Development of a ToxR-based Loop-mediated Isothermal Amplification Assay for Detecting Vibrio Parahaemolyticus

Overview

Journal BMC Microbiol

Publisher Biomed Central

Specialty Microbiology

Date 2010 Feb 12

PMID 20146814

Citations 29

Authors

Siyi Chen

Beilei Ge

Affiliations

Soon will be listed here.

Abstract

Background: Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Sensitive and specific detection methods are needed to better control V. parahaemolyticus infections. This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting V. parahaemolyticus in oysters. A set of five LAMP primers, two outer, two inner, and one loop were designed based on the published V. parahaemolyticus toxR sequence. Specificity of the assay was evaluated using a panel of 36 V. parahaemolyticus and 39 other strains. The assay sensitivity was determined using serial dilutions of V. parahaemolyticus ATCC 27969 culture ranging from 10(8) CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples.

Results: The toxR-based LAMP assay was able to specifically detect all of the 36 V. parahaemolyticus strains without amplification from 39 other strains. The detection limit was 47-470 cells per reaction in pure culture, up to 100-fold more sensitive than that of toxR-PCR. When applied in spiked oysters, the assay was able to detect 1.1 x 10(5) V. parahaemolyticus cells per gram of oyster without enrichment, up to 100-fold more sensitive than that of toxR-PCR. Standard curves generated for detecting V. parahaemolyticus in both pure culture and spiked oyster samples showed good linear relationship between cell numbers and the fluorescence or turbidity signals.

Conclusions: The toxR-based LAMP assay developed in this study was sensitive, specific, and quantitative, holding great potential for future field detection of V. parahaemolyticus in raw oysters.

Citing Articles

Colorimetric and electrochemical analysis of DNAzyme-LAMP amplicons for the detection of Escherichia coli in food matrices.

Sewid A, Dylewski H, Ramos J, Morgan B, Gelalcha B, DSouza D Sci Rep. 2024; 14(1):28942.

PMID: 39578633 PMC: 11584896. DOI: 10.1038/s41598-024-80392-4.

Vibrio parahaemolyticus Epidemiology and Pathogenesis: Novel Insights on an Emerging Foodborne Pathogen.

Gavilan R, Caro-Castro J, Blondel C, Martinez-Urtaza J Adv Exp Med Biol. 2023; 1404:233-251.

PMID: 36792879 DOI: 10.1007/978-3-031-22997-8_12.

Visual LAMP method for the detection of Vibrio vulnificus in aquatic products and environmental water.

Tian Z, Yang L, Qi X, Zheng Q, Shang D, Cao J BMC Microbiol. 2022; 22(1):256.

PMID: 36271365 PMC: 9585733. DOI: 10.1186/s12866-022-02656-1.

Point-of-care system for rapid real-time detection of SARS-CoV-2 virus based on commercially available Arduino platforms.

Ngoc H, Quyen T, Vinayaka A, Bang D, Wolff A Front Bioeng Biotechnol. 2022; 10:917573.

PMID: 35992344 PMC: 9385952. DOI: 10.3389/fbioe.2022.917573.

Fluorescent Detection of Methicillin Resistant by Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum Dots.

Aliasgharian A, Gill P, Ahanjan M, Rafati A Avicenna J Med Biotechnol. 2022; 14(1):79-88.

PMID: 35509356 PMC: 9017464. DOI: 10.18502/ajmb.v14i1.8546.

References

Zhang X, Meaden P, Austin B . Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi. Appl Environ Microbiol. 2001; 67(7):3161-7. PMC: 92995. DOI: 10.1128/AEM.67.7.3161-3167.2001. View

. Vibrio parahaemolyticus infections associated with consumption of raw shellfish--three states, 2006. MMWR Morb Mortal Wkly Rep. 2006; 55(31):854-6. View

Venkateswaran K, Dohmoto N, Harayama S . Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp. Appl Environ Microbiol. 1998; 64(2):681-7. PMC: 106102. DOI: 10.1128/AEM.64.2.681-687.1998. View

Osorio C, Klose K . A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among Vibrio species. J Bacteriol. 2000; 182(2):526-8. PMC: 94307. DOI: 10.1128/JB.182.2.526-528.2000. View

DePAOLA A, Kaysner C, Bowers J, Cook D . Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Appl Environ Microbiol. 2000; 66(11):4649-54. PMC: 92362. DOI: 10.1128/AEM.66.11.4649-4654.2000. View

Butt A, Aldridge K, Sanders C . Infections related to the ingestion of seafood Part I: Viral and bacterial infections. Lancet Infect Dis. 2004; 4(4):201-12. DOI: 10.1016/S1473-3099(04)00969-7. View

Gooch J, DePAOLA A, Kaysner C, Marshall D . Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters. Appl Environ Microbiol. 2001; 67(2):721-4. PMC: 92640. DOI: 10.1128/AEM.67.2.721-724.2001. View

Kim J, Lee G, Kim J, Kwon J, Kwon S . The development of rapid real-time PCR detection system for Vibrio parahaemolyticus in raw oyster. Lett Appl Microbiol. 2008; 46(6):649-54. DOI: 10.1111/j.1472-765X.2008.02368.x. View

Cook D, OLeary P, Hunsucker J, Sloan E, Bowers J, Blodgett R . Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: a national survey from June 1998 to July 1999. J Food Prot. 2002; 65(1):79-87. DOI: 10.4315/0362-028x-65.1.79. View

10.

Parida M, Posadas G, Inoue S, Hasebe F, Morita K . Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol. 2004; 42(1):257-63. PMC: 321710. DOI: 10.1128/JCM.42.1.257-263.2004. View

11.

Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K . Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification. BMC Microbiol. 2008; 8:94. PMC: 2446398. DOI: 10.1186/1471-2180-8-94. View

12.

DePaola A, Nordstrom J, Bowers J, Wells J, Cook D . Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters. Appl Environ Microbiol. 2003; 69(3):1521-6. PMC: 150055. DOI: 10.1128/AEM.69.3.1521-1526.2003. View

13.

Lin Z, Kumagai K, Baba K, Mekalanos J, Nishibuchi M . Vibrio parahaemolyticus has a homolog of the Vibrio cholerae toxRS operon that mediates environmentally induced regulation of the thermostable direct hemolysin gene. J Bacteriol. 1993; 175(12):3844-55. PMC: 204801. DOI: 10.1128/jb.175.12.3844-3855.1993. View

14.

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N . Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000; 28(12):E63. PMC: 102748. DOI: 10.1093/nar/28.12.e63. View

15.

Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M . Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol Lett. 2005; 253(1):155-61. DOI: 10.1016/j.femsle.2005.09.032. View

16.

Monis P, Giglio S, Saint C . Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem. 2005; 340(1):24-34. DOI: 10.1016/j.ab.2005.01.046. View

17.

Altekruse S, Bishop R, Baldy L, Thompson S, Wilson S, Ray B . Vibrio gastroenteritis in the US Gulf of Mexico region: the role of raw oysters. Epidemiol Infect. 2000; 124(3):489-95. PMC: 2810935. DOI: 10.1017/s0950268899003714. View

18.

Croci L, Suffredini E, Cozzi L, Paniconi M, Ciccaglioni G, Colombo M . Evaluation of different polymerase chain reaction methods for the identification of Vibrio parahaemolyticus strains isolated by cultural methods. J AOAC Int. 2008; 90(6):1588-97. View

19.

Han F, Walker R, Janes M, Prinyawiwatkul W, Ge B . Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters. Appl Environ Microbiol. 2007; 73(21):7096-8. PMC: 2074966. DOI: 10.1128/AEM.01116-07. View

20.

Oliver J, Warner R, Cleland D . Distribution and ecology of Vibrio vulnificus and other lactose-fermenting marine vibrios in coastal waters of the southeastern United States. Appl Environ Microbiol. 1982; 44(6):1404-14. PMC: 242203. DOI: 10.1128/aem.44.6.1404-1414.1982. View