Thermo Fisher Scientific BTVNS3 Instruction Manual

INSTRUCTIONS FO R USE

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:

denaturation, then amplification, during the real-time RT-PCR.

−

VetMAX™ BTV NS3 All Genotypes Kit

TaqMan™ real-time RT-PCR for detection of BTV (Bluetongue Virus)

Catalog Number BTVNS3

Doc. Part No. 100020325 Pub. No. MAN0008222 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 ruminants (sheep, goats)

Spleen or aborted fetus (spleen, liver, heart)

Blood (in EDTA tubes)

Individual

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
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™ BTV NS3 All Genotypes Kit is a molecular diagnostic tool for BTV. It serves to specifically
detect from 1 to 27 genotypes of the BTV using real-time reverse transcription PCR. It does not detect the EHDV (Epizootic
Hemorrhagic Disease Virus), a relative of BTV.

Each RNA sample is analyzed in a single well: the same well is used to specifically detect the viral RNA of BTV 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™ BTV NS3 All Genotypes Kit contains components that can be used for detecting both BTV 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

• The detection system for the BTV target: forward and reverse

3 - Mix BTVNS3
(Green tube)

4a - EPC BTVNS3
(Brown tube)

NOTE: We recommend storing 3 – Mix BTVNS3 in aliquots (50-µL minimum volume), to avoid more than 3 freeze-thaw cycles.

primers, as well as a TaqMan
(NFQ = Non-Fluorescent Quencher).

• The detection system for IPC: forward and reserve primers, as

well as a TaqMan

BTV positive control. It uses nucleic acid already extracted for

Description

™

probe labeled with FAM™ - NFQ

™

probe labeled with VIC™ - TAMRA™.

Volume

(100 tests)

2 × 1,000 µL −30°C to −10°C −30°C to −10°C

2 × 90 µL

Upon receipt After initial use

30°C to −10°C −30°C to −10°C

Storage

Extraction and amplification controls

The VetMAX™ BTV NS3 All Genotypes Kit contains one control used to validate the amplification of viral RNA:

4a - EPC BTVNS3: positive control of BTV

A positive control, already extracted, for amplification during 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 BTVNS3

5 µL

Negative extraction control (NCS)

Extracted and denatured NCS

5 µL

Negative amplification control (NC)

DNase/RNase-free water

5 µL

A positive result within the specified Ct range enables amplification validation of the BTV target by real-time RT-PCR.

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 BTV 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 BTV 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 follo win g fluorophores:

– FAM™ (maximum emiss ion: λ515 nm)
– VIC™ (maximum emission: λ554 nm)

• Optical-quality consumables compatible with the thermal cycler used:
– PCR 96-well pla tes , PCR strips (8 or 12 wells), microtubes or capillaries
– Suitable plate covers or caps for ca pping

Analysis procedure

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

• 3 - Mix BTVNS3: 20 µL per reaction

• Extracted RNA: 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™ BTV NS3 All Genotypes 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 conta in ing 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 BTVNS3 between +2°C and +8°C, on ice or on a refrigerated rack.

3. Homogenize the 3 - Mix BTVNS3 tube by gentle agitation, then centrifuge briefly.

4. Add 20 µL of 3 - Mix BTVNS3 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

™

BTV NS3 All Genotypes Kit Instructions for Use