|
KMID : 0545120210310121672
|
|
Journal of Microbiology and Biotechnology
2021 Volume.31 No. 12 p.1672 ~ p.1683
|
|
|
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
Huang Xian-Hui
Guo Li-Qing
Shen Zi-Chen
Lv Lin-Xue
Li Feng-Xia
Zhou Zan-Hu
Zhang Dan-Feng
|
|
|
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°C 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.
|
|
|
KEYWORD
|
|
|
Vibrio parahaemolyticus, loop-mediated isothermal amplification (LAMP), lateral flow dipstick (LFD), blaCARB-17 gene, rapid molecular detection, food safety
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|