Інституційний Репозитарій Білоцерківського НАУ
Будь ласка, використовуйте цей ідентифікатор, щоб цитувати або посилатися на цей матеріал:
http://rep.btsau.edu.ua/handle/BNAU/2806| Назва: | Визначення представників Salmonella spp. методом ПЛР у реальному часі |
| Інші назви: | Determination of representatives of Salmonella spp. by real-time PCR |
| Автори: | Облап, Руслан Васильович Oblap, Ruslan |
| Ключові слова: | Salmonella spp;ПЛР у реальному часі;харчові продукти;продовольча сировина;Real-time PCR;food and food raw materials;the sensitivity limitation;the optimization of amplification conditions |
| Дата публікації: | 2013 |
| Видавництво: | Білоцерківський національний аграрний університет |
| Бібліографічний опис: | Облап Р.В. Визначення представників Salmonella spp. методом ПЛР у реальному часі / Р.В.Облап // Технологія виробництва і переробки продукції тваринництва: збірник наукових праць .- Біла Церква: БНАУ, 2013 .- Вип.10(105) .- С. 60-64. |
| Короткий огляд (реферат): | Розроблено тест-систему для визначення бактерій роду Salmonella методом полімеразної ланцюгової реакції в режимі реального часу (Real-Time PCR). Система уможливлює ідентифікацію представників Salmonella spp. у харчових продуктах та продовольчій сировині. Діагностикум адаптовано для проведення аналізів на приладах найбільш відомих виробників (BioRad, Applied Biosystems, Corbett Research, Синтол, ДНК-технология) і може бути рекомендовано до застосування у лабораторній діагностиці. |
| Опис: | The diffusion of food origin diseases, caused by microorganisms, has become one of the most urgent human health problems facing the world. Every third inhabitant of industrialised countries suffers from food diseases. That’s why solving problems concerning the safety of food products and the assurance of their quality has been the primary task for the modern society. Salmonellosis is one of the widespread human and agricultural animal enteric infections. Main sources and transfer factors of disease agent are caused by contaminated food products, raw material, water. Salmonella spp. bacteria causes salmonellosis. Modern diagnostic of salmonellosis is based on bacteriological, serological and molecular-genetic methods of analysis. The traditional salmonella detection methods are quite laborious and require much time. During the last years new PCR ex-press-methods of diagnostic have been widely implemented in laboratories. Recent designs in PCR-technology, for example Real-Time PCR, allow to speed up the time of analysis, reducing its cost price and laborious tasks and assuring qualitative and quantitative pathogen detection in food products, raw material and feed. The aim of study was making a design and approbation of domestic diagnosticum on the basis of Real-Time PCR for de-tection Salmonella spp. and its identification in food products and plant and animal raw material. Material sources for bacterial DNA extraction were presented by strains of Salmonella, Lіsterіа, Shigella and Escherichia genus, and food products where Salmonella spp. presence was confirmed. We used TaqMan technology of Real-Time PCR because of its high sensitivity, specificity and velocity of performing during test-system designing. The designed system is multiplex, it allows running two individual reactions in one tube. One reaction detects genus-specific fragment of gene invA, which allows to detect Salmonella spp. representatives, the second reaction – detects the fragment of bacterial 16S rRNA as endogenous control of PCR running. The assessing actions aiming at increasing of test-system effectiveness (specificity, sensitivity, limitation of detection, re-peatability and reproducibility results of analysis) were performed according to the requirements for PCR test-systems design in comparison with the control samples. The optimization of amplification conditions was performed at the temperature of primers annealing, MgCl2 concentration, concentration and correlation of primers and probes. The experimental detection of specificity was performed by testing of S. enteritidis, S. typhimurium, S. infantis, S. agona, S. blegdam, S. dublin, E. coli, L. monocytogenes, L. innocua, L. ivanovii, S. sonnei, S. flexneri, S. Boydii DNAs. No cross-reactions were detected. The sensitivity limitation of the designed test-system was detected by preparation of dilution series of bacterial DNA (from 0,0001 to 100 ng of genomic DNA). The limitation of sensitivity for invA gene was 0,01 ng, for 16S rRNA gene - 0,001 ng. The designed diagnosticum was adapted for equipment of the most famous producers (BioRad, Applied Biosystems, Cor-bett Research, Синтол, ДНК-технология) which are implemented in diagnostic laboratories within Ukraine. Thus, functional amplification conditions were designed and proposed for applying this diagnostic test-system on Real-Time PCR basis, which allows to identify Salmonella spp. representatives in food raw material and food products. |
| URI (Уніфікований ідентифікатор ресурсу): | http://rep.btsau.edu.ua/handle/BNAU/2806 |
| ISSN: | 2310-9289 |
| УДК: | 619:614.31:637:577.2 |
| Розташовується у зібраннях: | Наукові публікації |
Файли цього матеріалу:
| Файл | Опис | Розмір | Формат | |
|---|---|---|---|---|
| Vyznachennia_predstavnykiv.pdf | 216,75 kB | Adobe PDF | Переглянути/Відкрити |
Усі матеріали в архіві електронних ресурсів захищені авторським правом, всі права збережені.