Establishment of a Helicase-Dependent Isothermal DNA Amplification Method for Rapid Detection of Lactobacillus plantarum in Fermented Milk

A rapid detection method based on helicase-dependent isothermal DNA amplification (HDA) was established for Lactobacillus plantarum in fermented milk. Specific primers were designed according to the scrB gene sequence of Lactobacillus plantarum (Genbank Accession NO. AJ579541.1), and the optimal concentrations of UvrD helicase and T4 gp32 in the reaction system were determined through experiments. The limit of detection (LOD), specificity, consistency and stability of the proposed method were evaluated by use of L. plantarum-spiked samples, amplification of various strains, and electrophoresis of amplified products and sequence alignment analysis, respectively. The results showed that the optimized of UvrD helicase and T4 gp32 in the reaction system were found to be 0.15 and 5.0 μg, respectively. The HDA method had high specificity with no amplification of other strains tested. The detection limit was 2.8 × 10¹ CFU/g. The amplified product was consistent with the designed sequence length (273 bp) and the sequence homology was 100%. In conclusion: this method is rapid, simple, sensitive and suitable for the detection of Lactobacillus plantarum in fermented milk.