Thermo Fisher Scientific VetMAX Swine Influenza A-09 Kit Operating instructions

For Veterinary Use Only. For In Vitro Use Only.

INSTRUCTIONS FOR USE

VetMAX™ Swine Influenza A-09 Kit

TaqMan® real-time RT-PCR for detection of the Influenza A virus

Catalog Number INFAPSWINE

Doc. Part No. 100020377 Pub. No. MAN0008325 Rev. A.0

Technology

Species

Nucleic acid isolated from matrices

Test type

Real-time RT-PCR (RNA)

–Duplex

–Endogenous IPC

Porcine

Nasal swabs

Organs

Bronchoalveolar lavage

Amniotic fluid

Cell culture supernatant

Individual

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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

The Applied Biosystems™ VetMAX™ Swine Influenza A-09 Kit is a molecular diagnostic tool for influenza type A virus.

The VetMAX™ Swine Influenza A-09 Kit serves to specifically detect a highly conserved region of the M gene which is specific to all

influenza A viruses, including the pandemic Influenza A/H1N1/2009 strain by real-time reverse transcriptase PCR. It does not detect

other strains of influenza (types B and C).

Each RNA sample is analyzed in a single well: the same well is used to specifically detect the viral RNA of influenza type A 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 nasal swabs, organs, bronchoalveolar lavage, amniotic fluid and cell culture

supernatant.

Complete protocols for viral RNA extraction from these matrices are available upon request from Technical Support.

Kit contents and storage

The VetMAX™ Swine Influenza A-09 Kit contains components that can be used for detecting both influenza type A and an IPC. Upon

receipt, the kit should be stored unopened at −30°C to −10°C. After initial use, follow the recommendations for storage of each

component in the following table:

Component Description Volume

(100 tests)

Storage

Upon receipt After initial

use

3 - Mix Influenza A

(Green tube)

Mix for TaqMan

RT-PCR. Contains:

•The detection system for influenza A target: forward and reverse

primers, as well as a TaqMan® probe labeled with FAM™ - NFQ

(NFQ = Non-Fluorescent Quencher).

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

a TaqMan® probe labeled with VIC™ - TAMRA™.

•Buffer, reverse transcriptase and PCR enzyme.

2 × 1000 µL −30°C to

−10°C

−30°C to

−10°C

4c - EPC Influenza A

(Blue tube)

External Positive Control:

Positive control for influenza A. It is an inactive biological agent to be

extracted and then amplified during real-time RT-PCR.

2 × 200 µL −30°C to

−10°C

−30°C to

−10°C

2 VetMAX™ Swine Influenza A-09 Kit Instructions for Use

Extraction and amplification controls

The VetMAX™ Swine Influenza A-09 Kit contains one control used to validate the extraction and amplification of viral RNA:

4a - EPC Influenza A: positive control for influenza A

A positive control to be extracted at the same time as the samples in the first analysis.

Extracted RNA can be stored below −16°C for subsequent RT-PCR analysis. Extracted RNA should be aliquoted to limit the number of

freeze-thaw cycles to a maximum of 3. A positive result within the specified Ct range is used to validate amplification of the

influenza A 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

every sample. A positive IPC result for a sample validates the extraction of this sample, whether the sample is positive or negative for

the pathogen being investigated, enabling elimination of false negatives and verification of inhibition, if present.

We recommend including two negative controls to confirm correct analysis:

NCS: negative extraction control

This control consists of reagents used in the extraction without addition of the sample (sample volume can be replaced by the buffer

used in the sample preparation or by DNase/RNase-free water) that undergoes the same treatment (nucleic acid extraction and

real-time RT-PCR) as the samples. A negative result for influenza A and IPC serves to confirm proper handling of the extraction and

the absence of contamination during extraction and real-time RT-PCR.

NC: negative amplification control

This control consists of 20 µL of real-time RT-PCR mix and 5 µL of DNase/RNase-free water that undergoes real-time RT-PCR. A

negative result for all targets (influenza A 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.

•Adjustable micropipettes (range of 1 µL to 1,000 µL) with DNase/RNase-free filtered tips

•DNase/RNase-free water

•1X TE buffer

•1X PBS buffer

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

–FAM™ (maximum emission: λ515 nm)

–VIC™ (maximum emission: λ554 nm).

•Optical-quality consumables compatible with the thermal cycler used:

–PCR 96-well plates, PCR strips (8 or 12 wells), microtubes or capillaries

–Suitable plate covers or caps for capping

Analysis procedure

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

•3 - Mix Influenza A: 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: For information about extraction methods that are compatible with and validated for the VetMAX™ Swine Influenza A-09 Kit,

please contact Technical Support.

Preparation of the real-time RT-PCR reactions

1. Create an analysis plan for distribution of the mixes and samples. If possible, keep the EPC away from the other samples.

2. Thaw 3 - Mix Influenza A at +2°C to +8°C on ice or in a refrigerated rack.

3. Mix 3 - Mix Influenza A by gentle agitation, then briefly centrifuge.

4. Add 20 µL of 3 - Mix Influenza A to each PCR plate well, PCR strip or capillary.

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

Type of analysis Component Sample volume

Sample for analysis

RNA extracted from sample 5 µL

Positive amplification control RNA extracted from 4c - EPC Influenza A 5 µL

Negative extraction control (NCS) Extracted NCS 5 µL

Negative amplification control (NC)

DNase/RNase-free water 5 µL

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

VetMAX™ Swine Influenza A-09 Kit Instructions for Use 3

Amplification by real-time RT-PCR

1. Set up the following detectors on the thermal cycler:

Reporter Quencher

INFA (Influenza A) FAM™ NFQ (Non-Fluorescent Quencher)

IPC INFA

VIC™ TAMRA™(1)

Passive reference: ROX

™(1)

(1) The TAMRA™ and ROX™ fluorophores must be entered for real-time PCR analysis if the thermal cycler is capable of detecting them. For all other thermal

cyclers, absence of the ability to detect these fluorophores does not compromise the analysis by real-time PCR.

2. Set up the INFA and the IPC INFA detectors for each well used in the analysis.

3. Set up the following real-time RT-PCR program for the analysis:

Number of cycles Temperature Duration

Step 1

1× 45°C 10 minutes (10'00")

Step 2

1× 95°C 10 minutes (10'00")

Step 3 45×

95°C

15 seconds (00'15")

60°C

(1)

45 seconds (00'45")

(1) Collection of fluorescence data during the 60°C – 45 seconds stage.

4. Place the PCR plate, PCR strips or capillaries in the thermal cycler and start the real-time RT-PCR.

Interpretation of results

Raw data analysis

Refer to the thermal cycler manufacturer’s recommendations for raw data analysis.

1. Set the threshold lines separately for each target.

2. Interpret the results based on the Ct values of the samples for each detector according to the following recommendations.

Validation

The test is validated if the following criteria are met:

“INFA” detector “IPC INFA” detector Validation

EPC (Influenza A)

Ct = Ct QC INFA of "4c - EPC Influenza A" ± 3Ct(1) Ct < 45 or Ct > 45(2) PCR validated

NCS

Ct > 45 Ct > 45 Extraction validated

> 45

PCR reagents validated

(1) Please refer to the values shown in Section 2.1, “EPC”, of the Certificate of Analysis of the batch used for the test.

(2) The IPC value in the EPC should not be used for validation.

Interpretation of results

For each sample analyzed, the results should be interpreted as shown below:

“INFA” detector

“IPC INFA” detector Interpretation

< 45

Ct < 45 or Ct > 45 Influenza A detected

> 45

< 45

Influenza A not detected

> 45

Ct > 45 Not validated(1)

(1) The sample will be returned as not validated due to the negative IPC value.

Procedure for handling non-validated samples

1. Dilute the RNA at a 1:10 dilution of RNA in 1X TE buffer.

2. Perform RT-PCR analysis on 5 µL of this dilution.

3. If the diluted RNA is positive for Influenza A or negative for Influenza A with an acceptable IPC result, the result obtained is then

validated.

4. If the diluted RNA is negative for Influenza A with an unacceptable IPC result, the result obtained is still not validated. In this case,

repeat the nucleic acid extraction using a sample diluted at 1:10 in 1X PBS buffer.

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14 July 2017

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you have any questions, please contact Life Technologies at thermofisher.com/support.

Laboratoire Service International (LSI) | 6 Allée des Écureuils | Parc Tertiaire du Bois-Dieu | 69380 Lissieu, France

Translated from the French Pub. No. MAN0007808 Rev. B.0.

The information in this guide is subject to change without notice.

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MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history of Pub. No. MAN0008325 (English)

Revision

Date

Description

A.0

14 July 2017

Updated to the current document template, with associated updates to the warranty, trademarks, and logos.

1.0

6 June 2013

Baseline for revision history

Limited Use Label License No. 460: PCR Veterinary Diagnostics. Notice to Purchaser: For veterinary diagnostic services including the reporting of such services for a fee and

for research purposes only. Human diagnostic uses require a separate license from Roche.