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<p>Recombinase polymerase amplification (RPA) has emerged over the past two decades as one of the most promising alternatives to the polymerase chain reaction (PCR) for nucleic acid detection. RPA exploits enzymatic machinery derived from bacteriophage T4 to achieve exponential DNA amplification at a constant, near-physiological temperature of 37–42&#xa0;°C, removing the need for thermal-cycling equipment entirely. The reaction typically produces detectable amplification products within 20–30&#xa0;min and shows useful tolerance of the matrix inhibitors common in complex biological samples. This review provides a critical account of RPA’s molecular mechanism, kit formats, assay optimisation, and applications across pathogen detection, antimicrobial-resistance (AMR) surveillance, food safety and authenticity testing, and as a pre-amplification step coupled to CRISPR-based detection. Key performance data are summarised in comparative tables to facilitate at-a-glance appraisal. A direct comparison with PCR and loop-mediated isothermal amplification (LAMP) is provided, together with practical guidelines for primer/probe design and multiplexing. Persistent challenges, notably nonspecific amplification, limited quantification accuracy, and reagent cost, are critically evaluated alongside emerging solutions. This review supports the United Nations Sustainable Development Goals by contributing to SDG 3 (Good Health and Well-being) through the promotion of rapid, accessible molecular diagnostics for improved disease detection and surveillance, and to SDG 9 (Industry, Innovation and Infrastructure) by advancing innovative, portable diagnostic technologies for decentralized healthcare and public health preparedness.</p>

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Recombinase polymerase amplification: principles, advances, and applications in modern molecular diagnostics

  • 期刊论文

  • 发表日期：

    2026年7月20日

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  • Sanchit Pal Singh,
  • Shruti Gupta,
  • Rohit Solanki,
  • Vivek Singh,
  • Amritanshu Upadhyay

摘要

Recombinase polymerase amplification (RPA) has emerged over the past two decades as one of the most promising alternatives to the polymerase chain reaction (PCR) for nucleic acid detection. RPA exploits enzymatic machinery derived from bacteriophage T4 to achieve exponential DNA amplification at a constant, near-physiological temperature of 37–42 °C, removing the need for thermal-cycling equipment entirely. The reaction typically produces detectable amplification products within 20–30 min and shows useful tolerance of the matrix inhibitors common in complex biological samples. This review provides a critical account of RPA’s molecular mechanism, kit formats, assay optimisation, and applications across pathogen detection, antimicrobial-resistance (AMR) surveillance, food safety and authenticity testing, and as a pre-amplification step coupled to CRISPR-based detection. Key performance data are summarised in comparative tables to facilitate at-a-glance appraisal. A direct comparison with PCR and loop-mediated isothermal amplification (LAMP) is provided, together with practical guidelines for primer/probe design and multiplexing. Persistent challenges, notably nonspecific amplification, limited quantification accuracy, and reagent cost, are critically evaluated alongside emerging solutions. This review supports the United Nations Sustainable Development Goals by contributing to SDG 3 (Good Health and Well-being) through the promotion of rapid, accessible molecular diagnostics for improved disease detection and surveillance, and to SDG 9 (Industry, Innovation and Infrastructure) by advancing innovative, portable diagnostic technologies for decentralized healthcare and public health preparedness.