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Abstract
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Article Information:
Construction of Method for Rapid Detection of Vibrio Parahaemolyticus Using the Quantitative Real-Time PCR Based on the ToxR Gene
Dayong Wang, 1Zhendong Fang, 2Chaoxin Xie and 1Yutong Liu
Corresponding Author: Dayong Wang
Submitted: March 28, 2013
Accepted: April 29, 2013
Published: August 05, 2013 |
Abstract:
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In this research, a sensitive, rapid and highly reproducible SYBR green based real-time PCR assay was developed for detection of toxR positive pathogenic Vibrio parahaemolyticus. To establish a real-time PCR assay for accurate and rapid detection of Vibrio parahaemolyticus. The special target sequence of toxR gene of Vibrio parahaemolyticus was amplified and characterized with a pair of primes. Reaction system and determination approach of real-time PCR were established for detection of Vibrio parahaemolyticus. The foodborne pathogen of Staphylococcus aureus, Salmonella were used as specificity reference for the method, the amplification curves were observed as a typical “S” curve, other pathogens were also tested and no amplification was observed. In addition, the results of melting curve analysis showed only a specific peak with a melting temperature of 86.33°C and no primer-dimers peak was observed. These findings indicated that the PCR primers had high specificity. Analysis of standard curves revealed excellent correlation between the number of copies (in the range of 4×109 to 4×101 and PCR threshold cycle (Ct) with a correlation coefficient of 0.992 (R2 = 0.992). It was found that the limit of this assay was 15 CFU/mL for pure culture and 1.535pg for genomic DNA. The total detection assay could be completed in 2 h. Results indicated that real-time PCR detection methods established in this research was accurate, sensitive, rapid, reproducible for the quantitative detection of environmental Vibrio parahaemolyticus.
Key words: Rapid detection, real-time PCR, toxR gene, Vibrio parahaemolyticus, , ,
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Cite this Reference:
Dayong Wang, 1Zhendong Fang, 2Chaoxin Xie and 1Yutong Liu, . Construction of Method for Rapid Detection of Vibrio Parahaemolyticus Using the Quantitative Real-Time PCR Based on the ToxR Gene . Advance Journal of Food Science and Technology, (08): 1022-1030.
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ISSN (Online): 2042-4876
ISSN (Print): 2042-4868 |
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