Molecular Beacons: A New Approach for Detecting Salmonella

Chapter 20

Molecular Beacons: A New Approach for Detecting Salmonella Species

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Wilfred Chen, Grisselle Martinez, and Ashok Mulchandani Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a new approach to detect the presence of Salmonella species using these fluorogenic reporter molecules and demonstrated their ability to discriminate between similar E. coli species in real-time P C R assays. A detection limit of 1 CFU per P C R reaction was obtained. The assays were carried out entirely in sealed P C R tubes, enabling fast and direct detection in semiautomated format.

Salmonella is an important food and water-borne pathogen associated with acute gastrointestinal illnesses around the world. The infective dose can be as low as 152 0 cells, depending on the age, health status of the host and differences between Salmonella strains. It is estimated that over 4 million cases of Salmonella infections and one thousand deaths occur in the United States (1). Human infection usually occurs from consuming raw or undercooked foods. The largest Salmonellosis outbreak in the United States, involved 16,000 cases and was caused by milk products from a Chicago dairy in 1985. Although no outbreaks associated with drinking water have been reported, recent studies found Salmonella widely distributed in Southern California surface water (2). Currently, Salmonella is one of many waterborne pathogens regularly screened by water authorities. Current detection methods involve primarily cell culturing in selective broth. Although a variety of selective media and protocols are available, most of these methods require a series of sequential enrichment and culturing steps. These are lengthy, labor-intensive procedures which require further confirmation analyses. In addition, false-negative results may be obtained when Salmonella is outgrown by other organisms. The need for rapid and selective methods for detection of Salmonella has prompted the development of several rapid detection methods including new media 292

© 2000 American Chemical Society

Mulchandani and Sadik; Chemical and Biological Sensors for Environmental Monitoring ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

293 formulations or modifications, miniature biochemical tests, nucleic acid-based assays, antibody-based assays and automated systems (3). Amplification of nucleic acid using the polymerase chain reaction (PCR) or reverse transcription-PCR (RTPCR) for the detection of m R N A is the current preferred method, which provides the best sensitivity. Typical detection methods of P C R products involve visual detection of an appropriately sized D N A band followed by specific hybridization with a labeled D N A probe, which could take up to 15 hrs and is approximately 50% of the total time requirement for detection. New improvements in both the sensitivity and speed of the final detection step, preferably real-time monitoring of P C R products within 1 or 2 hr, must be developed to realize this powerful P C R based method for practical applications. Molecular beacons are stem-loop shaped oligonucleotide probes labeled with a fluorophore at the 5' end and a quenching moiety at the 3' end. Minimal fluorescence is emitted when the beacons are self-hybridized due to the close proximity of the quencher to the fluorophore, which are held together by the stem structure (4). Hybridization of the loop to a complimentary D N A sequence forces the stem arms apart, separating the quencher from the fluorophore. This separation results in a significant increase in fluorescence (Figure 1). Molecular beacons incorporated into a PCR reaction mix become brightly fluorescent as they hybridize to complimentary target sequences amplified during the reaction. The fluorescent signal is proportional to the amount of amplicons synthesized. Detection of this signal provides a powerful tool for real-time monitoring of P C R amplification products. The interaction of molecular beacons with their targets is extraordinarily specific. No increase in fluorescence is observed even in the presence of a target strand that contains only a single nucleotide mismatch (5). This study describes the development of a real-time P C R assay employing molecular beacons for detection of Salmonella. This assay combined the speed and specificity of P C R with the specificity and high sensitivity of molecular beacons. A 122 bp region of the himA gene was used as the target (6). The real-time P C R assay is highly specific and a detection limit of one single cell was achieved. Experimental Bacterial strains, media and culture conditions. Salmonella typhimurium L T 2 and Escherichia coli W3110 were obtained from the American Type Culture Collection (ATCC). Seven Salmonella isolates were obtained from the County Sanitation Districts of Los Angeles County. These isolates were identified and labeled: 1) Salmonella OC1, 2) Salmonella Group C-Factor 7, 3) Salmonella choleracious, 4) Salmonella enteriditis, 5) Salmonella heidelberg, 6) Salmonella newport and 7) Salmonella thompson. A l l cultures were grown in Nutrient Broth (DIFCO Laboratories) overnight at 37°C. The cell count of overnight cultures was estimated by enumeration using the spread plate technique and expressed as the number of colony forming units (CFU). D N A Extraction. 1.5 ml of each culture was harvested and resuspended in 567 | i l of T E (10 m M Tirs and 1 m M E D T A ) buffer. 30ul of 10% SDS and 3 ul of 20 mg/ml proteinase K were added to each sample and incubated in a water bath at


294 37°C for 1 hour. 100 u l of 5 M NaCl and 80ul of a 10% C T A B (hexadecyltrimethyl ammonium chloride) in 0.7 M NaCl solution were added to each sample, followed by incubation in a water bath at 65°C for 10 minutes. The solution was then extracted with 0.8 ml of chloroform/isoamyl alcohol (24:1). The aqueous supernatants were removed and mixed with 400 u l of isopropanol until D N A precipitates were visible. The precipitates were pelleted by centrifuging. The pellets were washed with 200 u l of ice-cold 70% ethanol. After removing the ethanol, the pellets were allowed to dry in a vacuum oven at 50°C for 5 minutes and resuspended in 100 u l of T E buffer. D N A recovery was confirmed by gel electrophoresis.

Target

Molecular Beacon

1111111111~ Fluorophore

Hybrid

Quencher

Figure 1. Principle of operation of molecular beacons.

Design of Molecular Beacons. The molecular beacon ( B H M A 1 ) 5 ' - F A M C G C T A T C C - G G G G C G T A A C C C G T A G C G - 3 ' D A B C Y L was synthesized by M I D L A N D Certified Reagent Company. The target sequence was designed to be perfectly complementary to the himA gene of Salmonella, but contained two nucleotide mismatches relative to the same region in the himA gene of Escherichia coli. Beacons were resuspended in T E buffer, stored at -20°C and protected from light. 16 m M aliquots were prepared and used for subsequent studies. Thermal Denaturation Profiles. Thermal denaturation profile studies were conducted to determine the optimum annealing temperature for the real-time P C R . The changes in fluorescence of a 50 u l solution containing 0.3uM of the beacon probe with or without 0.9uM of a perfectly complimentary single stranded oligonucleotide. The samples were place in a Perkin-Elmer A B I Prism 7700


295 Sequence Detector System and heated to 90°C for 5 minutes. The temperature was then reduced at 1°C per minute increments to 20°C. Data were recorded at each temperature interval. The optimal hybridization temperatures for each beacon were determined from these plots. PCR Primers. Primers S H I M A F 5' - C G T G C T C T G G A A A A C G G T G A G - 3 ' and S H I M A R 5' - C G T G C T G T A A T A G G A A T A T C T T C A - 3 ' (Genosys) specific for a 122 bp fragment of the himA gene were previously reported by Bej et al. (6). To amplify the himA region of E. coli, a similar pair of primers EHIMA-Forwrad: 5'C G C G C T C T G G A A A A C G G - 3 ' and EHIMA-Reverse: 5 ' - C G T G C T G T A A T G G G A A T A T C - 3 ' were designed. PCR conditions. The Perkin-Elmer A B I Prism 7700 sequence Detection System was used for real-time analyses. Thermal cycling conditions were specified as follows: initial melting at 98°C for 10 minutes, followed by 40 cycles of melting at 95°C for 1 minute, annealing at 57°C for 1 minute and extension at 72°C for 1 minute. These reactions ended with a final extension step at 72°C x 5 minutes. Fluorescent measurements were recorded during each annealing step. A t the end of each P C R run, data were automatically analyzed by the system and amplification plots were obtained.

Results and Discussion Thermal Denaturation Profiles. In the absence of a complimentary target, the molecular beacon expressed approximately 40% fluorescence (relative percentage) at 80°C (Figure 2), indicating that the stem structure had been denatured and the beacons had assumed a coiled conformation. A s the temperature was decreased, stable hybridization of the stems was noted at 50°C, when the fluorescence had decreased to 0%. The melting temperature of the stem was determined to be 5055°C. Beacon-target hybrids also expressed 40% of total fluorescence at 80°C (Figure 2). As the temperature was decreased, a slow but steady increase in fluorescence was noted, indicating the re-naturation of the hybrids. The melting temperature of these hybrids was between 60-65°C. The optimal annealing temperature for the beacon, which allows a clear discrimination between randomly coiled beacons and beacon-target hybrids, was determined to be between 55-60°C. Initial studies in real-time P C R were conducted between this temperature range and the highest sensitivity was obtained at 57°C. This temperature was chosen as the annealing temperature for subsequent real-time P C R studies. Real-Time PCR. Ten fold serial dilutions of template D N A were used in the real time P C R assay. Figure 3 shows the resulting amplification plot. ARn is the normalized fluorescence of each sample. As expected, samples that contain a higher number of template molecules, produced higher signals. As few as one C F U was detected with the real-time assay. The critical cycle (Ct), the cycle at which a


296 significant increase in fluorescence is first recorded, increased as the initial number of template molecules D N A decreased. This was expected, as samples containing low concentrations of template D N A would require more P C R cycles in order to replicate enough copies to produce a significant increase in fluorescence. Since the critical cycle is inversely proportional to the logarithm of the initial number of target molecules (7), these data can be used to formulate a standard quantification curve for the detection of Salmonella. Target Specificity. The specificity of the beacons was evaluated. Real-time P C R assays were conducted with Salmonella and E. coli as the initial templates. Although a 122 bp PCR fragment was amplified from both the Escherichia coli and Salmonella templates, only the Salmonella template elicited very strong fluorescence. This result clearly demonstrates the ability of molecular beacons to discriminate between very similar sequences. (Figure 4).

8000 -i

S

Temperature ( C)

Figure 2. Thermal denaturation profile of molecular beacons.


297

Figure 3. Real-time PCR assay. Initial template concentrations ranging from 1 to 1800 CFU.

Figure 4. Real-time P C R amplification of E. coli and Salmonella himA genes. Beacon specific for Salmonella failed to detect the himA homologue in Escherichia coli.


298 Conclusion Molecular beacon assays are simple and fast. Reagents are mixed in one step and reactions are carried in closed tubes, thus preventing contamination. Data are recorded during each cycle and results are automatically analyzed immediately after the reaction is completed, usually within 2-3 hours. Due to their high specificity and high sensitivity, molecular beacons can be effectively incorporated into real-time P C R assays and provide a quick and accurate method for detection of specific nucleic acid sequences in homogeneous solutions. We envision that the speed and sensitivity of bacterial pathogen detection based on PCR-assay method can be greatly enhanced with the application of molecular beacons.

Acknowledgments This research was supported by the Water Environmental Research Foundation. We wish to thank Tammy Chen for her help on D N A sequencing and real-time P C R assays.

References 1.

2. 3. 4. 5. 6. 7.

U . S. Food and Drug Administration. 1998. Foodborne Pathogenic Organisms and Natural Toxins Handbook. Center for Food Safety and Applied Nutrition. http://vm.cfsan.fda.gov/list.html/ Moriginio, M. A . ; Cornax, R. and Munoz, P.R. Water Resource 1990, 24, 117 Feng, P. J. of Food and Drug Analysis 1993, 1, 119. Tyagi, S. and Kramer, F.R. Nature Biotechnology 1996, 14, 303. Tyagi, S.; Bratu, D . P. and Kramer, F. R. Multicolor molecular beacons for allele discrimination. Nature Biotechnology 1998, 16, 49. Bej, A . K . ; Mahbubani, M. H.; Boyce, M. J. and Atlas, R. M. Applied and Environmental Microbiology 1993, 60, 368. Heid, C.A.; Stevens, J.; Livak, K.J. and Williams, P . M . Genome Methods 1996, 6, 986.


Molecular Beacons: A New Approach for Detecting Salmonella

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