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<p>The prevalence of macrolide-resistant <i>Mycoplasma pneumoniae</i> (<i>M. pneumoniae</i>) is increasing, and rapid macrolide resistance detection assays are needed for clinical management. The A2063G mutation in the 23S rRNA gene of <i>M. pneumoniae</i> is the most common mutation and confers high macrolide resistance. Programmable nucleases have risen as new tools of molecular diagnosis, in which Argonaute is independent of the protospacer adjacent motif (PAM) manner and flexible in molecular diagnosis. By leveraging the specific recognition and cleavage capability of the programmable nuclease Argonaute, we developed a <i>Pyrococcus furiosus</i> Argonaute (<i>Pf</i>Ago)–based biosensor for detecting the macrolide resistance–associated A2063G mutation in the 23S rRNA gene of <i>M. pneumoniae</i>&#xa0;without flanking PAM. Through systematic optimization of the <i>Pf</i>Ago and guide DNA concentrations to maximize sensitivity and specificity, we achieved a detection limit of 1 fM for DNA single-base mutations, with no cross-reactivity observed against 11 other respiratory tract pathogens. When tested on 25 clinical specimens from patients infected with <i>M. pneumoniae</i>, our assay showed that 20 cases were positive with the A2063G mutation, and 5 cases were negative without the A2063G mutation. Compared to DNA sequencing analysis, our assay demonstrated 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic (area under the curve value = 1.00) (<i>n</i> = 25). We developed a platform for the highly sensitive and specific identification of the macrolide-resistance&#xa0;associated A2063G mutation. This can be used for the detection of the macrolide-resistant <i>M. pneumoniae</i> in clinical specimens and provides a new method for the identification of drug-resistant isolates.</p>

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Highly sensitive PfAgo-based biosensor for the detection of mutations in the 23S rRNA gene of macrolide-resistant Mycoplasma pneumoniae

  • 期刊论文

  • 发表日期：

    2025年10月30日

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  • Houqi Wang,
  • Huimin Liao,
  • Huiyun Xie,
  • Suhui He,
  • Xiang Xiao,
  • Haoliang Yin,
  • Zishi Chen,
  • Rong Wei,
  • Zhaoyang Xie,
  • Luxin Yu,
  • Zhangquan Chen

摘要

The prevalence of macrolide-resistant Mycoplasma pneumoniae (M. pneumoniae) is increasing, and rapid macrolide resistance detection assays are needed for clinical management. The A2063G mutation in the 23S rRNA gene of M. pneumoniae is the most common mutation and confers high macrolide resistance. Programmable nucleases have risen as new tools of molecular diagnosis, in which Argonaute is independent of the protospacer adjacent motif (PAM) manner and flexible in molecular diagnosis. By leveraging the specific recognition and cleavage capability of the programmable nuclease Argonaute, we developed a Pyrococcus furiosus Argonaute (PfAgo)–based biosensor for detecting the macrolide resistance–associated A2063G mutation in the 23S rRNA gene of M. pneumoniae without flanking PAM. Through systematic optimization of the PfAgo and guide DNA concentrations to maximize sensitivity and specificity, we achieved a detection limit of 1 fM for DNA single-base mutations, with no cross-reactivity observed against 11 other respiratory tract pathogens. When tested on 25 clinical specimens from patients infected with M. pneumoniae, our assay showed that 20 cases were positive with the A2063G mutation, and 5 cases were negative without the A2063G mutation. Compared to DNA sequencing analysis, our assay demonstrated 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic (area under the curve value = 1.00) (n = 25). We developed a platform for the highly sensitive and specific identification of the macrolide-resistance associated A2063G mutation. This can be used for the detection of the macrolide-resistant M. pneumoniae in clinical specimens and provides a new method for the identification of drug-resistant isolates.