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首页> 外文期刊>Medycyna Weterynaryjna >Application of the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes located on plasmids pXO1 and pXO2 as well as the specific chromosomal rpoB sequence of Bacillus anthracis strainsOriginal Title (non-English) Zastosowanie melody Real-time PCR opartej na fluorescencji barwnika SYBR Green I do wykrywania genow zlokalizowanych w DNA plazmidow pXO1 i pXO2 oraz specyficznej sekwencji chromosomalnej rpoB szczepow Bacillus anthracis [Polish]
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Application of the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes located on plasmids pXO1 and pXO2 as well as the specific chromosomal rpoB sequence of Bacillus anthracis strainsOriginal Title (non-English) Zastosowanie melody Real-time PCR opartej na fluorescencji barwnika SYBR Green I do wykrywania genow zlokalizowanych w DNA plazmidow pXO1 i pXO2 oraz specyficznej sekwencji chromosomalnej rpoB szczepow Bacillus anthracis [Polish]

机译：插入染料SYBR Green I实时PCR方法在炭疽芽孢杆菌菌株质粒pXO1和pXO2以及特定染色体rpoB序列基因检测中的应用原始名称（非英语）Zastosowanie旋律实时PCR opartej na fluorescencji barwnika SYBR Green我做wykrywania genow zlokalizowanych w DNA plazmidow pXO1 i pXO2 oraz specyficznej sekwencji chromosomalnej rpoB szczepow炭疽杆菌[波兰语]

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摘要

The aim of the study was to apply the real-time PCR method with the intercalating dye SYBR Green 11 for the detection of genes of Bacillus anthracis located on plasmids pXO1 and pXO2 and the specific chromosomal rpoB sequence. The research was conducted on one vaccinal and four field strains of Bacillus anthracis. The assessment of the specificity of the tests involved isolates of other species of the genus Bacillus as well as strains of six other species of microorganisms. The studies were conducted with the PCR QuantiTect kit (Qiagen) and primers specific for the gene pag coding PA protein, gene cap coding capsule, and primers for the amplification of the specific chromosomal sequence. PCR enabled the detection of all genes under examination by the observation of amplification curves. The specificity of real-time PCR was confirmed by estimating melting temperatures of PCR products. It was shown that the melting temperatures of amplicons obtained in the reaction were 78 degrees C, 79 degrees C and 76 degrees C in cases of detecting the chromosomal rpoB sequence, pag gene, and cap-C gene, respectively. The sensitivity and linearity of the reactions were determined using a regression coefficient. A high regression coefficient of 0.99 was demonstrated for all the reactions. The real-time tests were highly sensitive and specific.
机译：该研究的目的是将实时荧光定量PCR方法与嵌入染料SYBR Green 11一起用于检测质粒pXO1和pXO2上的炭疽芽孢杆菌基因以及特定的染色体rpoB序列。该研究针对炭疽芽孢杆菌的一株疫苗和四株田间菌株进行。对测试特异性的评估涉及芽孢杆菌属其他物种的分离株，以及其他六种微生物的菌株。研究使用PCR QuantiTect试剂盒（Qiagen）和特异于编码PA蛋白的基因pag的引物，基因帽编码胶囊以及用于扩增特定染色体序列的引物进行。通过观察扩增曲线，PCR可以检测所有受检基因。通过估计PCR产物的解链温度来证实实时PCR的特异性。结果表明，在检测染色体rpoB序列，pag基因和cap-C基因的情况下，反应中获得的扩增子的解链温度分别为78℃，79℃和76℃。使用回归系数确定反应的灵敏度和线性。对于所有反应，均显示出0.99的高回归系数。实时测试是高度敏感和特定的。

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