Borrelia burgdorferi: Methods and Protocols (Methods in Molecular Biology, 2742)

Preface
Contents
Contributors
Chapter 1: Real-Time Microfluidic PCRs: A High-Throughput Method to Detect 48 or 96 Tick-borne Pathogens in 48 or 96 Samples
	1 Introduction
	2 Materials
		2.1 Tick Washing
		2.2 DNA and RNA Extraction
		2.3 Pool 0.2x
		2.4 Preamplification
		2.5 Assay Plate Stock
		2.6 Microfluidic Chip
	3 Methods
		3.1 Ticks Washing
		3.2 DNA Extraction
		3.3 Pool 0.2x
		3.4 Preamplification
		3.5 Assay Plate Stock
		3.6 Microfluidic Chip
			3.6.1 Prime Chip
			3.6.2 Assay Plate Preparation
			3.6.3 Sample Plate Preparation
			3.6.4 Chip Loading
			3.6.5 Chip Run
		3.7 Analysis of Microfluidic Chip
		3.8 Analysis of the Excel File
		3.9 Chip Results Confirmation
	4 Notes
	References
Chapter 2: Direct Detection of Borrelia Species in Tissues
	1 Introduction
	2 Materials
		2.1 Immunofluorescence Assay
			2.1.1 Deparaffinization and Fixative
			2.1.2 Antigen Retrieval (See Note 1)
			2.1.3 Permeabilization
			2.1.4 Blocking and Staining
			2.1.5 Negative and Positive Controls Staining
			2.1.6 Preparation of Lipofuscin Autofluorescence Quencher
			2.1.7 DAPI Preparation
		2.2 DNA Isolation from the Tissue Samples
			2.2.1 Isolation of DNA from FFPE Tissue or Fresh-Frozen Tissue
			2.2.2 Conventional and Nested Polymer Chain Reaction (PCR)
		2.3 RNAscope (RNA In Situ Hybridization)
	3 Methods
		3.1 Immunofluorescence
			3.1.1 Deparaffinization
			3.1.2 Antigen Retrieval
			3.1.3 Permeabilization
			3.1.4 Blocking and Staining
			3.1.5 Negative and Positive Controls Used to Determine the Specificity of the Antibodies
			3.1.6 Staining of Fixed But Not Paraffin-Embedded Tissue
			3.1.7 Staining of Fresh-Frozen Tissue Slides
		3.2 DNA Isolation from the Tissue Samples
			3.2.1 Isolation of DNA from FFPE Tissue Sample
			3.2.2 Isolation of DNA from Fresh-Frozen Tissue Sample
			3.2.3 Conventional and Nested Polymer Chain Reaction (PCR)
		3.3 RNAscope (RNA In Situ Hybridization)
			3.3.1 Hybridization
			3.3.2 Amplification
			3.3.3 Detection
	4 Notes
	References
Chapter 3: Purification of Borrelia burgdorferi Outer Membrane Vesicles
	1 Introduction
	2 Materials
		2.1 BSK-II Culture Media for Borrelia burgdorferi
		2.2 OMV Preparation
		2.3 OMV Filtering and Concentration Steps
		2.4 OMV Purification
	3 Methods
		3.1 Preparation of the Culture Media BSK-II
		3.2 OMV Preparation and Purification
		3.3 OMV Filtering and Concentration Steps
		3.4 OMV Purification Step
	4 Notes
	References
Chapter 4: Constructing an ELISA for Detection of Anti-Borrelia in Wildlife and Agricultural Animals
	1 Introduction
	2 Materials
		2.1 Blood Collection
		2.2 Antigen Preparation for the ELISA
		2.3 ELISA Solutions and Supplies
		2.4 Immunoblots
	3 Methods
		3.1 Blood Collection of Cows and Horses
		3.2 Antigen Preparation
		3.3 Making ELISA Plates
		3.4 ELISA Procedure
		3.5 Quantification of ELISA Values
		3.6 Optimizing Secondary Antibody Concentration
		3.7 Sensitivity and Specificity Calculations
		3.8 Immuno/Western Blotting
	4 Notes
	References
Chapter 5: A Step-by-Step Guide to Selecting an Optimal Cut-Off Value Based on the Receiver Operating Characteristic and Youde...
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 6: Adaptive Immune Response Investigation in Lyme Borreliosis
	1 Introduction
	2 Materials
		2.1 ELISA Step (see Note 1)
		2.2 EliSpot Step
	3 Methods
		3.1 TICKPLEX ELISA
		3.2 EliSpot
			3.2.1 Procedure of Isolation of Peripheral Blood Lymphocytes
			3.2.2 Procedure to Perform the EliSpot
	4 Notes
	References
Chapter 7: CYTOSPOT: Intracellular Staining and Detection of Cytokines by Flow Cytometer, an Alternative to LYMESPOT
	1 Introduction
	2 Materials
		2.1 Reagents
	3 Methods
		3.1 Cell Preparation
		3.2 In Vitro Culture of Lymphocytes
		3.3 Staining for Flow Cytometry
		3.4 Flow Cytometry Acquisition and Analysis
	4 Notes
	References
Chapter 8: Use of Specific Borrelia Phages as a New Strategy for Improved Diagnostic Tests
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 PCR
	3 Methods
		3.1 DNA Extraction
		3.2 PCR
	4 Notes
	References
Chapter 9: Identification of Unambiguous Borrelia Peptides in Human Urine Using Affinity Capture and Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 Affinity Net Preparation
		2.2 Affinity Net Validation
		2.3 Urine Sample Incubation
		2.4 Mass Spectrometry Analysis
	3 Methods
		3.1 Affinity Net Preparation (Nylon Dyeing)
		3.2 Affinity Net Validation
		3.3 Urine Sample Processing
		3.4 Mass Spectrometry Analysis
		3.5 Peptide Authentication
	4 Notes
	References
Chapter 10: Preparation of Borrelia-Infected Mammalian Cells for Helium Ion Microscopy
	1 Introduction
	2 Materials
		2.1 Coverslip Preparation with Poly-L-Lysine
		2.2 Human Cell Infection with Borrelia
		2.3 Cell Preparation for Helium Ion Microscopy
	3 Methods
		3.1 Coverslip Preparation with Poly-L-Lysine
		3.2 Human Cell Infection with Borrelia
		3.3 Sample Preparation for Helium Ion Microscopy
		3.4 Some Notes for Imaging with HIM
	4 Notes
	References
Chapter 11: Establishing a Zebrafish Model for Borrelia burgdorferi Infection Using Immersion and Microinjection Methods
	1 Introduction
	2 Materials
		2.1 Zebrafish Breeding
		2.2 B. burgdorferi Culture
		2.3 Immersion Infection
		2.4 Microinjection
		2.5 DNA Extraction and PCR Amplification
		2.6 RNA Extraction and RT-PCR
		2.7 Tissue Embedding and FITC Staining
		2.8 Wholemount Immunohistochemistry
	3 Methods
		3.1 Zebrafish Breeding
		3.2 B. burgdorferi Culture
		3.3 Immersion Infection
		3.4 Microinjection Infection
		3.5 DNA Extraction and Nested PCR
		3.6 RNA Extraction and RT-PCR
		3.7 Live Fluorescence Imaging
		3.8 Tissue Embedding and FITC Staining
		3.9 Wholemount Immunohistochemistry
	4 Notes
	References
Chapter 12: Development of Organoids to Study Infectious Host Interactions
	1 Introduction
	2 Materials
		2.1 Primary Human Tissue Samples
		2.2 Organoids Culture
	3 Methods
		3.1 Wnt3a Conditioned Medium
		3.2 Rspondin 1 (RspoI) Conditioned Medium
		3.3 Noggin Conditioned Medium
		3.4 Digestion Buffer
		3.5 Chelation Buffer
		3.6 Organoid Expansion Medium
		3.7 ADF+++ Media
		3.8 Lung Organoid Medium
		3.9 Organoid Differentiation Medium
		3.10 Coating Solution
		3.11 Small Intestine Organoid Culture
		3.12 Lung Organoid Culture
		3.13 Infection of Organoids
	4 Notes
	References
Chapter 13: Content Analysis: An Approach to Exploring the Depiction of Tick-Borne Diseases
	1 Introduction
	2 Materials
		2.1 Examples of Text and Content Analysis
		2.2 Examples of Data/Text and Tick-Borne Diseases
	3 Methods
	References
Chapter 14: Applying BERT and ChatGPT for Sentiment Analysis of Lyme Disease in Scientific Literature
	1 Introduction
	2 Materials
		2.1 Data Collection Tools
		2.2 NLP Tools
	3 Methods
		3.1 Process
			3.1.1 Data Collection
			3.1.2 Data Cleaning
			3.1.3 Sentiment Analysis
	4 Notes
	References
Chapter 15: Building a Binary Classification Machine-Learning Model: A Guide to Predicting Participation in a Lyme Disease Pro...
	1 Introduction
	2 Materials
		2.1 Python
		2.2 Integrated Development Environment (IDE)
		2.3 Python Libraries
			2.3.1 NumPy
			2.3.2 Pandas
			2.3.3 Matplotlib
			2.3.4 Seaborn
			2.3.5 Scikit-Learn
		2.4 Data Science Datasets
	3 Methods
		3.1 Import Libraries
		3.2 Import Data
		3.3 Exploratory Data Analysis
		3.4 Divide Data into Training and Testing Sets
		3.5 Feature Engineering
		3.6 Feature Scaling
		3.7 Feature Selection
		3.8 Model Building, Hyperparameter Tuning, and K-fold Cross Validation
		3.9 Analyzing and Visualizing Model Performance
	4 Notes
	References
Chapter 16: Clinical Algorithm and Diagnostic Tools for Lyme Disease
	1 Introduction
	2 Methods
	3 Notes
	References
Chapter 17: Establishing a Pregnancy Lyme Disease Biobank
	1 Introduction
	2 Materials
	3 Methods
	References
Chapter 18: Developing a Prospective Gestational Lyme Disease Study
	1 Introduction
	2 Proposed Study Design
	3 Defining Infection
	4 Case Definitions for Lyme Disease
		4.1 Case Definition for Lyme Disease in Pregnant Women
		4.2 Case Definition for Lyme Disease in Fetus
		4.3 Case Definition for Lyme Disease in Neonates
	5 Serological Testing for Lyme Disease
	6 Non-serological Testing for Lyme Disease
	7 Microscopy and Culture
	8 Molecular Methods for Lyme Disease
	9 Serological Testing for Other Tick-Associated Infections
	10 Multiplex Molecular Testing for Other Tick-Associated Infections
	11 Specimens
	12 Clinical Assessment
	13 Maternal, Fetal, or Neonatal Infection
	14 Human Subjects Review
	15 Reality
	References
Index