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

Area of Science:

• Microbiology
• Molecular Biology
• Food Science

Background:

• Traditional Lactobacillus cell enumeration via cultivation is time-consuming (4-7 days).
• Strain-specific quantitative PCR (qPCR) offers rapid enumeration but struggles with difficult PCR targets like dimer-forming sequences.
• Developing rapid and specific methods for bacterial quantification is crucial for food safety and quality control.

Purpose of the Study:

• To develop a rapid and specific quantitative PCR (qPCR) method for enumerating Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101).
• To address the limitations of traditional qPCR methods when dealing with challenging PCR target sequences.
• To validate the efficacy of a novel "Ct contrast" approach for strain-specific bacterial quantification.

Main Methods:

• A strain-specific PCR targeting NTU 101 was developed using random amplification of polymorphic DNA (RAPD)-derived sequences.
• A two-component "Ct contrast" approach was implemented within the strain-specific qPCR system.
• The method was tested using pure cultures and complex food samples, including spike-in tests.

Main Results:

• The NTU 101 strain-specific qPCR system demonstrated a quantitative range of 3.0 × 10^1 to 3.0 × 10^5 copies in pure cultures.
• In multi-strain or unknown food samples, the quantitative range was 3.0 × 10^2 to 3.0 × 10^5 copies.
• Non-specific background signals did not compromise test specificity, confirming the system's potential for food sample analysis.

Conclusions:

• The "Ct contrast" approach effectively discriminates qPCR targets, even with unpredictable sequence variations.
• This method provides a simple and robust solution for challenging qPCR targets in bacterial enumeration.
• The developed NTU 101 strain-specific qPCR system is suitable for quantifying bacteria in food matrices.