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Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction
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Mengqin HUANG1, Yu XIN1, Danfeng FU2, Ziyang LIU1, Daoyun PENG3, Shu ZENG4, Yi WAN4, Qian CUI5, Fei CHEN6
Acta Microbiologica Sinica | 2026, 66(4) : 2007 - 2021
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Acta Microbiologica Sinica | 2026, 66(4): 2007-2021
• Technology and Method •
Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction
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Mengqin HUANG1, Yu XIN1, Danfeng FU2, Ziyang LIU1, Daoyun PENG3, Shu ZENG4, Yi WAN4, Qian CUI5, Fei CHEN6
Affiliations
  • 1.School of Marine Biology and Fisheries, Hainan University, Haikou, Hainan, China
  • 2.Hainan Provincial Aquatic Product Quality and Safety Inspection Center (Hainan Provincial Fisheries Technology Extension Station), Haikou, Hainan, China
  • 3.School of Biomedical Engineering, Hainan University, Haikou, Hainan, China
  • 4.School of Marine Sciences (State Key Laboratory of Marine Resource Utilization in South China Sea), Hainan University, Haikou, Hainan, China
  • 5.School of Life and Health Sciences, Hainan University, Haikou, Hainan, China
  • 6.School of Food Science and Engineering, Hainan University, Haikou, Hainan, China
Published: 2026-04-04 doi: 10.13343/j.cnki.wsxb.20260051
Outline
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Objective To develop a fluorescence method for Vibrio parahaemolyticus detection by the combination of CRISPR system and the hybridization chain reaction (HCR), thus achieving rapid, sensitive, and accurate detection of the pathogen. Methods Cascade probe (RP/I) and HCR hairpin structures were first designed according to a specific conserved sequences screened from V. parahaemolyticus. Subsequently, the feasibility, specificity, and sensitivity of the method were evaluated after the optimization of reaction conditions. Furthermore, V. parahaemolyticus-contaminated aquatic products were used to validate the interference resistance of the method. Results The cleavage of CRISPR/Cas13a was activated upon binding to the target RNA (T-RNA), leading to the trans-cleavage of the RP/I cascade probe and the release of I strand. Then, the released I strand subsequently triggered HCR, generating a significant fluorescence signal for target detection. The established method successfully distinguished target sequences with single-base, double-base, and triple-base mismatches and enabled the specific identification of V. parahaemolyticus against other non-target bacteria, including V. alginolyticus, V. vulnificus, V. harveyi, V. cholerae, and Escherichia coli, demonstrating excellent specificity. The assay showed a good linear correlation over a T-RNA concentration range of 25 pmol/L to 10 nmol/L. The corresponding linear regression equation was y=7 236.75×lg CT-RNA-8 590.11 (R2=0.99, C represents the T-RNA concentration and y represents the fluorescence intensity), with the LOD of 1.01 pmol/L. The proposed method enabled rapid detection of RNA extracted from V. parahaemolyticus in various aquatic products, yielding results consistent with those obtained by RT-qPCR. Conclusion The fluorescence method based on CRISPR/Cas13a-HCR established in this study realizes rapid detection of V. parahaemolyticus, demonstrating good sensitivity, specificity, and accuracy.

Vibrio parahaemolyticus  /  CRISPR/Cas13a  /  hybridization chain reaction  /  fuorescence detection
Mengqin HUANG, Yu XIN, Danfeng FU, Ziyang LIU, Daoyun PENG, Shu ZENG, Yi WAN, Qian CUI, Fei CHEN. Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction[J]. Acta Microbiologica Sinica, 2026 , 66 (4) : 2007 -2021 . DOI: 10.13343/j.cnki.wsxb.20260051
Funding
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  • National Natural Science Foundation of China(22204036)
  • Hainan Provincial Science and Technology Special Fund(ZDYF2025XDNY066)
Appendix
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Year 2026 volume 66 Issue 4
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8388
4197
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Article Info
doi: 10.13343/j.cnki.wsxb.20260051
  • Receive Date：2026-01-19
  • Online Date：2026-04-14
  • Published：2026-04-04
Article Data
Affiliations
History
  • Received：2026-01-19
  • Accepted：2026-03-05
Funding
National Natural Science Foundation of China(22204036)
Hainan Provincial Science and Technology Special Fund(ZDYF2025XDNY066)
Affiliations
    1.School of Marine Biology and Fisheries, Hainan University, Haikou, Hainan, China
    2.Hainan Provincial Aquatic Product Quality and Safety Inspection Center (Hainan Provincial Fisheries Technology Extension Station), Haikou, Hainan, China
    3.School of Biomedical Engineering, Hainan University, Haikou, Hainan, China
    4.School of Marine Sciences (State Key Laboratory of Marine Resource Utilization in South China Sea), Hainan University, Haikou, Hainan, China
    5.School of Life and Health Sciences, Hainan University, Haikou, Hainan, China
    6.School of Food Science and Engineering, Hainan University, Haikou, Hainan, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
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Genus		 种数
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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