A "Ct contrast"-based strain-specific real-time quantitative PCR system for Lactobacilllus paracasei subsp. paracasei NTU 101

Abstract

Background/purposes: Routine cell number determination for specific Lactobacillus strain by cultivation requires at least 4-7 days. Thus rapid and specific cell number determine methods such as strain-specific quantitative PCR (qPCR) are valuable. However, qPCR method is vulnerable to difficult PCR target such as dimer/secondary structure forming sequence.

Methods: In this study, a two-component, "Ct contrast" approach was applied to strain-specific qPCR system following the development of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) strain-specific PCR with random amplification of polymorphic DNA (RAPD)-derived strain-specific sequences.

Results: The quantitative range of the NTU 101 strain-specific qPCR system was 3.0 × 10¹ to 3.0 × 10⁵ copies for pure cultures, and 3.0 × 10² to 3.0 × 10⁵ copies for multi-strain or unknown food samples. The results of spike in test and real sample testing suggested that non-specific weak background signals did not compromise test specificity, and demonstrated the potential of the NTU 101 strain-specific qPCR system in food samples.

Conclusion: The two-component, "Ct contrast" approach is useful for qPCR discrimination when no ideal PCR target is available or the variance of the target site is unpredictable. The Ct contrast approach might provide a simple and robust solution for other challenging qPCR targets.

Keywords: Lactobacillus; Probiotics; RAPD; Strain-specific; qPCR.