Code NRLAI_PCR_4V1 Page 1 of 5. Real-time RT-PCR for the detection of Influenza A Virus of Subtype H7 (Method AIV-H7.2)

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1 Code NRLAI_PCR_4V1 Page 1 of 5 Title: Real-time RT-PCR for the detection of Influenza A Virus of Code: NRLAI_PCR_4V1 Valid for: NRL AI Generated by B. Hoffmann on: Modified by T. Harder on: Valid as of: Replacing./. Issued by T. Harder on:

2 Code NRLAI_PCR_4V1 Page 2 of 5 1. Introduction This assay can be used to detect viral RNA isolated from various sample matrices, e.g. blood, tissues, swabs. This real-time RT-qPCR aims at detecting nucleic acids of Avian Influenza Virus (AIV) Subtype H7 and will generate test results even in the absence of replicative-competent virus. The AIV-H7-2 method was developed by the Friedrich-Loeffler-Institute and was validated for Eurasian and some North American AIV H7 strains, available in the stock selection of the reference laboratory for Influenza. The assay will detect a broad variety of AIV subtype H7 strains. However, a few H10 strains gave weak positive signals as well. Thus, a positive sample should be verified by additional analyses including conventional RT-PCR and sequencing. 2. RNA-Extraction This assy ist o be used with sample RNA that has already been tested positive for AIV RNA by the generic AIV-M-1.2-assay. Therefore, an internal control system can be omitted. 3. Real-time RT-PCR 3.1 General information a) Amplification is based on SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (# ; Invitrogen) in 25 µl total volume. If different kits are intended to be used, separate validation runs need to confirm comparable sensitivity and specificity. b) Controls include RNA-isolation controls (RIC), a no template control (NTC) as well as at least one positive control (PC-H7). In order to prevent/minimize cross contaminations, the concentration of the positive control AIV-H7-RNA should be low, with a Cq-threshold of approximately 33 in the M1.2 assay. c) After preparation of the mastermix (see table below), 20 µl will be used for each reaction, and 5 µl template RNA are to be added Preparation of reaction mix: a) The volume of the master mix depends on the number of extracted samples and the number of controls (RIC, PC-IAV-H7; NTC) plus an additional volume reserve correcting for possible pipetting errors. b) After preparing and mixing, the master mix can be stored on ice for up to two hours until use. c) After adding template RNA disclosure of reaction tubes/racks is followed by a short centrifugation of the vessels.

3 Code NRLAI_PCR_4V1 Page 3 of 5 Progression Mastermix-components Volume 1. RNase free water 4,5 µl 2. 2x reaction mix 12,5 µl 3. Enzyme mix 1,0 µl 4. Primer-probe mix: AIV-H7.2-mix-FAM 2,0 µl Total volume mastermix 20 µl Addition of template RNA 5. Samples RIC NTC (SDW) PC-H7 (low RNA concentration Cq = ca.33) 5 µl, resp. Total volume of reaction mixture 25 µl 3.4 Cycler-program Reverse Transkription 50 C 30 min Inactivation RT /activation Taq 94 C 2 min Denaturing 94 C 30 sec Annealing 56 C 30 sec 42 cycles Elongation 68 C 30 sec # measurement of fluorescence is recorded in the FAM-channel. # measurements are recorded during phase of annealing 3.5. Analysis of fluorescence data FAM-channel:: a) For the PC/standards a predefined Cq-FAM value should be obtained. Cq values of 33 are considered ideal with respect to reduced cross-contamination risks. b) The analysis of field samples is valid if all controls are responding as expected. c) A field sample is considered positive if a Cq-FAM-value <40 is detected and if the FAMfluorescence increases significantly over the base level.

4 Code NRLAI_PCR_4V1 Page 4 of 5 4. Preparation of primer/probe mix 4.1 AIV-H7-2-Mix-FAM (For internal use only!!) 20 µl AIV-HA7_1593-F (100 pmol/µl): 5 -AYA GAA TAC AGA TWG ACC CAG T µl AIV-HA7_1740-R (100 pmol/µl): 5 -TAG TGC ACY GCA TGT TTC CA -3 2,5 µl AIV-HA7_1649-FAM (100 pmol/µl): 5 -FAM- TGG TTT AGC TTC GGG GCA TCA TG-BHQ ,5 µl 0,1 x TE (ph 8,0) 200 µl AIV-H7-2-Mix-FAM 10 pmol Primer/µl + 1,25 pmol probe/µl

5 Code NRLAI_PCR_4V1 Page 5 of 5 Real-time RT-PCR run #:. AIV-H7.2 Assay Processed by:... Date:... Uhrzeit:... Remarks:... Processing steps Mastermix (SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (# ; Invitrogen)) 1. RNase free water 4,5 µl 2. 2x Reaction mix 12,5 µl 3. Enzyme mix 1,0 µl 4. AIV-H7.2-Mix-FAM 2 µl Total volume mastermix 20 µl 6. Addition of template-rna -Field samples, RIC, -PC-H7, NTC AIV-H7.2 1 x N x 5 µl, resp. Total volume reaction 25 µl 96-well-plate RT 30min 50 C Activation Taq 2min 94 C 42 cycles Denat. 30sec 94 C Annealing 30sec 56 C Elongation 30sec 68 C Name... Date... Valid Yes No... Results:..

Annex 6.2 CDC real-time measles RT-PCR protocol targeting measles N gene (75 nt region)

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