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VIRNPP0001.02

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United States Department of Agriculture 

Center for Veterinary Biologics 

Nucleic Acid Production Protocol 

Production of Chicken Anemia Virus (CAV) DNA Positive Control

Date: 

Number: 

Supersedes: 

Contact: 

September 1, 2015 

VIRNPP0001.02 

VIRNPP0001.01, July 13, 2011 

Sandra K. Conrad, (515) 337-7200 

Approvals: 

/s/Geetha B. Srinivas Date: 02Oct15 

Geetha B. Srinivas, Section Leader 

Virology 

United States Department of Agriculture 

Animal and Plant Health Inspection Service 

P. O. Box 844 

Ames, IA 50010 

INTERNAL USE ONLY 

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.

Entered into CVB Quality Management System

by: /s/Linda S. Snavely 08Oct15 

Linda S. Snavely Date

Quality Management Program Assistant
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for Production

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

4. Production of Reagent

4.1 DNA extraction

4.2 Amplification of CAV DNA

4.3 Analysis of amplified CAV DNA

4.4 PCR purification

5. Preparation and Packaging of the Product

5.1 Determination of dilution of DNA to use for reagent

5.2 Bottling and filling

6. Testing

6.1 Sequencing

6.2 Final reagent inhibition study

7. Summary of Revisions
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1. Introduction

This Nucleic Acid Production Protocol (NPP) describes the production of the CAV DNA control 

used for CAV extraneous agent testing using PCR analysis. 

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Laminar Flow Biological Safety Cabinets (BSCs) (NuAire Inc., Labgard)

Note: Use good laboratory practices to perform the DNA template 

preparation of the CAV positive control in an area not related to the testing 

of other poultry viruses. Also, BSCs should be designated as “Clean” and 

“Dirty.” The clean BSC is only used to prepare master mix. DNA should only 

be worked with in the dirty BSC.

2.1.2 Thermocycler (Applied Biosystems, GeneAmp PCR System 9700 or 

Veriti 96-well thermocycler)

2.1.3 Thermo EC Electrophoretic gel system

2.1.4 Eppendorf Adjustable Volume Research micro-pipettes, 2.5-μL, 10-μL, 

20-μL, 100-μL, 200-μL, 1000-μL

Note: Separate micro-pipettes are assigned to the individual BSCs and 

should not be interchanged between designated hoods to avoid 

contamination.

2.1.5 Pipette-aid automatic pipettor (Drummond Scientific)

2.1.6 Microcentrifuge (Eppendorf 5415 C)

2.1.7 Gel imaging system (Carestream, Gel Logic 2200)

2.1.8 Micro-spectrophotometer (Thermo, Nanodrop 2000)

2.1.9 Automated 250-μL 12 channel pipette (Matrix, Catalog # 2012)

2.1.10 8-channel screw cap decapper (Thermo, Catalog # 4105MAT)
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2.2.11 500μL screw top tubes and rack, (Matrix, Catalog # 3744)

2.2.12 Qiacube Instrument (Qiagen, Catolog # 9001292) – optional, can be used 

for extraction.

2.2 Reagents/supplies

Equivalent reagents may be substituted for any brand name listed below.

2.2.1 DNeasy Blood and Tissue Kit (Qiagen, Catalog # 6504)

2.2.2 CAV virus, Del-Ross strain, Lot 9-2-88, harvested and diluted 1:100

2.2.3 HotStar Taq Master Mix Kit (Qiagen, Catalog # 203443)

Note: Although a different brand name of Taq polymerase can be used, it 

does need to be a hot start enzyme.

2.2.4 CAV primers, cav-1 and cav-2 at approximately 50 pmol/μL each

Reconstitution of the primers is determined by the synthesis data sheet. The 

primer batch yield is recorded on the sheet. Convert the nmol amount to 

pmol by multiplying the amount in nmols by 1000 and dividing the result by 

50 to determine amount in microliters of water (PCR grade) to add for a 

final concentration of 50 pmol per 1 μL. 

Primer Sequences:

cav-1, 5’-CTAAGATCTGCAACTGCGGA-3’

cav-2, 5’-CCTTGGAAGCGGATAGTCAT-3’

2.2.5 PBS for HI (National Centers for Animal Health (NCAH), Media #30102)

2.2.6 Ethanol 200 proof molecular grade (Sigma-Aldrich, Product #E7023)

2.2.7 2% E-gel with ethidium bromide (Invitrogen, Catalog # G5018-02)

Caution: Ethidium bromide is a suspected carcinogen. Use gloves when 

handling any reagents containing ethidium bromide. Refer to Material Safety 

Data Sheet (MSDS) and the NCAH Environmental Health and Safety Unit 

guidelines for safe handling, usage, and disposal.

2.2.8 Screw cap glass bottle or flask
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2.2.9 PCRSizer 100 base pair (bp) ladder (Norgen, Catalog # 11400)

2.2.10 PCR reaction tube caps (Applied Biosystems, MicroAmp, 

Part # N8010535)

2.2.11 96-well PCR reaction tube tray/retainer (Applied Biosystems, MicroAmp, 

Part # N8015530)

2.2.12 96-well PCR reaction tube base (Applied Biosystems, MicroAmp, 

Part # N8015531)

2.2.13 Cap Installing Tool (Applied Biosystems, Part # N8010438)

2.2.14 Plastic 96-well Microtiter Plate (Dynatech Laboratories, Catalog # 001-

010-2801)

2.2.15 Aerosol resistant filter tips for micropipettes, various sizes

2.2.16 DNase/RNase Free water appropriate for use in PCR master mix

2.2.17 Latex, vinyl, or nitrile powder free disposable gloves 

2.2.18 1.5-mL centrifuge tubes

2.2.19 Kimwipes

2.2.20 QIAquick PCR Purification Kit (Qiagen, Catalog # 28104)

2.2.21 200-μL snap-cap microcentrifuge tubes

2.2.22 Liquid reservoir (Matrix, Catalog # 8086)

3. Preparation for Production

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques, including the preparation, the proper handling 

and disposal of biological agents, reagents, tissue culture samples, and chemicals. 
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3.2 Preparation of equipment/instrumentation

Operate all equipment/instrumentation according to manufacturers’ instructions and 

monitor for compliance with current corresponding standard operating 

policies/procedures (SOPs). Wear non-powdered disposable gloves. Program the 

thermocycler (see thermocycler manual) with the following steps:

Amplification of CAV DNA

Pre-PCR Heat Start Hold: 5 minutes at 95°C

Cycles 1 - 35: 30 seconds at 95°C

45 seconds at 51.5°C

1 minute at 72°C, with a 1 second increase following each cycle

Post-PCR Hold: 10 minutes at 72°C

Soak/Storage: 4°C

4. Production of Reagent

4.1 DNA extraction

Wear disposable, powder free vinyl, nitrile, or latex gloves for all portions of the test 

procedure. Perform the extraction in a dirty BSC.

4.1.1 Thaw CAV virus suspension. 

4.1.2 Transfer 200 μL of virus suspension to a 1.5-mL centrifuge tube. 

Note: At this point, the Qiacube Instrument (Qiagen, Catolog # 9001292) can 

be used for extraction using the program “CVB DNA Blood and Tissue.”

4.1.3 Continue extraction following the protocol that is provided in the Qiagen 

DNeasy Blood and Tissue Kit, starting with the addition of 20 μL of proteinase K 

to the sample, and continue from there. The Qiagen Kit will use the heating block 

or water bath, the microcentrifuge, and the 200 proof ethanol.

4.1.4 Once the Qiagen purification and extraction is completed, store the tubes 

at -20°C until ready to continue to Section 4.2.
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4.2 Amplification of CAV DNA

4.2.1 In a clean BSC or area, prepare master mix for amplification of CAV 

DNA as follows:

1. Calculate amount of master mix needed by determining total number of 

tubes (reactions) needed plus one extra. 

2. Add the master mix ingredients in the order listed in the following 

section (volumes are for 1 reaction). 

Item μL

HotStar Taq Master Mix 2x 25.0

Nuclease Free Water 11.0

Coral Load 10x 2.0

Primer cav-1 50 pmol/μL 1

Primer cav-2 50 pmol/μL 1

Sample 10

Total Volume 50

Note: For producing CAV DNA reagent, running multiple repeats is 

needed. For the current lot of CAV DNA control, 20 repeated samples 

were amplified by CAV PCR.

4.2.2 Insert PCR reaction tube strips into two rows on the tray/retainer 

assembly. Snap top of assembly to secure strips. Place assembly on base. 

4.2.3 Transfer 40 of the master mix to each reaction tube either in the clean 

hood where the master mix was made, or, before any DNA template is opened, in 

the DNA/RNA template (dirty) BSC.

4.2.4 In the template BSC, add 10 μL of sample template to a PCR reaction 

tube. The total reaction volume will be 50 μL. 

4.2.5 Cap all the tubes. If necessary, use a capping instrument.

4.2.6 Place the tube tray in the thermocycler and close the heated cover.

4.2.7 Run the CAV amplification program (see Section 3.2). Make sure the 

reaction volume is set at 100 μL for the 9700 thermocycler and 50 μL for the 

Veriti thermocycler.

4.2.8 Once the program is completed, store the tubes at 4°C until ready to 

analyze PCR product(s).
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4.3 Analysis of amplified CAV DNA

Wear disposable, powder-free nitrile, vinyl, or latex gloves for all portions of 

analysis.

4.3.1 Loading gel

See the current version of VIRTWS1073, HotStarTaq Plus – PCR Assay, for the

gel worksheet.

1. Connect power base for E-gel to electrical plug.

2. The gel used is a 2% pre-cast agarose gel with 12 wells that hold 20 

μL per well. Remove gel cassette from packaging.

3. Insert gel cassette into base, making sure the gel cassette is pushed all 

the way in and the light at the top turns on.

4. Using a filtered micropipette tip, load 20 μL of the reaction product 

(from Section 4.2) into the 2nd well. Repeat for remaining wells. Leave the 

first well and last well empty. These are loaded with ladder. 

Note: Samples can be pooled, so twenty PCR samples can be pooled 

into two microcentrifuge tubes, and the pooled samples from those 

two tubes the DNA can by analyzed on the E-gel.

5. Record the order that the samples are loaded on the current version of 

the gel lane area on VIRTWS1073.

6. Pipette the 100 bp ladder to the first lane of the gel and to the last lane 

of gel, if needed. Record the addition of the ladder on the lane worksheet. 

4.3.2 Running gel

1. Press the button for the 30 minute run. Make sure the light turns green.

2. When the run is over, the light will be red and blinking and the 

apparatus will beep. Unplug the E-gel base and remove gel cassette.

4.3.3 Visualizing and documenting gel

1. Place gel in UV light box.

2. View and photograph gel according to available programs. The current 

program for recording pictures is the Kodak Gel Logic 2200 imaging 
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system, and the analysis program is Kodak Molecular Imaging Software,

version 4.0.4, or current version image analysis software. Attach a 

photograph or electronic picture to the current version of VIRTWS1074, 

PCR Report Out Worksheet, and retain the original copy with the batch 

records. After photographing, dispose of gel in a container designated for 

solid EtBr waste. 

4.4 PCR purification

4.4.1 Using the microcentrifuge instructions for the QIAquick PCR purification 

kit, start with adding 5 volumes of buffer “PB” to 1 volume of PCR product. 

Continue through to elution following the kit instructions.

Note: DNA that was pooled after the PCR run should be run in multiple 

tubes, using no more then 100-200 μL of DNA per reaction tube.

4.4.2 The purified DNA may be visualized using a gel to make sure DNA is still 

intact. Follow steps from Section 4.3.

4.4.3 The DNA concentration is measured using a Nanodrop 2000. From the 

DNA concentration, the total number of copies can be determined.

Note: Purified DNA is pooled again before Nanodrop reading.

4.4.4 After PCR purification, the DNA is stored at -20oC.

5. Preparation and Packaging of the Product

5.1 Determination of dilution of DNA to use for reagent

5.1.1 CAV DNA is tenfold serially diluted using nuclease free water.

5.1.2 Following the steps in Section 4.2, each dilution of CAV DNA is 

amplified.

5.1.3 Following the steps in Section 4.3, the dilutions of CAV DNA are 

visualized using a gel. Based on the results of the gel analysis, a dilution is picked 

that will work as the dilution for the final reagent. Ideally, this value should be at 

the dilution that is one or two folds more concentrated then the dilution with the 

last visible band on the gel.
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5.2 Bottling and filling

The volumes given in this section are for a concentration of 1 x 105copies/μL

based off of the dilutions made in Section 5.1.

5.2.1 500-μL screw cap Matrix tubes are labeled and placed in trays.

5.2.2 300 μL of 1 x 1011 copies/μL dilution is transferred to 29.7 mL of

nuclease free water, resulting in a final concentration of 1 x 105copies/μL.

5.2.3 The final reagent solution of 1 x 105copies/μL is transferred to a liquid 

reservoir.

5.2.4 Tubes are uncapped using a Thermo automated decapper.

5.2.5 Using a 12-channel automated pipette, 100 μL of the reagent was 

transferred to each labeled tube. Tubes are then capped using the automated 

decapper.

5.2.6 After filling and capping, the labeled tubes are stored at -20oC.

6. Testing

6.1 Sequencing

The CAV DNA standard is tested for sequence identity to Chicken Anemia Virus.

6.2 Final reagent inhibition study

The final reagent, after diluting and bottling, is tested using steps from Sections 4.2 and 

4.3 and following VIRPRO0118, Polymerase Chain Reaction Assay for Detection and 

Identify of Extraneous Chicken Anemia Virus (CAV), to make sure the product is 

inhibited by tissue samples, including chicken embryo fibroblasts, ground chicken bursa, 

chicken embryo, chicken liver and kidney. (Chicken bursa has shown to partially inhibit 

the amplification of CAV DNA.)

7. Summary of Revisions

Version .02

 Contact information has been updated.

 Master mix changed to HotStar Kit
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 Gel electrophoresis updated to E-gel 

 Section 6.2 updated to better describe criteria for reagent viability.