Exploring specific primers targeted against different genes for a multiplex PCR for detection of Listeria monocytogenes
Exploring specific primers targeted against different genes for a multiplex PCR for detection of Listeria monocytogenes
复制标题
DOI：
10.1007/s13205-014-0225-x
复制
发表时间：
2015-06-01
期刊：
影响因子：
2.8
通讯作者：
Batish, Virender Kumar
中科院分区：
文献类型：
--
作者：
Kumar, Ashwani;Grover, Sunita;Batish, Virender Kumar
在单独的PCR测定中评价了靶向16S rRNA和毒力基因如“iap”、“hly”和“prf”的六组不同引物的效力。针对16SrRNA的引物对导致扩增1.2kb的PCR产物。然而，针对'iap'不同区域的引物组分别产生371和660 bp的PCR产物。针对'prf'基因设计的引物可扩增出508 bp的产物。三个引物对靶向“hly”的不同区域，即，'hly'、'hly A'和'hly K9'分别能扩增出713、276和384 bp的产物。PCR条件也针对落入“iap”和“hly”基因内的两组内部引物进行优化，其扩增119和188 bp产物，以验证用相应的外部引物组获得的PCR结果。在相应的多重PCR测定中探索涉及基于16S rRNA的四组引物的三种不同组合，即“iap”、“hly”和“prf”，以选择合适的组合。组合1和3成功地起作用，如通过在琼脂糖凝胶上扩增预期大小的所有四个条带所揭示的。然而，在优化用于开发功能性多重PCR的不同参数时，观察到在这两种组合中，仅两种扩增产物，即，1.2 kb和713 bp的片段均能检测到。因此，这两个引物组合在多重PCR中，并优化了条件，应用于乳制品检测单核细胞增生李斯特菌。
The efficacy of six different sets of primers targeted against 16S rRNA and virulence genes such as 'iap', 'hly' and 'prf' was evaluated in separate PCR assays. The primer pairs targeted against 16S rRNA resulted into amplification of 1.2 kb PCR product. However, sets of primers targeted against different regions of 'iap' produced 371 and 660 bp PCR products, respectively. The primer pair targeted against 'prf' gene could produce 508 bp product. Three primer pairs targeted against different regions of 'hly', i.e., 'hly', 'hly A' and 'hly K9' were able to amplify 713, 276 and 384 bp products, respectively. The PCR conditions were also optimized in respect of two internal sets of primers falling within 'iap' and 'hly' genes that amplified 119 and 188 bp products to verify the PCR results obtained with respective external sets of primers. Three different combinations involving four sets of primers based on 16S rRNA, 'iap', 'hly' and 'prf' were explored in respective multiplex PCR assays in order to select a suitable combination. Combination 1 and 3 worked successfully as revealed by amplification of all the four bands of expected sizes on agarose gel. However, while optimizing the different parameters for developing a functional multiplex PCR, it was observed that in both these combinations, only two of the amplified products, i.e., 1.2 kb and 713 bp could be invariably detected. Hence, these two primers were combined in the multiplex PCR and the conditions were optimized for application in dairy foods for detection of Listeria monocytogenes.