Methods to Detect Microbes in the EnvironmentPart 2

ENVR 421Mark D. Sobsey

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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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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

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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

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Cryptosporidium parvumDifferential Interference Contrast Microscopy

Image courtesy of O.D. “Chip” Simmons, III

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Cryptosporidium parvum: Microscopic Analysis of NC field isolate

Differential Interference Contrast

DAPI stain

Immunofluorescence

Images courtesy of O.D. “Chip” Simmons, III

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Microscopic Analysis of BacteriaFluorescent 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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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

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Molecular Pathology and Diagnostics

•INHERITED DISEASES (GENETICS)– Cystic fibrosis– Sickle cell anemia– Predispositions to cancer

•INFECTIOUS DISEASES– Bacteria– Viruses– Fungi

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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

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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

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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

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Hydrogen Bonds

H

H

H H

O

O

H

C

C

C C

N

N

C

ThymineH

N

H

H

N

C C

C

C

N

N H

N

C

Adenine

H

O

N

H C

C C

N

N

C

Cytosine

H

H

H

N

C C

C

C

N

N H

N

C

Guanine

NH

O

H

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DNA: Helix

5’ 5’

3’

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.

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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

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Antiparallel Chains

5’p OH3’

3’OH p5’

Two strands of the DNA double helix are antiparallel and complementary to each other

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Gene

promoter Structural gene

flank flank

upstream downstream5’ 3’

•A gene is a unit of inheritance

•Carries the information for a:

-polypeptide

-structural RNA molecule

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Nucleases

Endonuclease

5’ Exonuclease 3’ Exonuclease

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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

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Restriction enzymes (cont’d)

• Example of a palindrome:GAATTCCTTAAG

• 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

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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.

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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

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Double-stranded

DNA

Denaturation

Single-stranded

DNA

InitialBase

pairing

Denaturation - Renaturation

Renatured DNA

Renaturation

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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)

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Solid Support Hybridization

• Solid support hybridization: DNA or RNA is immobilized on an inert support so that self-annealing 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

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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

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Southern Blot

Restriction enzyme

DNA of various sizes

Electrophorese on agarose gel

gel

Denature - transfer to filter paper.

blot

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Hybridize to probe

Visualize

Denature- transfer to filter paper.

blot

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Southern Blot

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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

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Slot Blot

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Some Methods for Molecular Genetic Detection & Typing of MicrobesMethod Basis Resolution Advantages Disadvantages

Gene Probes (Nucleic Acid Hybridization)

Probe Specificity

Variable: format-specific

Easy; Rapid; Low/Med. $

Insensitive Must amplify

Genome segment length polymorphism analysis: electropherotyping

Segment length

Subtypes Easy, Rapid, Low $ Does not detect mutations, Only applicable to segmented genomes (some viruses), Insensitive (needs high titer)

PCR and RT-PCR

Primer Specificity

Variable Moderate Difficulty; Flexible; Specific; Varuable Speed; Moderate $

Technical; Variable Time, Med. $; Product Confirmation Needed

RFLP Analysis, PFGE, Ribotyping

Restriction enzyme cuts

Variable Easy-Moderate, Rapid, Variable $

Need restriction sites and good enzymes. Consider variability of target NA

Oligonucleotide fingerprinting

RNAaseT1 cleavage

Subtypes Applicable to RNA; Detects point mutations

Technical electrophoresis method; Med. $

RNase protection assay

Probe specificity

Subtypes Applicable to RNA viruses & other RNA

Technical; Uses radioactive probe

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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 pathogensPros: 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)

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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.

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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 hybridizationOR:• 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 (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 fragments2. CVB3 , 145 bp3. negative control4. HAV, 244 bp5.negative control6. CVB3 and HAV7.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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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

Reverse transcribe

Polymerase Chain Reaction Amplification (PCR)

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

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

• 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

In Out

CellReceptor

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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

: Virus Particle

: sCAR

Culture + media; :sCAR produced

(RT-) PCR

sCARpurification

: Blocking protein

Amine Terminated Support Magnetic Bead : BioSpheres(Biosource)Pre-coated to provide available amine groups for covalent couplingof proteins or other ligands by glutaraldehyde-mediated coupling method

Covalent coupling to paramagnetic beads

Blocking post-coupling

Sample containing viruses

NA extraction

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Ligand capture: capture of CVB3 with magnetic beads coupled with purified sCARBead control : Reaction of CVB3 with BSA coated magnetic bead

Ligand Capture of CVB3 Followed by RT-PCR(Magnetic Bead-sCAR-CVB3)

Magnetic Bead : BioSpheres (Amine Terminated Support)Viral RNA extraction: QIAamp kit

200bp

110100103SM 0.1 PFU100103 +

Ligand capture

Bead control

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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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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