Real-Time PCR Detection and HRM Differentiation of Selected Brucella Species

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F70565813%3A_____%2F18%3AN0000035" target="_blank" >RIV/70565813:_____/18:N0000035 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Real-Time PCR Detection and HRM Differentiation of Selected Brucella Species

Popis výsledku v původním jazyce

Brucellosis still belongs to a significant cause of zoonosis in many countries in the world, e.g. Latin America or the Middle East. Pathogenic Brucella species still represent a considerable risk of human infection due to its natural reservoirs – farm animals, as well as insufficient vaccination. Moreover, these bacteria could be also misused as a bioterrorist weapon in the form of aerosol. Another factor according to that these agents are classified as highly risky is the low infectious dose of approximately ten to hundred bacteria. Here, we would like to present a new methodology for the detection and differentiation of main pathogenic Brucella species – B. abortus, melitensis and suis. This methodology is based on the real-time PCR followed by the high resolution melting analysis (HRM). The HRM analysis was chosen because of a high degree of genomic sequence similarity among Brucella species. The assay is consisted of four sets of primers separated into two reactions that are able to be run under the same cycling and melting conditions. The first reaction enables the detection of and separation between two groups of Brucella. B. abortus together with B. melitensis are found in group one, group two includes both B. suis and canis. According to the first reaction, target Brucella species are distinguished from other non-target Brucella. Only samples classified as the members of particular target group are further evaluated in the second reaction. The second reaction involves two additional sets of primers designed to differentiate inside both group one and two between B. abortus and melitensis or between B. suis and canis, respectively. Finally, all target Brucella are distinguished from each other on the basis of the HRM analysis. The assay is aimed to detect all B. abortus and melitensis biovars. In case of B. suis, the assay is designed to detect B. suis biovars 1-4. Biovar 5 of B. suis contains a higher portion of genomic sequence variability alike the rest of B. suis biovars. The whole methodology is validated in concert with the MIQE guidelines.

Název v anglickém jazyce

Real-Time PCR Detection and HRM Differentiation of Selected Brucella Species

Popis výsledku anglicky

Brucellosis still belongs to a significant cause of zoonosis in many countries in the world, e.g. Latin America or the Middle East. Pathogenic Brucella species still represent a considerable risk of human infection due to its natural reservoirs – farm animals, as well as insufficient vaccination. Moreover, these bacteria could be also misused as a bioterrorist weapon in the form of aerosol. Another factor according to that these agents are classified as highly risky is the low infectious dose of approximately ten to hundred bacteria. Here, we would like to present a new methodology for the detection and differentiation of main pathogenic Brucella species – B. abortus, melitensis and suis. This methodology is based on the real-time PCR followed by the high resolution melting analysis (HRM). The HRM analysis was chosen because of a high degree of genomic sequence similarity among Brucella species. The assay is consisted of four sets of primers separated into two reactions that are able to be run under the same cycling and melting conditions. The first reaction enables the detection of and separation between two groups of Brucella. B. abortus together with B. melitensis are found in group one, group two includes both B. suis and canis. According to the first reaction, target Brucella species are distinguished from other non-target Brucella. Only samples classified as the members of particular target group are further evaluated in the second reaction. The second reaction involves two additional sets of primers designed to differentiate inside both group one and two between B. abortus and melitensis or between B. suis and canis, respectively. Finally, all target Brucella are distinguished from each other on the basis of the HRM analysis. The assay is aimed to detect all B. abortus and melitensis biovars. In case of B. suis, the assay is designed to detect B. suis biovars 1-4. Biovar 5 of B. suis contains a higher portion of genomic sequence variability alike the rest of B. suis biovars. The whole methodology is validated in concert with the MIQE guidelines.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10600 - Biological sciences

Projekt

<a href="/cs/project/VI20172019063" target="_blank" >VI20172019063: Vývoj nových metodik pro detekci biologických agens v oblastech souvisejících s dodržováním Úmluvy o zákazu biologických zbraní</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů