Aerobiology

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Aerobiology

• Microbiology of the Atmosphere

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Aerobiology

• The interdisciplinary science that deals with the
  movement and dispersal of bioaerosols
• The movement of bioaerosols is generally passive
  and is greatly influenced by the environment
• The survival of viable bioaerosols is also
  dependent on the environmental conditions

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Bioaerosols

• Biological agents carried in the air as large
  molecules, volatile compounds, single particles,
  or clusters of particles that are living or were
  released from a living organism
• Particles sizes - 0.5?m to 100 ?m
• Capable of eliciting diseases that may be
  infectious, allergic, or toxigenic with the
  conditions being acute or chronic

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Bioaerosols in Our Environment

• Outdoor Sources
• Fungal Spores
• Pollen
• Bacteria

• Indoor Sources
• Viruses
• Bacteria
• Fungal Spores
• Dust mites
• Cockroaches
• Animal Dander - especially cats

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Outdoor Aerobiology

• Atmosphere
• Transport
• Fungal Spores
• Pollen

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Spores, Pollen and Gravity

• In still air spores (and pollen) fall to the
  ground at a rate (based on Stokes law) that is
  proportional to the square of its radius
• i.e. the bigger the spore the faster it will
  fall
• Aerodynamics also influenced by
• non-spherical shape or irregular shape
• ornamentation and aggregation
• these increase drag and delay deposition

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Stokes Law
2 s - r
V
gr2
9 m
V terminal velocity in cm/s s density of
sphere r density of air g acceleration due to
gravity m viscosity of air )1.8 x 10-4
g/cm/sec r radius of sphere
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0.5 - 14.0
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Settling rates of particles
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Airborne Transport
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The Aerobiological Pathway
Dispersal
Deposition
Take-off
Effects of environmental factors
Source
Impact
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Airborne Transport

• Perfectly still air seldom occurs
• Prevailing air currents delay deposition by
  gravity
• Air flow is complex allowing bioaerosols to be
  transported over short ranges to global distances
• Transport occurs in the turbulent layer of the
  atmosphere

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Turbulent Layer of Atmosphere

• Air movement shifting and unpredictable
• Depends on wind speed, direction, temperature,
  and local eddies caused by roughness of the
  terrain
• Fungal spores are a normal component of the
  turbulent layer possibly up to 200,000 spores/m3
  of air however, pollen levels normally two
  orders of magnitude lower

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Long distance transport

• Well documented in palynology literature as well
  as in aerobiology literature
• Typically these are reports of one time
  incursions of exotic pollen types or preseason
  pollen
• Exotic pollen in sediments in Canada
• Preseason birch pollen in Scandinavian countries
  from southern areas in Europe

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Well studied examples of long distance transport

• Wheat rust spores Starting in the 1920s the
  movement of Puccinia graminis spores has been
  demonstrated
• Tobacco blue mold Since late 1980s, the
  movement of Peronospora tabaccina spores has been
  followed
• Mountain cedar pollen My lab has been following
  the LDT since 1980
• African dust and associated microorganisms have
  been studied over past decade

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Juniperus ashei pollen

• Juniperus ashei (mountain cedar) has a limited
  distribution
• Arbuckle Mountains of Oklahoma
• Edwards Plateau of Texas
• Scattered areas of Ozarks
• Highly allergenic (cedar fever in Texas)
• Tulsa allergist reported 15 patient sensitivity
• Mid-winter pollination (Dec. and Jan.)
• Tulsa pollen season Feb through early Nov
• Evidence of pollen in Tulsa atmosphere
• Cedar pollen detected since 1980

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Distribution of Juniperus ashei
Juniperus ashei grows amongst the dissected
slopes of Cretaceous limestone of the Edwards
Plateau. The species co-occurs with J.
virginiana to the east and J. pinchotii other
Juniperus spp. to the west.
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Incursion of J. ashei pollen into Tulsa

• Each winter since 1980 J. ashei pollen has been
  registered by our Tulsa air samplers
• Pollen recorded on 20 to 60 of the days in Dec
  and Jan
• Concentrations typically low however very high
  concentrations have been registered on several
  occasions (based on NAB level of very high gt1500
  pollen grains/m3)

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Mountain Cedar Forecasting

• Started in Dec 1998
• Daily forecasts in Dec and Jan
• Forecasts posted on internet at
  http//pollen.utulsa.edu
• During past 6 season 812 forecasts issued
• Three sites in Texas Austin, Junction, San
  Angelo
• One site in Oklahoma Arbuckle Mountains
• One site in Arkansas Ozark Mountains (5 yrs)

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Forecast Components

• Release forecast based on meteorological
  conditions and phenology of the plant
• Temperatures above 45o F
• R.H. below 50
• Sunshine
• No rain in previous 24 hours
• Pollen cone maturity
• Downwind forecast based on model projections
  using
• HY-SPLIT model trajectories
• Meteorological conditions along the path

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1998-2004 Tulsa Cedar Profile

• During Dec and Jan of the 6 years cedar pollen
  present on 233 days
• Most days low levels
• Over 50 days moderate to very high levels
• 12 days with high levels
• One day had very high level (2,109 pollen/m3)
• Trajectories crossed Tulsa gt150 times with the
  majority of trajectories from the Arbuckle Mts.

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London, Ontario

• Air sampling data from Jim Anderson from London,
  Ontario
• Juniperus pollen recorded 22 times in Dec and Jan
  from 1998-2004
• Maximum was 58 pollen grains/m3

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Continental transport

• 27 Jan 99, Jim Anderson in London, Ontario
  reported atmospheric Juniperus pollen - 58 pollen
  grains/m3
• Trajectories show that the source of this pollen
  was Texas population of Juniperus ashei
• Our Jan 26 forecast indicated that the pollen
  has the potential to travel very long distances.

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Fungal Bioaerosols
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THE FUNGI
Kingdom Protista
Div Myxomycota Div Oomycota
Kingdom Eumycota
Div Chytridiomycota Div
Zygomycota Div
Ascomycota Div
Basidiomycota
Asexual Fungi
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FUNGAL SPORES

• Majority of spore types adapted for airborne
  dispersal
• Spores unicellular to multicellular from 1 to 100
  ?m
• Main sources in the environment
• Leaf surfaces (phylloplane fungi)
• Soil

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Spore Release Mechanisms

• Passive
• Wind - generally the most abundant airborne
  spores in the atmosphere - include members of the
  Dry Air Spora which peak in afternoon
• Rain - rain splash as well as tap and puff
• Active
• Generally require moisture
• Common mechanism for ascospores and basidiospores

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Wind Dispersed Spores

• Frequently related to wind speed and turbulence
• Typically borne on erect conidiophores or
  sporangiophores that elevate spores above the
  substrate
• Dry Air Spora Cladosporium, Alternaria,
  Epicoccum, Drechslera, Pithomyces, Curvularia,
  smut spores

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Dry Air Spora
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Diurnal Rhythm of Cladosporium
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Cladosporium spores peak hourly concentration of
gt120,000 spores/m3 during a spore plume
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Cladosporium

• Common fungal genus occurring both indoors and
  outdoors
• Most abundant outdoor spore type with a worldwide
  distribution
• Normally exists as a saprobe or weak plant
  pathogen
• Spores are known to be allergenic

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Cladosporium spp.
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Passive Discharge during Rain

• Vibration and shaking as raindrops hit leaf
• May explain increases in Cladosporium during
  rainfall
• Release of basidiospores by puffballs shows
  similar puffing when raindrop strikes mature
  fruiting body

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Rain Splash

• Generally spores surrounded by mucilage
• Mucilage protects from desiccation but also
  prevent dispersal by wind
• First raindrops dissolve mucilage
• Resulting spore suspension dispersed by
  subsequent raindrops
• Spores commonly thin, colorless, elongate

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Active Discharge - Ascospores

• Hygroscopic material within ascus absorbs
  moisture
• Ascus swells and develops high osmotic pressure
• Pressure causes ascus to burst, explosively
  shooting spores into the atmosphere
• Need for rainfall will often override diurnal
  rhythm of release

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• Basidiospore discharge from basidium
• Moisture condense around crystal of manitol
• Bullers drop enlarges
• Fuses with a film of water around spore
• Shifts center of gravity

• Result of discharge
• Spores shot only a fraction of a millimeter
• Spores freed from gill or pore and able to fall
  free and reach turbulent layer

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Basidiospore Rhythm

• Need for moisture confines spore release to
  periods of high humidity
• Typically peak levels in pre-dawn hours and low
  levels in afternoon

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Airborne Fungal Spore Concentrations in Tulsa
2002
2002
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23 Taxa identified Cladosporium, Ascospores,
Basidiospores, and Alternaria Comprised 90 of
Total
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Indoor Aerobiology

• Outdoor spores enter readily
• Many fungi can also amplify indoors anytime
  moisture is available fungi can grow on many
  indoor substrates
• Penicillium, Aspergillus, and Cladosporium are
  most common indoors
• Many can form mycotoxins Aspergillus spp and
  Stachybotrys

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Environmental factors that influence indoor
fungal contamination

• Outdoor concentration and type
• Type and rate of ventilation
• Activity levels
• Indoor moisture levels
• Modern building materials

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Typical Yearly Spore Levels 1998
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Spore plumes show the influence of environmental
conditions
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Water Leak
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Humidity

• Indoor relative humidity
• Below 30 R.H. no mold growth
• Above 70 optimal for mold
• Usually mold growth can occur above 50
• Humid air condenses on cool surfaces
• Cold windows in winter - molding and sills become
  wet and suitable for fungal growth
• Cold floors in winter
• Cooling coils in AC units in summer
• Humid air allows hygroscopic materials to absorb
  water

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High humidity in home where subslab ducts failed
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Moisture Problems

• Become worse in past 20 - 30 yrs
• increased use of washing machines, dishwashers
• vaporizers and humidifiers actively spray
  droplets into the air (often contaminated)
• tighter buildings for energy conservation trap
  moisture
• Anytime moisture available fungi will grow

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Cladosporium one of the most common indoor fungi
growing on a diffuser
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Penicillium

• One of the most common soil fungi in natural
  environment
• Over 250 species
• Well known allergen
• Some species produce mycotoxins
• Some species produce antibiotics
• Produce VOCs

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Penicillium in culture
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Aspergillus

• Also common soil fungi
• Cause rot of stored grain
• Over 150 species
• Well known allergens
• Several species form mycotoxins
• Some species can grow at high temperatures
• Several species cause infections in lung,
  sinuses, and hypersensitivity pneumonitis

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Aspergillus niger
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Aspergillus fumigatus
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Penicillium and Aspergillus

• Small spores passively aerosolized when spore
  clusters disturbed
• Spores extremely buoyant, remain airborne for
  extended time
• Penicillium and Aspergillus spores look alike
  distinguished in culture

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Stachybotrys chartarum

• Soil fungus in nature
• Commonly found indoors on wet materials
  containing cellulose, such as wallboard, jute,
  wicker, straw baskets, and paper materials
• Spores in slimy mass
• Thought to be allergenic although little is known
• May produce potent mycotoxins

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Indoor air sample
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Air Sampling Techniques
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Principal Collection Methods

• Gravity
• Impaction
• Impingement
• Filtration

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Gravity

• Simplest but least accurate method of collecting
  airborne biological samples
• Coated microscope slide or open petri dish
  containing agar exposed to atmosphere
• Non quantitative for atmospheric concentrations
• Affected by particle size and shape and air
  movement
• Biased deposition of large particles

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IMPACTION SAMPLERS

• Separate particles from the air stream by using
  inertia of particles
• Forces deposition onto a solid or agar surface
• Most commonly used method
• Instruments available for culturable sampling and
  total spore sampling

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Impaction SamplersTotal Spore Samplers

• Outdoor Samplers
• Hirst Spore Trap Burkard and others
• Rotorod Sampler
• Samplair MK-3 Sampler
• Indoor Samplers
• Burkard Continuous Recording Air Sampler
• Samplair MK-3 Sampler
• Burkard Personal Volumetric Sampler
• Air-O-Cell Sampler
• Cyclex-D Sampler

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Burkard Spore Trap
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Advantages of Burkard Spore Trap

• High efficiency down to less than 5 mm
• Allows for accuracy for small fungal spores such
  as basidiospores and small ascospores
• Time discrimination
• Permits analysis for diurnal rhythms
• Permanent slides for future reference

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Burkard 7-day sampler head

• Standard is the 7-day sampling head
• Sampler drum mounted on 7-day clock
• Drum moves by orifice at 2 mm per hr
• Melenex tape mounted on drum and greased
  (Lubriseal, High Vacuum Grease, other)
• Air is brought in at 10 l/min and impacts on
  greased Melenex tape
• Drum changed each week

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Seven Day Sampling Head
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Processing the 7-day drum

• Melenex tape removed from drum
• Tape cut into seven 24 hour segments each 48 mm
  long
• Segments mounted on microscope slides in 10
  gelvatol (polyvinyl alcohol) and dried
• Glycerin-jelly mounting medium added and a 50 mm
  cover slip
• Mounting medium contains pollen stain - either
  basic fuchsin or phenosafarin

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Melenex tape on cutting board
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24 hour sampling head
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Outdoor Air Sample from Burkard Spore Trap
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Rotorod Samplers
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Rotorod Samplers

• Models most often used have retracting rods for
  intermittent operation (10 sampling time
  typical)
• Head rotates at 2400 rpm
• Leading edge of rod coated with grease
• Pollen and spores impacted on greased surface
• Efficient for pollen and spores gt10 mm

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Rotorod Sampler
Rods spin at 2400 rpm. Particles (spores and
pollen) are impacted on leading face of the rod
which is greased. Efficiency decreases
dramatically below 10 mm. Intermittent head spins
10 of time.
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Allergenco Samplair
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Continuous Recording Burkard
24 hour sampling onto a coated microscope slide
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Burkard Personal Sampler
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Air-O-Cell Sampler
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Air-O-Cell Slide
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Cyclex-d
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Andersen (N-6) Single Stage Sampler
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Andersen 6-Stage and 2-Stage Impactors
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Biocassette Sampler
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Burkard Portable Air Sampler for Agar Plates
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Inpingement Samplers

• Separate particles from the air stream by using
  inertia of particles
• Forces deposition into liquid collection medium
  (usually a dilute buffer)
• Aggregates of cells can be broken apart
• Allows for several possible analytic
  applications culture, microscopy, biochemistry,
  immunoassays, PCR

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Impingers
Burkard Multi-stage
BioSampler
AGI-30
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Filtration

• Separates particles from airstream by passage
  through a porous substrate, usually a membrane
  filter
• Collection depends on filter pore size and flow
  rate
• Loss of viability may occur due to desiccation
• Adaptable for a variety of assays

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Sampler Performance

• Collection efficiency is the ability to capture
  the particles onto the collection medium
• Physical characteristics of the inlet and the
  airflow rate used to calculate d50
• Particle diameter at which 50 of particles are
  collected
• Because of sharp cut-off it is generally accepted
  that all particles above this size are collected

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d50 values for some samplers (CUT SIZE)
Buttner MP, Willeke K, Grinshpun SA. Sampling and
Analysis of Airborne Microorganisms. In Manual
of Environmental Microbiology 2nd ed. ASM Press,
Washington, 2002.
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Analytic Methods

• Microscopy
• Culture
• Biochemistry
• Molecular Biology

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Microscopy

• Identification of total spores (both culturable
  and non-culturable) along with pollen and other
  particulates
• Identification to species level usually not
  possible
• Identification of morphologically similar spores
  not possible

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Culture

• Only viable bacteria and fungi
• Limited to those taxa able to grow in culture on
  medium used
• Success based on medium and incubation time and
  temperature
• Permits speciation when required
• Results expressed as CFU/m3

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Biochemistry

• Detection of specific compounds
• May not be specific for a genus or species
• Assay examples
• Ergosterol
• B 1,3-glucan
• Endotoxins
• Mycotoxins Stachybotrys toxins and ochratoxin
• Various types of assays such as HPLC

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Immunochemistry

• Specific allergen molecules
• Not useful for routine air sampling
• Typically used for filter samples and impinger
  samples but have been used for spore trap samples
• Requires prior development of an antibody
• Widely used for dust mite, cockroach, cat, etc.
• Few fungal assays available

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Molecular Biology

• Detection of specific genetic elements
• Highly specific and sensitive
• Currently restricted to a few organisms