Real-time PCR assay for robust detection and global surveillance of the Mycobacterium tuberculosis Haarlem genotype
Tkachuk D. Vyazovaya A. Kostyukova I. Couvin D. Zhdanova S. Bakuła Z. Jiao W. Solovieva N. Eliseev P. Badleeva M. Kobesov N. Dzagoev V. Wuyep V.B. Ikeh E.I. Nayzabaeva D. Kondratov I. Bezruchko M. Chekrygin S. Polev D. Gorokhov I. Sergeev G. Chilikina A. Pasechnik O. Schwartz Y. Zhuravlev V. Ogarkov O. Mariandyshev A. Shen A. Jagielski T. Singh U. Valcheva V. Skiba Y. Mokrousov I.
December 2025Nature Research
Scientific Reports
2025#15Issue 1
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.
Haarlem genotype , Mycobacterium tuberculosis , Spoligotyping , Whole-genome sequencing
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St. Petersburg Pasteur Institute, St. Petersburg, Russian Federation
Clinical Tuberculosis Dispensary, Omsk, Russian Federation
Institut Pasteur de la Guadeloupe, Guadeloupe, Abymes, France
Institute of Epidemiology and Microbiology, Scientific Centre for the Family Health and Human Reproduction Problems, Irkutsk, Russian Federation
Department of Medical Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland
Beijing Key Laboratory of Core Technologies for the Prevention and Treatment of Emerging Infectious Diseases in Children, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Respiratory Diseases, Beijing Children’s Hospital, Beijing Pediatric Research Institute, Capital Medical University, National Center for Children’s Health, Beijing, 100045, China
St. Petersburg Research Institute of Phthisiopulmonology, St. Petersburg, Russian Federation
Northern State Medical University, Arkhangelsk, Russian Federation
National Medical Research Center for Phthisiopulmonology and Infectious Diseases, Moscow, Russian Federation
Department of Infectious Diseases, Dorji Banzarov Buryat State University, Ulan-Ude, Buryatia, Russian Federation
Republican Clinical Center of Phthisiopulmonology, North Ossetia-Alania, Vladikavkaz, Russian Federation
Plateau State College of Health Technology, Jos, Zawan, Nigeria
Department of Medical Microbiology, School of Medical and Health Sciences, College of Medical Sciences, University of Jos, Jos, Nigeria
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan
Almaty Branch of National Center for Biotechnology in Central Reference Laboratory, Almaty, Kazakhstan
Resource Center Bio-bank Center, Research Park of St. Petersburg State University, St. Petersburg, Russian Federation
Department of Public Health, Omsk State Medical University, Omsk, Russian Federation
Novosibirsk Tuberculosis Research Institute, Novosibirsk, Russian Federation
Northern Arctic Federal University, Arkhangelsk, Russian Federation
Henan Children’s Hospital, Children’s Hospital Affiliated to Zhengzhou University, Zhengzhou Children’s Hospital, Zhengzhou, China
All Indian Institute of Medical Sciences, Delhi and Head of National TB, Program, India
Laboratory of Molecular Genetics of Mycobacteria, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Department of Biotechnology, Al-Farabi Kazakh National University, Almaty, Kazakhstan
St. Petersburg Pasteur Institute
Clinical Tuberculosis Dispensary
Institut Pasteur de la Guadeloupe
Institute of Epidemiology and Microbiology
Department of Medical Microbiology
Beijing Key Laboratory of Core Technologies for the Prevention and Treatment of Emerging Infectious Diseases in Children
St. Petersburg Research Institute of Phthisiopulmonology
Northern State Medical University
National Medical Research Center for Phthisiopulmonology and Infectious Diseases
Department of Infectious Diseases
Republican Clinical Center of Phthisiopulmonology
Plateau State College of Health Technology
Department of Medical Microbiology
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry
Almaty Branch of National Center for Biotechnology in Central Reference Laboratory
Resource Center Bio-bank Center
Department of Public Health
Novosibirsk Tuberculosis Research Institute
Northern Arctic Federal University
Henan Children’s Hospital
All Indian Institute of Medical Sciences
Laboratory of Molecular Genetics of Mycobacteria
Department of Biotechnology
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