Methods to Detect Microbes in the Environment
Part 2
ENVR 421
Mark D. Sobsey
Pathogen Detection by Biochemical Methods
• Enzymatic activities unique to target microbe
• Signature Biolipid Analysis:
– Detection of unique biolipids by gas-chromatography,
mass spectrometry and other advanced organic
analytical methods
• Extract and purify from cells
• Analyze
• Other biochemical markers unique to a specific pathogen
or class of pathogens.
2
Microscopic and Imaging Detection of Pathogens
• Still widely used for parasites and bacteria
• Specific staining and advanced imaging to distinguish
target from non-target organisms
– Differential interference contrast microscopy
– Confocal laser microscopy
• Distinguish infectious from non-infectious organisms
– Combine with infectivity, viability or activity assays
• Overcome sample size limitation due to presence of nontarget particles
– Flow cytometry and other advanced imaging
techniques
– Advanced imaging methods require expensive
hardware
3
Microscopic Detection of Pathogens
Still Widely Used in Clinical Diagnostic Microbiology
• Preferred method
for parasites
– Example:
Cryptosporidium
parvum oocysts,
~5 um diam.
– Acid fast stain of
fecal preparation
4
Cryptosporidium parvum
Differential Interference Contrast Microscopy
Image courtesy of O.D. “Chip” Simmons, III
5
Cryptosporidium parvum:
Microscopic Analysis of NC field isolate
Immunofluorescence
Differential Interference
Contrast
DAPI stain
Images courtesy of O.D. “Chip” Simmons, III
6
Microscopic Analysis of Bacteria
Fluorescent In Situ Hybridization - FISH
• Bacteria of the
target group are red
• Other bacteria are
blue
(artificial colors)
7
FISH:
DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt)
Enhanced image with
artificial colors.
•Blue: DAPI stain
•Red: INT grains;
indicate respiratory
active bacteria.
8
MOLECULAR BIOLOGY TECHNIQUES
Utilize DNA, RNA, and enzymes
that interact with nucleic acids to
identify, understand, characterize
and quantify biological structures,
process, phenomena and
activities at a molecular leve
9
Molecular Pathology and
Diagnostics
•INHERITED DISEASES (GENETICS)
– Cystic fibrosis
– Sickle cell anemia
– Predispositions to cancer
•INFECTIOUS DISEASES
– Bacteria
– Viruses
– Fungi
10
NUCLEIC ACIDS
•
•
•
•
Genetic material of all known organisms
DNA: deoxyribonucleic acid
RNA: ribonucleic acid (e.g., some viruses)
Consist of chemically linked sequences of
nucleotides
• Nitrogenous base
• Pentose- 5-carbon sugar (ribose or
deoxyribose)
• Phosphate group
• The sequence of bases provides the genetic
information
11
Bases
• Two types of bases
• Purines are fused five- and six-membered rings
• Adenine A
DNA RNA
• Guanine G
DNA RNA
• Pyrimidines are six-membered rings
• Cytosine C
DNA RNA
• Thymine T
DNA
• Uracil
U
RNA
12
Base-pairing
• Hydrogen bonds are relatively weak bonds
compared to covalent bonds
• Hydrogen bonds can form between a pyrimidine
and a purine
• Watson-Crick base-pairing rules
•A T
•G C
13
Hydrogen Bonds
H
H
H
C
Thymine
H
H
O
C
H
C
C
N
C
N
H
C
N
Adenine
N
C
O
C
C
N
C
H
N
N
H
H
N
H
Cytosine
H
C
H
C
N
C
N
C
N
O
N
Guanine
N
C
H
C
C
C
H
C
H
N
O
N
H
14
DNA: Helix
3’
5’
5’
3’
In general, DNA is double-stranded.
Double-stranded (ds) DNA takes the
form of a right handed helix with
approximately 10 base pairs per turn of
the helix.
15
Complementarity
• In the DNA double helix, purines and pyrimidines
face each other
• The two polynucleotide chains in the double helix
are connected by hydrogen bonds between the
bases
• Watson-Crick base-pairing rules
•A T
•G C
• GC base pairs (bps)have more energy than AT bps
• Since one strand of DNA is complementary to the
other, genetic material can be accurately
reproduced; each strand serves as the template
for the synthesis of the other
16
Antiparallel Chains
5’p
OH3’
3’OH
p5’
Two strands of the DNA double helix are
antiparallel and complementary to each
other
17
Gene
•A gene is a unit of inheritance
•Carries the information for a:
-polypeptide
-structural RNA molecule
flank
5’
upstream
flank
promoter
Structural gene
3’
downstream
18
Nucleases
5’ Exonuclease
3’ Exonuclease
Endonuclease
19
Restriction enzymes
• Specific endonucleases
• Recognize specific short sequences of DNA and
cleave the DNA at or near the recognition
sequence
• Recognition sequences: usually 4 or 6 bases but
there are some that are 5, 8, or longer
• Recognition sequences are palindromes
• Palindrome: sequence of DNA that is the same
when one strand is read from left to right or the
other strand is read from right to left– consists of
adjacent inverted repeats
20
Restriction enzymes (cont’d)
• Example of a palindrome:
GAATTC
CTTAAG
• Restriction enzymes are isolated from bacteria
•
•
•
•
•
Derive names from the bacteria
Genus- first letter capitalized
Species- second and third letters (small case)
Additional letters from “strains”
Roman numeral designates different enzymes from the
same bacterial strain, in numerical order of discovery
• Example: EcoRI
– E Escherichia
– Co coli
– R R strain
– I first enzyme discovered from Escherichia coli R
21
Detection of Pathogens by Detection of Nucleic Acids
Potentially very useful, but problems and limitations:
•
•
•
•
•
High detection limits (about 100-1000 genomic targets or
more)
• Target microbes in environmental samples often at low
concentration
Large sample volumes; impractical for most hybridization
protocols without further concentration
– Concentrate target microbe/NA in environmental
sample
Hybridization reaction failures (false negatives)
Ambiguities (false positives) due to sample-related
interferences and non-specific reactions
Uncertainties about whether positive reactions are truly
indicative of infectious pathogens.
22
Hybridization
• Nucleic acid hybridization is the formation of a
duplex between two complementary sequences
• Intermolecular hybridization: between two
polynucleotide chains which have complementary
bases
– DNA-DNA
– DNA-RNA
– RNA-RNA
• Annealing is another term used to describe the
hybridization of two complementary molecules
23
Denaturation - Renaturation
Renaturation
Denaturation
Doublestranded
DNA
Singlestranded
DNA
Initial
Base
pairing
Renatured
DNA
24
Probes
• Probe is a nucleic acid that
– can be labeled with a marker which allows
identification and quantitation
– will hybridize to another nucleic acid on
the basis of base complementarity
• Types of labels
– Radioactive (32P, 35S, 14C, 3H)
– Fluorescent
• FISH: fluorescent in situ hybridization
– chromosomes
– Biotinylated (avidin-streptavidin)
25
Solid Support Hybridization
• Solid support hybridization: DNA or RNA is
immobilized on an inert support so that selfannealing is prevented
• Bound sequences are available for hybridization
with an added nucleic acid (probe).
• Filter hybridization is the most common
application:
– Southern Blots
– Dot/Slot Blots
– Northern Blots
• In-silica hybridization (glass slides)
– in situ hybridization (tissue)
– Chromosomal (FISH)
– Microarrays
26
Southern Blots
• Southern blotting is a procedure for transferring
denatured DNA from an agarose gel to a solid
support filter where it can be hybridized with a
complementary nucleic acid probe
• The DNA is separated by size so that specific
fragments can be identified
• Procedure:
– Restriction digest to make different sized fragments
– Agarose gel electrophoresis to separate by size
– Since only single strands bind to the filter, the DNA
must be denatured.
– Denaturation to permit binding to the filter (NaOH)
– Transfer to filter paper (capillary flow)
– Hybridization to probe
– Visualization of probe
27
Southern Blot
Restriction enzyme
DNA of
various sizes
Electrophorese on agarose gel
gel
Denature - transfer to
filter paper.
blot
28
Denature- transfer to
filter paper.
blot
Hybridize to probe
Visualize
29
Southern Blot
30
Dot/Slot Blots
• DNA or RNA is bound directly to a solid support
filter
• No size separation
• Ideal for multiple samples and quantitative
measurements
• Important to establish specificity of conditions
31
Slot Blot
32
Some Methods for Molecular Genetic Detection & Typing of Microbes
Method
Basis
Resolution
Advantages
Disadvantages
Gene Probes
(Nucleic Acid
Hybridization)
Genome segment
length
polymorphism
analysis:
electropherotyping
PCR and
RT-PCR
Probe
Specificity
Variable:
formatspecific
Subtypes
Easy; Rapid;
Low/Med. $
Insensitive
Must amplify
RFLP Analysis,
PFGE, Ribotyping
Oligonucleotide
fingerprinting
Restriction Variable
enzyme
cuts
RNAaseT1 Subtypes
cleavage
RNase protection
assay
Probe
specificity
Segment
length
Primer
Specificity
Variable
Subtypes
Easy, Rapid, Low $ Does not detect mutations,
Only applicable to
segmented genomes (some
viruses), Insensitive (needs
high titer)
Moderate Difficulty; Technical; Variable Time,
Flexible; Specific; Med. $; Product
Varuable Speed;
Confirmation Needed
Moderate $
Easy-Moderate,
Need restriction sites and
Rapid, Variable $
good enzymes. Consider
variability of target NA
Applicable to RNA; Technical electrophoresis
Detects point
method; Med. $
mutations
Applicable to RNA Technical; Uses radioactive
viruses & other
probe
RNA
33
Progress in Detection of Environmental Pathogens by
Nucleic Acid Hybridization
Cons: early 1990s
•
•
•
•
High detection limits (>1000 genomic targets)
Sample volumes too large without concentration
False (-) and false (+) due to sample interferences
Uncertain if positive reactions truly indicate infectious pathogens
Pros: late 1990s
• Confirm identity of PCR and RT-PCR products
– Oligoprobe hybridization
• Detect PCR products as they are generated
– Labeled primers
• Simultaneously genotype many gene targets with multiple probes
– Reverse Line Blot Hybridization Assay (caliciviruses)
34
Nucleic Acid Hybridization to
Genotype F+ RNA Coliphages
Lysis zone (plaque) hybridization
• Left: specificities of 6
oligonucleotide probes
– prototype F+ RNA
coliphages,
• Right: classification of 5 isolates
from environmental samples
– isolates P1 and P2 from
piglet feces, W1 and W2
from surface water, and E1
from secondary effluent.
• Pairs of membranes in each row
were hybridized with probes I, II,
III, IV, A, and B, respectively.
35
Agarose Gel Electrophoresis
• Separate nucleic acid fragments in
an agarose gel
• Resolves small DNA molecules:
0.1 to 50 kb
• % agarose determines resolution
of DNA size:
– 0.3% w/v: resolves 5 to 50 kb
– 2% w/v resolves 0.1 to 2 kb
• Resolving large molecules (up to
500 kb) requires specialized
methods
– Pulse-field gel electrophoresis
(PFGE)
DNA
marker
ladder
Specific
DNA
fragment
36
Direct Detection of Viruses and Other Microbes by
Nucleic Acid Amplification
For viruses not growing in lab hosts:
• Detect directly by in-vitro amplification of their nucleic
acids
• PCR (DNA viruses) or RT-PCR (RNA viruses)
• Amplify nucleic acids (105-106 times)
– Detect by oligoprobe hybridization
OR:
• Amplify nucleic acids and detect in real-time by
fluorescent signal as primers are incorporated during
amplification
– Taqman PCR with LightCycler
37
Nucleic Acid Amplification - PCR
38
Example: RT-PCR and Oligoprobe Detection of
Enteroviruses in Water
•Filter
•Elute
•Precipitate
•Extract RNA
•RT-PCR
•Oligoprobe
(10 ul sample)
39
Real-Time PCR and Quantitative Fluorogenic Detection
• Molecular beacon. Several 5'
bases form base pairs with
several 3' bases; reporter and
quencher in close proximity.
– If reporter is excited by light,
its emission is absorbed by
quencher & no fluorescence is
detected.
• Detection of PCR product by
molecular beacon.
– Beacon binds to PCR product
and fluoresces when excited
by the appropriate  of light.
– [Fluorescence] proportional to
[PCR product amplified]
40
Real-Time, Multiplex RT-PCR:
Hepatitis A Virus (HAV) and Enteroviruses (EV)
• Fluorescent probes to
simultaneously detect HAV and EV
(CVB3).
• HAV and EV primer pairs gave
predicted 244 and 145-bp products.
– Detect <10 genomic RNA copies
• Evaluated for virus detection in
spiked water concentrate.
• Fluorogenic reporter probes (FAMand ROX-labeled) specifically
detected HAV or enterovirus,
respectively.
• No amplified products from viruses
not belong to these group.
1 2 3 4 5 6 78
1. Std, 100 bp fragments
2. CVB3 , 145 bp
3. negative control
4. HAV, 244 bp
5.negative control
6. CVB3 and HAV
7.negative control
8. Std, 100 bp fragments
41
Assessing DNA Polymorphisms to Detect and
Characterize Specific Bacteria
• Molecular methods used to group or type bacteria based on
genomic homogeniety or diversity
• Identifies groups of closely-related isolates (presumed to
arise from a common ancestor in the same chain of
transmission) and divergent, epidemiologically unrelated
isolates arising from independent sources.
• Restriction fragment length polymorphisms: variable and
distinct size fragments of DNA detected by cutting DNA at
unique sites using specific restriction endonucleases
– Macrorestriction analysis
– Ribotyping: cutting DNA amplifies from 16S ribosomal RNA
– Restriction analysis of virulence-associated genes
• Arbitrary-primed PCR (Randomly Amplified Polymorphic DNA)
42
Restriction Endonucleases used in Molecular Biology
43
Restriction Fragment Polymorphisms
• Variations in DNA sequences are manifest as
changes in some recognition sites for specific
restriction endonuclease enzymes
• Alters size and number of DNA fragments
obtained from restriction enzyme digestion of
chromosomal DNA
• Whole genomic DNA: macrorestriction analysis
• Specific gene(s): ribotyping (rRNA operons)
44
Example: Macrorestriction Analysis of E. coli Isolates
45
RFLP Analysis Procedure
• Isolate chromosomal DNA
• Digest DNA with restriction endonuclease
• Agarose gel electrophoresis
– Macrorestriction analysis
• Southern blotting and hybridization
– Transfer DNA from gel to membrane (cellulose or
nylon)
– Hybridize with labeled probe to gene of interest
• e.g., rDNA
– Ribotype
46
DNA from electrophoresed gel (left) is transferred to membrane filter by
contact and DNA on membrane is hybridized with specific probe(s) (right)
47
Ribotyping
• Gene-specific RFLP for polymorphisms in rRNA genes
(rDNA)
• Identify rDNA fragments from electrophoresed chromosomal
restriction digests by Southern hybridization
• Use specific restriction enzymes with good discrimination
abilities to generate restriction patterns from rDNA
• rRNA is found in all bacteria
• Some sequences are highly conserved and are common in
broad groups (genera); can identify genus as first step with
broad rRNA probe
• rDNA has less but sufficient variability compared to other
genes to type specific species and strains of related bacteria
48
49
50
RFLP of Other Genes
• Species-specific genes as targets for RFLP
• Virulence genes
–
–
–
–
Toxins
Pili
Flagellar genes
Outer membrane protein genes
51
Arbitrarily-Primed PCR (Randomly Amplified
Polymorphic DNA or RAPID)
• Identifies strain-specific variations in DNA
• Use arbitrarily-chosen primers pairs (10- to 20-mers) to
amplify chromosomal DNA under non-stringent conditions
• Variations in DNA sequences of different strains will give
differences in numbers and sizes of their PCR products
• Provides a unique DNA fingerprint
• Limited number of patterns or groups per species of
bacterium
• Problems in reproducability and interpretation have occurred
52
Repetitive Element-PCR (Rep-PCR)
• PCR amplify specific fragments of chromosomal
DNA lying between known repeat motifs of the
chromosome
• Use two outwardly directed primers for the
repeat element at high stringency to generate
unique DNA products that are strain-specific.
53
Detecting Active or Viable Pathogens Using Nucleic Acid
Targets
Detect short-lived nucleic acids present in only
viable/infectious microbes:
– ribosomal RNA
– messenger RNA
– genomic RNA of viruses (large amplicons)
• Detect pathogen nucleic acid by fluorescent in-situ
hybridization (FISH)
– applied to bacteria, protozoan cysts and oocysts, as
well as viruses in infected cell cultures
• (see pictures in later slides)
54
Infectious Microbe Detection by Nucleic Acid Amplification
Target RNA (viral RNA or mRNA)
Viruses (and other microbes)
growing slowly or without
visible signs of growth:
Reverse transcribe 
• Detect rapidly by amplification
of nucleic acids produced in
cells or by vial nucleic acids in
host cells
Polymerase Chain
Reaction Amplification
(PCR)
– Integrated cell culture-PCR (or
RT-PCR) for viruses
– mRNA in viable cells
Nucleic acids in cells
or in virus-infected
infected cells
55
Detecting Infectious Viruses by Direct Nucleic
Acid Analysis - A Functional Approach
Infectious
• Direct nucleic acid analysis alone
does not assure detection of
infectious viruses
Noninfectious
– Nucleic acid still present in
inactivated viruses or free in the
sample (water, etc.)
• Infectious viruses have intact surface
chemistries (epitopes) that react with
host cells to initiate virus infection
– The presence of functional surface
epitopes for binding to cell receptors
is evidence of virus infectivity
Nucleic
acid
Cell
Receptor
In
Out
56
Virus Capture Plus RT-PCR to Detect Infectious
Viruses - The sCAR System
• The cell receptor gene for Coxsackieviruses and
Adenoviruses has been cloned and expressed,
producing a soluble protein receptor, sCAR
• Expressed, purified and bound sCAR to solid
phases to capture infectious Coxsackieviruses
from environmental samples
– The nucleic acid of the sCAR-captured viruses is RT-PCR
amplified for detection and quantitation
57
Application of sCAR with Para-Magnetic Beads for
Virus Particle Capture and then RT-PCR
sCAR
purification
Covalent coupling
to paramagnetic beads
Culture + media;
:sCAR produced
Blocking
post-coupling
: sCAR
: Virus Particle
: Blocking protein
Sample
containing
viruses
(RT-) PCR
NA
extraction
Amine Terminated Support Magnetic Bead : BioSpheres(Biosource)
Pre-coated to provide available amine groups for covalent coupling
of proteins or other ligands by glutaraldehyde-mediated coupling method
58
Ligand Capture of CVB3 Followed by RT-PCR
(Magnetic Bead-sCAR-CVB3)
200bp
SM
103 100
10
Ligand capture
1
0.1
103 100
+
PFU
Bead control
Ligand capture: capture of CVB3 with magnetic beads coupled with purified sCAR
Bead control : Reaction of CVB3 with BSA coated magnetic bead
Magnetic Bead : BioSpheres (Amine Terminated Support)
Viral RNA extraction: QIAamp kit
59
Microbe Nucleic Acid Detection by DNA Microarrays
or “Gene Chip” Technology
Generate/obtain DNA complimentary to genes
(sequences) of interest;
– 1000s of different ones
Apply tiny quantities of each different one onto
solid surfaces at defined positions
– “gene chip” or “DNA microarray”
Isolate or amplify target NA of interest and label
with a fluorescent probe
Apply sample NA to the “gene chip” surface
– Sample NA binds to specific DNA probes on
chip surface; wash away unbound NA
Detect bound DNA or RNA by fluorescence after
laser excitation
Analyze hybridization data using imaging
systems and computer software
Fluorescing Gene Chip or
DNA Microarray
60
Summary Detecting and Quantifying Microbes in the Environment
• Get representative samples
• Recover the microbes from the samples
– may have to separate, concentrate and purify
• very low numbers lots of other similar objects and other
stuff (interferences)
• Analyze for the recovered microbes:
– observe and count them - microscopy/imaging
– culture them on media or in live hosts
– detect them as antigens (immunoassays)
– detect their genetic material (nucleic acid assays)
– detect their unique or characteristic chemical properties or
other properties (e.g., antibiotic resistance)
61
Future Directions in Microbial Detection in the
Environment
• Rapid and Sensitive Pathogen Detection Methods
– Molecular detection for real-time or near real-time monitoring
of pathogens (BT agents, too).
• Real-time PCR
• Couple with methods to selectively recover and detect
potentially infectious microbes
• Enrich for virulence genes of microbes in environmental
media - early warning/alerts system
• Nucleic acid microarrays (“gene chips”) for 1000s at a time
– Culture plus molecular or immunodetection
• Detect pathogen nucleic acids or antigens early in microbial
proliferation in culture
62