Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using blaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD)

  • Hu, Yuan-qing (School of Biological Science and Biotechnology, Minnan Normal University) ;
  • Huang, Xian-hui (School of Biological Science and Biotechnology, Minnan Normal University) ;
  • Guo, Li-qing (Zhangzhou Center for Disease Control and Prevention) ;
  • Shen, Zi-chen (School of Biological Science and Biotechnology, Minnan Normal University) ;
  • LV, Lin-xue (School of Biological Science and Biotechnology, Minnan Normal University) ;
  • Li, Feng-xia (School of Biological Science and Biotechnology, Minnan Normal University) ;
  • Zhou, Zan-hu (Comprehensive Technical Service Center, Zhangzhou Customs) ;
  • Zhang, Dan-feng (School of Biological Science and Biotechnology, Minnan Normal University)
  • Received : 2021.07.12
  • Accepted : 2021.09.02
  • Published : 2021.12.28
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Abstract

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The blaCARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this blaCARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg2+, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63℃ for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10-4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

Keywords

Acknowledgement

This work was supported by the Project of Fujian Natural Science Foundation of China [Grant No. 2021J01995, 2020J01809]; the Open Program of Jiangsu Key Laboratory of Zoonosis [Grant No. R1402]; and the Project of Natural Science Foundation of General Customs Administration [Grant No. 2019HK103].

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