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

2021 ◽  
Author(s):  
Zeyu Jia ◽  
Yijing Dong ◽  
Heng Xu ◽  
Fengping Wang
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
False Positive ◽  
Signal Intensity ◽  
Sample Pretreatment ◽  
Extraction Methods ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Widespread Application

AbstractFluorescence 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 Methanococcoides 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.

Combination of Cytology, Fluorescence In Situ Hybridization for Aneuploidy, and Reverse-Transcriptase Polymerase Chain Reaction for Human Mammaglobin/Mammaglobin B Expression Improves Diagnosis of Malignant Effusions

2004 ◽  
Vol 22 (3) ◽  
pp. 474-483 ◽  
Author(s):  
Michael Fiegl ◽  
Margot Haun ◽  
Anita Massoner ◽  
Jens Krugmann ◽  
Elisabeth Müller-Holzner ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Tumor Cell ◽  
Fluorescence In Situ Hybridization ◽  
Cell Detection ◽  
Malignant Cells ◽  
Chain Reaction ◽  
Tumor Cell Detection ◽  
Polymerase Chain

Purpose The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. The aim of this study was to improve tumor cell detection in effusions by molecular approaches. Materials and Methods A total of 157 effusions from patients with tumors and 72 effusions from patients without a history or evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, fluorescence in situ hybridization (FISH) for aneuploidy, and reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of human mammaglobin (hMAM) and mammaglobin B (hMAM-B). Results In effusions from patients with tumors, the sensitivities of tumor cell detection by cytology, FISH, and hMAM and hMAM-B detection were 46.2%, 53.3%, 36.4%, and 57.7%, respectively. The corresponding specificities were 94.4%, 97.0%, 87.1%, and 88.6%. Notably, a high percentage of effusions containing malignant cells were in fact transudates, indicating the necessity for molecular diagnostic work-up of transudates collected from patients with tumors. Dependent on the tumor type, the use of appropriate marker combinations improved tumor cell detection in effusions significantly. By combining all four diagnostic tests, a positive test result indicating the presence of malignancy was achieved in 81.1%, with a fairly good specificity of 70.1%. Conclusion Molecular techniques are definitely useful to detect malignancy in cytologically negative effusions. Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. This finding should help to improve tumor staging, prognostic assessment, and treatment monitoring.

scholarly journals Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pei Zhuang ◽  
Huanqing Zhang ◽  
Ryan M. Welchko ◽  
Robert C. Thompson ◽  
Shunbin Xu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Mrna Detection
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