723 Journal of Food Protection, Vol. 78, No. 4, 2015, Pages 723–727 doi:10.4315/0362-028X.JFP-14-193 Copyright G, International Association for Food Protection
Isothermal Target and Probe Amplification Assay for the Real-Time Rapid Detection of Staphylococcus aureus HYEWON SHIN,1 MINHWAN KIM,2 EUNJU YOON,3 GYOUNGWON KANG,1 SEUNGYU KIM,2 AELEE SONG,1 JEONGSOON KIM1*
AND
1Food
Research and Development Center, Samsung Welstory, Inc., 2442-1 Yonggu-daero, Giheunggu, YongIn-si, Gyeonggi-do, South Korea; 2DxGene, Inc., 306 Ace Technotower 5th, 197-22 Guro-3-dong, Guro-gu, Seoul, South Korea; and 3Department of Food Science and Nutrition, Dong-A University, 550 Nakdong-daero, Sahagu, Busan, South Korea MS 14-193: Received 22 April 2014/Accepted 7 November 2014
ABSTRACT Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer–based signal probe under isothermal conditions at 61uC or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non–S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 101 CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods.
Foodborne pathogens are routinely detected by traditional culture methods involving homogenization of the food, inoculation into selective media, and incubation for a predetermined period. A major drawback of these methods is that they are labor intensive and require 2 to 3 days to obtain results and up to 7 to 10 days for confirmation. This conventional methodology is not sufficient for food safety testing of raw or ready-to-eat foods because of the fast turnaround time of food consumption. In the last decade, PCR and real-time PCR methods have been proposed for the detection of foodborne pathogens to replace the timeconsuming traditional culture-based techniques (2, 9). PCRbased methods are rapid, sensitive, and specific when used correctly. However, real-time PCR assay have the major disadvantages of requiring high-cost equipment, a high level of expertise, and expensive purification reagents. Sensitivity may also decline dramatically for direct analysis of food samples, and controversy exists about the detection limits of real-time PCR assays. To overcome these problems, isothermal target and probe amplification (iTPA) technology (Fig. 1) was developed (10) as a highly specific and * Author for correspondence. Tel: (z82)31-899-0587; Fax: (z82)31288-0871; E-mail: [email protected].
sensitive method for detecting foodborne pathogens by simultaneously amplifying the target gene and the signal probe under isothermal conditions after preenrichment (11). In Korea, staphylococcal food poisoning is one of the most commonly occurring foodborne diseases (10% [228] of total outbreaks (13)). Staphylococcus aureus is a common bacterium found on the skin and in the noses of healthy individuals and animals. Food poisoning caused by S. aureus is so common that its incidence causes significant economic loss ($120 million per year (21)). S. aureus–related food poisoning occurs after the consumption of foods and beverages containing staphylococcal enterotoxin, which causes vomiting and diarrhea (20). Food handlers and asymptomatic carriers are often the source of food contamination; therefore, the presence of S. aureus and its enterotoxins in food is usually indicative of poor hygiene in the food production line. In this study, a set of specific primers and a probe targeting the conserved regions of the nuclease (nuc) gene of S. aureus were designed. The nuc gene, which encodes thermonuclease, is highly specific to S. aureus, is not found in other Staphylococcus species, such as S. intermedius and S. hyicus (1), and is highly conserved within S. aureus species, with 99% homology (15). Therefore, targeting of
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75 nM FRET signal probe (25 nM was used for the assay of inoculated food samples), 3 U of Bst polymerase (NEB, Ipswich, MA), 6 U of RNase H (Epicentre, Madison, WI), 6 U of RNase inhibitor (Solgent, Daejeon, South Korea), and 10 ml of DNA template (replaced by 10 ml of sterilized water for the negative control). The iTPA reaction mix was incubated at 61uC for 90 min with real-time isothermal fluorescent equipment (iGene-S, Scinco, South Korea), and the fluorescent signal was acquired every minute. The results were interpreted as positive for S. aureus when the threshold time (Tt) was within 60 min, with a DRn value .600, where DRn is an expression of the magnitude of the normalized fluorescence signal generated by the reporter each minute during iTPA amplification. The point at which the amplification plot crosses the threshold is defined as Tt (Tables 3 and 4). The cutoff Tt was determined from preenrichment cultures of uninoculated food samples. Sterilized water was used for the negative control.
FIGURE 1. Scheme of the isothermal target and probe amplification (iTPA) method.
the nuc gene with a real-time iTPA assay is a highly sensitive and specific method for detecting S. aureus in food. Further development of this method can allow its application for the detection of other foodborne pathogens. MATERIALS AND METHODS Bacterial strains and DNA template preparation. All bacterial strains used in this study were maintained in LuriaBertani (LB) broth. S. aureus (ATCC 25923) was used for sensitivity testing. A total of 91 S. aureus strains and 51 non–S. aureus strains were used to evaluate inclusivity and exclusivity (Table 1). For preparation of the DNA template, a single bacterial colony grown on LB agar was suspended in 1 ml of Tris-EDTA (TE) buffer solution and centrifuged at ambient temperature at 10,770 | g for 1 min, and the supernatant was discarding. The pellet was then resuspended in 200 ml of TE buffer solution and placed in a heating block at 100uC for 10 min. The crude lysate was centrifuged at ambient temperature at 10,770 | g for 10 min, and the supernatant was stored at 220uC until it was used without further purification. iTPA primers, FRET signal probe, and assay conditions. The S. aureus nuc gene (GenBank V01281) was used as the target for iTPA primers and probe design. Four primers, two outer, two inner, and one fluorescence resonance energy transfer (FRET) signal probe that recognized five regions of the target sequence were designed using DNASTAR software (Madison, WI) and synthesized by IDT (San Diego, CA). Oligonucleotide sequences and the locations of the primers and the probe are given in Table 2. The basic local alignment search tool indicated a high level of specificity (no similarity with any other bacteria). The 20-ml iTPA reaction mix consisted of 10 mM Tris-HCl (pH 8.5), 22 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1 mg/ml acetylated bovine serum albumin, 6 mM dithiothreitol, 1.6 mM concentrations of the deoxynucleoside triphosphates (dNTPs), 0.11 mM concentrations of each outer primer, 1.1 mM concentrations of each inner primer,
PCR assay. For comparison, PCRs were performed using the iTPA outer primers. Each PCR (20 ml total volume) contained 1 U of i-Taq plus DNA polymerase (Intron Biotechnology, Seongnam, South Korea), 2.5 mM concentrations of the dNTPs, 2.5 mM MgCl2, 2.5 mM KCl, 10 pM concentrations of the forward and reverse primers, and 10 ml of DNA template. PCRs were conducted using an initial denaturation cycle at 95uC for 7 min followed by 40 cycles of denaturation at 94uC for 0.5 min, primer annealing at 61uC for 0.5 min, and extension at 72uC for 0.5 min (2720 thermal cycler, Applied Biosystems, Foster City, CA). Aliquots (5 ml) of the PCR products were analyzed via electrophoresis on 2% agarose gels in 0.5| Tris-borate buffer at 100 V for 25 min. The amplification products were stained with ethidium bromide and visualized by UV transillumination. Real-time iTPA inclusivity, exclusivity, and detection limits. A total of 142 bacterial strains (Table 1) were used to evaluate the performance of the real-time iTPA assay. Inclusivity and exclusivity tests were conducted three times. To determine the detection limits of this assay, serial 10-fold dilutions of a mid-logphase S. aureus culture (1.2 | 109 CFU) grown in LB broth were prepared in phosphate-buffered saline and quantified using the pour plate method. The detection limit tests were conducted four times. Real-time iTPA testing of experimentally inoculated food samples. Three kinds of foods were used for the study: kiwi dressing, soybean milk, and custard cream. Food samples were processed as described previously (12), with slight modifications. Plastic food bags with 225 ml of LB broth and 25 g of kiwi dressing were inoculated with 1 ml of serial dilutions (1:10 to 1:108) of the S. aureus ATCC 25923 stock solution (1.2 | 109 CFU/ml) in buffered peptone water and vigorously mixed with a homogenizer (Pro-media SH-001, ELMEX Ltd., Tokyo, Japan) for about 30 s to distribute the bacteria. Cultures were incubated at 37uC for 16 h, and then 1 ml of the preenriched solution was centrifuged at 10,770 | g for 1 min and the supernatant was discarded. The pellet was washed with 1 ml of 0.01 N NaOH solution by centrifuging at 10,770 | g for 1 min, and the supernatant was discarded. The pellet was then washed with 1 ml of 95% ethanol and 1 ml of TE buffer sequentially using the same procedure. The washed pellet was suspended in 200 ml of TE buffer solution and heated at 100uC for 10 min in a dry heating block. The crude cell lysate was centrifuged at 10,770 | g for 10 min, and 10 ml of the supernatant was used for the real-time iTPA assay. For negative samples, 10 ml of sterilized water was used instead of the sample supernatant. The tests using inoculated food samples were conducted three times.
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TABLE 1. Bacterial strains used in this study for testing of S. aureus inclusivity and exclusivity a Positive strains (n ~ 91) ID (DRn . 600)
Strain(s)
S. aureus
S. aureus z Salmonella
S. aureus z E. coli
S. aureus z Yersinia enterocolitica S. aureus z Listeria monocytogenes
Negative strains (n ~ 51) Strain
Salmonella Typhimurium
KCTC1621 KCTC1916 KCTC1927 KCTC1928 ATCC 6538 ATCC 13565 ATCC 14458 ATCC 19095 ATCC 23235 ATCC 25923 NCCP11470 NCCP11472 Cultures from KFDA (63)b
Salmonella Enteritidis Salmonella Heidelberg Salmonella Dublin Salmonella Braenderup Salmonella enterica Salmonella Choleraesuis S. bongori Bacillus cereus
Cultures from Samsung Welstory (2)c ATCC 25923 z KCTC2515 ATCC 25923 z KCTC2421 KCTC1927 z KCTC2515 KCTC1927 z KCTC 2421 ATCC 25923 z ATCC 43894 ATCC 25923 z KCCM41657 KCTC1927 z ATCC 43894 KCTC1927 z KCCM41657 ATCC 25923 z ATCC 23715 KCTC1927 z ATCC 23715 ATCC 25923 z ATCC 35152 ATCC 25923 z ATCC 19113 KCTC1927 z ATCC 35152 KCTC1927 z ATCC 19113
Escherichia coli
E. coli (O55:K59(B5):H2) E. coli (EPEC_eaeA) (ETEC_LT) (ETEC_ST) (EIEC_inV) (EHEC_VT1) (EHEC_VT2) (EAEC) Vibrio parahaemolyticus
V. vulnificus V. mimicus Campylobacter jejuni Shigella sonnei
S. flexneri S. boydii Yersinia enterocolitica Listeria monocytogenes Clostridium perfringens Staphylococcus cohnii S. caprae S. lentus
ID (DRn , 400)
NCCP10812 NCCP12241 KCTC2515 ATCC 14028 ATCC 13076 KCCM12021 NCCP10322 NCCP10860 NCCP12232 ATCC BAA614 ATCC 14028 KCCM41575 KCCM41758 NCCP10623 NCCP10715 NCCP14579 ATCC 11775 ATCC 43887 ATCC BAA1706 ATCC 12014 NCCP13715 NCCP13717 NCCP13718 NCCP13719 NCCP13720 NCCP13721 NCCP14039 ATCC 10511 ATCC 17802 ATCC 27969 ATCC 27562 ATCC 33653 KCCM41773 KCTC2009 KCTC2518 Unknown (4)d KCTC2517 Unknown (2) Unknown (2) KCCM41657 ATCC 23715 ATCC 700545 Unknown Unknown Unknown Unknown
a
ATCC, American Type Culture Collection, Manassas, VA; KCCM, Korean Culture Center of Microorganisms, Seoul, Korea; KCTC, Korean Collection for Type Cultures, Daejeon, Korea; NCCP, National Culture Collection for Pathogens, Ohsong, Korea; KFDA, Korean Food and Drug Administration, Seoul, Korea. DRn, an expression of the magnitude of the normalized fluorescence signal generated by the reporter each minute during iTPA amplification: Rn – average Rn of the first 10 min. b From food samples generated in the KFDA laboratory. c S. aureus strains from the hands of food workers at Samsung Welstory food court. d Unknown, strains from food samples from Samsung Welstory Food R&D Center.
RESULTS Inclusivity and exclusivity of the real-time iTPA assay. The S. aureus nuc-based real-time iTPA assay
successfully detected all 91 S. aureus strains and produced negative results for the 51 non–S. aureus strains (Table 1), validating the high specificity of the nuc-based real-time iTPA assay for S. aureus. All of the S. aureus strains had
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TABLE 2. Oligonucleotide primers and probe sequences used in this study Name
Sequence (59–39)
Position (bp)a
Outer forward Outer reverse Inner forward Inner reverse FRET probe
AAA GCG ATT GAT GGT GAT ACG GT ACT CGA CTT CAA TTT TCT TTG CAT TTT CTA CAG ACA TCC AGC TGrG rArGrA TCA ACC AAT GAC ATT CAG ACT ATT GTC GAG CAG TAA CTC TrGrAr GrGA GGA CCA TAT TTC TCT ACA CCT T FAM-TGG TTG ATA CAC CTrG rArArA CAA AGC ATC CT-BHQ1b
508–530 659–688 549–572 608–630 574–603
a b
GenBank no. V01281. BHQ1, black hole quencher 1.
DRn values .600, and the non–S. aureus strains had DRn values of ,400 (data not shown). The PCR assay using the iTPA outer primers yielded amplicons of the expected size (181 bp) for all 91 S. aureus strains without spurious priming and cross-reactivity with the non–S. aureus strains (data not shown). For a rigorous comparison of exclusivity, low concentrations of genomic DNA (ca. 103 CFU/ml) from the positive strains and very high concentrations of genomic DNA (ca. 107 CFU/ml) from the negative strains were used as the template. Neither false-positive nor false-negative results were observed for any of the 142 bacterial strains with the real-time iTPA assay using two primer sets and the FRET signal probe, indicating a high level of specificity. Detection limits of the real-time iTPA assay. The detection limits of the real-time iTPA assay using 10-fold serial dilutions of S. aureus ATCC 25932 after overnight incubation (approximately 1.2 | 109 CFU/ml) were determined (Table 3). The lowest level of cells detected was 103 CFU/ml (with an DRn value .600 within 60 min). Validity of the iTPA assay for food samples. The detection limits for S. aureus in three kinds of food samples were determined using the iTPA assay after enrichment (Table 4). Inocula of 101 CFU in the preenriched samples of kiwi dressing, custard cream, and soybean milk, with final populations of 108 to 109 CFU/ml after enrichment, were consistently detected. The lowest detection limit achieved in this study was 101 CFU per 25 g of preenriched food sample. Without enrichment of the bacterial culture, the detection limit was 103 CFU/ml. The same detection limits
were obtained for the serial dilutions of S. aureus ATCC 25923 (data not shown). DISCUSSION The PCR is one of the most widely used techniques in diagnostic applications because it allows sensitive and rapid diagnosis. However, this technique is not suitable for routine food safety testing because of the need for expensive thermal cycler equipment and complex operations. To overcome such limitations, several nucleic acid amplification methods have been developed for which thermal cycling is not required and operation is simple. For example, transcription-mediated amplification (14), nucleic acid sequence–based amplification (3), strand displacement amplification (23, 24), helicase-dependent amplification (22), rolling-circle amplification (5, 16), and loop-mediated isothermal amplification (17–19) are efficient nucleic acid amplification techniques under isothermal conditions, although each method has advantages and disadvantages (7). In contrast, the iTPA method both amplifies the target DNA sequences and increases the FRET probe signal because the probe is also amplified. As a result, iTPA increases the level of sensitivity compared with other isothermal methods. iTPA also achieves a high level of specificity through the use of two pairs of primers and an TABLE 4. Detection limits for S. aureus in inoculated food samples by iTPA assay Food sample
Kiwi dressing TABLE 3. Sensitivity for detection of S. aureus ATCC 25923 using real-time iTPA assay Level (CFU/ml)
Avg Tt (min)a
CV of Tt (%)b
Avg DRnc
60.6 33.3 29 26.2 22.5 20.2 17.3 16.1
46 14.9 10.2 10.8 8.4 6.7 7.3 22.4
400 2,339 2,800 2,931 2,773 2,876 2,732 2,704
Soybean milk 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 a b c
| | | | | | | |
102 103 104 105 106 107 108 109
Tt, threshold time. CV, coefficient of variation. DRn ~ Rn – average Rn of the first 10 min.
Custard cream
a b
S. aureus (CFU)
1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2
| | | | | | | | | | | | | | |
101 102 103 104 105 101 102 103 104 105 101 102 103 104 105
Tt (min)a
Avg DRnb
¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡
955 1,511 1,375 1,408 1,339 1,148 1,525 1,542 1,603 1,449 1,492 1,056 1,604 1,555 1,195
27.0 16.0 16.9 17.1 18.3 21.4 16.0 16.3 16.9 18.1 18.0 27.3 16.2 17.4 21.2
9.0 1.4 2.9 3.0 5.3 1.3 1.1 1.7 0.9 1.7 2.2 4.3 2.0 1.6 1.8
Tt, threshold time. DRn ~ Rn – average Rn of the first 10 min.
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amplicon-specific FRET signal probe based on cycling probe technology (4, 6, 8). We previously found that the iTPA-based assay was highly effective for the detection of Salmonella contamination in food samples (11). In the present study, a novel and rapid real-time DNA detection method was developed using real-time isothermal fluorescence equipment, which is much less expensive and much simpler to operate than real-time PCR equipment because thermal cycling is not required. A set of DNA-RNA-DNA chimeric primers and a FRET signal probe were designed to specifically target the nuc gene of S. aureus for use in this novel and rapid real-time DNA detection method without expensive equipment. We found that the real-time iTPA assay can be used to detect S. aureus inocula with a detection limit of 103 CFU/ml within 1 h. The four primers and one FRET probe designed herein from five regions of the S. aureus nuc gene are highly specific to S. aureus. The testing of 91 S. aureus strains and 51 non–S. aureus strains on a rapid fluorescent detection platform resulted in 100% inclusivity and 100% exclusivity for the detection of S. aureus (Table 1). Compared with endpoint iTPA detection using a water bath and a fluorescent reader, the real-time iTPA assay is much faster and more convenient because no sample tube transition is required between the amplification and detection steps. The cost and routine maintenance requirements of a real-time isothermal fluorescent reader (e.g., the iGene-S) are much less than those for current commercial real-time PCR equipment because the expensive and specialized thermal cycling module is not needed. In conclusion, a real-time isothermal DNA detection system was developed that has great potential for applications in pathogen testing for foods. The nuc-based real-time iTPA assay is a specific, sensitive, and rapid method for the detection of S. aureus in food samples. This simple method should allow rapid risk assessment of staphylococcal food poisoning at a low cost, which would be more practical for the many food manufacturers and stakeholders interested in food safety. REFERENCES 1. Brakstad, O. G., K. Aasbakk, and J. A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654–1660. 2. Candrian, U. 1995. Polymerase chain reaction in food microbiology. J. Microbiol. Methods 23:89–103. 3. Compton, J. 1991. Nucleic acid sequence-based amplification. Nature 350:91–92. 4. Duck, P., G. Alvarado-Urbina, B. Burdick, and B. Collier. 1990. Probe amplifier system based on chimeric cycling oligonucleotides. BioTechniques 9:142–149. 5. Fire, A., and S.-Q. Xu. 1995. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92:4641–4645. 6. Fong, W. K., Z. Modrusan, J. P. McNevin, J. Marostenmaki, B. Zin, and F. Bekkaoui. 2000. Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology. J. Clin. Microbiol. 38:2525–2529. 7. Gill, P., and A. Ghaemi. 2008. Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids 27: 224–243.
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8. Harvey, J. J., S. P. Lee, E. K. Chan, J. H. Kim, E. S. Hwang, C. Y. Cha, J. R. Knutson, and M. K. Han. 2004. Characterization and applications of CataCleave probe in real-time detection assays. Anal. Biochem. 333:246–255. 9. Hill, W. E. 1996. The polymerase chain reaction: applications for the detection of foodborne pathogens. Crit. Rev. Food Sci. Nutr. 36:123– 173. 10. Jung, C. H., J. W. Chung, U. O. Kim, M. H. Kim, and H. G. Park. 2010. An isothermal target and probe amplification (iTPA) method, based on a combination of an isothermal chain amplification (ICA) technique and a FRET cycling probe technology (CPT). Anal. Chem. 82:5937–5943. 11. Kim, J. S., M. S. Jahng, G. G. Lee, K. J. Lee, H. K. Chae, J. H. Lee, J. H. Lee, and M. H. Kim. 2011. Rapid and simple detection of the invA gene in Salmonella spp. by isothermal target and probe amplification (iTPA). Lett. Appl. Microbiol. 52:399–405. 12. Kim, J. S., G. G. Lee, J. S. Park, Y. H. Jung, H. S. Kwak, S. B. Kim, Y. S. Nam, and S. T. Kwon. 2007. A novel multiplex PCR assay for rapid and simultaneous detection of five pathogenic bacteria: Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus. J. Food Prot. 70:1656–1662. 13. Korea Food and Drug Association. 2009. Annual outbreaks of Korea. Available at: http://www.mfds.go.kr/e-stat/index.do?nMenuCode~33. Accessed January 2015. 14. Kwoh, D. Y., G. R. Davis, K. M. Whitfield, H. L. Chappelle, L. J. DiMichele, and T. R. Gingeras. 1989. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc. Natl. Acad. Sci. USA 86:1173–1177. 15. Lee, N., K. Y. Kwon, S. K. Oh, H. J. Chang, H. S. Chun, and S. W. Choi. 2014. A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food. Foodborne Pathog. Dis. 11:574–580. 16. Lizardi, P. M., X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, and D. C. Ward. 1998. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nature Genet. 19:225– 232. 17. Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 15:e63. 18. Ohtsuka, K., K. Yanagawa, K. Takatori, and Y. Hara-Kudo. 2005. Detection of Salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates. Appl. Environ. Microbiol. 71:6730–6735. 19. Osawa, R., A. Yoshida, Y. Masakiyo, S. Nagashima, T. Ansai, H. Watari, T. Notomi, and T. Takehara. 2007. Rapid detection of Actinobacillus actinomycetemcomitans using a loop-mediated isothermal amplification method. Oral Microbiol. Immunol. 22:252– 259. 20. Rosec, J. P., J. P. Guiraud, C. Dalet, and N. Richard. 1997. Enterotoxin production by staphylococci isolated from foods in France. Int. J. Food Microbiol. 35:213–221. 21. Shin, H., S. Lee, J. S. Kim, J. Kim, and K. H. Han. 2010. Socioeconomic costs of food-borne disease using the cost-of-illness model: applying the QALY method. J. Prev. Med. Public Health 43:352–361. (In Korean.) 22. Vincent, M., Y. Xu, and H. Kong. 2004. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5:795–800. 23. Walker, G. T., M. S. Fraiser, J. L. Schram, M. C. Little, J. G. Nadeau, and D. P. Malinowski. 1992. Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20:1691–1696. 24. Walker, G. T., M. C. Little, J. G. Nadeau, and D. D. Shank. 1992. Isothermal in vitro amplification of DNA by a restriction enzyme/ DNA polymerase system. Proc. Natl. Acad. Sci. USA 89:392–396.
Isothermal Target and Probe Amplification Assay for the Real-Time Rapid Detection of Staphylococcus aureus HYEWON SHIN,1 MINHWAN KIM,2 EUNJU YOON,3 GYOUNGWON KANG,1 SEUNGYU KIM,2 AELEE SONG,1 JEONGSOON KIM1*
AND
1Food
Research and Development Center, Samsung Welstory, Inc., 2442-1 Yonggu-daero, Giheunggu, YongIn-si, Gyeonggi-do, South Korea; 2DxGene, Inc., 306 Ace Technotower 5th, 197-22 Guro-3-dong, Guro-gu, Seoul, South Korea; and 3Department of Food Science and Nutrition, Dong-A University, 550 Nakdong-daero, Sahagu, Busan, South Korea MS 14-193: Received 22 April 2014/Accepted 7 November 2014
ABSTRACT Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer–based signal probe under isothermal conditions at 61uC or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non–S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 101 CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods.
Foodborne pathogens are routinely detected by traditional culture methods involving homogenization of the food, inoculation into selective media, and incubation for a predetermined period. A major drawback of these methods is that they are labor intensive and require 2 to 3 days to obtain results and up to 7 to 10 days for confirmation. This conventional methodology is not sufficient for food safety testing of raw or ready-to-eat foods because of the fast turnaround time of food consumption. In the last decade, PCR and real-time PCR methods have been proposed for the detection of foodborne pathogens to replace the timeconsuming traditional culture-based techniques (2, 9). PCRbased methods are rapid, sensitive, and specific when used correctly. However, real-time PCR assay have the major disadvantages of requiring high-cost equipment, a high level of expertise, and expensive purification reagents. Sensitivity may also decline dramatically for direct analysis of food samples, and controversy exists about the detection limits of real-time PCR assays. To overcome these problems, isothermal target and probe amplification (iTPA) technology (Fig. 1) was developed (10) as a highly specific and * Author for correspondence. Tel: (z82)31-899-0587; Fax: (z82)31288-0871; E-mail: [email protected].
sensitive method for detecting foodborne pathogens by simultaneously amplifying the target gene and the signal probe under isothermal conditions after preenrichment (11). In Korea, staphylococcal food poisoning is one of the most commonly occurring foodborne diseases (10% [228] of total outbreaks (13)). Staphylococcus aureus is a common bacterium found on the skin and in the noses of healthy individuals and animals. Food poisoning caused by S. aureus is so common that its incidence causes significant economic loss ($120 million per year (21)). S. aureus–related food poisoning occurs after the consumption of foods and beverages containing staphylococcal enterotoxin, which causes vomiting and diarrhea (20). Food handlers and asymptomatic carriers are often the source of food contamination; therefore, the presence of S. aureus and its enterotoxins in food is usually indicative of poor hygiene in the food production line. In this study, a set of specific primers and a probe targeting the conserved regions of the nuclease (nuc) gene of S. aureus were designed. The nuc gene, which encodes thermonuclease, is highly specific to S. aureus, is not found in other Staphylococcus species, such as S. intermedius and S. hyicus (1), and is highly conserved within S. aureus species, with 99% homology (15). Therefore, targeting of
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75 nM FRET signal probe (25 nM was used for the assay of inoculated food samples), 3 U of Bst polymerase (NEB, Ipswich, MA), 6 U of RNase H (Epicentre, Madison, WI), 6 U of RNase inhibitor (Solgent, Daejeon, South Korea), and 10 ml of DNA template (replaced by 10 ml of sterilized water for the negative control). The iTPA reaction mix was incubated at 61uC for 90 min with real-time isothermal fluorescent equipment (iGene-S, Scinco, South Korea), and the fluorescent signal was acquired every minute. The results were interpreted as positive for S. aureus when the threshold time (Tt) was within 60 min, with a DRn value .600, where DRn is an expression of the magnitude of the normalized fluorescence signal generated by the reporter each minute during iTPA amplification. The point at which the amplification plot crosses the threshold is defined as Tt (Tables 3 and 4). The cutoff Tt was determined from preenrichment cultures of uninoculated food samples. Sterilized water was used for the negative control.
FIGURE 1. Scheme of the isothermal target and probe amplification (iTPA) method.
the nuc gene with a real-time iTPA assay is a highly sensitive and specific method for detecting S. aureus in food. Further development of this method can allow its application for the detection of other foodborne pathogens. MATERIALS AND METHODS Bacterial strains and DNA template preparation. All bacterial strains used in this study were maintained in LuriaBertani (LB) broth. S. aureus (ATCC 25923) was used for sensitivity testing. A total of 91 S. aureus strains and 51 non–S. aureus strains were used to evaluate inclusivity and exclusivity (Table 1). For preparation of the DNA template, a single bacterial colony grown on LB agar was suspended in 1 ml of Tris-EDTA (TE) buffer solution and centrifuged at ambient temperature at 10,770 | g for 1 min, and the supernatant was discarding. The pellet was then resuspended in 200 ml of TE buffer solution and placed in a heating block at 100uC for 10 min. The crude lysate was centrifuged at ambient temperature at 10,770 | g for 10 min, and the supernatant was stored at 220uC until it was used without further purification. iTPA primers, FRET signal probe, and assay conditions. The S. aureus nuc gene (GenBank V01281) was used as the target for iTPA primers and probe design. Four primers, two outer, two inner, and one fluorescence resonance energy transfer (FRET) signal probe that recognized five regions of the target sequence were designed using DNASTAR software (Madison, WI) and synthesized by IDT (San Diego, CA). Oligonucleotide sequences and the locations of the primers and the probe are given in Table 2. The basic local alignment search tool indicated a high level of specificity (no similarity with any other bacteria). The 20-ml iTPA reaction mix consisted of 10 mM Tris-HCl (pH 8.5), 22 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1 mg/ml acetylated bovine serum albumin, 6 mM dithiothreitol, 1.6 mM concentrations of the deoxynucleoside triphosphates (dNTPs), 0.11 mM concentrations of each outer primer, 1.1 mM concentrations of each inner primer,
PCR assay. For comparison, PCRs were performed using the iTPA outer primers. Each PCR (20 ml total volume) contained 1 U of i-Taq plus DNA polymerase (Intron Biotechnology, Seongnam, South Korea), 2.5 mM concentrations of the dNTPs, 2.5 mM MgCl2, 2.5 mM KCl, 10 pM concentrations of the forward and reverse primers, and 10 ml of DNA template. PCRs were conducted using an initial denaturation cycle at 95uC for 7 min followed by 40 cycles of denaturation at 94uC for 0.5 min, primer annealing at 61uC for 0.5 min, and extension at 72uC for 0.5 min (2720 thermal cycler, Applied Biosystems, Foster City, CA). Aliquots (5 ml) of the PCR products were analyzed via electrophoresis on 2% agarose gels in 0.5| Tris-borate buffer at 100 V for 25 min. The amplification products were stained with ethidium bromide and visualized by UV transillumination. Real-time iTPA inclusivity, exclusivity, and detection limits. A total of 142 bacterial strains (Table 1) were used to evaluate the performance of the real-time iTPA assay. Inclusivity and exclusivity tests were conducted three times. To determine the detection limits of this assay, serial 10-fold dilutions of a mid-logphase S. aureus culture (1.2 | 109 CFU) grown in LB broth were prepared in phosphate-buffered saline and quantified using the pour plate method. The detection limit tests were conducted four times. Real-time iTPA testing of experimentally inoculated food samples. Three kinds of foods were used for the study: kiwi dressing, soybean milk, and custard cream. Food samples were processed as described previously (12), with slight modifications. Plastic food bags with 225 ml of LB broth and 25 g of kiwi dressing were inoculated with 1 ml of serial dilutions (1:10 to 1:108) of the S. aureus ATCC 25923 stock solution (1.2 | 109 CFU/ml) in buffered peptone water and vigorously mixed with a homogenizer (Pro-media SH-001, ELMEX Ltd., Tokyo, Japan) for about 30 s to distribute the bacteria. Cultures were incubated at 37uC for 16 h, and then 1 ml of the preenriched solution was centrifuged at 10,770 | g for 1 min and the supernatant was discarded. The pellet was washed with 1 ml of 0.01 N NaOH solution by centrifuging at 10,770 | g for 1 min, and the supernatant was discarded. The pellet was then washed with 1 ml of 95% ethanol and 1 ml of TE buffer sequentially using the same procedure. The washed pellet was suspended in 200 ml of TE buffer solution and heated at 100uC for 10 min in a dry heating block. The crude cell lysate was centrifuged at 10,770 | g for 10 min, and 10 ml of the supernatant was used for the real-time iTPA assay. For negative samples, 10 ml of sterilized water was used instead of the sample supernatant. The tests using inoculated food samples were conducted three times.
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TABLE 1. Bacterial strains used in this study for testing of S. aureus inclusivity and exclusivity a Positive strains (n ~ 91) ID (DRn . 600)
Strain(s)
S. aureus
S. aureus z Salmonella
S. aureus z E. coli
S. aureus z Yersinia enterocolitica S. aureus z Listeria monocytogenes
Negative strains (n ~ 51) Strain
Salmonella Typhimurium
KCTC1621 KCTC1916 KCTC1927 KCTC1928 ATCC 6538 ATCC 13565 ATCC 14458 ATCC 19095 ATCC 23235 ATCC 25923 NCCP11470 NCCP11472 Cultures from KFDA (63)b
Salmonella Enteritidis Salmonella Heidelberg Salmonella Dublin Salmonella Braenderup Salmonella enterica Salmonella Choleraesuis S. bongori Bacillus cereus
Cultures from Samsung Welstory (2)c ATCC 25923 z KCTC2515 ATCC 25923 z KCTC2421 KCTC1927 z KCTC2515 KCTC1927 z KCTC 2421 ATCC 25923 z ATCC 43894 ATCC 25923 z KCCM41657 KCTC1927 z ATCC 43894 KCTC1927 z KCCM41657 ATCC 25923 z ATCC 23715 KCTC1927 z ATCC 23715 ATCC 25923 z ATCC 35152 ATCC 25923 z ATCC 19113 KCTC1927 z ATCC 35152 KCTC1927 z ATCC 19113
Escherichia coli
E. coli (O55:K59(B5):H2) E. coli (EPEC_eaeA) (ETEC_LT) (ETEC_ST) (EIEC_inV) (EHEC_VT1) (EHEC_VT2) (EAEC) Vibrio parahaemolyticus
V. vulnificus V. mimicus Campylobacter jejuni Shigella sonnei
S. flexneri S. boydii Yersinia enterocolitica Listeria monocytogenes Clostridium perfringens Staphylococcus cohnii S. caprae S. lentus
ID (DRn , 400)
NCCP10812 NCCP12241 KCTC2515 ATCC 14028 ATCC 13076 KCCM12021 NCCP10322 NCCP10860 NCCP12232 ATCC BAA614 ATCC 14028 KCCM41575 KCCM41758 NCCP10623 NCCP10715 NCCP14579 ATCC 11775 ATCC 43887 ATCC BAA1706 ATCC 12014 NCCP13715 NCCP13717 NCCP13718 NCCP13719 NCCP13720 NCCP13721 NCCP14039 ATCC 10511 ATCC 17802 ATCC 27969 ATCC 27562 ATCC 33653 KCCM41773 KCTC2009 KCTC2518 Unknown (4)d KCTC2517 Unknown (2) Unknown (2) KCCM41657 ATCC 23715 ATCC 700545 Unknown Unknown Unknown Unknown
a
ATCC, American Type Culture Collection, Manassas, VA; KCCM, Korean Culture Center of Microorganisms, Seoul, Korea; KCTC, Korean Collection for Type Cultures, Daejeon, Korea; NCCP, National Culture Collection for Pathogens, Ohsong, Korea; KFDA, Korean Food and Drug Administration, Seoul, Korea. DRn, an expression of the magnitude of the normalized fluorescence signal generated by the reporter each minute during iTPA amplification: Rn – average Rn of the first 10 min. b From food samples generated in the KFDA laboratory. c S. aureus strains from the hands of food workers at Samsung Welstory food court. d Unknown, strains from food samples from Samsung Welstory Food R&D Center.
RESULTS Inclusivity and exclusivity of the real-time iTPA assay. The S. aureus nuc-based real-time iTPA assay
successfully detected all 91 S. aureus strains and produced negative results for the 51 non–S. aureus strains (Table 1), validating the high specificity of the nuc-based real-time iTPA assay for S. aureus. All of the S. aureus strains had
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TABLE 2. Oligonucleotide primers and probe sequences used in this study Name
Sequence (59–39)
Position (bp)a
Outer forward Outer reverse Inner forward Inner reverse FRET probe
AAA GCG ATT GAT GGT GAT ACG GT ACT CGA CTT CAA TTT TCT TTG CAT TTT CTA CAG ACA TCC AGC TGrG rArGrA TCA ACC AAT GAC ATT CAG ACT ATT GTC GAG CAG TAA CTC TrGrAr GrGA GGA CCA TAT TTC TCT ACA CCT T FAM-TGG TTG ATA CAC CTrG rArArA CAA AGC ATC CT-BHQ1b
508–530 659–688 549–572 608–630 574–603
a b
GenBank no. V01281. BHQ1, black hole quencher 1.
DRn values .600, and the non–S. aureus strains had DRn values of ,400 (data not shown). The PCR assay using the iTPA outer primers yielded amplicons of the expected size (181 bp) for all 91 S. aureus strains without spurious priming and cross-reactivity with the non–S. aureus strains (data not shown). For a rigorous comparison of exclusivity, low concentrations of genomic DNA (ca. 103 CFU/ml) from the positive strains and very high concentrations of genomic DNA (ca. 107 CFU/ml) from the negative strains were used as the template. Neither false-positive nor false-negative results were observed for any of the 142 bacterial strains with the real-time iTPA assay using two primer sets and the FRET signal probe, indicating a high level of specificity. Detection limits of the real-time iTPA assay. The detection limits of the real-time iTPA assay using 10-fold serial dilutions of S. aureus ATCC 25932 after overnight incubation (approximately 1.2 | 109 CFU/ml) were determined (Table 3). The lowest level of cells detected was 103 CFU/ml (with an DRn value .600 within 60 min). Validity of the iTPA assay for food samples. The detection limits for S. aureus in three kinds of food samples were determined using the iTPA assay after enrichment (Table 4). Inocula of 101 CFU in the preenriched samples of kiwi dressing, custard cream, and soybean milk, with final populations of 108 to 109 CFU/ml after enrichment, were consistently detected. The lowest detection limit achieved in this study was 101 CFU per 25 g of preenriched food sample. Without enrichment of the bacterial culture, the detection limit was 103 CFU/ml. The same detection limits
were obtained for the serial dilutions of S. aureus ATCC 25923 (data not shown). DISCUSSION The PCR is one of the most widely used techniques in diagnostic applications because it allows sensitive and rapid diagnosis. However, this technique is not suitable for routine food safety testing because of the need for expensive thermal cycler equipment and complex operations. To overcome such limitations, several nucleic acid amplification methods have been developed for which thermal cycling is not required and operation is simple. For example, transcription-mediated amplification (14), nucleic acid sequence–based amplification (3), strand displacement amplification (23, 24), helicase-dependent amplification (22), rolling-circle amplification (5, 16), and loop-mediated isothermal amplification (17–19) are efficient nucleic acid amplification techniques under isothermal conditions, although each method has advantages and disadvantages (7). In contrast, the iTPA method both amplifies the target DNA sequences and increases the FRET probe signal because the probe is also amplified. As a result, iTPA increases the level of sensitivity compared with other isothermal methods. iTPA also achieves a high level of specificity through the use of two pairs of primers and an TABLE 4. Detection limits for S. aureus in inoculated food samples by iTPA assay Food sample
Kiwi dressing TABLE 3. Sensitivity for detection of S. aureus ATCC 25923 using real-time iTPA assay Level (CFU/ml)
Avg Tt (min)a
CV of Tt (%)b
Avg DRnc
60.6 33.3 29 26.2 22.5 20.2 17.3 16.1
46 14.9 10.2 10.8 8.4 6.7 7.3 22.4
400 2,339 2,800 2,931 2,773 2,876 2,732 2,704
Soybean milk 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 a b c
| | | | | | | |
102 103 104 105 106 107 108 109
Tt, threshold time. CV, coefficient of variation. DRn ~ Rn – average Rn of the first 10 min.
Custard cream
a b
S. aureus (CFU)
1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2
| | | | | | | | | | | | | | |
101 102 103 104 105 101 102 103 104 105 101 102 103 104 105
Tt (min)a
Avg DRnb
¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡
955 1,511 1,375 1,408 1,339 1,148 1,525 1,542 1,603 1,449 1,492 1,056 1,604 1,555 1,195
27.0 16.0 16.9 17.1 18.3 21.4 16.0 16.3 16.9 18.1 18.0 27.3 16.2 17.4 21.2
9.0 1.4 2.9 3.0 5.3 1.3 1.1 1.7 0.9 1.7 2.2 4.3 2.0 1.6 1.8
Tt, threshold time. DRn ~ Rn – average Rn of the first 10 min.
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amplicon-specific FRET signal probe based on cycling probe technology (4, 6, 8). We previously found that the iTPA-based assay was highly effective for the detection of Salmonella contamination in food samples (11). In the present study, a novel and rapid real-time DNA detection method was developed using real-time isothermal fluorescence equipment, which is much less expensive and much simpler to operate than real-time PCR equipment because thermal cycling is not required. A set of DNA-RNA-DNA chimeric primers and a FRET signal probe were designed to specifically target the nuc gene of S. aureus for use in this novel and rapid real-time DNA detection method without expensive equipment. We found that the real-time iTPA assay can be used to detect S. aureus inocula with a detection limit of 103 CFU/ml within 1 h. The four primers and one FRET probe designed herein from five regions of the S. aureus nuc gene are highly specific to S. aureus. The testing of 91 S. aureus strains and 51 non–S. aureus strains on a rapid fluorescent detection platform resulted in 100% inclusivity and 100% exclusivity for the detection of S. aureus (Table 1). Compared with endpoint iTPA detection using a water bath and a fluorescent reader, the real-time iTPA assay is much faster and more convenient because no sample tube transition is required between the amplification and detection steps. The cost and routine maintenance requirements of a real-time isothermal fluorescent reader (e.g., the iGene-S) are much less than those for current commercial real-time PCR equipment because the expensive and specialized thermal cycling module is not needed. In conclusion, a real-time isothermal DNA detection system was developed that has great potential for applications in pathogen testing for foods. The nuc-based real-time iTPA assay is a specific, sensitive, and rapid method for the detection of S. aureus in food samples. This simple method should allow rapid risk assessment of staphylococcal food poisoning at a low cost, which would be more practical for the many food manufacturers and stakeholders interested in food safety. REFERENCES 1. Brakstad, O. G., K. Aasbakk, and J. A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654–1660. 2. Candrian, U. 1995. Polymerase chain reaction in food microbiology. J. Microbiol. Methods 23:89–103. 3. Compton, J. 1991. Nucleic acid sequence-based amplification. Nature 350:91–92. 4. Duck, P., G. Alvarado-Urbina, B. Burdick, and B. Collier. 1990. Probe amplifier system based on chimeric cycling oligonucleotides. BioTechniques 9:142–149. 5. Fire, A., and S.-Q. Xu. 1995. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92:4641–4645. 6. Fong, W. K., Z. Modrusan, J. P. McNevin, J. Marostenmaki, B. Zin, and F. Bekkaoui. 2000. Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology. J. Clin. Microbiol. 38:2525–2529. 7. Gill, P., and A. Ghaemi. 2008. Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids 27: 224–243.
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