Abstract
Goal of the study: to define the design of primers and probes specific to DNA of non-tuberculous mycobacteria and evaluate their diagnostic value in case of simultaneous detection of non-tuberculous mycobacteria and M. tuberculosis complex by real time PCR. Materials and methods. Primer 3, Primer BLAST, Ugene Uni Pro were used to design primers and probes. Preliminary assessment of specificity and sensitivity of detection of non-tuberculous mycobacteria DNA was performed on cultures belonging to 18 types of non-tuberculous mycobacteria, 16 strains of M. tuberculosis complex and 14 types of microorganisms being none Mycobacterum. Analytic sensitivity was tested on 284 cultures of non-tuberculous mycobacteria and diagnostic sensitivity was tested on 124 sputum samples. The kit ofM-Sorb-Tub-Avtomat (ZAO Sintol) was used for DNA isolation. Cultures were subcultured on the liquid medium of Middlebrook 7H9 in Bactec MGIT 960. Cultures were identified with the use of standard microbiological techniques. Analysis of DNA isolated from cultures was performed by the reagent kit of GenoTypeCM/AS (Hain Lifescience, Germany). Results. 100% specificity and sensitivity of PCR was demonstrated in mycobacterial cultures and 100% specificity and 69-70% sensitivity was demonstrated in diagnostic material analysis.
Highlights
Цель исследования: определить дизайн праймеров и зондов, специфичных к ДН К нетуберкулезных микобактерий (Н Т М Б), и оценить их диагностическую значимость при одновременном выявлении НТМБиM. tuberculosis comp1ex (М БТ К ) методом ПЦ Р в режиме реального времени
Pre1iminary assessment of specificity and sensitivity of detection of non-tubercu1ous m ycobacteria DNA was performed on cu1tures be1onging to 18 types of non-tubercu1ous mycobacteria, 16 strains of M. tuberculosis comp1ex and 14 types of microorganisms being none M ycobacterum
Ana1ytic sensitivity was tested on 284 cu1tures of non-tubercu1ous mycobacteria and diagnostic sensitivity was tested on 124 sputum samp1es
Summary
Микобактериальные штаммы Для предварительной оценки специфично сти и чувствительности ПЦР на выявление ДНК НТМБ использовали культуры НТМ Б (18 видов: M. avium, M. abscessus spp. abscessus, M. abscessus spp. chelonae, M. fortuitum spp. peregrinum, M. fortuitum spp. fortuitum, M. gastri, M. gordonae, M. intracellulare, M. interjectum, M. kansasii, M. malmoense, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. smegmatis, M. simiae, M. szulgai, M. xenopi), МБТК (16 штаммов: 5 штаммов M. bovis Bovinus, 1 штамм M. bovis Vallee, 5 штаммов M. bovis BCG, 5 штаммов M. tuberculosis) и микроорганизмов, не относящихся к роду Mycobacterium (14 видов: Achromobacter xylosoxidans, Bukholderia cenocepacia, Candida ablicans, Corynebacterium sp , Gordonia polyisoprenivorans, Nocardia farcinica, Klebsiella pneumoniae, Moraxella catarrhalis, Proteus vulgaris, Pseudomonas aeruginosa, Rhodococcus bronchialis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae), хранящиеся в коллек ции отдела микробиологии ФГБНУ «ЦНИИТ». Sequences of primers and probes, specific to gene of metH M. avium, M. abscessus spp. abscessus, M. abscessus spp. chelonae, M.fortuitum spp. fortuitum, M.fortuitum spp. peregrinum, M. gordonae, M. intracellulare, M. interjectum, M. mucogenicum, M. nonchromogenicum, M. scorfulaceum, M. simiae, M. smegmatis, M. szulgai and gene oftufM. Table 1. sequences of primers and probes, specific to gene of metH M. avium, M. abscessus spp. abscessus, M. abscessus spp. chelonae, M.fortuitum spp. fortuitum, M.fortuitum spp. peregrinum, M. gordonae, M. intracellulare, M. interjectum, M. mucogenicum, M. nonchromogenicum, M. scorfulaceum, M. simiae, M. smegmatis, M. szulgai and gene oftufM. gastri, M. kansasii, M. malmoense, M. xenopi
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