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<p>The Haarlem genotype is a significant yet understudied part of the Euro-American lineage of <i>Mycobacterium tuberculosis</i>, characterized by unique pathogenetic features. Spoligotyping is a primary method for its detection, but it is not suitable for isolates with long blocks of deleted spacers. We have developed a simple and robust method to detect the Haarlem genotype. The real-time PCR (RT-PCR) assay with LNA probes was designed to detect the <i>Rv0282</i> 211&#xa0;C &gt; T mutation that was shown to be specific for the Haarlem genotype. The developed RT-PCR assay was optimized with 68 isolates with known whole-genome sequencing (WGS) data and applied to the geographically and genetically diverse collection of 428 isolates. As a result, 396 isolates were concordantly assigned to either Haarlem or non-Haarlem genotypes by both methods, whereas 32 isolates were discrepant cases. Twenty-two isolates of “unknown genotype” (Russia, Belarus, and Poland) were assigned to Haarlem by SNP-based assay. WGS of these isolates confirmed the results of the RT-PCR assay. To conclude, the developed RT-PCR method provides a reliable detection of the Haarlem genotype in retrospective collections and under prospective epidemiological surveillance. Its actual proportion in <i>M. tuberculosis</i> populations in certain world regions is higher than previously thought. Abridged spoligoprofiles with large deleted blocks of spacers require caution in interpretation.</p>

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Real-time PCR assay for robust detection and global surveillance of the Mycobacterium tuberculosis Haarlem genotype

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月11日

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  • Daria Tkachuk,
  • Anna Vyazovaya,
  • Irina Kostyukova,
  • David Couvin,
  • Svetlana Zhdanova,
  • Zofia Bakuła,
  • Weiwei Jiao,
  • Natalia Solovieva,
  • Platon Eliseev,
  • Maria Badleeva,
  • Nikolai Kobesov,
  • Valentin Dzagoev,
  • Valentine B. Wuyep,
  • Eugene I. Ikeh,
  • Dinara Nayzabaeva,
  • Ilya Kondratov,
  • Mikhail Bezruchko,
  • Sergei Chekrygin,
  • Dmitrii Polev,
  • Ivan Gorokhov,
  • Grigorii Sergeev,
  • Alena Chilikina,
  • Oksana Pasechnik,
  • Yakov Schwartz,
  • Viacheslav Zhuravlev,
  • Oleg Ogarkov,
  • Andrei Mariandyshev,
  • Adong Shen,
  • Tomasz Jagielski,
  • Urvashi Singh,
  • Violeta Valcheva,
  • Yuriy Skiba,
  • Igor Mokrousov

摘要

The Haarlem genotype is a significant yet understudied part of the Euro-American lineage of Mycobacterium tuberculosis, characterized by unique pathogenetic features. Spoligotyping is a primary method for its detection, but it is not suitable for isolates with long blocks of deleted spacers. We have developed a simple and robust method to detect the Haarlem genotype. The real-time PCR (RT-PCR) assay with LNA probes was designed to detect the Rv0282 211 C > T mutation that was shown to be specific for the Haarlem genotype. The developed RT-PCR assay was optimized with 68 isolates with known whole-genome sequencing (WGS) data and applied to the geographically and genetically diverse collection of 428 isolates. As a result, 396 isolates were concordantly assigned to either Haarlem or non-Haarlem genotypes by both methods, whereas 32 isolates were discrepant cases. Twenty-two isolates of “unknown genotype” (Russia, Belarus, and Poland) were assigned to Haarlem by SNP-based assay. WGS of these isolates confirmed the results of the RT-PCR assay. To conclude, the developed RT-PCR method provides a reliable detection of the Haarlem genotype in retrospective collections and under prospective epidemiological surveillance. Its actual proportion in M. tuberculosis populations in certain world regions is higher than previously thought. Abridged spoligoprofiles with large deleted blocks of spacers require caution in interpretation.