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A portable detection method for Staphylococcus aureus based on RPA-CRISPR/dCas9 and NanoBiT
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Shenjunjie LU1, Guijie BAI1, Yanle WANG1, Xin HUANG1, Yanning ZHANG1, Duo LIU2, 3, *, Hanjie WANG1, 2, 3, 4, *
Acta Microbiologica Sinica | 2026, 66(10) : 5294 - 5310
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Acta Microbiologica Sinica | 2026, 66(10): 5294-5310
• Technology and Method •
A portable detection method for Staphylococcus aureus based on RPA-CRISPR/dCas9 and NanoBiT
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Shenjunjie LU1, Guijie BAI1, Yanle WANG1, Xin HUANG1, Yanning ZHANG1, Duo LIU2, 3, *, Hanjie WANG1, 2, 3, 4, *
Affiliations
  • 1.School of Life Sciences, Faculty of Medicine, Tianjin University, Tianjin, China
  • 2.Key Laboratory of Tropical Biological Resources of Ministry of Education and Hainan Engineering Research Center for Drug Screening and Evaluation, School of Pharmaceutical Sciences, Hainan University, Haikou, Hainan, China
  • 3.State Key Laboratory of Synthetic Biology, Tianjin University, Tianjin, China
  • 4.Engineering Research Center for the Prevention and Control of Animal Original Zoonosis of Fujian Province University, College of Life Science, Longyan University, Longyan, Fujian, China
About Author:

These authors contributed equally to this work.

Published: 2026-10-04 doi: 10.13343/j.cnki.wsxb.20260214
Outline
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[Objective] To address the current issue that pathogen detection methods rely on specialized personnel and precision instruments and are thus unsuitable for on-site rapid testing, this study established a portable and rapid detection method for Staphylococcus aureus (SA) based on the recombinase polymerase amplification (RPA)-CRISPR/dCas9 system and NanoLuc binary interaction technology (NanoBiT). [Methods] Firstly, targeting the highly conserved thermostable nuclease-encoding gene (nuc) of SA, RPA primers and single guide RNA (sgRNA) with high sensitivity were selected. Secondly, reaction parameters including the concentration ratio of sgRNA to dCas9 protein and the detection time were optimized, and detection signals were read by a self-developed portable photoelectric biosensor. Finally, with the optimized reaction system and conditions, this method was evaluated in terms of the sensitivity, specificity, adaptability under different environmental temperatures, and practical sample detection performance. [Results] The optimized method can complete the detection within 1 h. With the mean signal of the negative control plus three times the standard deviation (mean+3SD) as the threshold, the limit of detection (LOD) reached 102 copies/µL. The method was specifically sensitive only to the target bacterium SA, demonstrating high specificity, sensitivity, and environmental adaptability. [Conclusion] A highly sensitive, specific, portable, and low-cost detection method for SA was successfully established based on RPA-CRISPR/dCas9 and NanoBiT. This method is particularly suitable for on-site rapid testing in resource-limited settings and holds broad application prospects.

CRISPR  /  RPA  /  nucleic acid detection  /  NanoBiT  /  Staphylococcus aureus  /  photoelectric biosensor
陆沈焌杰, 白桂杰, 王焱乐, 黄鑫, 张燕宁, 刘夺, 王汉杰. 基于RPA-CRISPR/dCas9与NanoBiT技术的金黄色葡萄球菌便携式检测方法. 微生物学报, 2026 , 66 (10) : 5294 -5310 . DOI: 10.13343/j.cnki.wsxb.20260214
Shenjunjie LU, Guijie BAI, Yanle WANG, Xin HUANG, Yanning ZHANG, Duo LIU, Hanjie WANG. A portable detection method for Staphylococcus aureus based on RPA-CRISPR/dCas9 and NanoBiT[J]. Acta Microbiologica Sinica, 2026 , 66 (10) : 5294 -5310 . DOI: 10.13343/j.cnki.wsxb.20260214
Funding
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  • National Key Research and Development Program of China(2024YFC3407000)
  • National Natural Science Foundation of China(32571637)
  • Tianjin Natural Science Foundation(23JCJQJC00210)
  • Beijing-Tianjin-Hebei Basic Research Cooperation Project of Beijing Natural Science Foundation(23JCZXJC00370)
  • Shanghai “Pioneer Program” Bio-Hybrid Robot Project(25XF3200800)
Appendix
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Year 2026 volume 66 Issue 10
208
86
Cite this Article
BibTeX
Article Info
doi: 10.13343/j.cnki.wsxb.20260214
  • Receive Date：2026-03-13
  • Online Date：2026-10-13
  • Published：2026-10-04
Article Data
Affiliations
History
  • Received：2026-03-13
  • Accepted：2026-06-02
Funding
National Key Research and Development Program of China(2024YFC3407000)
National Natural Science Foundation of China(32571637)
Tianjin Natural Science Foundation(23JCJQJC00210)
Beijing-Tianjin-Hebei Basic Research Cooperation Project of Beijing Natural Science Foundation(23JCZXJC00370)
Shanghai “Pioneer Program” Bio-Hybrid Robot Project(25XF3200800)
Affiliations
    1.School of Life Sciences, Faculty of Medicine, Tianjin University, Tianjin, China
    2.Key Laboratory of Tropical Biological Resources of Ministry of Education and Hainan Engineering Research Center for Drug Screening and Evaluation, School of Pharmaceutical Sciences, Hainan University, Haikou, Hainan, China
    3.State Key Laboratory of Synthetic Biology, Tianjin University, Tianjin, China
    4.Engineering Research Center for the Prevention and Control of Animal Original Zoonosis of Fujian Province University, College of Life Science, Longyan University, Longyan, Fujian, China

Corresponding:

*E-mail: WANG Hanjie,
LIU Duo,
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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
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
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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