Quantitative real-time PCR is widely used to determine absolute abundance of microbes in food fermentation.However,it remains challenges in the application for quantification at the species level due to the difficulty...Quantitative real-time PCR is widely used to determine absolute abundance of microbes in food fermentation.However,it remains challenges in the application for quantification at the species level due to the difficulty in designing species-specific primer sets.This work,using Lactobacillaceae,a dominant family within the lactic acid bacteria that involved in diversity food fermentations,as a case,presents an extendable strategy to design species-specific primer sets for microbial quantita-tive analysis.136,257 species-specific genes were obtained from all 307 species within Lactobacillaceae family through comparative genomics analysis.A total of 130,521 primer sets were designed using species-specific genes.Among them,81,710 primer sets had 100%interspecific specificity and 100%intraspecific coverage,and were reserved to quantify all 307 individual Lactobacillaceae species.These primer sets had uniform melting temperature(57–63℃)and product size(100–300 bp),that allowed simultaneously quantify different Lactobacillaceae species with the same qPCR condition.We then established a Lactobacillaceae species quantitation primer database(LSQP-DB,http://lsqp-db.com)containing all 81,710 species-specific primer sets.The database would facilitate a fast and easy absolute quantitation analysis of all indi-vidual Lactobacillaceae species.This work represented the first ever large-scale integration of species-specific primer sets for microorganism,it can be extended to other bacterial and fungal genera to advance development of microbial absolute quantification.展开更多
基金supported by the National Natural Science Foundation of China(32172175)National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)+1 种基金the Priority Academic Program Development of Jiangsu Higher Education Institutions,the 111 Project(No.111-2-06)Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX21-2029).
文摘Quantitative real-time PCR is widely used to determine absolute abundance of microbes in food fermentation.However,it remains challenges in the application for quantification at the species level due to the difficulty in designing species-specific primer sets.This work,using Lactobacillaceae,a dominant family within the lactic acid bacteria that involved in diversity food fermentations,as a case,presents an extendable strategy to design species-specific primer sets for microbial quantita-tive analysis.136,257 species-specific genes were obtained from all 307 species within Lactobacillaceae family through comparative genomics analysis.A total of 130,521 primer sets were designed using species-specific genes.Among them,81,710 primer sets had 100%interspecific specificity and 100%intraspecific coverage,and were reserved to quantify all 307 individual Lactobacillaceae species.These primer sets had uniform melting temperature(57–63℃)and product size(100–300 bp),that allowed simultaneously quantify different Lactobacillaceae species with the same qPCR condition.We then established a Lactobacillaceae species quantitation primer database(LSQP-DB,http://lsqp-db.com)containing all 81,710 species-specific primer sets.The database would facilitate a fast and easy absolute quantitation analysis of all indi-vidual Lactobacillaceae species.This work represented the first ever large-scale integration of species-specific primer sets for microorganism,it can be extended to other bacterial and fungal genera to advance development of microbial absolute quantification.
