1 ФГБНУ «Федеральный центр токсикологической, радиационной и биологической безопасности», г. Казань, РФ 2 ФГАОУ ВО «Казанский (Приволжский) федеральный университет», г. Казань, РФ 3 Казанская государственная медицинская академия -филиал ФГБОУ ДПО «РМАНПО» МЗ РФ, г. Казань, РФ Цель исследования: анализ видов и генетических линий туберкулезных микобактерий, выделенных от человека и крупного рогатого скота, методом сполиготипирования. Материалы и методы. Объектом исследования служил биологический материал от крупного рогатого скота, а также работников животноводческих ферм (ежегодно проходивших флюорографическое исследование) и больных туберкулезом пациентов. Для идентификации микобактерий использовали метод сполиготипирования. Результаты. Представлена оценка вариабельности участков внутри прямых повторов (спейсеров), которые в лабораторной диагностике используются при сполиготипировании микобактерий и идентификации возбудителей туберкулеза. Сопоставление встречаемости комбинаций различных спейсеров у анализируемых микобактерий в исследуемом материале, идентифицированных как Mycobacterium tuberculosis complex, со сполигопрофилем известных микобактерий устанавливает принадлежность их к конкретной генетической линии. Исследованные изоляты изучены микроскопическими, бактериологическими и молекулярно-генетическими (ПЦР) методами. По результатам сполиготипирования определена их принадлежность к генетическим линиям M. tuberculosis Beijing, LAM и Haarlem.
Objective of this work was to develop the algorithms for differential PCR indication of Brucella genus strains using databases of their genomes. Materials and methods .Resources of the National Center for Biotechnology Information (NCBI) and BLAST and Vector NTI 9.1.0 software utilities. For PCR amplification, B. suis, B. abortus, B. melitensis nucleic acids, as well as plasmid DNA with marker insertions were used. Results and conclusions. We assessed brucella gene sequences, some of which are found in Brucella genus bacteria, others only in representatives of B. melitensis, and the third ones – only in representatives of B. abortus. As a result of primers and probes designing for indication of Brucella genus bacteria and representatives of B. melitensis and B. abortus species, criteria for marker sequence amplification have been established. These criteria provide for simultaneous differentiation in a single reaction. The determination of strain differences within one species of Brucella is described in multilocus VNTR assay technique, and the profiles of tandem repeats of various B. melitensis and B. abortus strains are available in the public domain. To monitor the progress of amplification, a positive control has been developed that has the nucleotide sequence of all marker regions. The text of the paper discloses all the nucleotide sequences of primers, probes and positive control, which makes it possible to independently acquire them in competent organizations.
Based on the identified genes, the design of specific primers and probes for qPCR was made, which makes it possible to differentiate B. melitensis and B. abortus in a single reaction. For the differentiation of Brucella strains of the same species, the PCR method of multilocus VNTR analysis, optimized for uniform temperature conditions, is described. In addition, MLVA profiles for B. abortus and B. melitensis genomes are presented. All methods of indication and differentiation of Brucella species and strains are described in detail, which allows them to be used in the everyday work of any PCR laboratory dealing with brucellosis. Control of amplification in the indication of bacteria of the genus Brucella and determination of their belonging to B. abortus and B. melitensis species is recommended to be carried out using the plasmid DNA designed in this work.
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