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TitleDiagnostic Bacteriology: Methods and Protocols
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Table of Contents
Chapter 1: Whole-Genome Enrichment Using RNA Probes and Sequencing of Chlamydia trachomatis Directly from Clinical Samples
	1 Introduction
	2 Materials
		2.1 DNA Extraction and Quantification
		2.2 DNA Shearing
		2.3 Post Shearing Sample Purification
		2.4 End Repair, A-Tailing, and Adapter Ligation
		2.5 Amplification of Adapter-Ligated Library
		2.6 Hybridization of Adapter-Ligated Library to RNA Baits
		2.7 Addition of Index Tags by Post-­hybridization Amplification
		2.8 Pooling Samples for Multiplexed Sequencing
		2.9 Illumina Sequencing
		2.10 Data Analysis
	3 Methods
		3.1 DNA Extraction and Quantification
		3.2 DNA Shearing
		3.3 Post Shearing Sample Purification
		3.4 End Repair, A-Tailing, and Adapter Ligation
		3.5 Amplification of Adapter-Ligated Library
		3.6 Hybridization of Adapter-Ligated Library to RNA Baits
		3.7 Addition of Index Tags by Post-­hybridization Amplification
		3.8 Pooling Samples for Multiplexed Sequencing
		3.9 Illumina Sequencing
		3.10 Data Analysis
	4 Notes
Chapter 2: Characterization of Sinus Microbiota by 16S Sequencing from Swabs
	1 Introduction
	2 Materials
		2.1 Sample Collection
		2.2 DNA Extraction from Low Biomass Swabs
		2.3 DNA Extraction from High Biomass Swabs
		2.4 16S rDNA qPCR
		2.5 Molecular Barcoding
		2.6 Agarose Gel Electrophoresis
		2.7 DNA Normalization Reagents
		2.8 Sample Concentration and DNA Purification
		2.9 Fluorometric DNA Quantification
	3 Methods
		3.1 Swab Collection and Storage
		3.2 DNA Isolation of Low Biomass Swabs
		3.3 DNA Extraction of High Biomass Swabs
			3.3.1 Cell Lysis
			3.3.2 Precipitation and DNA Isolation
		3.4 Measuring Total Bacterial 16S DNA by qPCR
		3.5 Molecular Barcoding
		3.6 Confirmation of Amplification with Gel Electrophoresis
		3.7 DNA Concentration Normalization and Sample Pooling
		3.8 Purify and Measure DNA Concentration
	4 Notes
Chapter 3: Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR)
	1 Introduction
	2 Materials
		2.1 DNA Isolation
		2.2 Oligonucleotide Master and Working Stocks
		2.3 Multiplex Oligonucleotide Ligation
		2.4 Singleplex PCR with Universal Primers
		2.5 Hybridization to MagPlex-TAG Beads and Incubation with SAPE
	3 Methods
		3.1 DNA Isolation
		3.2 Oligonucleotide Master and Working Stocks
		3.3 Multiplex Oligonucleotide Ligation
		3.4 Singleplex PCR with Universal Primers
		3.5 Hybridization to MagPlex-TAG Beads and Incubation with SAPE
		3.6 Analysis on MAGPIX
		3.7 Data Interpretation
	4 Notes
Chapter 4: Detection of Helicobacter pylori DNA in Formalin-Fixed Paraffin-­Embedded Gastric Biopsies Using Laser Microdissection and qPCR
	1 Introduction
	2 Materials
		2.1 Sample Fixation and Histotechnical Process
		2.2 Tissue Embedding and Processing
		2.3 Selection and Cut of Bacteria in Tissue Sections
		2.4 DNA Isolation and Amplification by qPCR
	3 Methods
		3.1 Sample Fixation and Histotechnical Process
		3.2 Tissue Embedding and Processing
		3.3 Selection and Tissue Sectioning
		3.4 DNA Isolation and Amplification by qPCR
	4 Notes
Chapter 5: Mycobacterial Load Assay
	1 Introduction
	2 Materials
		2.1 Instruments
		2.2 Additional Equipment, Consumables, and Plasticware
		2.3 Reagents
	3 Methods
		3.1 Sample Preservation
		3.2 Preparation of RT-qPCT Mastermix
		3.3 Preparation of Primers and Probes
		3.4 RNA Extraction (See Note 8)
		3.5 DNase Treatment (Turbo DNA-Free Kit, Ambion AM1907)
		3.6 Reverse Transcriptase—Quantitative PCR (RT-qPCR)
			3.6.1 Samples
			3.6.2 Preparing Standard Samples for Standard Curve
			3.6.3 Master Mix Preparation
			3.6.4 Thermocycler Set Up Using RotorGene Q
			3.6.5 Result Interpretation and qPCR Output Data Analysis
			3.6.6 Troubleshooting
		3.7 Construction of Standard Curves for the MBL Assay
			3.7.1 Principle
			3.7.2 Standard Curves Construction
			3.7.3 Standard Curve Data Analysis
			3.7.4 Importing the Standard Curve for MBLA Analysis
	4 Notes
Chapter 6: Defining Diagnostic Biomarkers Using Shotgun Proteomics and MALDI-TOF Mass Spectrometry
	1 Introduction
	2 Materials
	3 Methods
		3.1 Colony Transfer and Treatment
		3.2 Experimental Data Acquisition by Mass Spectrometry
			3.2.1 Acquisition of MALDI-TOF MS Spectra
			3.2.2 Low Molecular Weight Shotgun Proteomics
		3.3 Mass Spectrometry Data Processing
			3.3.1 MALDI-TOF Data Processing
			3.3.2 Processing of Low Molecular Weight Shotgun Data
			3.3.3 Identifying Proteins Responsible of MALDI-TOF m/z Signals with the Help of the Low Molecular Weight Shotgun Data
			3.3.4 Selecting and Validating with a Representative Panel of Bacteria
	4 Notes
Chapter 7: Detection and Typing of “Candidatus Phytoplasma” spp. in Host DNA Extracts Using Oligonucleotide-Coupled Fluorescent Microspheres
	1 Introduction
	2 Materials
		2.1 Oligonucleotide Probe Design
		2.2 cpn60 Universal Target Amplicon Generation
		2.3 Coupling Oligonucleotides to Fluorescent Beads
		2.4 Hybridization of PCR Product to Oligonucleotide-­Coupled Beads
	3 Methods
		3.1 Oligonucleotide Probe Design
		3.2 cpn60 Amplicon Generation
		3.3 Coupling Oligonucleotides to Fluorescent Beads and Hybridization of PCR Product to Oligonucleotide-­Coupled Beads
		3.4 Hybridization of PCR Product to Oligonucleotide-­Coupled Beads
		3.5 Assessment of Capture Probe Target Specificity
	4 Notes
Chapter 8: Detection of Helicobacter pylori in the Gastric Mucosa by Fluorescence In Vivo Hybridization
	1 Introduction
	2 Materials
		2.1 Probe Design
		2.2 Probe Synthesis and Administration
		2.3 Probe Synthesis and Administration
		2.4 FIVH Analysis
	3 Methods
		3.1 Probe Design
		3.2 Fluorescence in Vivo Hybridization (FIVH) Probe Synthesis
			3.2.1 Synthesis
			3.2.2 Purification and Characterization of FIVH Probe
		3.3 Fluorescence In Vivo Hybridization (FIVH)
			3.3.1 FIVH in Mice
			3.3.2 Analysis of the Samples by Epifluorescence Microscopy
	4 Notes
Chapter 9: Rapid Antibiotic Susceptibility Testing for Urinary Tract Infections
	1 Introduction
	2 Materials
		2.1 Sample Pretreatment
		2.2 Padlock Probe Phosphorylation
		2.3 Padlock Probe Ligation, Amplification, and Detection
	3 Methods
		3.1 Sample Pretreatment
		3.2 Padlock Probe Phosphorylation
		3.3 Padlock Probe Ligation, Amplification, and Detection
	4 Notes
Chapter 10: Detection and Differentiation of Lyme Spirochetes and Other Tick-Borne Pathogens from Blood Using Real-­Time PCR with Molecular Beacons
	1 Introduction
	2 Materials
		2.1 DNA Isolation
		2.2 Quantitative Real-Time PCR
	3 Methods
		3.1 DNA Isolation
			3.1.1 DNA Isolation from Patient Samples
			3.1.2 DNA Isolation from Infected Mouse Tissue
		3.2 Quantitative Real-Time PCR
			3.2.1 Molecular Beacon Design
			3.2.2 Preparation of B. burgdorferi and Mouse DNA Standards
			3.2.3 Real-Time PCR Assays
		3.3 Testing of Samples
	4 Notes
Chapter 11: Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform
	1 Introduction
	2 Materials
		2.1 Laboratory Organization
		2.2 Sample Processing
		2.3 Material for DNA Extraction
		2.4 Assembly of the PCR Plate and Amplification
	3 Methods
		3.1 Sample Processing
		3.2 DNA Extraction
		3.3 Preparation of the PCR Controls
		3.4 Preparation of the PCR Mix, Design, and Assembly of the PCR Microplate and Amplification
		3.5 Interpretation of the Results
	4 Notes
Chapter 12: Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood
	1 Introduction
	2 Materials
		2.1 Specialist Equipment
		2.2 Bacterial Strains and Bacteriological Media
		2.3 Real-Time PCR
	3 Methods
		3.1 Handling and Preparation of Oligonucleotide Primers and Probes
		3.2 Handling and Preparation of PCR Mastermix and Solutions
		3.3 Incubation of Blood Cultures (See Note 4)
		3.4 Culture of Reference and Control Bacterial Strains
		3.5 Preparation of Bacterial DNA (Method 1: From Reference and Control Bacterial Strains in BHI Broth)
		3.6 Preparation of Bacterial DNA (Method 2: From Blood Culture Bottles)
		3.7 Biological Controls (See Note 5)
		3.8 Preparing and Loading the 96-Well Plate
		3.9 Operating the LightCycler® 480 Real-Time PCR Instrument
	4 Notes
Chapter 13: Multiplex Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) for Diagnosis of Bacterial Vaginosis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of FISH Samples
			3.1.1 Preparation of Vaginal Samples
		3.2 FISH Hybridization Procedure
		3.3 Microscopic Visualization
		3.4 Bacteria Count and BV Diagnosis
	4 Notes
Chapter 14: A Closed-tube Loop-Mediated Isothermal Amplification Assay for the Visual Endpoint Detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis
	1 Introduction
	2 Materials
		2.1 General Considerations
		2.2 Equipment
		2.3 Reagents and Solutions
	3 Methods
		3.1 Preparation of 10× LAMP Primers Mix
		3.2 Preparation of 2× LAMP Mix
		3.3 DNA Extraction from Culture
		3.4 LAMP Operating Procedure
		3.5 LAMP Reaction and End Point Detection
	4 Notes
Chapter 15: Highly Specific Ligation-dependent Microarray Detection of Single Nucleotide Polymorphisms
	1 Introduction
	2 Materials
		2.1 Washing Buffer
		2.2 Silanization of Glass Slide
		2.3 Spotting
		2.4 Ligation and Detection
	3 Methods
		3.1 Cleaning of Glass Slides
		3.2 Silanization of Glass Slides
		3.3 Ligation and Detection
	4 Notes
Chapter 16: Multilocus Sequence Typing (MLST) for Cronobacter spp.
	1 Introduction
	2 Materials
		2.1 PCR Amplification of the Seven MLST Genes
		2.2 Agarose Gel Electrophoresis
		2.3 PCR Purification
	3 Methods
		3.1 Polymerase Chain Reaction
		3.2 Agarose Gel Electrophoresis
		3.3 PCR Purification and Sequencing
		3.4 Sequence Analysis
		3.5 Allele and Sequence Type Designation
		3.6 Submission of Newly Identified Alleles and Sequence Types
	4 Notes
Chapter 17: Diagnostic Bacteriology: Raman Spectroscopy
	1 Introduction
		1.1 Raman Spectroscopy and Surface-Enhanced Raman Spectroscopy (SERS)
		1.2 SERS and Raman Spectroscopy in the Clinical Setting
		1.3 Future Use
		1.4 Conclusions
	2 Materials
		2.1 Bacterial Culture Components
		2.2 Silver Nanoparticle Components
		2.3 Raman Spectroscopy Components
	3 Methods
		3.1 Growth of Bacterial Culture
		3.2 Silver Nanoparticle Preparation (See Note 1)
		3.3 Slide Preparation
		3.4 Spectrum Collection
	4 Notes
Document Text Contents
Page 1


Kimberly A. Bishop-Lilly Editor

Methods and Protocols

Methods in
Molecular Biology 1616

Page 2

M e t h o d s i n M o l e c u l a r B i o l o g y

Series Editor
John M. Walker

School of Life and Medical Sciences
University of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

Page 132


5. T7 exonuclease (New England Biolabs).
6. 0.5 M ethylenediaminetetraacetic acid (EDTA) solution, pH

7. Two workstations reserved for PCR work fitted with ultra-

violet bulbs (e.g., PCR Cleanspot, Coy Laboratory Products).
See Note 1 for cautionary steps to be taken to avoid

8. Quant-iT DNA quantification kit (Invitrogen) or analogous
fluorescence-based DNA quantification system and fluorome-
ter (e.g., Qubit fluorometer, Invitrogen).

1. EDC 1-ethyl-3-(3-dimethylamiopropyl) carbodiimide HCl.
2. 5.6 μm polystyrene Bio-Plex beads, various spectral signatures

(colors). Magnetic beads may also be used.
3. Target-specific capture oligonucleotides, 5-amino-C12 modi-

fied (IDT, Invitrogen, Eurofins, or other suppliers).
4. 0.1 M MES pH 4.5 (protect this solution from light).
5. 0.02% Tween 20.
6. 0.1% sodium dodecyl sulfate.
7. TE buffer (10 mM Tris–Cl pH 8.0; 1 mM EDTA).

1. Bio-Plex or analogous instrument (e.g., Luminex).
2. Streptavidin-R-phycoerythrin (SAPE), 1 mg/ml. High-purity

SAPE solution is essential, e.g., Life Technologies cat no.

3. Thermowell 96-well PCR plates, low profile.
4. Thermowell silicone sealing mat. Can be reused.
5. 5 M tetramethyl ammonium chloride (TMAC), 10% sarkosyl,

1 M Tris–Cl, pH 8.0.

2.3 Coupling
to Fluorescent Beads

2.4 Hybridization
of PCR Product
to Oligonucleotide-
Coupled Beads

Table 1
Sequences of the cpn60 amplification primers

Primer name [16] Primer sequence (5′–3′)a

H279-phyto-lum 5′-biotin-[*C][*G][*A][*C]GATIIIGCAGGIGATGGAACMACIAC


D0317-lum 5′-biotin-[*C][*G][*A][*C]GATIIIKCIGGIGAYGGIACIACIAC


Note that there are two upstream and two downstream primers that can be mixed to various molar ratios in order to
amplify the cpn60 UT from a diverse array of Phytoplasma spp. We have determined that a 1:7 molar ratio of H279p/
H280p:D0317/D0317 successfully amplifies the cpn60 UT from a wide range of “Ca. Phytoplasma” spp. [16]
aI = inosine; Y = C or T; R = A or G; K = T or G; S = C or G; [*] = phosphorothioate-modified nucleotide

Edel Pérez-López et al.

Page 133


3 Methods

The objective of this analysis is to identify regions that are likely to
contain sequences specific to the target sequence of interest. The
output is a series of ranges that contain “signature” regions that
are capable of discerning the target sequence from all of the other
sequences in the “outgroup.” Other strategies are equally valid
but this is our typical approach. Capture oligonucleotide design
for a previously described cpn60 UT sequence from Bois Noir
(strain BN45660; GenBank accession no. KJ939981) is used as an

1. Place the sequence files (in Fasta format) to be analyzed in an
appropriately labeled folder (for the current example, this
folder is labeled, “phyto”).

2. Within this folder, arrange the sequences so that the sequence
to be examined for signatures (in this case, BN-cpn60.fasta) is
at the same level as another folder (here called, “outgroup”)
containing all of the sequences to be discerned from the target
sequence. In this case, the “outgroup” folder contains cpn60
sequences from all of the non-BN phytoplasmas that are known
to date [16]. These can be retrieved from GenBank (ncbi. or cpnDB (

3. Execute the sigoli command from a folder one level above the
“phyto” folder, as follows:
/path-to-sigoli-1-1/sigoli -operation = ranges
-sequence-directory = phyto -oligo-size = 20
-diff=yes > BN-sigs.txt
This specifies that the software should return the ranges and
their mid-point (nucleotide positions) of signatures within the
sequence directory, “phyto,” with an oligonucleotide size of
20. The “diff=yes” flag specifies that the ranges it finds must
differ in more than one place from all of the other sequences.
The output of the program is directed to a new file called

4. Examine the output of sigoli; see Fig. 1a for an example. The
output will show the ranges of nucleotide positions within
BN- cpn60.fasta that contain at least two differences to all of
the sequences in outgroup within a sliding window of 20
nucleotides. These areas are suitable for specific capture oligo-
nucleotide design.

5. Enter all of the sequences into PrimerPlex. Execute a search
for capture oligonucleotides (under Analyze > Capture Probe
Search), ensuring that the “Design Anti-Sense Probe” radio
button is selected. The number of capture probe sequences
returned can be adjusted.

3.1 Oligonucleotide
Probe Design

Detection and Typing of “Candidatus Phytoplasma” spp. in Host DNA Extracts…

Page 264


28. Maquelin K, Dijkshoorn L, van der Reijden TJ,
Puppels GJ (2006) Rapid epidemiological anal-
ysis of Acinetobacter strains by Raman spectros-
copy. J Microbiol Methods 64:126–131

29. Maquelin K et al (2000) Raman spectroscopic
method for identification of clinically relevant
microorganisms growing on solid culture
medium. Anal Chem 72:12–19

30. Kalasinsky KS et al (2007) Raman chemical
imaging spectroscopy reagentless detection
and identification of pathogens: signature
development and evaluation. Anal Chem

31. Zeiri L, Bronk BV, Shabtai Y, Czege J, Efrima
S (2002) Silver metal induced surface enhanced
Raman of bacteria. Colloids Surf A Physicochem
Eng Asp 208:357–362

32. Ghebremedhin M, Yesupriya S, Luka J, Crane
NJ (2015) Validation of hierarchical cluster
analysis for identification of bacterial species
using 42 bacterial isolates. Proc SPIE

33. Premasiri WR, Sauer-Budge AF, Lee JC,
Klapperich CM, Ziegler LD (2012) Rapid

bacterial diagnostics via surface enhanced Raman
microscopy. Spectroscopy (Springf) 27:s8–s31

34. Ozaki Y, Kneipp K, Aroca R (2014) Frontiers
of surface-enhanced Raman scattering: single
nanoparticles and single cells. Wiley, West

35. Rosch P et al (2005) Chemotaxonomic identi-
fication of single bacteria by micro-Raman
spectroscopy: application to clean-room-
relevant biological contaminations. Appl
Environ Microbiol 71:1626–1637

36. Premasiri WR, Moir DT, Klempner MS,
Zeigler LD (2007) In: Kneipp K, Aroca R,
Kneupp H, Wentrup-Byrne E (eds) New
approaches in biochemical spectroscopy.
Oxford University Press, New York, p 164

37. Crane NJ, Elster EA (2012) Profiling wound
healing with wound effluent: Raman spectro-
scopic indicators of infection. Proc SPIE

38. National Academies of Sciences, Engineering,
and Medicine (2015) Improving diagnosis in
health care. The National Academies Press,
Washington, DC

Raman Spectroscopy

Page 265


Kimberly A. Bishop-Lilly (ed.), Diagnostic Bacteriology: Methods and Protocols, Methods in Molecular Biology, vol. 1616,
DOI 10.1007/978-1-4939-7037-7, © Springer Science+Business Media LLC 2017


Antibiotic susceptibility testing ��������������������������������147–152


Bacterial vaginosis ������������������������������������������������������������218
Biomarkers ������������������������������������������������������� 107–119, 253
Blood ����������������������������������������������24, 28, 85, 144, 155–169,

183–206, 253
Brucella �����������������������������������������������������������������������������229


Candidatus Phytoplasma �������������������������������������������121–135

abortus �����������������������������������������������������������������171–180
pneumoniae ���������������������������������������������������������� 171–180
psittaci ������������������������������������������������������������������ 171–180
trachomatis ������������������������������������������1–21, 171, 172, 176

Cronobacter ����������������������������������������������������������������241–247


Fluorescence in vivo hybridization ���������������������������137–144
Fluorescent microspheres �����������������������������������������121–135
Formalin-fixed paraffin-embedded (FFPE) �����������������71–87


Gastric biopsy ���������������������������������������������������������������73, 84


Helicobacter pylori ������������������������������������������ 71–87, 137–144


Laser microdissection ���������������������������������������������������71–87
Loop-mediated isothermal amplification �����������������221–229
Lyme disease ������������������������������������������������������������155–157


Matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) mass spectrometry �������������107–119

Microarray ������������������������������������������������ 122, 123, 231–239

beacons ���������������������������������������������������������������155–169
subtyping ����������������������������������������������������������������39–68

Multilocus sequence typing (MLST) �����������������������241–247

avium ��������������������������������������������������������������������������229
paratuberculosis ������������������������������������������������������������� 229
tuberculosis ����������������������������������������2, 3, 72, 89, 103, 147


Panton-Valentine leukocidin (PVL) ������������������������ 184, 185,
187, 199, 200

Peptide nucleic acid fluorescence in situ hybridization
(PNA-FISH) ������������������������������������������������209–218

Polymerase chain reaction (PCR)
multiplex oligonucleotide ligation-PCR

(MOL-PCR) ����������������������������������������������������39–68
quantitative (qPCR) ����������������������������������������� 25, 97–99
real-time ����������������������27, 90, 99, 155–169, 171–180, 227

Protein A ��������������������������������������������������������������������������184
Proteomics ����������������������������������������������������������������107–119


Raman spectroscopy
surface enhanced (SERS) �����������������������������������250–253


Salmonella Typhimurium ����������������������������������������������39–68

16S������������������������������������������ 23–37, 84, 86, 87, 92, 122,
137, 138, 140, 173, 216, 241

whole genome ��������������������������������������������������� 1–21, 242
Sinus �����������������������������������������������������������������������������23–37
Spirochetes ���������������������������������������������������������������155–169

aureus ����������������������������������������������������������������������24, 29
coagulase-negative ����������������������������������������������184, 185
methicillin resistant ������������������������������������������������������29

Swabs ������������������������������������� 3, 7, 23–37, 176, 211–213, 217


Urinary tract infections ���������������������������������������������147–152


Virulence �����������������������������������73, 83, 86, 87, 108, 183–206


Whole genome enrichment ��������������������������������������������1–21


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