Analytical Biochemistry 280, 166–172 (2000) doi:10.1006/abio.2000.4518, available online at http://www.idealibrary.com on Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay for Detecting Salmonella Wilfred Chen,*,1 Grisselle Martinez,*,† and Ashok Mulchandani* *Department of Chemical and Environmental Engineering and †Microbiology Graduate Program, University of California, Riverside, California 92521 Received November 30, 1999 selective enrichment and confirmation steps (2). Im- Molecular beacons are oligonucleotide probes that munological assays have also been used to detect the become fluorescent upon hybridization. We developed presence of Salmonella. These methods generally lack a real-time PCR assay to detect the presence of Salmo- specificity and can take up to 5 days to complete (3). nella species using these fluorogenic reporter mole- The advent of molecular biology has led to the devel- cules. A 122-base-pair section of the himA was used as opments of new tools such as the polymerase chain the amplification target. Molecular beacons were de- reaction for pathogen detection. A number of PCR as- signed to recognize a 16-base-pair region on the am- says specific for Salmonella have been developed (4– plicon. As few as 2 colony-forming unit (CFU) per PCR 6). Typical detection methods of PCR products involve reaction could be detected. We also demonstrated the visual detection of an appropriately sized DNA band ability of the molecular beacons to discriminate be- followed by specific hybridization with a labeled DNA tween amplicons obtained from similar species such probe, which could take up to 15 h and is very difficult as Escherichia coli and Citrobacter freundii in real- time PCR assays. These assays could be carried out to automate. New improvements in both the sensitivity entirely in sealed PCR tubes, enabling fast and direct and speed of the final detection step, preferably real- detection of Salmonella in a semiautomated format. time monitoring of PCR products within 1 or 2 h, must © 2000 Academic Press be developed to realize this powerful PCR-based method for practical applications. Recently, a new technique, known as molecular bea- con (MB),2 has been reported for the construction of Salmonella is an important food- and water-borne probes that are useful for real-time detection of nucleic pathogen associated with acute gastrointestinal ill- acids (7). These probes are based on single-stranded nesses around the world. Disease is caused by the nucleic acid molecules that possess a stem-and-loop penetration of Salmonella organisms from the gut lu- structure (Fig. 1). The loop portion contains sequence men into the epithelium of the small intestine and complementary to a target, and the stem is formed by enterotoxin production. The infective dose can be as annealing of two complementary arm sequences not low as 15–20 cells (1). It is estimated that over 4 related to the target. A fluorescent moiety is attached million cases of Salmonella infections and 1000 deaths to the end of one arm and a nonfluorescent quenching occur in the United States annually. Salmonellosis ac- moiety is attached to the other end. No fluorescence is counts for 60% of all bacterial disease outbreaks in the produced when the two arms are in close proximity, United States (1). due to the quenching action. When the probe encoun- Rapid methods for detecting Salmonella in food ters a single-strand target, it forms a hybrid with the products and water resources have been hindered by target, undergoing a spontaneous conformation change the requirement that they be extremely sensitive. Con- that forces the arm sequences apart and causes fluo- ventional cell-culturing methods for detection and iso- rescence to occur. The interaction of MBs with their lation involve several time-consuming, labor-intensive targets is extraordinarily specific. No increase in fluo- 1 To whom correspondence should be addressed. Fax: 909-787- 2 Abbreviations used: MB, molecular beacon; CFU, colony-forming 2425. Email: [email protected]. unit; MWD, Los Angeles Metropolitan Water District. 166 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. REAL-TIME POLYMERASE CHAIN REACTION ASSAY FOR DETECTING Salmonella 167 plate technique and expressed as the number of colony- forming units. DNA Extraction One and one-half milliliters of each culture was transferred into Eppendorff tubes and centrifuged at 1400 rpm for 2 min. The pellets were resuspended in 567 ␮l of TE buffer. Thirty microliters of 10% SDS and ␮ FIG. 1. (a) Molecular beacons anneal to complementary template. 3 l of 20 mg/ml proteinase K were added to each This results in separation of the quencher from the fluorophore and sample. After thorough mixing, the samples were in- increases fluorescence. (b) Molecular beacons do not form stable cubated in a water bath at 37°C for 1 h. After incuba- hybrids with mismatched template sequences. The proximity of the tion, 100 ␮l of 5 M NaCl was added and mixed quencher to the fluorophore when the stem portion is hybridized thoroughly. Eighty microliters of a 10% CTAB results in very low background fluorescence. (hexadecyltrimethyl ammonium chloride) in 0.7 M NaCl solution was added to each sample, followed by incubation in a water bath at 65°C for 10 min. Then 0.8 rescence is observed even in the presence of a target ml of chloroform:isoamyl alcohol (24:1) was added after strand that contains only a single nucleotide mismatch incubation. The samples were briefly vortexed and cen- (7). Since unhybridized beacons do not have to be sep- trifuged for 5 min. The aqueous supernatants were arated, they can be included in PCR reactions, permit- transferred into clean tubes. This was followed by a ting the progress of the reaction to be followed in real second extraction. Four hundred microliters of isopro- time. In real-time PCR, MBs as well as the primers panol was added and each sample was gently mixed hybridize to the templates during the annealing stage. until DNA precipitates were visible. The precipitates Data are collected at each annealing stage, when MBs were pelleted by centrifuging at 1400 rpm for 10 min. are hybridized to the target. When the temperature is The pellets were washed with 200 ␮l of ice-cold 70% increased during the extension stage, MBs dissociate ethanol. After removing the ethanol, the pellets were from the templates and do not interfere with polymer- allowed to dry in a vacuum oven at 50°C for 5 min and ization. As the target strands synthesized in a reaction resuspended in 100 ␮l of TE buffer. DNA recovery was accumulate, the fraction of MBs that is bound to tar- confirmed by gel electrophoresis. PCR reactions were gets will increase, causing a brighter fluorescent sig- carried out with each sample to confirm the DNA qual- nal. Recent studies have successfully employed MB for a variety of PCR applications (8–11). ity. PCR products were verified by gel electrophoresis. In this paper, we described the detection of Salmo- This method provided good yields of PCR quality DNA nella by real-time PCR assay based on MB. A 122-bp and was used for subsequent DNA extractions. region of the himA gene was used as the target. We demonstrate that the real-time assay is highly specific Design of Molecular Beacons with the capability to discriminate even a single base mismatch. As few as 2 colony-forming units (CFU) can MB (BHMA1) 5Ј-FAM-CGCTATCCGGGGCGTA- be detection with the MB-based PCR assay. ACC-CGTAGCG-3Ј-DABCYL was designed to be per- fectly complementary to the himA gene of Salmonella. MATERIALS AND METHODS The target sequence contained two nucleotide mis- matches, relative to the same region in the himA gene Bacterial Strains, Media, and Culture Conditions of E. coli. Salmonella typhimurium LT2, Escherichia coli MBs were synthesized by MIDLAND Certified Re- W3110, Citrobacter freundii ATCC 8090, and Enter- agent Company. BHMA1 was labeled at the 5Ј end obacter cloacae ATCC13047 were obtained from the with fluorescein (6-FAM) and the quencher 4-(4Ј-di- American Type Culture Collection (ATCC). Seven Sal- methylaminophenylazo)benzoic acid (DABCYL) at the monella isolates were obtained from the Los Angeles 3Ј end. The stem sequence was selected so that they County Sanitation Districts. These isolates were iden- would not complement the sequences within the loop tified and labeled: (1) Salmonella OC1, (2) Salmonella region. The length of the beacon was selected so that Group C-Factor 7, (3) S. choleracious, (4) S. enteriditis, the annealing temperature is slightly higher than the (5) S. heidelberg, (6) S. newport, and (7) S. thompson. annealing temperature of the PCR primers. The bea- All cultures were grown in Nutrient Broth (DIFCO cons were resuspended in TE buffer, stored at Ϫ20°C, Laboratories) overnight at 37°C. The cell count of over- and protected from light. Aliquots (16 mM) were pre- night cultures was estimated by enumeration of spread pared and used for subsequent studies. 168 CHEN, MARTINEZ, AND MULCHANDANI melting at 95°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min. Fluorescent measure- ments were recorded during each annealing step. At the end of each PCR run, data were automatically analyzed by the system and amplification plots were obtained. For each PCR, 5 ␮l of DNA template was added to 45 ␮l of PCR master (5 ␮lof1ϫ PCR buffer II [Perkin–Elmer], 3.5 mM MgCl2, 500 nM of each primer, 200 nM dNTPs, 0.5 U of AmpliTaq DNA poly- merase, 0.5 mM of beacons, and 25 ␮l of water) were added to the PCR tubes. The PCR buffer contained ROX (60 nmol) as the reference dye for normalization of the reactions.