Methods to Detect Microbes in the Environment ENVR 133 – Part 2

1
Methods to Detect Microbes in the
EnvironmentENVR 133 Part 2

• Mark D. Sobsey

2
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.

3
Some Methods for Molecular Genetic Detection
Typing of Microbes
4
Progress in Detection of Environmental Pathogens
by Nucleic Acid Hybridization

• Cons early 1990s
• High detection limits (gt1000 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)

5
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
6
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

7
Nucleic Acid Amplification - PCR
8
Example RT-PCR and Oligoprobe Detection of
Enteroviruses in Water

• Filter
• Elute
• Precipitate
• Extract RNA
• RT-PCR
• Oligoprobe
• (10 ul sample)

9
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

10
Real-Time, Multiplex RT-PCRHepatitis 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 lt10 genomic RNA copies
• Evaluated for virus detection in spiked water
  concentrate.
• Fluorogenic reporter probes (FAM- and
  ROX-labeled) specifically detected HAV or
  enterovirus, respectively.
• No amplified products from viruses not belong to
  these group.

1 2 3 4 5 6 7 8
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
11
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)

12
Restriction Endonucleases used in Molecular
Biology
13
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)

14
Example Macrorestriction Analysis of E. coli
Isolates
15
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

16
DNA from electrophoresed gel (left) is
transferred to membrane filter by contact and DNA
on membrane is hybridized with specific probe(s)
(right)
17
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

18
(No Transcript)
19
(No Transcript)
20
RFLP of Other Genes

• Species-specific genes as targets for RFLP
• Virulence genes
• Toxins
• Pili
• Flagellar genes
• Outer membrane protein genes

21
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

22
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.

23
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)

24
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
• Detect rapidly by amplification of nucleic acids
  produced in cells or by vial nucleic acids in
  host cells
• Integrated cell culture-PCR (or RT-PCR) for
  viruses
• mRNA in viable cells

Reverse transcribe ?
Polymerase Chain Reaction Amplification (PCR)
Nucleic acids in cells or in virus-infected
infected cells
25
Detecting Infectious Viruses by Direct Nucleic
Acid Analysis - A Functional Approach

• Direct nucleic acid analysis alone does not
  assure detection of infectious viruses
• 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

Infectious Non-infectious Nucleic acid
Cell Receptor
In Out
26
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

27
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
(RT-) PCR
sCAR
NA extraction
Sample containing viruses
Virus Particle
Blocking protein
Amine Terminated Support Magnetic Bead
BioSpheres(Biosource) Pre-coated to provide
available amine groups for covalent coupling of
proteins or other ligands by glutaraldehyde-mediat
ed coupling method
28
Ligand Capture of CVB3 Followed by
RT-PCR (Magnetic Bead-sCAR-CVB3)
Ligand capture
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
29
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
30
Microscopic Detection of Pathogens Still Widely
Used in Clinical Diagnostic Microbiology
C. parvum oocysts 5 um diam. Acid fast stain of
fecal preparation
31
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 non-target particles
• Flow cytometry and other advanced imaging
  techniques
• Advanced imaging methods require expensive
  hardware

32
Fluorescent In Situ Hybridization - FISH

• Bacteria of the target group are red
• Other bacteria are blue
• (artificial colors)

33
FISH DAPI-stained Bacteria Incubated with INT
(Tetrazolium Salt)

• Enhanced image with artificial colors.
• Blue DAPI stain
• Red INT grains indicate respiratory active
  bacteria.

34
Cryptosporidium parvumDifferential Interference
Contrast Microscopy
Image courtesy of O.D. Chip Simmons, III
35
Cryptosporidium parvum Microscopic Analysis of
NC field isolate
Immunofluorescence
Differential Interference Contrast
DAPI stain
Images courtesy of O.D. Chip Simmons, III
36
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.

37
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)

38
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