实时荧光定量PCR检测大曲中两种克罗彭施泰特氏菌的方法与应用  

Establishment and application of real-time quantitative PCR for detecting two Kroppenstedtia species in Daqu

作　　者：唐胜硕 张晓娟[1,2] 柴丽娟 史劲松 王松涛 张宿义 沈才洪 陆震鸣 许正宏[3] TANG Shengshuo;ZHANG Xiaojuan;CHAI Lijuan;SHI Jinsong;WANG Songtao;ZHANG Suyi;SHEN Caihong;LU Zhenming;XU Zhenghong(Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China;National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;College of Biomass Science and Engineering,Sichuan University,Chengdu 610065,China;School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,China;National Engineering Research Center of Solid-State Brewing,Luzhou 646000,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,生物工程学院,无锡214122 [2]江南大学粮食发酵与食品生物制造国家工程研究中心,无锡214122 [3]四川大学轻工科学与工程学院,成都610065 [4]江南大学生命科学与健康工程学院,无锡214122 [5]国家固态酿造工程技术研究中心,泸州646000

出　　处：《应用与环境生物学报》2025年第1期121-130,共10页Chinese Journal of Applied and Environmental Biology

基　　金：国家重点研发计划项目(2022YFD2101204-01)资助。

摘　　要：象牙色克罗彭施泰特氏菌(Kroppenstedtia eburnea)与血液克罗彭施泰特氏菌(K. sanguinis)是大曲细菌群落中的核心功能物种.为实现对大曲中这两个物种的快速检测与定量,从K. eburnea与K. sanguinis基因组中筛选了特异性强的基因序列作为模板设计特异性引物,通过近缘菌株和不同类型大曲样品分别验证引物的特异性与有效性,并建立基于实时荧光定量PCR(RT-qPCR)的特异性检测方法.结果表明,以K. eburnea与K. sanguinis的DNA旋酶B亚单位基因(gyrB基因)为参考序列分别设计的引物KE-P-1与KS-P-1具有良好的特异性,其扩增目的片段长度分别为277 bp和287 bp.根据引物KE-P-1和KS-P-1建立的RT-qPCR方法特异性强、灵敏度高且准确性高,扩增效率分别为101.8%和97.70%,检测范围分别为2.089-9.089 lg(copies/μL)和1.088-9.088 lg(copies/μL),成功应用于大曲样品中K. eburnea与K. sanguinis的检测.对高温大曲发酵过程的研究表明,高温大曲中的K. eburnea与K. sanguinis含量均表现出先增加后降低再增加的变化规律,发酵结束时含量分别为9.20±0.11 lg(copies/g)和9.71±0.22 lg(copies/g).本研究所建立的RT-qPCR方法可对各类大曲发酵过程中K. eburnea与K. sanguinis进行特异性鉴定和快速定量.(图4表4参37)To establish a real-time quantitative PCR(RT-qPCR)method for the detection of two core functional bacteria(Kroppenstedtia eburnea and Kroppenstedtia sanguinis)in Daqu samples,specific genes in K.eburnea and K.sanguinis genomes were screened to design PCR sequence-speciffc primers.The speciffcity and accuracy of the specific primers were verified by PCR using the isolated strains and samples of Daqu,respectively.The RT-qPCR method was established to analyze the contents of K.eburnea and K.sanguinis in different types of Daqu and samples during the fermentation process of high-temperature Daqu.The results showed that specific primers KE-P-1 and KS-P-1 were designed for K.eburnea and K.sanguinis based on their gyrase B subunit gene(gyrB gene),respectively,with product lengths of 277 and 287 bp.The RT-qPCR methods established using KE-P-1 and KS-P-1 showed strong speciffcity,high sensitivity,and good accuracy.The RT-qPCR quantitative standard curves for primers KE-P-1 and KS-P-1 were y=-0.3050x+1.12(R2=0.9990)and y=-0.2960x+10.63(R2=0.9998),respectively.The amplification efficiency was 101.8%and 97.70%and the detection ranges were 2.089-9.089 lg(copies/μL)and 1.088-9.088 lg(copies/μL),respectively.The methods can be applied to detect K.eburnea and K.sanguinis in high-,medium-,and low-temperature Daqu.A study of the high-temperature Daqu fermentation process indicated that K.eburnea and K.sanguinis exhibited an initial increase,followed by a decrease and then an increase.At the end of fermentation,the content of K.eburnea and K.sanguinis was 9.20±0.11 lg(copies/g)and 9.71±0.22 lg(copies/g),respectively.In conclusion,the RT-qPCR method established in this study enabled the rapid identiffcation and quantiffcation of K.eburnea and K.sanguinis in Daqu.

关 键 词：大曲 象牙色克罗彭施泰特氏菌 血液克罗彭施泰特氏菌 特异性引物 RT-QPCR 

分 类 号：R73[医药卫生—肿瘤]