Rapid and accurate on-site identification of Lactobacillus delbrueckii subspecies in dairy products using direct polymerase chain reaction with microfluidic chip

Abstract

Lactobacillus delbrueckii subspecies are important microorganisms for cheese or yogurt production, and timely and effective monitoring is required to improve dairy product quality. In this study, an ultrafast polymerase chain reaction (PCR) assay with a microfluidic device was developed to detect three L. delbrueckii subspecies, namely L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, and L. delbrueckii subsp. lactis, in pure culture and spiked milk samples without the need for complicated genomic DNA isolation. Based on genetic markers derived from comparative genomics, primer pairs for the identification of the subspecies were designed. The specificity of the primer pairs was confirmed using 65 reference strains. In pure culture and milk samples, this assay exhibited sensitivity of 1 × 102 CFU/ml. Twenty-nine probiotic or dairy products were evaluated to confirm the applicability of direct ultrafast PCR assay with a simple lysis step. The developed assay enabled the rapid identification and quantification of L. delbrueckii subspecies in foods from sampling to results within 30 min. Therefore, our assay might be applied as a convenient portable system for the on-site detection of L. delbrueckii subspecies in the dairy industry.