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Cleavable molecular beacon-based loop-mediated isothermal amplification assay for the detection of adulterated chicken in meat

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Abstract

A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for chicken authentication detection. LAMP and CMB were used for DNA amplification and amplicon analysis, respectively. Targeting the mitochondrial cytochrome b gene of chickens, five primers and one CMB probe were designed, and their specificity was validated against nine other animal species. The structure of CMB and concentrations of dNTPs, MgSO4, betaine, RNase H2, primers and CMB were optimized. The CMB-LAMP assay was completed within 17 min, and its limit of detection for chicken DNA was 1.5 pg μL−1. Chicken adulteration as low as 0.5% was detected in beef, and no cross-reactivity was observed. Finally, this assay was successfully applied to 20 commercial meat products. When combined with our developed DNA extraction method (the extraction time was 1 min: lysis for 10 s, washing for 20 s and elution for 30 s), the entire process (from DNA extraction to results analysis) was able to be completed within 20 min, which is at least 10 min shorter than other LAMP-based methods. Our method showed great potential for the on-site detection of chicken adulteration in meat.

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Acknowledgments

The National Natural Science Foundation of China (No. 32102060), Key R&D Program of Zhejiang (Nos. 2021C02059-4 and 2022C02028), Natural Science Foundation of Zhejiang Province (No. LQ21B050003), Science and Technology Programs of Ningbo (Nos. 202003N4127 and 20211ZDYF020179) and State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (No. 2021DG700024-KF202112) are gratefully acknowledged for financial support.

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Authors and Affiliations

  1. State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, China

    Song Yan, Hangzhen Lan, Zhen Wu, Yangying Sun, Maolin Tu & Daodong Pan

  2. Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang Province and College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315800, China

    Song Yan, Hangzhen Lan, Zhen Wu, Yangying Sun, Maolin Tu & Daodong Pan

  3. National R&D Center for Freshwater Fish Processing, Jiangxi Normal University, Nanchang, 330022, Jiangxi, China

    Daodong Pan

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  1. Song Yan
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Yan, S., Lan, H., Wu, Z. et al. Cleavable molecular beacon-based loop-mediated isothermal amplification assay for the detection of adulterated chicken in meat. Anal Bioanal Chem 414, 8081–8091 (2022). https://doi.org/10.1007/s00216-022-04342-7

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