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<p>The development of rapid, sensitive, and user-friendly diagnostic methods for <i>Chlamydia psittaci</i> (<i>C. psittaci</i>) is critical for early diagnosis and effective control of infections. Current techniques, such as qPCR and next-generation sequencing, face limitations in accessibility and practicality for point-of-care testing (POCT). In this study, we present the WPTTS platform (one-step recombinase polymerase amplification (RPA)-CRISPR/Cas12b assay using <i>w</i>eak <i>p</i>rotospacer adjacent motif (PAM) recognition with <i>t</i>wo-<i>t</i>emperature <i>s</i>hifting), which leverages the temperature-dependent PAM stringency of <i>Alicyclobacillus acidophilus</i> Cas12b (AapCas12b) to overcome the challenges of one-step CRISPR detection. By employing weak PAM sequences and a two-temperature protocol (37&#xa0;°C for amplification, 60&#xa0;°C for detection), WPTTS minimizes interference between RPA amplification and CRISPR cleavage, achieving a sensitivity of 10<sup>2</sup> copies/reaction—equivalent to two-step methods—while maintaining operational simplicity. The platform also demonstrates enhanced specificity, enabling single-base resolution at critical target positions. Clinical validation with 40 samples confirmed its superior performance, with 95% positive agreement and 100% negative agreement compared to qPCR. This study highlights the potential of WPTTS as a robust, rapid, and highly sensitive tool for POCT applications in <i>C. psittaci</i> detection and broader infectious disease diagnostics.</p> Graphical abstract <p></p>

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One-step detection of Chlamydia psittaci using recombinase polymerase amplification and CRISPR/Cas12b

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月2日

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  • Jingyi Jiang,
  • Jian Xu,
  • Li Gong,
  • Qiong Li,
  • Ping Yao,
  • Rong Wang,
  • Jinyi Jiang,
  • Lei Xu,
  • Fengming Wang,
  • Xujian Mao

摘要

The development of rapid, sensitive, and user-friendly diagnostic methods for Chlamydia psittaci (C. psittaci) is critical for early diagnosis and effective control of infections. Current techniques, such as qPCR and next-generation sequencing, face limitations in accessibility and practicality for point-of-care testing (POCT). In this study, we present the WPTTS platform (one-step recombinase polymerase amplification (RPA)-CRISPR/Cas12b assay using weak protospacer adjacent motif (PAM) recognition with two-temperature shifting), which leverages the temperature-dependent PAM stringency of Alicyclobacillus acidophilus Cas12b (AapCas12b) to overcome the challenges of one-step CRISPR detection. By employing weak PAM sequences and a two-temperature protocol (37 °C for amplification, 60 °C for detection), WPTTS minimizes interference between RPA amplification and CRISPR cleavage, achieving a sensitivity of 102 copies/reaction—equivalent to two-step methods—while maintaining operational simplicity. The platform also demonstrates enhanced specificity, enabling single-base resolution at critical target positions. Clinical validation with 40 samples confirmed its superior performance, with 95% positive agreement and 100% negative agreement compared to qPCR. This study highlights the potential of WPTTS as a robust, rapid, and highly sensitive tool for POCT applications in C. psittaci detection and broader infectious disease diagnostics.

Graphical abstract