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Recombinase polymerase amplification-lateral flow (RPA-LF) assay combined with immunomagnetic separation for rapid visual detection of Vibrio parahaemolyticus in raw oysters

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Abstract

This study was the first attempt to optimize a recombinase polymerase amplification (RPA) and lateral flow (LF) assay combined with immunomagnetic separation (IMS) for the detection of Vibrio parahaemolyticus in raw oysters. The newly developed IMS-RPA-LF assay effectively combines sample preparation, amplification, and detection into a single platform. Under optimal conditions, the average capture efficiency (CE) for 104 colony forming units (CFU)/mL of four V. parahaemolyticus strains with 0.4 mg of immunomagnetic beads within 45 min was 80.3%. After optimization, the RPA-LF assay was able to detect V. parahaemolyticus within 15 min, comprising DNA amplification with RPA for 10 min at 37 °C and visualization of the amplicons through LF strips for 5 min. The RPA-LF assay exhibited good specificity by showing a test line for eight V. parahaemolyticus strains with different serotypes but no cross-reaction with 12 non-V. parahaemolyticus bacteria. RPA-LF assay was found to be sensitive and detected as low as 10 pg genomic DNA of V. parahaemolyticus. For spiked oyster samples, the detection sensitivity of V. parahaemolyticus was improved to 2 CFU/g by IMS-RPA-LF after enrichment for 4 h; in contrast, the IMS-PCR method required 8 h. Hence, even when V. parahaemolyticus was present in very low numbers in samples, the IMS-RPA-LF assay could be completed within half a workday. Because of the high sensitivity, specificity, and speed of the IMS-RPA-LF assay, this newly developed method opens a novel pathway for rapid diagnostic screening of V. parahaemolyticus in seafood, which is an increasingly important health issue worldwide.

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Funding

This study was financially supported by the National Key Research and Development Programs of China (no. 2017YFC1200201), the Shanghai Natural Science Foundation of China (nos. 17ZR1437200 and 18ZR1413400), and the National Natural Science Foundation of China (no. 31702277).

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Authors and Affiliations

  1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang, Shanghai, 200241, China

    Wei Jiang, Xiangan Han, Tongling Shan, Zhaoguo Chen & Quan Wang

  2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China

    Yaling Ren & Yongjie Liu

  3. Shanghai Customs, Shanghai, 200135, China

    Junxin Xue

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  1. Wei Jiang
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  2. Yaling Ren
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  3. Xiangan Han
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  6. Zhaoguo Chen
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  7. Yongjie Liu
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Correspondence to Yongjie Liu or Quan Wang.

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Jiang, W., Ren, Y., Han, X. et al. Recombinase polymerase amplification-lateral flow (RPA-LF) assay combined with immunomagnetic separation for rapid visual detection of Vibrio parahaemolyticus in raw oysters. Anal Bioanal Chem 412, 2903–2914 (2020). https://doi.org/10.1007/s00216-020-02532-9

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