advancement of pcr testing for the san luis...
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Advancement of PCR Testing for the San Luis Valley
Greg HessDisease Lab Supervisor
Potato Certification Service
Colorado State University
0249 E. Rd. 9N.
Center, CO 8114
Advancement of PCR Testing for the San Luis Valley
Advancement of PCR Testing for the San Luis Valley
PYYn SURVEY FOR COMMERCIAL GROWERSTo gather more information regarding the extent of PVYn in the San Luis valley the CPAC committee is working with CCPGA, and PCS to conduct a PVYn survey in commercial potato fields this summer. CPAC and CCPGA are providing ¾ of the cost for this testing program. The cost to participate for growers is $2.50/acre. This survey is completely confidential and individual grower results will be kept anonymous.
Main focus will be to
- establish a Polymerase Chain Reaction (PCR) testing facility for our potato industry
- develop efficient and effective molecular testing protocols for various pathogens
Expected outcomes of PCR disease testing include:
1. Improved diagnostic tests to differentiate the PVY strains.
2. Comparing current methods to those newly developed, provide alternatives to current post harvest test methods.
3. Knowledge of varieties' susceptibility to potato tuber necrotic ringspotdisease caused by the invasive necrotic and recombinant strains of PVY.
4. Establishment of regional tests to evaluate PVY strain susceptibility and symptom expression in varieties and advanced breeding lines.
Advancement of PCR Testing for the San Luis Valley
Recent Timeline:
- Greg Hess hired and setting up PCR applications Sep 2014
- PCR reagents and standard PCR purchase/validate Oct 2014
- Visit Karasev Virology Lab, Moscow, Univ. Idaho Nov 2014
- Validation of pathogen tests (std PCR) Nov - Dec
- SCRI grant approved and processed Dec 2014
- qPCR apparatus quote & invoices submitted Dec 2014
- qPCR apparatus shipped and received Jan 2015
Colorado Potato Certification ServiceDisease Testing:
I. SerologicalEnzyme-linked immunosorbent assay (ELISA) Detection of pathogens using ELISA relies on the interaction between the antigen and specific antibodies.
II. Molecular
Polymerase Chain Reaction (PCR) The polymerase chain reaction is
molecular biology used to amplify a single copy or a few copies of a piece of DNAgenerating thousands to millions of copies of a particular DNA sequence.
Background
DNA
RNA
During a RT-PCR specific target RNA sequences are amplified exponentially into DNA copies.
Advancement of PCR Testing for the San Luis Valley
Cycle Copies
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
Advancement of PCR Testing for the San Luis Valley
1/10th dilution soakate samples (CMS cores: asymptomatic cores)
dil
Ag+
1:1
49
1:1
24
1:9
9
2:9
8
3:9
7
4:9
6
5:9
5
6:9
4
7:9
3
8:9
2
9:9
1
10
:90
Advancement of PCR Testing for the San Luis Valley
Colorado Potato Certification ServiceDisease Testing:
qPCR (quantitatative, also called real-time
A Real-Time PCR Assay for the Detection of Clavibacter michiganensis subsp. sepedonicusBased on the Cellulase A Gene Sequence Neil C. Gudmestad, Ipsita Mallik, Julie S. Pasche, Nolan R. Anderson, and Kasia Kinzer
Advancement of PCR Testing for the San Luis Valley
I. Serological
Enzyme-linked immunosorbent assay (ELISA) Detection of pathogens using
interaction between the antigen and specific antibodies.
Teresa Almeida
Advancement of PCR Testing for the San Luis Valley
Advancement of PCR Testing for the San Luis Valley
Chikh-Ali
Putative
RT-PCR
typing
PVYNP N N P N P PVYN
PVY-NE11N N N P N N PVY-NE11
PVYNA-NN N N N N P PVYNA-N
PVYZNTNN P N P N P PVYNTN
PVYNTNN P N P N P PVYNTN
PVYEN P N P N N PVYE
PVYN:ON P N P P N PVYN:O
PVYN-WiN P N N P N PVYN-Wi
PVYO-O5N N P N P N PVYO
PVYON N P N P N PVYO
Band Size in Base Pairs398 441 532 633 853 1,307
PVY multi-plex IC-RT-PCR Key
PVY positive tubers’ cores sampled directly under sprouts, gradient with asymptomatic cores IC-multi-plex PCR
Cold cores’ extract of tubers ELISA-PVY positive when testing sprouts. Strain Key (bp): N= 398, 633, 1307bp; NTN= 441, 633, 1307bp; N:O= 441, 633, 853bp; N-Wi= 441, 853bp; O= 532, 853bp; and NE11= 633. [Δ = liq.N, iScript enzyme, cold homogenize]
-853bp-633bp-532bp-441bp
-853bp---441bp
-853bp
-532bp
1037bp
-633bp-441bp
PVYN-Wi
PVYNTN
PVYO
Temporary Lab Setup:
Plant Virology Protocols: New Approaches to Detect Viruses and Host Responses 3rd Ed., Hokkaido Univ.,Sapporo, Japan 2015
Advancement of PCR Testing for the San Luis Valley
Colorado Potato Certification ServiceOutlook:
PCR TEST
Cost/400
tubers
When
offered?
CMS $125 Feb, 2015
PVY (sprouted) $520 Mar, 2015
PVY (Unsprout) TBD TBD
Other Path TBD Mar, 2015
Colorado Potato Certification ServiceOutlook:
PCR SUMMARYSecure electric and temp lab space (mobile-office) Feb 2015Vendor install and training qPCR, onsite at AES Feb 2015qPCR Lab operational Mar 2015Offer CMS/BRR PCR (Idaho) 1 week test NOW!Potential offer tuber core PVY PCR test (need equip) Mar 2015 Offer spectrum of tests to industry/commercial stakeholders SpringLate-blight, Mop Top, and others’ tests SummerOffer regular and early-kill post-harvest tuber PVY testing FallProvide broad survey for San Luis Valley
Thank You!
Please offer input and feedback