Optimizing the hybridization chain reaction-fluorescence in situ hybridization(HCR-FISH)protocol for detection of microbes in sediments  

作　　者：Zeyu Jia Yijing Dong Heng Xu Fengping Wang 

机构地区：[1]State Key Laboratory of Microbial Metabolism,Joint International Research Laboratory of Metabolic and Developmental Sciences,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China [2]School of Physics and Astronomy,Shanghai Jiao Tong University,Shanghai 200240,China [3]Institute of Natural Science,Shanghai Jiao Tong University,Shanghai 200240,China [4]School of Oceanography,Shanghai Jiao Tong University,Shanghai 200240,China

出　　处：《Marine Life Science & Technology》2021年第4期529-541,共13页海洋生命科学与技术（英文）

基　　金：We thank for the funding:National Key R&D Program of China(grant numbers 2018 YFC0310800,2018YFC0310803);COMRA Project DY135-B2-12;the National Natural Science Foundation of China(grant numbers 41525011,91751205,11774225);the Recruitment Program of Global Experts(Program for Young Professionals),and the Natural Science Foundation of Shanghai(grant no.18ZR1419800);This is also a contribution to the Center for Ocean Mega-Science,Chinese Academy of Sciences,the Senior User Project of RV KEXUE(KEXUE2019GZ06);the International Center for Deep-Life Investigation(IC-DLI).We thank Gunter Wegener for providing the ANME enrichment sample.

摘　　要：Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.

关 键 词：Fluorescence in situ hybridization Hybridization chain reaction HCR-FISH Microbial detection Sediment 

分 类 号：X172[环境科学与工程—环境科学]