Accepted Manuscript Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains Peiyan He , Zhongwen Chen , Jianyong Luo , Henghui Wang , Yong Yan , Lixia Chen , Wenjie Gao PII:
S0890-8508(14)00035-8
DOI:
10.1016/j.mcp.2014.06.001
Reference:
YMCPR 1080
To appear in:
Molecular and Cellular Probes
Received Date: 7 March 2014 Accepted Date: 1 June 2014
Please cite this article as: He P, Chen Z, Luo J, Wang H, Yan Y, Chen L, Gao W, Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains, Molecular and Cellular Probes (2014), doi: 10.1016/j.mcp.2014.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains
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Peiyan He, Zhongwen Chen*, Jianyong Luo, Henghui Wang, Yong Yan, Lixia Chen, Wenjie Gao
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*Correspondence author. Tel: 86-573-83685806. E-mail: [email protected].
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Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China
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ABSTRACT Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, a EvaGreen -based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (speciesspecific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.
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Keywords: Vibrio parahaemolyticus; Multiplex real-time PCR; toxR; tdh; trh
ACCEPTED MANUSCRIPT 1. Introduction
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Vibrio parahaemolyticus, a Gram-negative halophilic bacterium, which naturally inhabits marine and estuarine environment, has become an important food-borne pathogen throughout the world. Consumption of raw, undercooked or mishandled seafood contaminated with this bacterium can cause gastroenteritis. V. parahaemolyticus has accounted for large part of food poisoning cases in coastal provinces of China [1] and many other countries, such as the USA and Japan [2, 3]. Therefore, rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. Unfortunately, the classical approach for V. parahaemolyticus detection is culture-based [4]. It contains complex procedure, including liquid enrichment, selective isolation and biochemical identification. Apart from being laborious and time consuming, the culture-based method is subjective and low sensitive and requires experience in interpretation. Furthermore, V. parahaemolyticus represents pathogenic strains (virulence genes positive) and non-pathogenic strains (virulence genes negative), and the culture-based approach is not able to distinguish pathogenic strains from non-pathogenic strains. In order to overcome the limitations of the culture-based approach, several alternative methods have been developed, such as conventional PCR and real-time PCR. Compared with conventional PCR, real-time PCR allows the quantitation of amplicons in a closed-tube setting. The procedure is simple, fast and suitable for high-throughput routine testing. Several reports have shown the feasibility of using real-time PCR for pathogenic V. parahaemolyticus detection [5, 6, 7]. Real-time PCR can be carried out using fluorescent probes that bind specifically to particular DNA sequences or fluorescent dyes that intercalate with any double-stranded DNA [8]. Most of real-time PCR assays for pathogenic V. parahaemolyticus detection reported to date are Taqman probe-based. Though Taqman probe-based real-time PCR have many advantages, the relatively high cost of this method limits its use in routine testing [9]. In the present study, we established a cost-effective multiplex real-time PCR for the detection of pathogenic V. parahaemolyticus by using melting-curve analysis employing a fluorescent dye EvaGreen. This multiplex real-time PCR can specifically detect three distinct genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh).
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2. Material and methods
2.1. Bacterial strains and DNA extraction A total of 53 V. parahaemolyticus strains were used in this study, including one reference strain ATCC 33847 and 52 isolates. The latter isolates of V. parahaemolyticus were obtained from food or patients with diarrhoea in Jiaxing, China, and identified by culture-based approach. The presence of virulence genes (tdh and trh) of ATCC 33847 and isolates were tested by the Taqman probe-based real-time PCR assay reported by Nordstrom et al. [10]. Two isolates were non-pathogenic strains (virulence genes negative), 50 isolates and ATCC 33847 were pathogenic strains (virulence genes positive). Among 51 pathogenic strains, 3 isolates were test-positive for virulence gene tdh and trh, 47 isolates and the reference strain ATCC 33847 were test-positive for virulence gene tdh but negative for trh. Besides 53 V. parahaemolyticus strains, 19 other bacterial strains were also analyzed in this study (Table 1). All strains were cultured and maintained on
ACCEPTED MANUSCRIPT Luria-Bertani (LB) plates. Genomic DNA of bacterial strains was extracted with KAPA Express Extract Kits (KAPA Biosystems, Woburn, USA) according to the manufacturer’s instruction. The concentration of extracted DNA was measured in a Nano-100 spectrophotometer (Medclub Scientific, Taoyuan, China). 2.2. Oligonucleotide primers
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The DNA sequences of toxR, tdh and trh genes of V. parahaemolyticus were retrieved from GenBank and aligned using Clustal X. Based on the sequences alignment, the conserved regions were selected for primer design using the program Primer Premier 5.0. Three primer pairs of target genes are listed in Table 2.
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2.3. Multiplex real-time PCR assay
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The PCR mix consisted of 0.11 µmol/l toxR primers, 0.8 µmol/l tdh primers, 0.4 µmol/l trh primers, half of the total volume of EvaGreen real-time PCR mix (Bio-Rad Laboratories, Hercules, USA) and appropriate volume of sterile distilled water, finally DNA template or control was added to the PCR mix. The PCR amplification conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 5 s, and annealing/extension at 59 °C for 25 s. Fluorescence signals were measured once each cycle after annealing/extension step. A melting-curve analysis was conducted after PCR amplification. The PCR products were cooled to 75 °C and then heated to 85 °C at a rate of 0.1 °C per second. Fluorescence signals were continuously monitored during the whole process of melting-curve analysis. All reactions were performed using a Bio-Rad CFX 96TM real-time PCR system. 2.4. Specificity and sensitivity of the multiplex real-time PCR assay
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Specificity of the real-time PCR assay was evaluated by amplifying genomic DNA extracted from all of the strains listed in Table1. For sensitivity, genomic DNA of V. parahaemolyticus strain JX13021, which was an isolate that was PCR test-positive for virulence genes tdh and trh, was ten-fold serially diluted in sterile distilled water and tested by multiplex real-time PCR assay. The limit of detection was calculated with probit-analysis with 95% probability. 2.5. Reproducibility of multiplex real-time PCR assay To evaluate the reproducibility of real-time PCR assay, ten-fold serially diluted genomic DNA of V. parahaemolyticus was tested repeatedly. Three separate dilution series were assayed in the same run to evaluate intra-assay variability, and inter-assay variability was measured by testing each dilution in three different runs. The intra- and inter-assay variabilities were calculated as coefficient of variation (CV) based on Cq values of each testing. 2.6. Detection of V. parahaemolyticus in clinical samples Anal swab samples were obtained from 65 patients with diarrhoea. Two samples were
ACCEPTED MANUSCRIPT obtained from each patient and enriched in alkaline peptone water containing 3.5% of sodium chloride. One sample was enriched for 5 h and tested by multiplex real-time PCR. The other was enriched for 18 h and tested with culture-based approach in parallel. The presence of tdh and trh of V. parahaemolyticus strains isolated from swab samples by culture-based approach was tested by the real-time PCR assay reported by Nordstrom et al. [10].
3.1. Simplex and multiplex Real-time PCR amplification
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3. Results
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The toxR primers produced PCR products with melting peak at 82±0.1 °C (Fig. 1a), the tdh primers produced PCR products with melting peak at 79.5±0.1 °C (Fig. 1b) and the trh primers produced PCR products with melting peak at 77.5±0.1 °C (Fig. 1c) from genomic DNA of V. parahaemolyticus strain JX13021. When multiplex real-time PCR was performed using genomic DNA of V. parahaemolyticus strain JX13021, three distinguishable melting peaks could be observed (Fig. 1d). 3.2. Specificity of the multiplex real-time PCR
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Fifty three strains of V. parahaemolyticus and 19 strains of other bacteria were tested using the newly established multiplex real-time PCR assay. All V. parahaemolyticus strains showed positive amplification in the multiplex real-time PCR assay. The melting-curve analysis after PCR amplification showed that 2 strains were test-positive only for toxR, 48 strains for toxR and tdh but negative for trh; 3 strains were test-positive for toxR, tdh and trh. The results for tdh and trh were consistent with our previous results achieved by a different real-time PCR assay [10]. For 19 other bacterial strains and negative template control, there was no obvious amplification curve observed in the multiplex real-time PCR assay.
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3.3. Sensitivity of the multiplex real-time PCR
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Various concentrations of extracted genomic DNA of V. parahaemolyticus strain JX13021 were tested using the multiplex real-time PCR assay. Cq values increased with decreasing DNA concentration, and a good linear correlation of the samples with positive amplification was observed between Cq values and the concentration of DNA (R2=1.000, slope= -3.368) (Fig. 2). The probit-analysis showed that the limit of detection of the multiplex real-time PCR assay in 95% cases was 1.4 pg genomic DNA per reaction. 3.4. Reproducibility of multiplex real-time PCR assay Reproducibility of real-time PCR assay was assessed based on intra- and inter-assay variabilities. Intra-assay variability ranged from 0.18% to 0.27%, while inter-assay variability ranged from 0.60% to 0.91% (Table 3), which indicated that the multiplex real-time PCR assay was highly reproducible.
ACCEPTED MANUSCRIPT 3.5. Detection of V. parahaemolyticus in clinical samples
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Among the 65 samples tested by the multiplex real-time PCR assay following 5 h cultureenrichment, 21 samples (32.31%) showed positive amplification. All of these 21 samples were test-positive for toxR and tdh but negative for trh. The culture-based approach performed in parallel also isolated V. parahaemolyticus strains from these 21 test-positive samples, but V. parahaemolyticus was not isolated from other samples. The results of the real-time PCR assay reported by Nordstrom et al. [10] also showed all of these isolates were test-positive for tdh but negative for trh. 4. Discussion
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In this study, we established a multiplex real-time PCR assay for the detection and identification pathogenic V. parahaemolyticus in a single reaction tube. The detection limit of this assay is 1.4 pg genomic DNA per reaction (concentration which gives a test-positive result for 95% of samples). Three genetic markers of V. parahaemolyticus (toxR, tdh and trh) were selected as targets for this multiplex PCR. For total V. parahaemolyticus detection, gene toxR was targeted, a gene that has been widely used for V. parahaemolyticus identification [11, 12, 13]. For pathogenicity determination, genes tdh and trh were targeted because of the strong correlation between the presence of these two virulence genes and pathogenic of V. parahaemolyticus [14, 15]. Rapid detection and the identification of pathogenic V. parahaemolyticus are essential from a public health viewpoint. Therefore, many multiplex PCR assays have been developed for this purpose. For example, a multiplex PCR assay targeting tlh, tdh and trh [10] and another using groEL, tdh and trh [16]. However, the multiplex PCR assays reported to date are conventional PCR assays or Taqman probe-based real-time PCR assays. A conventional PCR assay is cost-effective, but it requires a post-PCR processing step for PCR products analysis, which is a low throughput assay and is laborious and susceptible to carryover contamination [17, 18]. Although Taqman probe-based real-time PCR overcomes these limitations of conventional PCR, unique fluorescence labeled probes are required for each target and synthesis of such probes is expensive. Furthermore, multiplex PCR assays for detection and identification of pathogenic V. parahaemolyticus usually target three genes (one species-specific gene and two virulence genes tdh and trh). Such Taqman probe-based multiplex real-time PCR assays require the use of instruments that contain at least a three colour channel. However, some instruments, such as Bio-Rad MyiQ TM 2 are based on two colour channel. The multiplex real-time PCR assay that we established combines the advantages of conventional PCR assay and Taqman probe-based real-time PCR assay. First, PCR amplification and amplicon analysis are accomplished in a closed tube format, which is practical, suitable for high-throughput routine testing and reduces the risk of contamination. Second, expensive fluorescently-labelled probes are not required. Thus, the cost is low and similar to that of conventional PCR assay. Third, our assay is suited to most instruments, as it requires only one colour channel, FAM/SYBR Green channel, which is the commonest channel in real-time PCR instruments, for the detection of three targets. Our multiplex real-time PCR assay was designed by using melting-curve analysis. During melting-curve analysis, after PCR amplification, a sudden decrease in the fluorescence of
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fluorescent dye was observed as the sample is heated through to the melting temperature (Tm) of PCR products [19]. Different PCR products have different Tm values as the difference in GC content, length and sequence, and thus they can produce different melting peaks, making multiplexing feasible. However, not all multiplex PCR assays are suitable to identify their amplicons by melting-curve analysis. A previous study [20] showed that amplicons with 1.2 °C differences in Tm value only produced a single melting peak. Many studies [19, 20, 21] reported amplicons with 2 °C differences in Tm value were reliable for producing distinguishable melting peaks. Therefore, primer design of three targets is critical for the development of our assay. The strategy for primer design of our assay was not only to achieve specificity and sensitivity of primers, but also to produce PCR products that could be distinguished by melting-curve analysis. In this study, a series of primers were designed to three targets using the program Primer Premier 5.0. Then, these primers were tested by simplex and multiplex assays. Finally, a set of primers suitable for the multiplex real-time PCR assay were selected. Tm values of PCR products of each primer pairs were greater than 2 °C away from each other. The validation of the newly established multiplex real-time PCR assay with reference strains, isolated strains and clinical samples in this study confirmed the sensitivity and specificity of the selected primer sets. In conclusion, our data suggest that the newly established multiplex real-time PCR assay was practical, cost-effective, sensitive and capable of specific high-throughput detection of pathogenic V. parahaemolyticus. Therefore, this multiplex real-time PCR might be useful for the routine detection of pathogenic V. parahaemolyticus and as a tool for responding to sudden outbreaks of food poisoning cases caused by V. parahaemolyticus.
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Acknowledgements We are grateful to Zhejiang Center for Disease Control and Prevention, P. R. China, for providing reference strains. This study was supported by the Science and Technology Program of Jiaxing City (No. 2013AY21051-1) and the Open Fund of Key Laboratory of Emergency Detection for Public Health of Zhejiang Province (No. 201302).
ACCEPTED MANUSCRIPT References
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[1] Wang S, Duan H, Zhang W, Li JW. Analysis of bacterial foodborne disease outbreaks in China between 1994 and 2005. FEMS Immunol Med Microbiol 2007; 51: 8-13. [2] Iwamoto M, Ayers T, Mahon BE, Swerdlow DL. Epidemiology of seafood-associated infections in the United States. Clin Microbiol Rev 2010; 23: 399-411. [3] Mahmud ZH, Neogi SB, Kassu A, Wada T, Islam MS, Nair GB, Ota F. Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in Japan. Int J Food Microbiol 2007; 118: 92-6. [4] Peeler JT, Houghtby GA. and Rainosek, A.P. Themost probable number technique. In: Downes FP, Ito K, editors. Compendium of Methods for the Microbiological Examination of Food ed, Washington, DC: American Public Health Association; 1992, p. 105-120. [5] Robert-Pillot A, Copin S, Gay M, Malle P, Quilici ML. Total and pathogenic Vibrio parahaemolyticus in shrimp: fast and reliable quantification by real-time PCR. Int J Food Microbiol 2010; 143: 190-7. [6] Kim JS, Lee GG, Kim J, Kwon JY, Kwon ST. The development of rapid real-time PCR detection system for Vibrio parahaemolyticus in raw oyster. Lett Appl Microbiol 2008; 46: 649- 54. [7] Rizvi AV, Panicker G, Myers ML, Bej AK. Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes. FEMS Microbiol Lett 2006; 262:185-92. [8] Wilhelm J, Pingoud A. Real-time polymerase chain reaction. Chembiochem 2003; 4: 1120-8. [9] Chen J, Tang J, Liu J, Cai Z, Bai X. Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens. J Appl Microbiol 2012; 112: 823-30. [10] Nordstrom JL, Vickery MC, Blackstone GM, Murray SL, DePaola A. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl Environ Microbiol 2007; 73: 5840-7. [11] Nakaguchi Y. Contamination by Vibrio parahaemolyticus and Its Virulent Strains in Seafood Marketed in Thailand, Vietnam, Malaysia, and Indonesia. Trop Med Health 2013; 41: 95-102. [12] Khouadja S, Suffredini E, Spagnoletti M, Croci L, Colombo MM, Amina B. Presence of pathogenic Vibrio parahaemolyticus in waters and seafood from the Tunisian Sea. World J Microbiol Biotechnol 2013; 29: 1341-8. [13] Rosec JP, Simon M, Causse V, Boudjemaa M. Detection of total and pathogenic Vibrio parahaemolyticus in shellfish: comparison of PCR protocols using pR72H or toxR targets with a culture method. Int J Food Microbiol 2009; 129: 136-45. [14] Alam MJ, Tomochika KI, Miyoshi SI, Shinoda S. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol Lett 2002; 208: 83-7. [15] Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Rev Med Microbiol 1993; 4: 106-13. [16] Hossain MT, Kim YO, Kong IS. Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL, tdh and trh genes. Mol Cell Probes 2013; 27(5-6):
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171- 5. [17] Huang J, Zhu Y, Wen H, Zhang J, Huang S, Niu J, Li Q. Quadruplex real-time PCR assay for detection and identification of Vibrio cholerae O1 and O139 strains and determination of their toxigenic potential. Appl Environ Microbiol 2009; 75: 6981-5. [18] Yang S, Lin S, Kelen GD, Quinn TC, Dick JD, Gaydos CA, Rothman RE. Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. J Clin Microbiol 2002; 40: 3449-54. [19] Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997; 245: 154-60. [20] Hernández M, Rodríguez-Lázaro D, Esteve T, Prat S, Pla M. Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection. Anal Biochem 2003; 323: 164-70. [21] Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D, Radel MQ, Goldman D. DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem 2001; 47: 635-44.
ACCEPTED MANUSCRIPT Fig. 1 Melting curves produced by the melting-curve analysis. (a) Melting curve of the toxR gene. (b) Melting curve of the tdh gene. (c) Melting curve of the trh gene. (d) Melting curve following multiplex PCR targeting toxR, tdh and trh genes. (e) Melting curve following multiplex PCR with negative template control.
Table 1. Strains used in this study. Table 2. Primer sequences used in this study.
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Table 3. Reproducibility of multiplex real-time PCR assay.
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Fig. 2 Multiplex real-time PCR detection of V. parahaemolyticus. (a) Amplification profiles of different concentrations of V. parahaemolyticus DNA template. (b) Standard curve.
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No. of strains
Source
V. parahaemolyticus
1
ATCC 33847
52
Isolated
1
ATCC 17749
2
Isolated
V. cholerae
5
Isolated
V. mimicus
1
V. furnissii
1
V. vulnificus
1
V. metschnikovii
1
Salmonella Typhi
1
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ATCC 33653
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V. alginolyticus
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Species
NCTC 11218 ATCC 27562 Isolated ATCC 50097
1
ATCC 12022
1
ATCC 25923
Listeria monocytogenes
1
CMCC 54006
Escherichia coli
1
ATCC 25922
Pseudomonas aeruginosa
1
ATCC 27853
Bacillus cereus
1
ATCC 11778
Shigella flexneri
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Staphylococcus aureus
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Primer
Sequence
toxR
toxR-F
5’-TCTTCTGACGCAATCGTTGAACCA-3’
toxR-R
5’-CTGATACTCACCAATCTGACGGAACTG-3’
tdh-F
5’-CTTCCATCTGTCCCTTTTCCTGCC-3’
tdh-R
5’-CCTGACGTTGTGAATACTGATTGACCATA-3’
trh-F
5’-TACCTTTTCCTTCTCCAGGTTCGG-3’
trh-R
5’-TCGTTTTATGTTTCGGTTTGTCCAGT-3’
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trh
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tdh
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Target gene
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Intra value
Inter value
50ng
0.20%
0.87%
5ng
0.22%
0.91%
500pg
0.23%
0.83%
50pg
0.27%
0.79%
5pg
0.18%
0.60%
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DNA concentration
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S0890-8508(14)00035-8
DOI:
10.1016/j.mcp.2014.06.001
Reference:
YMCPR 1080
To appear in:
Molecular and Cellular Probes
Received Date: 7 March 2014 Accepted Date: 1 June 2014
Please cite this article as: He P, Chen Z, Luo J, Wang H, Yan Y, Chen L, Gao W, Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains, Molecular and Cellular Probes (2014), doi: 10.1016/j.mcp.2014.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains
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Peiyan He, Zhongwen Chen*, Jianyong Luo, Henghui Wang, Yong Yan, Lixia Chen, Wenjie Gao
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*Correspondence author. Tel: 86-573-83685806. E-mail: [email protected].
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Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China
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ABSTRACT Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, a EvaGreen -based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (speciesspecific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.
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Keywords: Vibrio parahaemolyticus; Multiplex real-time PCR; toxR; tdh; trh
ACCEPTED MANUSCRIPT 1. Introduction
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Vibrio parahaemolyticus, a Gram-negative halophilic bacterium, which naturally inhabits marine and estuarine environment, has become an important food-borne pathogen throughout the world. Consumption of raw, undercooked or mishandled seafood contaminated with this bacterium can cause gastroenteritis. V. parahaemolyticus has accounted for large part of food poisoning cases in coastal provinces of China [1] and many other countries, such as the USA and Japan [2, 3]. Therefore, rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. Unfortunately, the classical approach for V. parahaemolyticus detection is culture-based [4]. It contains complex procedure, including liquid enrichment, selective isolation and biochemical identification. Apart from being laborious and time consuming, the culture-based method is subjective and low sensitive and requires experience in interpretation. Furthermore, V. parahaemolyticus represents pathogenic strains (virulence genes positive) and non-pathogenic strains (virulence genes negative), and the culture-based approach is not able to distinguish pathogenic strains from non-pathogenic strains. In order to overcome the limitations of the culture-based approach, several alternative methods have been developed, such as conventional PCR and real-time PCR. Compared with conventional PCR, real-time PCR allows the quantitation of amplicons in a closed-tube setting. The procedure is simple, fast and suitable for high-throughput routine testing. Several reports have shown the feasibility of using real-time PCR for pathogenic V. parahaemolyticus detection [5, 6, 7]. Real-time PCR can be carried out using fluorescent probes that bind specifically to particular DNA sequences or fluorescent dyes that intercalate with any double-stranded DNA [8]. Most of real-time PCR assays for pathogenic V. parahaemolyticus detection reported to date are Taqman probe-based. Though Taqman probe-based real-time PCR have many advantages, the relatively high cost of this method limits its use in routine testing [9]. In the present study, we established a cost-effective multiplex real-time PCR for the detection of pathogenic V. parahaemolyticus by using melting-curve analysis employing a fluorescent dye EvaGreen. This multiplex real-time PCR can specifically detect three distinct genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh).
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2. Material and methods
2.1. Bacterial strains and DNA extraction A total of 53 V. parahaemolyticus strains were used in this study, including one reference strain ATCC 33847 and 52 isolates. The latter isolates of V. parahaemolyticus were obtained from food or patients with diarrhoea in Jiaxing, China, and identified by culture-based approach. The presence of virulence genes (tdh and trh) of ATCC 33847 and isolates were tested by the Taqman probe-based real-time PCR assay reported by Nordstrom et al. [10]. Two isolates were non-pathogenic strains (virulence genes negative), 50 isolates and ATCC 33847 were pathogenic strains (virulence genes positive). Among 51 pathogenic strains, 3 isolates were test-positive for virulence gene tdh and trh, 47 isolates and the reference strain ATCC 33847 were test-positive for virulence gene tdh but negative for trh. Besides 53 V. parahaemolyticus strains, 19 other bacterial strains were also analyzed in this study (Table 1). All strains were cultured and maintained on
ACCEPTED MANUSCRIPT Luria-Bertani (LB) plates. Genomic DNA of bacterial strains was extracted with KAPA Express Extract Kits (KAPA Biosystems, Woburn, USA) according to the manufacturer’s instruction. The concentration of extracted DNA was measured in a Nano-100 spectrophotometer (Medclub Scientific, Taoyuan, China). 2.2. Oligonucleotide primers
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The DNA sequences of toxR, tdh and trh genes of V. parahaemolyticus were retrieved from GenBank and aligned using Clustal X. Based on the sequences alignment, the conserved regions were selected for primer design using the program Primer Premier 5.0. Three primer pairs of target genes are listed in Table 2.
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2.3. Multiplex real-time PCR assay
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The PCR mix consisted of 0.11 µmol/l toxR primers, 0.8 µmol/l tdh primers, 0.4 µmol/l trh primers, half of the total volume of EvaGreen real-time PCR mix (Bio-Rad Laboratories, Hercules, USA) and appropriate volume of sterile distilled water, finally DNA template or control was added to the PCR mix. The PCR amplification conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 5 s, and annealing/extension at 59 °C for 25 s. Fluorescence signals were measured once each cycle after annealing/extension step. A melting-curve analysis was conducted after PCR amplification. The PCR products were cooled to 75 °C and then heated to 85 °C at a rate of 0.1 °C per second. Fluorescence signals were continuously monitored during the whole process of melting-curve analysis. All reactions were performed using a Bio-Rad CFX 96TM real-time PCR system. 2.4. Specificity and sensitivity of the multiplex real-time PCR assay
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Specificity of the real-time PCR assay was evaluated by amplifying genomic DNA extracted from all of the strains listed in Table1. For sensitivity, genomic DNA of V. parahaemolyticus strain JX13021, which was an isolate that was PCR test-positive for virulence genes tdh and trh, was ten-fold serially diluted in sterile distilled water and tested by multiplex real-time PCR assay. The limit of detection was calculated with probit-analysis with 95% probability. 2.5. Reproducibility of multiplex real-time PCR assay To evaluate the reproducibility of real-time PCR assay, ten-fold serially diluted genomic DNA of V. parahaemolyticus was tested repeatedly. Three separate dilution series were assayed in the same run to evaluate intra-assay variability, and inter-assay variability was measured by testing each dilution in three different runs. The intra- and inter-assay variabilities were calculated as coefficient of variation (CV) based on Cq values of each testing. 2.6. Detection of V. parahaemolyticus in clinical samples Anal swab samples were obtained from 65 patients with diarrhoea. Two samples were
ACCEPTED MANUSCRIPT obtained from each patient and enriched in alkaline peptone water containing 3.5% of sodium chloride. One sample was enriched for 5 h and tested by multiplex real-time PCR. The other was enriched for 18 h and tested with culture-based approach in parallel. The presence of tdh and trh of V. parahaemolyticus strains isolated from swab samples by culture-based approach was tested by the real-time PCR assay reported by Nordstrom et al. [10].
3.1. Simplex and multiplex Real-time PCR amplification
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3. Results
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The toxR primers produced PCR products with melting peak at 82±0.1 °C (Fig. 1a), the tdh primers produced PCR products with melting peak at 79.5±0.1 °C (Fig. 1b) and the trh primers produced PCR products with melting peak at 77.5±0.1 °C (Fig. 1c) from genomic DNA of V. parahaemolyticus strain JX13021. When multiplex real-time PCR was performed using genomic DNA of V. parahaemolyticus strain JX13021, three distinguishable melting peaks could be observed (Fig. 1d). 3.2. Specificity of the multiplex real-time PCR
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Fifty three strains of V. parahaemolyticus and 19 strains of other bacteria were tested using the newly established multiplex real-time PCR assay. All V. parahaemolyticus strains showed positive amplification in the multiplex real-time PCR assay. The melting-curve analysis after PCR amplification showed that 2 strains were test-positive only for toxR, 48 strains for toxR and tdh but negative for trh; 3 strains were test-positive for toxR, tdh and trh. The results for tdh and trh were consistent with our previous results achieved by a different real-time PCR assay [10]. For 19 other bacterial strains and negative template control, there was no obvious amplification curve observed in the multiplex real-time PCR assay.
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3.3. Sensitivity of the multiplex real-time PCR
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Various concentrations of extracted genomic DNA of V. parahaemolyticus strain JX13021 were tested using the multiplex real-time PCR assay. Cq values increased with decreasing DNA concentration, and a good linear correlation of the samples with positive amplification was observed between Cq values and the concentration of DNA (R2=1.000, slope= -3.368) (Fig. 2). The probit-analysis showed that the limit of detection of the multiplex real-time PCR assay in 95% cases was 1.4 pg genomic DNA per reaction. 3.4. Reproducibility of multiplex real-time PCR assay Reproducibility of real-time PCR assay was assessed based on intra- and inter-assay variabilities. Intra-assay variability ranged from 0.18% to 0.27%, while inter-assay variability ranged from 0.60% to 0.91% (Table 3), which indicated that the multiplex real-time PCR assay was highly reproducible.
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Among the 65 samples tested by the multiplex real-time PCR assay following 5 h cultureenrichment, 21 samples (32.31%) showed positive amplification. All of these 21 samples were test-positive for toxR and tdh but negative for trh. The culture-based approach performed in parallel also isolated V. parahaemolyticus strains from these 21 test-positive samples, but V. parahaemolyticus was not isolated from other samples. The results of the real-time PCR assay reported by Nordstrom et al. [10] also showed all of these isolates were test-positive for tdh but negative for trh. 4. Discussion
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In this study, we established a multiplex real-time PCR assay for the detection and identification pathogenic V. parahaemolyticus in a single reaction tube. The detection limit of this assay is 1.4 pg genomic DNA per reaction (concentration which gives a test-positive result for 95% of samples). Three genetic markers of V. parahaemolyticus (toxR, tdh and trh) were selected as targets for this multiplex PCR. For total V. parahaemolyticus detection, gene toxR was targeted, a gene that has been widely used for V. parahaemolyticus identification [11, 12, 13]. For pathogenicity determination, genes tdh and trh were targeted because of the strong correlation between the presence of these two virulence genes and pathogenic of V. parahaemolyticus [14, 15]. Rapid detection and the identification of pathogenic V. parahaemolyticus are essential from a public health viewpoint. Therefore, many multiplex PCR assays have been developed for this purpose. For example, a multiplex PCR assay targeting tlh, tdh and trh [10] and another using groEL, tdh and trh [16]. However, the multiplex PCR assays reported to date are conventional PCR assays or Taqman probe-based real-time PCR assays. A conventional PCR assay is cost-effective, but it requires a post-PCR processing step for PCR products analysis, which is a low throughput assay and is laborious and susceptible to carryover contamination [17, 18]. Although Taqman probe-based real-time PCR overcomes these limitations of conventional PCR, unique fluorescence labeled probes are required for each target and synthesis of such probes is expensive. Furthermore, multiplex PCR assays for detection and identification of pathogenic V. parahaemolyticus usually target three genes (one species-specific gene and two virulence genes tdh and trh). Such Taqman probe-based multiplex real-time PCR assays require the use of instruments that contain at least a three colour channel. However, some instruments, such as Bio-Rad MyiQ TM 2 are based on two colour channel. The multiplex real-time PCR assay that we established combines the advantages of conventional PCR assay and Taqman probe-based real-time PCR assay. First, PCR amplification and amplicon analysis are accomplished in a closed tube format, which is practical, suitable for high-throughput routine testing and reduces the risk of contamination. Second, expensive fluorescently-labelled probes are not required. Thus, the cost is low and similar to that of conventional PCR assay. Third, our assay is suited to most instruments, as it requires only one colour channel, FAM/SYBR Green channel, which is the commonest channel in real-time PCR instruments, for the detection of three targets. Our multiplex real-time PCR assay was designed by using melting-curve analysis. During melting-curve analysis, after PCR amplification, a sudden decrease in the fluorescence of
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fluorescent dye was observed as the sample is heated through to the melting temperature (Tm) of PCR products [19]. Different PCR products have different Tm values as the difference in GC content, length and sequence, and thus they can produce different melting peaks, making multiplexing feasible. However, not all multiplex PCR assays are suitable to identify their amplicons by melting-curve analysis. A previous study [20] showed that amplicons with 1.2 °C differences in Tm value only produced a single melting peak. Many studies [19, 20, 21] reported amplicons with 2 °C differences in Tm value were reliable for producing distinguishable melting peaks. Therefore, primer design of three targets is critical for the development of our assay. The strategy for primer design of our assay was not only to achieve specificity and sensitivity of primers, but also to produce PCR products that could be distinguished by melting-curve analysis. In this study, a series of primers were designed to three targets using the program Primer Premier 5.0. Then, these primers were tested by simplex and multiplex assays. Finally, a set of primers suitable for the multiplex real-time PCR assay were selected. Tm values of PCR products of each primer pairs were greater than 2 °C away from each other. The validation of the newly established multiplex real-time PCR assay with reference strains, isolated strains and clinical samples in this study confirmed the sensitivity and specificity of the selected primer sets. In conclusion, our data suggest that the newly established multiplex real-time PCR assay was practical, cost-effective, sensitive and capable of specific high-throughput detection of pathogenic V. parahaemolyticus. Therefore, this multiplex real-time PCR might be useful for the routine detection of pathogenic V. parahaemolyticus and as a tool for responding to sudden outbreaks of food poisoning cases caused by V. parahaemolyticus.
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Acknowledgements We are grateful to Zhejiang Center for Disease Control and Prevention, P. R. China, for providing reference strains. This study was supported by the Science and Technology Program of Jiaxing City (No. 2013AY21051-1) and the Open Fund of Key Laboratory of Emergency Detection for Public Health of Zhejiang Province (No. 201302).
ACCEPTED MANUSCRIPT References
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[1] Wang S, Duan H, Zhang W, Li JW. Analysis of bacterial foodborne disease outbreaks in China between 1994 and 2005. FEMS Immunol Med Microbiol 2007; 51: 8-13. [2] Iwamoto M, Ayers T, Mahon BE, Swerdlow DL. Epidemiology of seafood-associated infections in the United States. Clin Microbiol Rev 2010; 23: 399-411. [3] Mahmud ZH, Neogi SB, Kassu A, Wada T, Islam MS, Nair GB, Ota F. Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in Japan. Int J Food Microbiol 2007; 118: 92-6. [4] Peeler JT, Houghtby GA. and Rainosek, A.P. Themost probable number technique. In: Downes FP, Ito K, editors. Compendium of Methods for the Microbiological Examination of Food ed, Washington, DC: American Public Health Association; 1992, p. 105-120. [5] Robert-Pillot A, Copin S, Gay M, Malle P, Quilici ML. Total and pathogenic Vibrio parahaemolyticus in shrimp: fast and reliable quantification by real-time PCR. Int J Food Microbiol 2010; 143: 190-7. [6] Kim JS, Lee GG, Kim J, Kwon JY, Kwon ST. The development of rapid real-time PCR detection system for Vibrio parahaemolyticus in raw oyster. Lett Appl Microbiol 2008; 46: 649- 54. [7] Rizvi AV, Panicker G, Myers ML, Bej AK. Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes. FEMS Microbiol Lett 2006; 262:185-92. [8] Wilhelm J, Pingoud A. Real-time polymerase chain reaction. Chembiochem 2003; 4: 1120-8. [9] Chen J, Tang J, Liu J, Cai Z, Bai X. Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens. J Appl Microbiol 2012; 112: 823-30. [10] Nordstrom JL, Vickery MC, Blackstone GM, Murray SL, DePaola A. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl Environ Microbiol 2007; 73: 5840-7. [11] Nakaguchi Y. Contamination by Vibrio parahaemolyticus and Its Virulent Strains in Seafood Marketed in Thailand, Vietnam, Malaysia, and Indonesia. Trop Med Health 2013; 41: 95-102. [12] Khouadja S, Suffredini E, Spagnoletti M, Croci L, Colombo MM, Amina B. Presence of pathogenic Vibrio parahaemolyticus in waters and seafood from the Tunisian Sea. World J Microbiol Biotechnol 2013; 29: 1341-8. [13] Rosec JP, Simon M, Causse V, Boudjemaa M. Detection of total and pathogenic Vibrio parahaemolyticus in shellfish: comparison of PCR protocols using pR72H or toxR targets with a culture method. Int J Food Microbiol 2009; 129: 136-45. [14] Alam MJ, Tomochika KI, Miyoshi SI, Shinoda S. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol Lett 2002; 208: 83-7. [15] Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Rev Med Microbiol 1993; 4: 106-13. [16] Hossain MT, Kim YO, Kong IS. Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL, tdh and trh genes. Mol Cell Probes 2013; 27(5-6):
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171- 5. [17] Huang J, Zhu Y, Wen H, Zhang J, Huang S, Niu J, Li Q. Quadruplex real-time PCR assay for detection and identification of Vibrio cholerae O1 and O139 strains and determination of their toxigenic potential. Appl Environ Microbiol 2009; 75: 6981-5. [18] Yang S, Lin S, Kelen GD, Quinn TC, Dick JD, Gaydos CA, Rothman RE. Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. J Clin Microbiol 2002; 40: 3449-54. [19] Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997; 245: 154-60. [20] Hernández M, Rodríguez-Lázaro D, Esteve T, Prat S, Pla M. Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection. Anal Biochem 2003; 323: 164-70. [21] Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D, Radel MQ, Goldman D. DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem 2001; 47: 635-44.
ACCEPTED MANUSCRIPT Fig. 1 Melting curves produced by the melting-curve analysis. (a) Melting curve of the toxR gene. (b) Melting curve of the tdh gene. (c) Melting curve of the trh gene. (d) Melting curve following multiplex PCR targeting toxR, tdh and trh genes. (e) Melting curve following multiplex PCR with negative template control.
Table 1. Strains used in this study. Table 2. Primer sequences used in this study.
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Table 3. Reproducibility of multiplex real-time PCR assay.
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Fig. 2 Multiplex real-time PCR detection of V. parahaemolyticus. (a) Amplification profiles of different concentrations of V. parahaemolyticus DNA template. (b) Standard curve.
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No. of strains
Source
V. parahaemolyticus
1
ATCC 33847
52
Isolated
1
ATCC 17749
2
Isolated
V. cholerae
5
Isolated
V. mimicus
1
V. furnissii
1
V. vulnificus
1
V. metschnikovii
1
Salmonella Typhi
1
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ATCC 33653
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V. alginolyticus
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Species
NCTC 11218 ATCC 27562 Isolated ATCC 50097
1
ATCC 12022
1
ATCC 25923
Listeria monocytogenes
1
CMCC 54006
Escherichia coli
1
ATCC 25922
Pseudomonas aeruginosa
1
ATCC 27853
Bacillus cereus
1
ATCC 11778
Shigella flexneri
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Staphylococcus aureus
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Primer
Sequence
toxR
toxR-F
5’-TCTTCTGACGCAATCGTTGAACCA-3’
toxR-R
5’-CTGATACTCACCAATCTGACGGAACTG-3’
tdh-F
5’-CTTCCATCTGTCCCTTTTCCTGCC-3’
tdh-R
5’-CCTGACGTTGTGAATACTGATTGACCATA-3’
trh-F
5’-TACCTTTTCCTTCTCCAGGTTCGG-3’
trh-R
5’-TCGTTTTATGTTTCGGTTTGTCCAGT-3’
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trh
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tdh
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Target gene
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Intra value
Inter value
50ng
0.20%
0.87%
5ng
0.22%
0.91%
500pg
0.23%
0.83%
50pg
0.27%
0.79%
5pg
0.18%
0.60%
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DNA concentration
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