Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad, Iran.
J Sci Food Agric. 2012 Oct;92(13):2652-6. doi: 10.1002/jsfa.5681. Epub 2012 May 4.
The objective of this study was optimisation of multiplex polymerase chain reaction (PCR) by a new primer set for simultaneous detection of ropiness agents as the main bacterial spoilage of Iranian bread. After inoculation of bread dough with activated Bacillus licheniformis (ATCC 9789) and Bacillus subtilis (ATCC 6633), DNA was extracted from the dough and subjected to PCR. Then simplex and multiplex PCR tests were optimised.
From the results obtained, the optimum PCR conditions for simultaneous detection of the target genes in Bacillus species were an annealing temperature of 59 °C and an MgCl(2) concentration of 2.5 mmol L(-1) . To design primers for these two bacteria, owing to close homology and the existence of similar conserved sequences in their 16S rRNA genes, lpaL and aprE genes (497 and 744 bp target sequences) respectively were chosen. Finally, by sequencing of PCR products, accurate and specific detection of the two desired Bacillus species was confirmed. The results were registered with GenBank under accession numbers HQ877873 and HQ871154.
Compared with culture-dependent methods, this procedure offers significantly higher accuracy and speed, which are crucial criteria when it comes to food safety and high volumes of referred samples respectively.
本研究的目的是通过新的引物组优化多重聚合酶链反应（PCR），以同时检测作为伊朗面包主要细菌腐败的粘性剂。在面团中接种活性地衣芽孢杆菌（ATCC 9789）和枯草芽孢杆菌（ATCC 6633）后，从面团中提取 DNA 并进行 PCR。然后优化单重和多重 PCR 试验。
从获得的结果来看，同时检测芽孢杆菌属目标基因的最佳 PCR 条件为退火温度为 59°C 和 MgCl(2)浓度为 2.5 mmol L(-1)。为了设计这两种细菌的引物，由于它们 16S rRNA 基因的高度同源性和相似的保守序列的存在，分别选择了 lpaL 和 aprE 基因（497 和 744 bp 靶序列）。最后，通过对 PCR 产物进行测序，证实了两种所需芽孢杆菌的准确和特异性检测。该结果已在 GenBank 中注册，登录号分别为 HQ877873 和 HQ871154。
与基于培养的方法相比，该方法具有更高的准确性和速度，这是食品安全和大量参考样本的关键标准。
