Download Methods to Detect Microbes in the Environment ENVR 133 – Lecture

Methods to Detect Microbes in the Environment
ENVR 133 – Part 2
Mark D. Sobsey
Detection of Pathogens by Detection and
Amplification of Nucleic Acids
Nucleic Acid Hybridization: potentially very useful, but:
(i) high detection limits (about 100-1000 genomic targets or
more)
(ii) large sample volumes; impractical for most hybridization
protocols without further concentration
(iii) hybridization reaction failures (false negatives)
and ambiguities (false positives) due to sample-related
interferences and non-specific reactions, and
(iv) uncertainties about whether positive reactions are truly
indicative of infectious pathogens.
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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
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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)
4
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
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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
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Nucleic Acid Amplification - PCR
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Example: RT-PCR and Oligoprobe Detection of
Enteroviruses in Water
•Filter
•Elute
•Precipitate
•Extract RNA
•RT-PCR
•Oligoprobe
(10 ul sample)
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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]
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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
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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)
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Restriction Endonucleases used in Molecular Biology
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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)
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Example: Macrorestriction Analysis of E. coli Isolates
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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
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DNA from electrophoresed gel (left) is transferred to membrane filter by
contact and DNA on membrane is hybridized with specific probe(s) (right)
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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
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18
19
RFLP of Other Genes
• Species-specific genes as targets for RFLP
• Virulence genes
–
–
–
–
Toxins
Pili
Flagellar genes
Outer membrane protein genes
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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
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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.
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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)
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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
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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
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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
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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
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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
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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
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Microscopic Detection of Pathogens:
Still Widely Used in Clinical Diagnostic Microbiology
C. parvum
oocysts ~5 um
diam.
Acid fast stain of
fecal preparation
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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
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Fluorescent In Situ Hybridization - FISH
• Bacteria of the
target group are red
• Other bacteria are
blue
(artificial colors)
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FISH:
DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt)
Enhanced image with
artificial colors.
•Blue: DAPI stain
•Red: INT grains;
indicate respiratory
active bacteria.
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Cryptosporidium parvum
Differential Interference Contrast Microscopy
Image courtesy of O.D. “Chip” Simmons, III
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Cryptosporidium parvum:
Microscopic Analysis of NC field isolate
Immunofluorescence
Differential Interference
Contrast
DAPI stain
Images courtesy of O.D. “Chip” Simmons, III
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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.
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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)
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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
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