Bovine Virus Diarrhea RNA Test Kit

VetMAX-Gold BVDV Detection Kit

Diagnostic test kit for the detection of Bovine Virus Diarrhea Virus RNA
Document PN 4425603 Rev. B

Product information

Required materials not supplied

Name, intended use, and principle of the procedure

Table2 Materials for RT-PCR

The Bovine Virus Diarrhea RNA Test Kit (VetMAX-Gold BVDV Detection
Kit, PN 4413938) is intended for use in the rapid, in vitro detection of
bovine virus diarrhea virus (BVDV) RNA extracted from ear notch samples in acute and persistently infected cattle.
BVDV is an approximately 12-kb single-stranded, positive RNA enveloped virus in the genus Pestivirus of the Flaviviridae family. Based on
antigenic and genomic sequence variation, BVDV is divided into two
subtypes: 1 and 2. BVDV infection in cattle is a worldwide problem that
results in significant economic losses in the dairy and beef industries.
It can cause respiratory disease, enteritis, still-birth, abortion, and
mucosal disease. In utero BVDV infection can induce immunotolerance,
causing animals to be persistently infected (PI) for life. PI animals continuously shed the virus and are the main source of BVDV infection in
herds. Rapid detection of PI cattle is essential for BVDV control.
The assay is a single-tube, real-time, reverse transcription-polymerase
chain reaction (RT-PCR) in which RNA is reverse transcribed into cDNA,
and BVDV RNA and Xeno RNA Control targets are amplified and
detected in real time using fluorescent TaqMan probes (hydrolysis
probe chemistry). BVDV Control RNA and Xeno RNA Control are provided to serve as positive controls for the RT-PCR reaction components.
Xeno RNA Control also serves as an internal positive control for the
RNA purification process. The 25 BVDV Primer Probe Mix provided is
optimized for multiplex real-time RT-PCR amplification of both Xeno
RNA Control and BVDV RNA targets.

Item

Source

96-well plates or tubes appropriate

for the real-time PCR instrument
used

At www.appliedbiosystems.com, select
Products Real-Time PCR 
Reaction Plates & Adhesive Films 
Plastic Consumables Compatibility
Chart

Nuclease-free pipettors and tips

Major laboratory supplier

Reagent reservoirs or tubes for

preparing master mixes

Major laboratory supplier

Real-time PCR thermal cycler

Applied Biosystems 7500 Fast RealTime PCR System

Applied Biosystems 7500 Real-Time
PCR System

Procedure
Isolate RNA from samples
Table3 Sample handling recommendations
Step or process

Transport/storage of samples Approximately 80 C to 20 C (transport on

dry ice).
Preparation of ear notch
supernatant

Limitations

3. Centrifuge at 10,000 g for 30 sec.

Samples should be handled as recommended in Table 3 to prevent

degradation of any BVDV RNA that is present.

Follow Good PCR practices on page 4 to prevent false positive

amplifications due to contamination of test samples with PCR products.

Kit contents and storage conditions

Reagents for 100 25-L RT-PCRs are supplied.
Table1 VetMAX-Gold BVDV Detection Kit components
Component
2 RT-PCR Buffer

Volume

Storage

1.375 mL 30 C to 10 C

25 RT-PCR Enzyme Mix

110 L

30 C to 10 C

25 BVDV Primer Probe Mix

110 L

30 C to 10 C

Xeno RNA Control (10,000 copies/L)

110 L

30 C to 10 C

15 L

30 C to 10 C

Nucleic Acid Dilution Solution

500 L

30 C to 10 C

Nuclease-free Water

1.75 mL 30 C to 25 C

25 BVDV Control RNA (10,000 copies/L)

1. Place one dry ear notch sample into a

2-mL tube, and add 1 mL of 1 Phosphate
Buffered Saline (1 PBS).
2. Vortex at high speed for 10 min at room
temperature.

The kit is not intended for discriminating viral subtypes.

RNA extraction methods should yield RNA free of RT-PCR inhibitors,

which can prevent amplification of target RNA.

Recommendation

4. Remove an appropriate volume of

supernatant for RNA isolation.
Preparation of mock-purified
samples (for use in extraction
control PCRs)

Prepare duplicate mock-purified samples,

utilizing 1 PBS as the starting material.
Process with the same RNA isolation method
as for test samples.

Proposed RNA isolation

method

MagMAX-96 Viral RNA Isolation Kit

(PN AM1836, AMB1836-5) or an equivalent
RNA isolation method.

Required modifications to
RNA isolation method

Add 0.8 L undiluted Xeno RNA Control

(8,000 copies) per isolation to the lysis
solution used for RNA isolation.
Add carrier RNA to the lysis solution
according to the manufacturers
recommendation. (Add carrier RNA in
addition to Xeno RNA Control; carrier RNA
is included in the MagMAX-96 Viral RNA
Isolation Kit.)

Instructions
08/2010

 TaqMan probe reporter dyes and quenchers:

Perform real-time RT-PCR

For all reagents, read the Safety Data Sheet, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and gloves.

1.

Target

Plan the reactions and thaw the reagents.

Reporter

Quencher

BVDV RNA

FAM dye

BHQ-1 dye

Xeno RNA Control

Cal Fluor Orange 560 dye

BHQ-1 dye

Cal Fluor Orange 560 dye has an absorbance maximum of 538 nm and an
emission maximum of 559 nm. When using an Applied Biosystems Real-Time
PCR System, calibrate with Cal Fluor Orange 560 dye. If that is not possible,
use the VIC dye detector.

a. Plan the reactions: on each plate, include the following control

reactions (see step 4).
Positive control (prepare duplicate reactions): use
10,000 copies each of BVDV Control RNA and Xeno RNA
Control per reaction.
No-template control (prepare duplicate reactions): use
Nuclease-free Water in place of sample RNA.
Extraction controls: use the mock-purified samples (two
samples).

Thermal cycler settings:

Stage

b. Thaw all reagents on ice, gently vortex each tube to mix the
contents thoroughly, then briefly centrifuge to collect the solution at the bottom of the tube. Keep reagents on ice.

Reps.

Temp.

Reverse transcription

45 C

10 min

Time

RT inactivation/ initial
denaturation

95 C

10 min

Amplification

40

95 C
60 C

15 sec
45 sec

b. Run the thermal cycler program and collect real-time amplification data during stage 3.

2.

Prepare RT-PCR master mix on ice.

Include 10% overage to ensure that you have enough RT-PCR master mix for all the samples.
RT-PCR master mix component
2 RT-PCR Buffer

Vol./Rxn.
12.5 L

25 BVDV Primer Probe Mix

1 L

25 RT-PCR Enzyme Mix

1 L

Nuclease-free Water

2.5 L

Total volume RT-PCR master mix per 25 L reaction

17 L

Data analysis
Refer to the user guide for your real-time PCR instrument for instructions on how to analyze your data, using the recommendations below.
Table4 Recommendations for data analysis
Recommendation

Details

Use the Auto CT

setting.

This setting minimizes subjectivity when setting the

threshold cycle (CT) for BVDV RNA and Xeno RNA
Control amplifications.

Check the raw

fluorescence data.

Verify that fluorescence increases seen in the

normalized data are evident without mathematical
data processing.

3.

Dispense 17 L of RT-PCR master mix to the wells of a PCR plate

or PCR tubes on ice.

4.

Add sample RNA, control RNA samples, or Nuclease-free Water to

each reaction well or tube.

Interpretation of test results

a. Add sample as described in the following table.

Verify that your RT-PCR run is valid before analyzing test sample
results.

Reaction type
Test sample

Sample
Sample RNA

Vol./Rxn.

No-template control (NTC) Nuclease-free Water

8 L

Extraction control

Mock-purified sample

8 L

Positive control

25 BVDV Control RNA

Xeno RNA Control
Nucleic Acid Dilution Solution

1.0 L
1.0 L
6.0 L

b. Seal each reaction vessel, mix, then centrifuge briefly to bring

the contents to the bottom.

5.

Set up and run the real-time PCR instrument for BVDV RNA and
Xeno RNA Control amplification.
Follow the manufacturers instructions to set up the instrument.

a. Set up the run using the following parameters.

Reaction volume: 25 L.
ROX passive reference dye: included in the RT-PCR Buffer.

Table5 Criteria for valid RT-PCR run

8 L
Reaction type
Positive control
NTC
Extraction control

CT value for
BVDV RNA

CT value for
Xeno RNA Control

< 30

< 32

40 (no

signal)

40 (no signal)

40 (no

signal)

3138

The run is invalid if the CT value for BVDV is < 38. If the CT value is 3840, the RT-PCR
may be contaminated. See Troubleshooting on page 3.
The run is invalid if the CT value for Xeno RNA Control is < 38. If the CT value is 3840,
the RT-PCR may be contaminated. See Troubleshooting on page 3.

Table6 Interpretation of test results

CT value for
BVDV RNA

CT value for Xeno

RNA Control

Interpretation

<38

3138

BVDV-positive sample

no signal

3138

BVDV-negative sample

3840

3138

Suspect result (see Table7 on

page 3)

Bovine Virus Diarrhea RNA Test Kit Instructions

08/2010

Table7 Assessment of suspect results

Analyze suspect RNA samples (BVDV CT 3840) for the presence/absence of RT-PCR inhibitors by calculating the Xeno RNA CT shift:
Xeno RNA CT shift = Xeno RNA CT SUSPECT SAMPLE Avg. Xeno RNA CT MOCK-PURIFIED SAMPLES (EXTRACTION CONTROLS)
Xeno RNA CT shift 1.5
Xeno RNA CT shift <1.5
Repeat the RT-PCR with 2 L of the suspect RNA sample. (RT-PCR inhibitors
may be present in the RNA.)

1. Repeat the RNA purification on triplicate aliquots of the original diagnostic

sample.

If BVDV CT < 38: BVDV positive

2. Repeat the RT-PCR with 8 L of purified RNA from step 1.

If BVDV CT 38:
1. Dilute the original diagnostic sample 1:4.

3. Determine the number of samples with BVDV CT < 40. The standard deviation
of the triplicates should be 1 CT.
0 of 3: BVDV negative.
1 of 3: Presumptive positive; confirm with secondary test method.
2 of 3: BVDV positive.

2. Repeat the RNA purification on triplicate aliquots of the diluted sample.

3. Repeat the RT-PCR with 8 L of purified RNA from step 2.
4. Determine the number of samples with BVDV CT < 40. The standard deviation
of the triplicates should be 1 CT.
0 of 3: BVDV negative.
1 of 3: Presumptive positive; confirm with secondary test method.
2 of 3: BVDV positive.

Troubleshooting
Observation

Possible cause

Solution or troubleshooting strategy

The BVDV Control RNA or Xeno RNA

Control was improperly handled,
resulting in RNA degradation.

Use appropriate precautions against RNase contamination when handling the control
RNAs. For example, wear clean gloves and use nuclease-free barrier pipette tips.

The 25 RT-PCR Enzyme Mix was

stored or handled improperly, and it
lost activity.

Repeat the RT-PCR with fresh reagents.

The thermal cycler was not properly

set up.

See step 5 on page 2.

The RT-PCR master mix was

prepared incorrectly.

See step 2 on page 2.

PCR contamination.

Repeat the RT-PCR with fresh reagents and freshly decontaminated pipettors.
Set up the RT-PCR in an area separate from areas used for RNA isolation and PCR
product analysis.

The Xeno RNA Control primers and

probe are at limiting concentrations
in the RT-PCR. High levels of BVDV
RNA in a sample can reduce
amplification of Xeno RNA Control.

No or low signal from Xeno RNA Control is expected in a reaction that has a strong
signal for BVDV RNA.

Test samples:

Poor RNA recovery.

Xeno RNA Controlno signal

-or-

BVDV RNAno signal or suspectrange signal

The RNA samples contain inhibitors

of RT-PCR.

Check the CT values of Xeno RNA Control in the mock-purified samples.

CT 38: indicates that Xeno RNA Control was omitted or that RNA recovery was poor.
Check that Xeno RNA Control was added to the lysis solution as described in
Isolate RNA from samples on page 1.
Check the recovery of carrier RNA used in RNA isolation. If it is poor, troubleshoot
your RNA preparation method.

Positive control reaction:

BVDV Control RNAno signal
Xeno RNA Controlno signal

No-template control reaction:

Signal detected
Test samples:
Xeno RNA Controlno signal
BVDV RNAhigh signal

CT 3138: indicates that RNA recovery is adequate, therefore the test samples may
have RT-PCR inhibitors. See Table7, Assessment of suspect results.
All samples:
Atypical non-sigmoidal
amplification curves when using the
Auto CT setting

Bovine Virus Diarrhea RNA Test Kit

Instructions 08/2010

Atypical data (usually due to

experimental error)

Repeat the RT-PCR according to the Assessment of suspect results workflow in

Table 7.

Good PCR practices

Safety information

PCR/RT-PCR assays require special laboratory practices to avoid false

positive amplifications. When preparing samples for PCR/RT-PCR
amplification:

Obtaining SDSs

Use positive-displacement pipettes or aerosol-resistant pipette tips.

Follow proper pipette-dispensing techniques to prevent aerosols.

At www.appliedbiosystems.com, select Support  MSDS. Search

by chemical name, product name, product part number, or MSDS
part number. Right-click to print or download the MSDS of interest.
At www.ambion.com, go to the web catalog page for the product of
interest. Select MSDS, then right-click to print or download.
E-mail (MSDS_Inquiry_CCRM@appliedbiosystems.com), telephone
(650-554-2756; USA), or fax (650-554-2252; USA) your request, specifying the catalog or part number(s) and the name of the product(s).
The associated SDSs will be e-mailed unless you request fax or
postal delivery. Requests for postal delivery require 1 to 2 weeks for
processing.

Wear clean gloves and a clean lab coat (not previously worn while
handling amplified PCR/RT-PCR products or used during sample
preparation).
Change gloves whenever you suspect that they are contaminated.
Maintain separate areas and dedicated equipment and supplies for:
Sample preparation and PCR/RT-PCR setup
PCR amplification and analysis of PCR/RT-PCR products
Never bring amplified PCR/RT-PCR products into the PCR/RT-PCR
setup area.

To obtain Safety Data Sheets (SDSs; previously known as MSDSs) for

any chemical product supplied by Applied Biosystems or Ambion:

Open and close all sample tubes carefully. Centrifuge tubes before
opening. Try not to splash or spray PCR/RT-PCR samples.

Note:For the SDSs of chemicals not distributed by Applied Biosystems or Ambion, contact the chemical manufacturer.

Keep reactions and components capped as much as possible.

Clean lab benches and equipment periodically with 10% bleach solution. Use DNAZap Solution (PN AM9890).

Manufactured by:
Applied Biosystems LLC, 2130 Woodward St., Austin, TX 78744, USA
US Veterinary License No. 432
Information in this document is subject to change without notice.
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Literature Citation: When describing a procedure for publication using this product, please refer to it as the VetMAX-Gold BVDV Detection Kit.
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Copyright 2010, Life Technologies Corporation. All rights reserved.

Part Number 4425603 Rev. B 08/2010

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