Detection and Differentiation of Several Food-Spoilage Lactic Acid Bacteria by Multiplex Polymerase Chain Reaction, Capillary Gel Electrophoresis, and Laser-Induced Fluorescence

• Published in: Journal of agricultural and food chemistry 2004-09, Vol.52 (18), p.5583-5587
• Main Authors: García-Cañas, Virginia, Macián, MariCarmen, Chenoll, Empar, Aznar, Rosa, González, Ramón, Cifuentes, Alejandro
• Format: Article
• Language: English
• Subjects: Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Biological and medical sciences; capillary electrophoresis; Carnobacterium; Carnobacterium divergens; Carnobacterium gallinarum; Carnobacterium maltaromicum; Electrophoresis, Capillary; Fluorescence; Food industries; Food Microbiology; food spoilage; Fundamental and applied biological sciences. Psychology; gel electrophoresis; General aspects; Lasers; Leuconostoc; Leuconostoc - classification; Leuconostoc - genetics; Leuconostoc - isolation & purification; Methods of analysis, processing and quality control, regulation, standards; microbial detection; Polymerase Chain Reaction
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf049298t

In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria. To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed. The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C. divergens, C. gallinarum, and C. maltaromicum). The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested. These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained. The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges. Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively. Keywords: CGE; LIF; Carnobacterium; Leuconostoc; foods; food spoilage; bacteria; multiplex PCR