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Phytophthora ramorum (sudden oak death) ramorum
leaf blight and ramorum dieback.
Since the early 1990s, oaks and tanoaks have been
dying in the coastal counties of California. Throughongoing surveys APHIS -PPQ continues to define the
extent of the pathogens distribution in the U S and
limit its artificial spread beyond infected areas
through quarantine and a public education program.
Regulations were published February 14 2002, to
control the artificial movement of sudden oak death
from infested counties in California and an area
under eradication in Oregon. Research service, US
forest service, university and others is under way to
better identify hosts, methods of detection, and
effective treatments. There are no chemical
treatments currently available to eliminate the
disease in nursery stock.
RISK ANALYSIS FOR P. RAMORUM:
Risk associated with the importation of plants.
Risk associated with domestic movement of the
pathogen through plants, plant products, soil, other
growing media, compost and water.
Mitigation measures to prevent the movement and
spread of P. ramorum to non- infested areas in the
United States.
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The risk presented by P. ramorum is high and based
on six elements: climate- host, interaction, host
range, dispersal potential, economic impact,
environmental impact, and pest opportunity.LABORATORY GUIDE.
Designed to outline basic components (facility
equiptment, and standard procedures) needed to
process and report sample results using the current
USDA approved protocols for P. ramorum diagnostics.
CONTAMINATION OF SAMPLES.
Cross contamination of samples can occur in either
ELISA, culture ID, or nested PCR from the initial
point of contact when the sampling takes place,
because the components being detected are
different for each test steps that prevent cross
contamination for one assay. Each diagnostic stage
must be physically separated from previous stage toreduce the risk of contamination.
SAMPLING PROCEDURES.
As per USDA -APHIS -PPQ Trace forward protocol
includes how the information that will be recorded
for each sample, how the sample will be stored prior
to shipment, and how the sample will be shipped.All diagnostic lab personnel will be familiar with the
sampling procedure so that they are aware of
treatment of the sample prior to its arrival in the
lab. The sampling will usually performed by trained
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field inspectors. Personnel involved in field collections
will never enter the diagnostic testing laboratory
because of potential P. ramorum contaminants on
clothes, shoes, hair and other personal items. At thepoint of shipment reception, a log will note the time
of arrival, who the package was from, who received
the package, and the shipper with the tracking
number. The parcel will remain unopened and
secured in a cool, dry place away from direct
sunlight, and a phone call made to a trained
member of the diagnostic testing lab to receive the
package to continue the chain of custody. The
trained lab personnel will take the parcel directly to
the lab for processing. The package will be opened
according to the procedures outlined in the state
and federal pest permit for p. ramorum, and will
include as a minimum that the samples will remain
isolated from the natural environment outside the lab
facility until a final disposition is reached. At the end
of the testing and once the final disposition is
reached, the samples and packaging will be disposed
of appropriately according to permit conditions and
will include autoclave destruction of the plant
samples and all packaging materials.
BASIC INFRASTRUCTURE REQUIREMENTS: The rooms needed for diagnosis are:
A receiving room that will contain a class 11, Type
A Bio- Safety Cabinet.
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Storage room or dedicated refrigerator (A) with the
capability of being locked or not available to the
public.
Sample processing room.
DNA preparation / sample extraction room.
PCR preparation room or PCR workstation.
Small area for the PCR machines physically
separated from the PCR set up area.
Separate room for PCR gel analysis.
PCR machines and PCR products WILL NEVER enter
the area where the samples are taken, prepare, or
where PCR reaction components are diluted or
distributed. P. ramorum culturing at the same time
lab would have at least one more room with a
transfer hood for isolation and growth of the
organism. All of the rooms will be ventilated andphysically separated enough to prevent airflow from
one area to another. Changing lab coats will be
highly implemented.
GENERAL CONSIDERATIONS FOR THE TESTING
LABORATORY:
Organization and log in of samples.
Following delivery, the diagnostic laboratory will
immediately notified and trained lab personal sent to
claim the package and take it to the sample
receiving / log - in room of the lab. The package
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will be open according to permit instructing cabinet.
The reasoning behind opening parcel in a separate
room is to minimize the chance of contaminating
uninfected samples from other shipments, if aninfected sample container is leaking or broken the
package contents will be examined to determine if
the appropriate paperwork accompanies each sample.
Sample numbering and paper work should match
exactly. Samples that are degraded or in poor
condition, or package with broken or leaky sample
bags will be rejected and disposed of by autoclaving
immediately. The sample receiving room will be
clean with adequate bench space for organization of
samples and paperwork.
STORAGE OF SAMPLES:
After samples are determined to contain the
appropriate paperwork and are in good condition, the
appropriate trained lab personnel are now
responsible for the chain of custody of the sample
that began at sample reception and will conclude
with reporting test results and destruction of the
sample and packaging materials. The sample will be
processed immediately or packaged for storage in a
refrigerator or a cold room at about 4 degree cent
that is not used to store cultures of P. ramorum.
INITIAL PROCESSING:
The initial processing will be conducted in a room
separate from both the initial examination and the
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following extraction and PCR analysis procedures. A
Type 11, Class A Bio - safety cabinet will be used
for this purpose. The hood will be properly prepared
before and after sampling by wiping out any residuewith a 10% household bleach solution or 70% ETOH
followed by running the UV sterilization lamps for
fifteen minutes. Medical quality matting paper will be
laid down in the hood and samples are cut in weigh
boats changed between each sample. Gloves will be
worn to select leaves and cut samples and changed
between each sample to avoid contaminating the
next sample.
ELISA:
ELISA sub samples should be selected and prepared
as directed in the ELISA protocol on the USDA
-APHIS -PPQ website.
DNA EXTRACTION:Only ELISA positive samples will be further tested
by PCR. Further testing of ELISA positives require
sorting and sampling of the previously generated sub
samples for DNA extraction The recommended
method for DNA extraction using the USDA - APHIS -
PPQ protocol is the Qlagen D Neasy plant mini kit,
although another more labor intensive protocol isalso currently available. Samples in 100mg aliquots
placed in 1.5 ml microcentrifuge tubes are now
ready for DNA extraction. This procedure should take
place in a clean lab, a standard molecular biology
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equipped lab not exposed to plant material or
microbial cultures equipment needed for for DNA
extractions should include : a full set of standard
micropipettes with appropriate aerosol resistantbarriers tips, autoclaved microcentrifuge tubes,
microcentrifuge, liquid-nitrogen, mini- bead beater or
appropriate tissue maceration system, sterile distilled
water, reagent grade ethanol ice bucket, refrigerator
and freezer. Always use microfuge tube openers to
avoid direct contact with the top rim of the
microfuge tubes. It is a good practice to store plant
samples or extracts in a separate freezer
compartment from PCR reaction components. If leaf
samples are contaminated with soil rinse them in
sterile water and pat- dry them with a hand towel
before sampling. Gloves will be worn and changed
often particularly between different segments of the
DNA extraction procedures. Disposable lab mats will
be used to cover bench areas and changed between
materials culture. Plates, or soil in an autoclave used
to sterilize buffers, glass-ware, or plastic- ware used
in the SOD DNA extraction because of potential
contamination from aerosols within the auto clave.
All racks, tubes openers, and other plastic materials
used in the procedure will be decontaminated
between each set of extractions by soaking in a10% bleach solution for 30 minutes followed by two
rinses with water to remove the bleach solution.
Bench areas, pipettes, centrifuge rotors, lab chairs,
drawer handles, and other knobs, etc in the
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environment of the bench used to do DNA
extractions will be wiped down every couple of days
with a DNA elimination solution such as DNA Away.
PCR SET - UP
The PCR will be done in a separate room than the
DNA extraction, especially if aerosol controlled
microcetrifuges are not used. Minimum requirements
are that the PCR will be set up on a lab bench that
has been cleaned and the bench paper replaced if it
also used for DNA extraction. Another option will be
to use clean bench top PCR enclosure cabinets. In
addition, it is also essential that are not used for
DNA extraction be used to set up the PCR. All
preparation of stock solutions will occur as far away
from any samples as possible preferably in a laminar
flow hood. Adding DNA templates to the PCR
reactions will be done outside the clean PCR area
using a dedicated pipette not pipettes used for
making and aliquotting the PCR mix or involved with
post PCR analysis.
GENERAL LIST OF EQUIPTMENT / PERSONNEL
NEEDEDE:
INITIAL RECEIPT AT LABORATORY:
Appropriate storage.
Office personnel ( aware of deliveries and informs
staff )
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ORGANIZATION AND LOG - IN:
Trained member of the diagnostic lab.Dry lab.
Disinfection supplies.
STORAGE:
Cold room or dedicated refrigerator (A).
SUB SAMPLING:
Isolated room with bio- safety cabinet.
Disinfection and implement flame source disposable
gloves, kim wipes.
Sterile forceps, single edge razor or scalpel
laboratory personnel in sterile techniques with
additional training in avoiding DNA contamination.
ELISA:
Standard wet lab separate grinding and assay areas.
Plant tissue maceration system, (appropriate cleaning
of system between samples if necessary).
ELISA plates washing supplies, ELISA plate reader.
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Agdia kit.
Laboratory personnel trained in ELISA.
DNA EXTRACTION :
Isolated clean lab set up for molecular biology.
Standard set of calibrated micropipettes,
microcentrifuge, source of liquid nitrogen, plant tissue
maceration system, disposal aerosol barrier tips,
sterile microcentrifuge tubes, reagent grade ethanol,Qiagen Dneasy plant mini kit, dis. Gloves, lab coat,
decontamination supplies.
Laboratory personnel proficient in molecular biology (
with additional specific training for SOD)
POL YMERASE CHAIN REACTION:
Molecular biology lab away from DNA extraction area
( this could be a separate lab bench, but ideally a
separate room).
Standard set of calibrated micropipettes (different
than ones used for DNA extraction ) disposal aerosol
barrier tips, sterile microcentrifuge tubes, thin wallPCR tubes, disp gloves, lab coat, decontamination
supplies.
Appropriate thermocycler.
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Laboratory personnel proficient in molecular biology
(with additional specific training for SOD )
GEL ELETROPHORESIS AND DOCUMENTATION:
Can be part of molecular biology lab or photographic
dark room.
Standard or commercial gel electrophoresis
equipment and power supply.
Gel documentation or photography equipment.
Laboratory personnel proficient in DNA extraction and
Trained and authorized laboratory personnel to read
results and record determinations.
CURRICULUM VITAE:
SAM JEFFERSON
UNDER- GRADUATE: UNIVERSITY OF GEORGIA.
Comprehensive major in Biology B. S (HONORS)
GRADUATE: Valdosta State University.
MAJOR: Biological science.
HONORS AND FELLOWSHIP
University Fellowship, University Of Georgia.
Sears Longwell Award.
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N S F National Needs Postdoctoral Award.
RESEARCH EXPERIENCE:
POSTDOCTORAL: Alabama State University.Burns Institution Of Washington.
Research Scientist, International Plant Research
Institute.
Research Geneticist, USDA- ARS, Plant Gene
Expression Center.
NELSON MILLS.
EDUCATION: University Of Wales P h D.
Department Of Integrative Biology.
North Arizona University. B, A
MAJOR: Biology science.
RESEARCH EXPERIENCE:
Post Doctoral Scholar, Flint Agroecology laboratory.
Doctoral dissertation research, university of North
Carolina.
Graduate student research assistant, university of
North Carolina.
Under-graduate honors thesis, West Brown College.
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PETER GABRIAL:
EDUCATION: Ph. D (Biology), Texas southernUniversity.
M.S (Biology ). Texas southern
University.
B.S (Biology) Texas Southern
University.
EXPERIENCE: Assistant professor University of Boston.
Research Mycologist USDA-
Agricultural Research Services, Systematic Botany and
Mycology laboratory. Houston.
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