Thermo Fisher Scientific BTV4 User Manual

Technology

Species

Nucleic acid isolated from matrices

Test type

Real-time RT-PCR ( RNA)

- IPC Endogenous

Mix for TaqMan™ RT-PCR. Contains:

• Buffer, reverse transcriptase, and real-time PCR enzyme.

External Positive Control:

INSTRUCTIONS FO R USE

VetMAX™ BTV4 IAH Typing Kit

TaqMan™ real-time RT-PCR for detection of BTV4 (Bluetongue Virus type 4)

Catalog Number BTV4GIAH50

Doc. Part No. 100020331 Pub. No. MAN0008695 Rev. C.0

IMPORTANT! Product registered with the French National Reference Laboratory – Anses Maisons-Alfort. Specific requirements apply
for the use of the kit in diagnostic procedures in France, see Appendix A.

- Duplex

Bovine

Small rumina nts (sheep, goats)

Spleen or aborted fetus (spleen, liver, heart)

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

Blood (in EDTA tubes)

Individual

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

WARNING! POTENTIAL BIOHAZARD. Read the biological hazard safety information at this product’s page at
thermofisher.com. Wear appropriate protective eyewear, clothing, and gloves.

Information about the product

Description of the product

Bluetongue disease is a non-contagious, insect-borne infectious disease in sheep, included on the A list of the International Office of
Epizootics. It is caused by infection with Bluetongue virus (BTV), a virus in the Reoviridae family, Orbivirus genus. To date, 35
different BTV serotypes have been identified.

BTV is basically considered dangerous for sheep: it leads to major morbidity and high mortality. The virus also infects cattle, goats and
other wild ruminants, but only rarely leads to clinical manifestations in these species (Lefèvre and Desoutter, 1998).

The virus is nearly always transmitted by the infectious bite of a small, blood-sucking fly belonging to the Ceratopogonidae family,
Culicoides genus. There are more than 1,400 species of Culicoides, but not all are capable of transmitting the virus. Culicoides becomes
infected after feeding on the blood of an infected animal, then the virus reproduces until a sufficient titer is reached, enabling
transmission to other susceptible animals.

The Applied Biosystems™ VetMAX™ BTV4 IAH Typing Kit is a BTV type 4 molecular diagnostic tool. This is a second-line kit that,
after BTV analysis of the positive group, enables specific detection of the BTV4 virus through RT-PCR technology in real time.

Each RNA sample is analyzed in a single well: the same well is used to specifically detect the viral RNA of BTV4 and an IPC (Internal
Positive Control). A positive IPC signifies both successful extraction and the absence of PCR inhibitors in the sample.

This kit can be used on viral RNA extracted from whole blood (in EDTA tubes), spleen, or organs of aborted animals.
Complete protocols for viral RNA extraction from these matrices are available upon request from Technical Support.

Kit contents and storage

The VetMAX™ BTV4 IAH Typing Kit contains components that can be used for detecting both BTV4 and an IPC. Upon receipt, the kit
should be stored unopened at −30°C to −10°C. After the initial use, follow the recommendations for storage of each component in the
following table:

Component Description

• The detec tion s ystem for the BTV4 target, incl uding a TaqMan™ probe labeled

3 - Mix BTVEUG4
(Blue tube)

4a - EPC BTVEUG4
(Blue tube)

™

FAM

– NFQ (Non-Fluore scent Que ncher).

• The detection system for the IPC, including a TaqMan

™

– NFQ (Non-Fluorescent Quencher).

VIC

BTV4 positive control. It uses nucleic acid already extracted for
denaturation, then amplification, during the real-time RT-PCR.

™

probe labeled

Volume

(50 tests)

1,000 µL −30°C to −10°C −30°C to −10°C

90 µL −30°C to −10°C −30°C to −10°C

Upon receipt After initial use

Storage

Extraction and amplification controls

The VetMAX™ BTV4 IAH Typing Kit contains one control used to validate the amplification of the viral RNAs:

4a - EPC BTVEUG4: positive control of BTV4

A positive control, already extracted, for amplification during real-time RT-PCR.
A positive result within the specified Ct range enables amplification validation of the BTV4 target by real-time RT-PCR.

For Veterinary Use Only. For In Vitro Use Only.

In France, follow the additional requirements described in Appendix A.

Type of analysis

Component

Sample vo lume

Sample for analysis

RNA extracted from the sample and denatured

5 µL

Positive amplification control

Denatured 4a - EPC BTVEUG4

5 µL

Negative extraction control (NCS)

Extracted and denatured NCS

5 µL

Negative amplification control (NC)

DNase/RNase-free water

5 µL

Validation of nucleic acid extraction for each sample is done by detection of an endogenous IPC (Internal Positive Control), present in
each sample.

A positive IPC result for a sample validates the extraction of that sample, whether the sample is positive or negative for the pathogen
being investigated, eliminating false negatives due to PCR inhibition.

We recommend including two negative controls to confirm correct analysis:

NCS: negative extraction control

This control consists of DNase/RNase-free water or a sample known to be free of target pathogen that undergoes the same treatment
(nucleic acid extraction and real-time RT-PCR) as the samples.

A negative result of BTV4 and endogenous IPC confirms the absence of contamination during the extraction and the real-time RT-PCR.

NC: negative amplification control

This control consists of 20 µL of real-time RT-PCR mix and 5 µL DNase/RNase-free water that undergoes real-time RT-PCR.
A negative result of BTV4 and IPC confirms the absence of contamination during real-time RT-PCR reaction preparation.

Materials required but not provided

Unless otherwise indicated, all materials are available through thermofisher.com.

• Adjustab le micropipettes (range of 1 µL to 1,000 µL) with DNas e/RNase-free filtered tips

• DNase/RNase-free water

• 1X TE buffer

• 1X PBS buffer

• Heating block capable of reaching 95°C

• A real-time PCR thermal cycler capable of detectin g the following fluorophores:

– FAM
– VIC

• Optical-quality consumables compatible with the thermal cycler used: PCR 96-well plates, PCR strips (8 or 12 wells), microtubes or

™

(maximum emission : λ515 nm)

™

(maximum emission: λ554 nm)

capillar ies; suitable plate covers or caps for capping

Analysis procedure

The reaction volume of the real-time RT-PCR is 25 µL:

• 3 - Mix BT VEUG4: 20 µL per reaction

• Extracted RN A: 5 µL per reaction

Extraction of viral RNA

RNA must be extracted from the samples prior to real-time RT-PCR analysis.

NOTE: To learn about compatible and validated extraction methods for the VetMAX™ BTV4 IAH Typing Kit, please contact
Technical Support.

Denaturation of RNA

1. Add the RNA to be denatured into the wells of a PCR plate or strip. Include 10% overage of the extracted RNA for all reactions to

ensure that sufficient RNA is present after denaturation.

2. Cap the wells containing the RNA.

3. Heat for 3 minutes to between +92°C and +98°C in a thermal cycler or heating block.

4. Store the denatured RNA at between +2°C and +8°C on crushed ice or on a refrigerated block until use.

Preparation of the real-time RT-PCR

1. Create an analysis plan for distribution of the mixes and samples. Keep the positive control (EPC) away from the other samples if

possible.

2. Thaw 3 - Mix BTVEUG4 between +2°C and +8°C, on ice or on a refrigerated rack.

3. Homogenize the 3 - Mix BTVEUG4 tube by gentle agitation, then centrifuge briefly.

4. Add 20 µL of 3 - Mix BTVEUG4 to each well on the PCR plate, PCR strip or capillary used.

5. Add RNA from samples and controls to the real-time RT-PCR mix solution according to the following preset analysis plan:

6. Cover the PCR plate, PCR strips or capillaries with an adhesive plate cover or suitable caps.

2 VetMAX™ BTV4 IAH Typing Kit Instructions for Use