title: Evaluation of a low-density hydrogel microarray technique for mycobacterial species identification.
abstract: In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation z@biochip.ru.
authors.forename: Danila V
authors.initials: DV
authors.lastname: Zimenkov
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kulagina
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Antonova
authors.affiliation: Moscow Scientific and Clinical Antituberculosis Center, Department of Health of Moscow, Moscow, Russian Federation.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Krasnova
authors.affiliation: Research Institute of Phthisiopulmonology, St. Petersburg, Russian Federation St. Petersburg State University, St. Petersburg, Russian Federation.
authors.forename: Ekaterina N
authors.initials: EN
authors.lastname: Chernyaeva
authors.affiliation: Research Institute of Phthisiopulmonology, St. Petersburg, Russian Federation.
authors.forename: Vyacheslav Y
authors.initials: VY
authors.lastname: Zhuravlev
authors.affiliation: Research Institute of Phthisiopulmonology, I. M. Sechenov First Moscow State Medical Academy, Moscow, Russian Federation.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Kuz'min
authors.affiliation: Research Institute of Phthisiopulmonology, I. M. Sechenov First Moscow State Medical Academy, Moscow, Russian Federation.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Popov
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Zasedatelev
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Gryadunov
mesh_terms: D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D058889:Molecular Typing / Q000379:methods*; D009161:Mycobacterium / Q000235:genetics*; D009164:Mycobacterium Infections / Q000175:diagnosis* / Q000382:microbiology*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate