Advancement in LH-PCR methodology for multiple microbial species detections in fermented foods

摘要

The length-heterogeneity PCR is a low throughput molecular biology methods explored to monitor bacteria populations in different environments. It could be more used in food microbiology analysis, not only for fingerprinting analysis, but it has been hampered until now by a limiting factor which relates to the high percentage of secondary peaks. With the aim to overcome this problem, different experiments were performed focusing on changing PCR parameters in order to obtain more specific amplicon patterns and also to reduce the complexity of community patterns. With this purpose, different annealing temperatures were tested on complex fermented food matrices taken from both animal and vegetable origin and also on the bacteria isolated from the same food source. In particular, the optimal annealing temperature identified for the fermented food samples is 59 °C and the optimal for bacterial strains varied between 63 °C and 65 °C. The approach allowed the modification of the LH-PCR protocol increasing the amplification efficiency and therefore the bacteria species discrimination. These temperatures also allowed the implementation of the previous LH-PCR published database. The modification in the level of accuracy of the LH-PCR technique could also allow an improvement in the relative species quantification by the peak area evaluation.