A CRISPR-Cas12a-Based Assay for Efficient Quantification of <i>Lactobacillus panis</i> in Chinese Baijiu Brewing Microbiome

Abstract

Although the quantification of key microorganisms in fermentation microbiomes is important for monitoring and regulating fermented food production, conventionally used methods are generally time-consuming, involve complicated operations, and have a high level of equipment dependence. We developed a CRISPR-Cas12a-based quantitative Chinese Baijiu brewing microorganism assay (CQAOB) for Lactobacillus panis, the most important lactic acid-producing fermentative microorganism. We initially verified the feasibility of CQAOB for detecting L. panis, and then optimized the reaction parameters to enhance Cas12a detection sensitivity. The specificity of the developed method was verified via the accurate distinction of L. panis nucleic acids from those of other lactic acid bacteria. The quantitative range and detection time for L. panis were 106–109 copies/μL and 40 min, respectively. Finally, we successfully applied CQAOB for quantifying L. panis count in fermented grains. Given its rapid detection and low level of equipment dependence, CQAOB may make an important contribution to quantifying key microorganisms in brewing processes.

Keywords

• quantification of microorganisms
• CRISPR
• Cas12a
• <i>Lactobacillus panis</i>
• Chinese Baijiu brewing microorganisms