containing reverse transcription and PCR reagents with no template; lane 4, negative control containing PCR reagents with no template; lane 5, MPCR products, showing the presence of the rfbE (419 bp) gene of V. cholerae O1, the tyv (329 bp) gene of Salmonella Typhi, and rfbE (239 bp) and fliC (179 bp) genes of E. coli O157:H7.

detection limit
To examine the limit of detection, we used various concentrations of total RNA (100, 50, 20, and 5 nM) to perform reverse transcription and MPCR. As shown in Fig. 3 , all four target genes were clearly detectable at 100 nM and 50 nM of total RNA (lanes 2 and 3). Although rfbE of E. coli O157:H7, rfbE of V. cholerae O1, and tyv of Salmonella Typhi were detected at concentrations as low as 5 nM, the fliC gene of E. coli O157:H7 was not visible at concentrations <50 nM. Thus, the minimum RNA concentration is 50 nM to confirm the presence of all three pathogens.



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  		Figure 3. Agarose gel electrophoresis of MPCR products obtained from various amounts of total RNA (5–100 nM).

Lane 1, 100-bp DNA ladder; lane 2, 100 nM total RNA; lane 3, 50 nM total RNA; lane 4, 20 nM total RNA; lane 5, 5 nM total RNA.

specificity
The specificity of the RT-MPCR assay was confirmed with seven unknown RNA samples. All RNA samples were prepared in a blinded manner by a different researcher. These samples were randomly assigned a number from 1 to 7. The results of gel electrophoresis analysis of the RT-MPCR products of the seven unknown samples are shown in Fig. 4Up . All seven samples were positively identified to contain the correct combination of these three bacteria. The identification of the seven samples was 100% correct. This demonstrates the potential application of the assay to accurately identify all four genes in unknown samples.

analysis of clinical isolates
To further test whether other bacteria interfere with the detection and identification of the target E. coli O157:H7 and Salmonella Typhi, we used this technique to analyze several bacterial mixtures that were prepared by mixing the clinical isolates obtained from patient stool samples. Two groups of bacteria were used to prepare the mixture, but no clinical sample of V. cholerae O1 was available for inclusion in this test. Group 1 mixture contained five bacteria: E. coli O157:H7, Salmonella Typhi, L. monocytogenes, Y. enterocolitica, and a nonpathogenic E. coli strain (ATCC 25922). Group 2 contained only the nonpathogenic E. coli, L. monocytogenes, and Y. enterocolitica. Samples containing various numbers of bacterial cells from these two groups were prepared by serial dilution. The RNA from these bacterial mixtures was extracted and subjected to reverse transcription and MPCR. The products were analyzed by gel electrophoresis. The results from samples containing 30–75 000 cells of the group 1 mixture of bacteria are shown in Fig. 5Up . The target bands of 319, 239, and 179 bp are clearly visible, corresponding to the tyv gene of Salmonella Typhi, and the rfbE and fliC genes of E. coli O157:H7, respectively. This demonstrates that the RT-MPCR specifically detects the target genes and that there is no interference from other bacteria. Therefore, the assay can simultaneously detect E. coli O157:H7 and Salmonella Typhi in mixed bacterial samples without interference.

No band was observed in seven samples containing 30–75 000 cells of nonpathogenic E. coli, L. monocytogenes, and Y. enterocolitica. Thus, no false positives resulted from bacterial samples that did not contain the targets.

The sensitivity of the assay was evaluated by use of the serially diluted bacterial samples. As shown in Fig. 5Up , the rfbE and fliC genes of E. coli O157:H7 and the tyv gene of Salmonella Typhi were visible in all dilutions from 75 000 to 30 cells. The RT-MPCR assay thus is capable of simultaneously detecting ~30 cells of Salmonella Typhi and E. coli O157:H7.

dead cell control
To confirm that the RT-MPCR assay detects only viable and replicating bacteria and that DNA does not cause a false-positive result, we prepared a sample containing ~107 E. coli O157:H7 cells, which was then boiled for 10 min to kill all bacterial cells. The same procedures as described above for the analysis of bacterial mixtures, including extraction of total RNA and RT-MPCR, were carried out on the dead cell control (data not shown). After agarose gel electrophoresis of the products, no band was visible, suggesting the absence of target RNA in the dead cell control. This demonstrates that the RT-MPCR assay specifically detects viable E. coli O157:H7.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Worldwide, E. coli O157:H7, V. cholerae O1, and Salmonella Typhi are an important public health concern. All three bacteria are extremely virulent and can cause severe gastrointestinal illness. As a result, detection techniques are required to monitor food and water supplies. Traditional detection techniques are time-consuming and lack specificity. The MPCR method is more specific because it amplifies unique DNA sequences in the specific genes of the target bacteria.

We chose three LPS genes and one flagella gene that are specific for the detection of the three target bacterial pathogens. The rfbE gene of E. coli O157, the rfbE gene of V. cholerae O1, and the tyv gene of Salmonella Typhi transcribe O-lipopolysaccharides. The surface O antigen plays an important role in the infectivity of the pathogens(30). For example, the wild-type V. cholerae O1 is 100-fold more pathogenic than its O antigen mutants, as measured by the median lethal dose(31). The rfbE gene of E. coli O157 encodes the O157 LPS and is therefore unique to the E. coli O157 serogroup(32). This gene has been identified as a good viability marker because it is transcribed in all growth phases from early exponential to late stationary phase(33). In addition, the rfbE mRNA is present only in live cells and degrades quickly after cell death(33).

The fliC gene of E. coli O157:H7 encodes the H7 flagellum, specifically indicating the presence of a functional flagellum(34). E. coli O157 without a functional flagellum (designated as E. coli O157:H–) has been suggested to be less pathogenic because it lacks motility(35). Detection of both rfbE and fliC enables us to distinguish E. coli O157:H7 (presence of both rfbE and fliC) from E. coli O157:H– (absence of fliC).

The distinction between viable (replicating) and nonviable (nonreplicating) bacteria is important because only viable bacteria produce toxins that cause human illness(36). Several studies have demonstrated mRNA as a potential viability marker(3)(17)(37). When reverse transcription is coupled with MPCR, simultaneous detection of several viable bacteria can be achieved.

In conclusion, the described RT-MPCR assay is capable of simultaneously detecting E. coli O157:H7 and Salmonella Typhi at concentrations as low as 30 cells. This sensitivity is comparable to other PCR-based methods for the detection of one type of bacterium at a time, which can usually detect bacteria present at 4–100 cells(12). The estimated oral infective dose of Salmonella Typhi is ~104 cells(38). The detection limit of the RT-MPCR assay thus is sufficient for screening the pathogen below the infective dose. When proper RNA extraction methods are used, this assay may be useful for detecting and identifying these pathogens in clinical samples or water and food samples. Further research is warranted to develop techniques that can efficiently extract bacterial RNAs in different samples.


   Acknowledgments
 
We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and Alberta Health and Wellness for funding this study. We also thank Drs. Glen Armstrong and James Xing, George Mulvey, and Julie Zhu for bacterial cultures and technical assistance. N. Morin would like to acknowledge the University of Alberta for a Graduate Research Assistance Award, and X.F. Li acknowledges NSERC for a University Faculty Award.


   Footnotes
 
1 Nonstandard abbreviations: LPS, lipopolysaccharide; MPCR, multiplex PCR; RT-MPCR, reverse transcription-multiplex PCR; and Tm, melting temperature.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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