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