Detection of Eurasian H5 avian influenza virus and High Pathogenicity H5 subtype virus by real time-PCR
This protocol is a copy of the standard operating procedure used by the OIE/FAO International Reference laboratory for AI at the Animal and Plant Health Agency. If you have any technical queries please contact AIWRL@apha.gov.uk
WOAH/FAO international reference laboratory for AI
Last updated: 11 February 2022
1. Introduction
1.1 Purpose/Scope of this SOP
1.1.1 This SOP provides the details required to detect Eurasian isolates of H5 avian influenza (AI) virus (AIV) by real-time reverse transcription (RRT)-PCR.
1.1.2 This SOP also describes the application of a ‘high pathogenicity AIV (HPAIV) H5 RRT-PCR’ (‘HP H5 detection’) assay to specifically detect the presence of a multi-basic cleavage site (MBCS) sequence indicative of an HPAIV. This assay is used once a sample has been confirmed as positive by the generic influenza A virus screening or H5 HA2 RRT-PCR assays. In most instances, the HP H5 detection RRT-PCR assay will be run alongside the H5 HA2 RRT-PCR assays.
1.2 Background information
1.2.1 Two real-time reverse transcription PCR (RRT-PCR) assays are described in this SOP. The first is a rapid, sensitive and specific method for the detection of H5 AIV. Validation at APHA has revealed the test to be effective in detecting H5 AIV directly from clinical specimens, i.e. there is no need to first isolate the suspect H5 isolate in embryonated eggs by growth to a high titre.
H5 RRT-PCR has been described previously (Spackman et al, 2002), but validation was restricted to a largely N American collection of H5 AIV isolates. For the Eurasian H5 RRT-PCR described in this SOP, haemaglutinnin gene sequences from Eurasian H5 isolates (mainly 1996-2004) were aligned. Here the H5 primer and hydrolysis probe sequences of Spackman et al (2002) were modified to ensure effective detection of recent Eurasian H5 isolates and this method together with its validation has been published by Slomka et al (2007).
1.2.2 The second RRT-PCR describes the specific detection of H5 HPAIV. AIVs of subtype H5 can be HPAIV or low pathogenicity AIVs (LPAIVs). The pathogenicity can be determined genetically, based on the presence of a single basic (LPAIV) or MBCS (HPAIV). The HP H5 detection RRT-PCR assay specifically detects the presence of a MBCS, indicative of a HPAIV.
Detection of H5 HPAIV by RRT-PCR has been described (Naguib et al, 2017) with further development and validation performed by APHA and described in James et al (2022) to provide the HP H5 detection RRT-PCR assay described in the SOP.
This assay is a useful tool to obtain a rapid pathotyping result that pre-empts the need to wait for confirmation of high pathogenicity-status from Sanger CS sequencing. However, it does not replace the need for definitive Sanger sequencing confirmation. Note also that a negative result from this assay does not necessarily mean that the virus strain is a LPAIV.
1.2.3 The ‘H5 RRT-PCR’ (see Appendix 1) uses the same chemistry, thermal profile and quality processes as the ‘HP H5 RRT-PCR’ (see Appendix 2). The individual recipes are detailed in the respective appendices.
2. Safety
2.1 Areas within this procedure which refer to Safety Critical activities are denoted in the paragraph number column with the sign to highlight these areas to users.
3. Materials
3.1 Documentation and software
3.1.1 Testing of submission cases (e.g. OIE/FAO Reference Laboratory submissions or UK Report Cases) by this method are recorded in a lab book and registered folder. Investigations of a suspect H5 outbreak and associated screening work may require a distinct recording system with dedicated paperwork and / or electronic data recording. It is important to cross-reference any other related H5 investigative work, e.g. ongoing wild bird surveys (WBS) for AIV, amplification of the same H5 isolate by conventional H5 PCR for statutory cleavage site sequencing etc. Other uses of the H5 RRT-PCR in e.g. other AI research projects are recorded in the relevant lab book.
3.1.2 All scientists who are trained to perform RRT-PCRs on either Agilent Mx3000/5p series PCR systems or AriaMx platforms must be sufficiently familiar with the accompanying software in order to run an experiment. Again, all potential operators must be able to at least use the software to demonstrate completion of a valid competency panel, where they are able to identify a specimen as being positive or negative. This includes the ability to determine the fluorescence threshold value for a given RRT-PCR test after its completion, i.e. to check the vertical axis on the amplification plot graph presented as a logarithmic scale. Advice from a more experienced colleague may be sought in order to set fluorescence threshold by this means. Those empowered as test signatories must be able to use the software to conduct further data analysis. Please refer to instruction manual for Mx3000/5p PCR system and to the relevant SOP and/or manual for AriaMx system for further guidance if required.
3.1.3 Other procedures to be read in conjunction with this SOP: RNA extraction from biological samples.
Extraction of nucleic acids from swabs using the QIAGEN BioRobot Universal
Agilent AriaMX – operation of the Real-Time PCR System
Worksheet for Real Time plate layout
Worksheet for Real Time PCR mastermix preparation Instruction manual for MX3000/MX3005/AriaMx.
3.2 Chemicals and reagents
3.2.1 Primers & probe (note: degenerate nucleotides shown in bold).
Primers and Probe for Eurasian H5 HA2 RRT-PCR are:
H5LH1 ACA TAT GAC TAC CCA CAR TAT TCA G
H5RH1 AGA CCA GCT AYC ATG ATT GC
H5PRO FAM-TCW ACA GTG GCG AGT TCC CTA GCA-TAMRA
Primers and Probe for HP H5 detection RRT-PCR are:
2344b_H5_F3 CCT TGC GAC TGG GCT CAG
2344b_H5_R2 ATC AAC CAT TCC CTG CCA
2344b_DE_Sonde FAM-AGA AGA AAR AGA GGG CTG TTT GGG GCT-BHQ-1
3.2.2 RRT PCR master mix
Both assays utilise the Qiagen OneStep RT-PCR kit (Cat No. 210212). Reagents not contained in the kit can be obtained from other suppliers.
The specific recipes including volumes are given in
Appendix 1 Real-time reverse transcription PCR (RRT-PCR) for the detection of Eurasian H5 avian influenza
Appendix 2 Real-time reverse transcription PCR (RRT-PCR) for the detection of HPAIVs of H5 subtype
3.2.3 DEPC-treated water (i.e. Rnase-free) – Ambion or similar (x5) Qiagen 1 step RT-PCR buffer
Rox ref dye (pre-diluted 1/500 from Agilent stock in DEPC-water) – Agilent or similar
Qiagen dNTP mix
Forward primer – Eurogentec or similar Reverse primer – Eurogentec or similar
Probe – Eurogentec or similar
25mM magnesium chloride – Promega or similar RNAsin (40U/µl) – Promega or similar
Qiagen 1 step RT-PCR enzyme mix
3.2.4 RRT-PCR controls:
These fall into the following categories:
Negative:
No template control (NTC)
Positive: Only the initial Eurasian H5 HA2 AIV RRT-PCR assay requires a positive extraction control
Standards: Dilution series (ten-fold) of suitable H5 RNA. See Appendix 3 for preparation of new batches.
3.2.5 Negatives:
Include at least one “no template control” (NTC) where either 2l or 5l volume is used (depending on assay performed see appendices 1 & 2) RNase free water are added.
3.2.6 Include at least one negative extraction control where either 2l or 5l volume is used (depending on assay performed see appendices 1 & 2)
3.2.7 H5 extraction control (positive):
For the Eurasian H5 HA2 RRT-PCR, use e.g. an aliquot of inactivated A/chicken/Scotland/59 H5N1 from the Avian Virology repository. This serves as an RNA extraction control (manual or robotic). The Ct/Cq value of this extracted RNA has been predetermined earlier: This must be within the Ct/Cq range 26-32. This control is included in the same RNA extraction procedure alongside the test samples. Higher titre (i.e. lower Ct/Cq) controls may risk contaminating neighbouring reaction wells.
NB A positive control must be included for all clinical specimens where haemagglutination activity is unknown. However, No positive control is required for manual extractions from egg amplified material where positivity has been demonstrated by haemagglutination as these act as positive controls in their own right.
3.2.8 H5 AIV RNA dilution series (“standards”):
For the Eurasian H5 HA2 RRT-PCR, use an RNA preparation from extracted egg fluid as x10 fold dilution series (at least 4 dilutions) of e.g. A/chicken/Scotland/59 H5N1 or that available.
For the HP H5 detection RRT-PCR, it is noted that the above standard will not work. The standard to use for this assay needs to be a more contemporary isolate as the assay was developed to identify recently emerging clades. An example of an isolate to use for standard preparation is H5N8 A/chicken/England/030786/2020. As this virus is very similar to strains circulating in the United Kingdom, it may potentially be difficult to distinguish between this standard and field samples, the use of a synthetic virus standard control will be investigated as an alternative for future use.
This series (entered appropriately as “standards” with relative quantities indicated by using the software while setting up the RRT-PCR experiment) is used to determine the RRT-PCR efficiency of the assay. It is important to include weak positive H5 controls which correspond to approximately 10 EID50/ml and 100 EID50/ml i.e. approx. Ct/Cq value 36.00 of H5 AIV in the sample prior to RNA extraction – this will relate to defining the positive cut-off for both H5 RRT PCR assays.
3.2.9 Quantification of H5 positive controls:
For testing scenarios where a qualitative result is required, i.e. specimens from Report Cases, International Reference Laboratory (IRL) submissions and outbreak / screening investigations), both the H5 positive extraction control and the H5 specimen used to prepare the ten-fold RNA dilution series do not require accurate quantification by virus titre determination. However, for certain research settings it may be necessary to quantify both these H5 positive controls which will permit the H5 RRT-PCR results to be interpreted semi-quantitatively. Here classical virus titre determination in embryonated chickens’ eggs may be used.
3.3 Equipment
3.3.1 QIAGEN BioRobot Universal
-70⁰ or lower freezer Pipettes
Sterile Rnase-free pipette tips with aerosol barrier AriaMx Real-Time PCR system
Mx3000/5p series real time RT-PCR quantitative PCR system
QIAGEN BioRobot Universal and extraction kit
Mx3000 real time or AriaMx RT-PCR plates/strips Microcentrifuge tubes (1.5ml)
Microcentrifuge (with rotor for 2ml tubes) Vortex mixer
4. Procedure: method
4.1 Test reliability
4.1.1 Process of assuring ongoing test reliability:
A positive control (where used) and a standard dilution series are included in each extraction and PCR test. The Ct/Cq values obtained from the extraction control and standards must fall within the range stated in the results section below. If not, the reason behind an out of range result being obtained must be investigated. In the event of a test falling out of the required range, the samples are retested. When further failure occurs or a trend becomes apparent, an investigation of possible causes must be made.
The Ct/Cq value obtained from the positive extraction control and standards efficiency are compared over time to identify trends that may indicate poor performance of any component of the test. If a trend is identified its cause must be investigated.
RRT-PCR performance must be gauged by monitoring the (i) Ct/Cq value from the positive extraction control and the (ii) Ct/Cq values and standard curve obtained from the 10-fold dilution series (RNA standards) used to determine the efficiency of the RRT-PCR. This monitoring is crucial for the routine use of the AIV RRT-PCRs in Reference / Diagnostic Laboratory work. The MRM team and Test Consultant will meet at 6 monthly intervals to review the performance of these AIV RRT-PCRs where quantified positive extraction controls and RNA standards have been used. For the positive extraction control, an appropriate AIV specimen will have been quantified prior to extraction, and then used in an RRT-PCR run as (i) the positive extraction control and (ii) act as the template for the preparation of the 10-fold dilution series (RNA standards). The outcome of this 6-monthly meeting will be recorded by the MRM team in the registered lab-book which documents “Ongoing Validation” experiments. The quantified AIV preparation for use as extraction control/RNA standard may be obtained from work group colleagues using pre-determined quantified virus for research or other activity. This will correlate the observed Ct/Cq values for these samples in all the AIV RRT-PCRs with the viral titre, thereby monitoring both consistent test performance and fitness-for-purpose for the AIV RRT-PCRs.
The fitness-for-purpose (diagnostic sensitivity and specificity) of the H5 RRT-PCR assay is also monitored annually by the running of the test on the EU Proficiency Panel samples and extensive feedback on performance is provided in the official report following the completion of the panel by the participating laboratories. While the running of the HP H5 detection RRT-PCR assay is not currently requested in the EU Proficiency panel, the assay is used to test the panel samples for internal fitness-for-purpose monitoring and the results can be evaluated from the official report along with the H5 RRT-PCR results.
H5 HA2 RRT-PCR test validation document reference (APHA TV)
HP H5 detection RRT-PCR test validation document reference (APHA TV)
There is an uncertainty of measurement document covering molecular detection which must be read in conjunction with this SOP.
External quality assessment (EQA) is carried out through completion of annual ring trials circulated by the EU Reference Laboratory for Avian Influenza and Newcastle Disease (Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) () and OFFLU (CSIRO-AAHL, Australia). The results from both ring trials are disseminated externally for review.
Frequency:
In order to maintain competence for quality assurance, staff must perform the test once annually and complete a blind ring trial every two years. The results from any appropriate EQA schemes may also serve to ensure that individual competencies and the robustness of this test are being maintained.
The exception to this applies to staff that carry out this test on a regular basis and can provide evidence of on-going competency in accordance with ‘AD.085-Staff Competence - Training, Assessment and Recording’. Primers undergo batch-to-batch fit for purpose testing using known H5 AIV-positive RNA.
4.2 Preparation of PCR master mix and loading
4.2.1 Preparation of PCR master mix and loading of real-time plate/strip(s) to be carried out in the PCR clean room.
4.2.2 Make up master mix sufficient for the number of samples to be tested according to the assay required and by referring to relevant recipe in either Appendix 1 or Appendix 2 for the H5 HA2 or HP H5 detection RRT-PCR, respectively.
4.2.3 Loosely place the plate caps on the portion of the plate to be used.
4.2.4 Bring the plate/strip(s) out of the clean room and place in an ice rack or in the fridge before addition of sample RNA and controls to the master mix. Care should be taken to avoid the ice coming directly into contact with the plastic of the wells, this could have an adverse effect on the PCR background readings.
4.3 Preparation of AIV RNA standards
4.3.1 Thaw an aliquot of the appropriate standard for the assay to be performed (see 3.2.8) All new batches of AIV RNA to be used to prepare AIV RNA standards are first checked for fitness-for-purpose as described in Appendix 3.
4.3.2 AIV RNA is prepared in batches with a designated arbitrary value of 107 and stored at -70C. Take out an aliquot and thaw. Carefully prepare ten-fold dilutions of RNA in DEPC-water as follows:
Ten-fold dilution | Designated value (label tubes with these values) |
10-1 | 106 |
10-2 | 105 |
10-3 | 104 |
10-4 | 103 |
10-5 | 102 |
10-6 | 101 |
It does not matter whether, in the software, you label the standards 1e6 to 1e2 or 1e-1 to 1e-5. The efficiency will be calculated in the same way.
Mix each dilution thoroughly by e.g. triturate / agitation / flicking / vortex. Make sufficient of each dilution so that enough is made to use for all the RRT-PCR runs for that day. Store the aliquots on ice. Discard at the end of the working day.
4.3.3 Each standard to be run is indicated on the worksheet using the designated value.
4.4 Manual addition of samples and standards
4.4.1 In order to minimise the risk of contamination, the different types of controls and test specimens are added in the following chronological order. It is important to keep wells covered after addition of control / sample, and also to add in such a manner to minimise any carry-over contamination risk into open wells to which control / sample has not yet been added. Change gloves frequently and do not hold tips containing RNA above the incorrect wells.
Referring to plate layout, add DEPC-water as a no template control (NTC), of the negative extraction control, Replace caps on tubes. Then add the sample RNA to the master mix. Replace caps on tubes.
4.4.2 Add the extracted RNA samples as per worksheet layout. Fitting the caps back on to the wells as you go.
4.4.3 Finally add the RNA standards (from weakest to strongest) and positive RNA extraction control to appropriate wells based on your worksheet layout.
4.4.4 It is important that the caps are fitted firmly and correctly onto the wells before insertion into the real-time instrument.
4.4.5 If the real-time plate/strip(s) are not to be loaded into the instrument immediately, keep the plate/strip(s) on a rack on ice or in the fridge until ready to test.
4.5 Reverse transcription (RT) and PCR
4.5.1 Place the real time plate/strip(s) in the appropriate real time machine.
4.5.2 Note that the following thermocycling profile H5 RRT-PCR is identical for both the Eurasian H5 HA2 and HP H5 detection RRT-PCR. This enables both H5 RRT-PCR assays to be run simultaneously on the same plate if required.
RT step: 50°C for 30 mins
Activation Taq 95°C for 15 mins PCR step (x40 cycles) :
Denaturation 95°C for 10 secs
Annealing 54°C for 30 secs
Elongation 72°C for 10 secs
NB: Collect fluorescence data at end of 54°C step using the ROX and FAM filters. Rox is the reference dye. Use endpoint read option with 1 read.
4.5.3 If an AriaMx PCR system is used for the experiment refer to GP.035 Agilent AriaMX – operation of the Real-Time PCR System for set-up
4.5.4 If using a Mx3000/5p Real-Time system perform the following:
4.5.5 Note that the lamp on Mx 3000/5p real time machines requires 20 minutes to warm up. Open a new file on the machine and use the real-time quantitative PCR (multiple standards) option for the experiment. The thermoprofile and plate setup can be re-entered for each experiment or imported from previous experiments if desired.
In the MxPro software it is possible to change the ‘Filter Set Gain Settings’ – click Instrument, then filter Set Gain Settings.
For our use Cy5 Rox and Hex are all set to x1, Fam is set to x2.
After servicing or repair this setting is often changed back to the factory default of Fam x8 and must be corrected.
4.5.6 When the plate setup and thermoprofile windows have been entered, select run to start thermocycling. A storage window will automatically open. Save the file in an appropriate folder on the hard drive of the computer.
4.5.7 The complete run takes approximately 2 hours. If the machine is not being used by anyone else after you, select the option to turn the lamp off at the end of the run as the lamps have a limited lifespan.
5. Results
5.1 Analysis and display of results
5.1.1 If the experiment has been run using an AriaMx refer to the relevant SOP Agilent AriaMX – operation of the Real-Time PCR System for guidance on how to access the results and analyse the data.
5.1.2 If the experiment has been run using a Mx3000/5p system follow 5.1.3 to 5.2.6
5.1.3 The fluorescence data can be viewed during and / or after the H5 HA2 RRT- PCR using the raw data plots tab in the “Run” section.
The filter set gain setting multiplies up the amount of raw fluorescence seen by the machine, if the raw fluorescence is too high then the accuracy of the readings is reduced. Raw fluorescence above 35,000 is too high.
5.1.4 When the run is complete make a record of the file name and its storage location and save a backup of the file in a separate location.
5.1.5 To analyse the data select the Analysis section button and select the wells to be examined in the Analysis/Setup window.
5.1.6 To view the results click on the Results tab in the Analysis section and view the amplification plots. There are four options for analysing the fluorescence:
R (Multicomponent view) displays the raw fluorescence in arbitrary units.
dR displays the baseline-corrected fluorescence. As all reactions and wells will start with a slightly different fluorescence readings this option sets a baseline value of 0 to all plots. This correction is determined by the fluorescence values obtained during the initial rounds of the PCR. The adaptive baseline algorithm calculates the best baseline for each plot individually.
Rn displays the fluorescence normalised to the passive reference dye (ROX). This allows for fluctuations in fluorescence, which are not due to cleavage of the Taqman probe.
dRn displays the baseline-corrected normalised fluorescence.
The dRn option with the ROX channel switched on is the most appropriate option for analysis of the data.
5.2 Interpretation of results
5.2.1 Analyse the data by (i) first checking the fluorescence threshold cut-off (3.1.2) and (ii) comparing the results obtained for the negative (NTC) and H5-positive controls.
5.2.2 Negative controls (NTCs)
5.2.3 All NTCs must give “No Ct/Cq” as their final result. High Ct/Cqs in all NTC wells with a linear character (e.g. >38) & giving a very low level final fluorescence (i.e. little greater than the initial “flare” fluorescence values at early cycles) suggest that probe degradation may have occurred, e.g. the probe has been excessively frozen and thawed. Although such observations may not invalidate the experiment, it is advised to consider discarding the aliquot & thaw-out a fresh aliquot of the relevant H5 probe for subsequent experiments and note result.
5.2.4 If the late Ct/Cq value (>38) has a logarithmic / sigmoidal character where clear final fluorescence values are observed, then contamination of the NTC wells with H5 RNA may be considered. Such very late Ct/Cq signals may occasionally occur spuriously. Repeat the H5 HA2 or HP H5 detection RRT-PCR.
5.2.5 Positive controls, which include reference to extraction controls (and the RNA dilution series (i.e. “standards”).
5.2.6 H5 extraction control
Controls must reproducibly amplify with a Ct/Cq value +/- 2 Cts either side of their predetermined Ct/Cq value. Increases above the intended Ct/Cq value range suggest that the following might have occurred:
5.2.7 • RNA extraction from the control has been suboptimal. Repeat.
5.2.8 • The H5 positive control may have degraded if stored incorrectly
e.g. 4°C rather than frozen, excessive freeze-thawing etc.….
5.2.9 Deviation (+/- 2 Ct/Cqs) may occur where software settings on the instrument are inaccurate / inappropriate and the fluorescence cut-off level excessively high / low. It may be possible to reset the software after the RRT-PCR run to restore the expected Ct/Cq value provided the negative extraction and NTC controls, and the standard curve are in order. To re-analyse data refer to 5.1.2 for data generated on Mx3000/5p systems. For data generated on AriaMx systems, refer to GP.035. Otherwise consult with the Molecular Resource Manager or Test Consultant.
5.2.10 Construct a standard curve from the RNA standard Ct/Cq values. A minimum of three standard values is required to generate a standard curve i.e. up to a maximum of two standard values can be removed for the analysis.
The curve must comply with the following criteria:
Efficiency: >80% and <120%
Slope between –3.1 and –3.8
R2 value of the straight line: >0.980 H5 RNA dilution series “standards” (3.2.5, paragraph 4)
However, deviation from these conditions indicates a non-valid test. This may be due to a number of factors such as (i) inaccurate volume of RNA standard(s) / pipetting errors (ii) degradation of the RNA standards by inappropriate storage or (iii) inefficient thermocycling / fluorescence reading due to an instrument / electrical fault.
5.2.11 Criteria for (i) assessing results and (ii) decision making steps for any repeat testing of samples are as follows in both H5 and H5 Pathotyping RRT PCR assays:
The H5 RNA standard in the ten-fold dilution series which corresponds to a titre of approximately 10 EID50/ml typically has a Ct/Cq value in the range
36.00 in both RRT-PCRs, i.e. this is the positive/negative cut-off point. Hence any Ct/Cq below this value is clearly positive by either test while “No Ct/Cq” results are clearly negative in both cases.
“Indeterminate” results which amplify at a Ct/Cq value above the positive cut-off Ct/Cq value (36.00) can be resolved by:
5.2.12 1. Examine the H5 RNA standard dilution series: Note the Ct/Cq value of the control RNA extracted from a Eurasian H5 sample at a titre of approximately 10 EID50/ml. This should yield a Ct/Cq value in the range 34 – 37 by both H5 and HP H5 detection RRT-PCRs.
5.2.13 2. Samples with Ct/Cq values >36 in the H5 or HP H5 detection RRT-PCR, but with a logarithmic / sigmoidal character giving clear final fluorescence greater than that observed during early cycles: These are generally considered as “inconclusive”, however, strategies for confirmation as either “H5 [very low] positive” or “H5 negative”
5.2.14 3. Repeat testing of extracted RNA:
Simply repeat the same H5 HA2 or HP H5 detection RRT-PCR test with the same original RNA samples in duplicate and compare to the corresponding M gene RRT-PCR result(s). If the repeat result is “No Ct/Cq” by all repeat tests, then it is highly likely that the original inconclusive result may have been spurious, or possibly due to a low level of contamination. However, if the same inconclusive result is observed upon repeat testing of the original extracted RNA, then proceed to re-extraction of the clinical specimen for further testing:
5.2.15 4. Re-extraction of RNA:
Check the Ct/Cq value for the (i) original and (ii) re-extracted RNA by the M gene RRT-PCR: A Ct/Cq value of 34-37 by M gene RRT--PCR suggests that this may be a very weak positive H5 or H5 Pathotyping RRT-PCR result. A reproducible result obtained after re-extraction of RNA from the same clinical specimen by H5 HA2/ HP H5 detection / M gene RRT-PCRs tends to confirm this. It is possible that such consistent inconclusive results may e.g. be due to the presence of a RRT-PCR inhibitor in a low AIV titre clinical specimen, which cannot be removed by repeated RNA extraction. It is unlikely that such “late Ct/Cq risers” (particularly those from field submissions) would yield positive results by other tests e.g. virus isolation or conventional H5 PCRs due to their lower sensitivity. However, it is also possible that removal of an inhibitor by re-extraction of the clinical specimen may reduce the Ct/Cq values sufficiently to provide a clear positive RRT-PCR result. If possible, it is recommended to conduct all such repeat testing of both the original (paragraph 3 above) and re-extracted RNA in duplicate in the same AIV RRT-PCR experiment.
5.2.16 However, if H5 HA2 / HP H5 detection / M gene RRT-PCR testing of re-extracted RNA (each test conducted in duplicate) yields a “No Ct/Cq” result then such specimens are considered negative.
5.2.17 Other considerations for the H5 and/or HP H5 detection RRT-PCRs:
The Eurasian H5 HA2 RRT-PCR was designed & validated to detect currently-circulating Eurasian H5 isolates. However, there is some evidence that certain American lineage H5s may be detected by this procedure. However, it must be stressed that detection of American H5 isolates by this Eurasian H5 HA2 RRT-PCR has not been validated.
Primer & probe design for Eurasian H5 isolates:
As these viruses continue to evolve, it is possible that new isolates will emerge which will be less efficiently detected by the above method, or even not detected at all. Any further revision of primer / probe sequences may be necessary as H5 AIVs continue to evolve.
5.2.18 Results for both RRT-PCR assays are currently reported to the customer on a submission-basis either on the official report released on LIMS via the ‘AND DP Outbreak’ (statutory notifiable avian disease investigations) portal, ‘AI Wildlife’ portal (wild bird surveillance) or via an official (non-LIMS) report released through the OIE/FAO International Reference Laboratory for Avian Influenza.
6. References
Spackman, E., Senne, D.A, Myers, T.J, Bulaga, L.L., Garber, L.P.,
Perdue, M.L., Lohman, K., Daum, L.T., Suarez, D.L, 2002. Development of a real time reverse transcriptase PCR for type A influenza virus and the avian H5 and H7 haemagglutination subtypes. J. Clin. Microbiol. 40: 3256-3260.
Slomka, M. J., Pavlidis, T., Banks, J., Shell, W., McNally, A., Essen, S., Brown, I. H., 2007. Validated H5 Eurasian real-time reverse transcriptase–polymerase chain reaction and its application in H5N1 outbreaks in 2005–2006. Avian Dis. 51, 373-377.
Naguib, M.M., Graaf, A., Fortin, A., Luttermann, C., Wernery, U., Amarin, N., Hussein, H.A., Sultan, H., Al Adhadh, B., Hassan, M.K., Beer, M., Monne, I. and Harder, T.C., 2017. Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017. Euro Surveill 22, 30435.
James, J., Seekings, A.H., Skinner, P., Purchase, K., Mahmood, S., Brown, I.H., Hansen, R.D.E., Banyard, A.C. and Reid, S.M. Rapid and sensitive detection of high pathogenicity Eurasian clade 2.3.4.4b avian influenza viruses in wild birds and poultry. J Virol Methods. 2022 Jan 6;301:114454. doi: 10.1016/j.jviromet.2022.114454. Epub ahead of print. PMID: 34998830.
Appendix 1
Testing new batches of reagents
New batches of probe and primers are tested when they are first re-suspended. Probe and primers are aliquoted once re-suspended to avoid repeated freeze thawing.
Test new reagents in the same real time PCR as the current reagents using the positive standards and NTC according to this SOP. If the new reagents are fit for purpose both sets of reagents give equivalent results.
Appendix 2
Preparation of TaqMan® real-time RT-PCR positive extraction controls and RNA standards
These can be prepared from either live or inactivated egg-grown antigen and can be supplied either as an aliquot of freeze-dried material or an aliquot from a wet ‘antigen stock.
NB - For live antigen follow safety procedures in place for the virus type being produced. Work at the appropriate containment level for the virus being used – refer to the virus categorisation risk document
To prepare positive extraction controls
1. If the antigen is supplied freeze-dried, reconstitute the vial with 1ml molecular grade water. Ensure the solution is mixed thoroughly by gentle vortexing.
2. Prepare a 10-fold dilution series of the antigen in PBS e.g. from 10-1 - 10-6 dilutions inclusive.
3. Freeze 200 µl aliquots of each dilution at -70 ºC.
4. Thaw-out an aliquot of each dilution and extract RNA from each aliquot either manually or utilising bio-robots.
5. Test the RNA from each member of the dilution series by the appropriate real-time PCR assay. Choose the optimal 10-fold dilution from the resulting Ct values. The acceptable Ct/Cq value range is documented in the relevant SOP for the real-time PCR assay used.
6. Prepare the chosen dilution of the antigen in bulk using PBS, assign a batch number, dispense into 200µl aliquots, label and freeze at -70 ºC.
7. Remove an aliquot from the freezer, extract the RNA and test by the appropriate real-time RT-PCR to confirm the Ct/Cq values from the dilution fall within the required range.
8. Record full preparation details in the Reagent Buffers Preparation Logbook
To prepare RNA PCR standards
1. If the antigen is supplied freeze–dried, reconstitute the vial with 1ml molecular grade water. Ensure the solution is mixed thoroughly by gentle vortexing.
2. Manually extract the RNA from the whole reconstituted antigen from 1ml of wet stock according to the relevant SOP, RNA extraction from biological samples.
3. Pool together the RNA extractions.
4. Freeze a 5µl aliquot at-70 ºC
5. Thaw this aliquot and prepare a 10-fold dilution series in molecular grade water from as described above from 10-1 - 10-7
6. Test the dilution series by the appropriate real-time RT-PCR. Test the current ‘in-use’ RNA standard batch in parallel on the same PCR test run.
7. Directly compare the results of the two batches. The Ct values and test efficiencies must be similar i.e. Ct/Cq values +/- 2.0 per corresponding dilution. If there is an apparent significant difference, adjust the dilution of the new batch of RNA (e.g. make a 1/10 dilution) and repeat stages 3-7 above.
8. Once the required dilution is achieved, prepare 5µl aliquots, assign a batch number, label the tubes and store at -70 ºC.
9. Record full preparation details in a Reagent Buffers Preparation Logbook
To prepare AIV RNA standards, divide the standards into aliquots for frozen storage. When a new batch of AIV RNA is taken to prepare standards for the first time, an aliquot is thawed and used to prepare a 10-fold dilution series. This is tested in the same H5 (HA2) RRT-PCR protocol alongside the previous AIV RNA batch, each as a 10-fold dilution series. Each AIV RNA batch is assessed for fitness-for-purpose.
Appendix 3
Real-time reverse transcription PCR (RRT-PCR) for the detection of Eurasian H5 avian influenza virus
Primers and probes
Oligo
Sequence (5- 3)
Forward primer H5LH1 ACA TAT GAC TAC CCA CAR TAT TCA G
Reverse primer H5RH1 AGA CCA GCT AYC ATG ATT GC
H5PRO FAM-TCW ACA GTG GCG AGT TCC CTA GCA-TAMRA
RT-qPCR reaction mix
Ingredients – all except primers & probe from Qiagen OneStep RT-PCR kit (cat # 210212) & other stated suppliers Volumes for 1x reaction recipe Volumes for 10x reaction recipe
DEPC-treated (i.e. RNAse-free)-H2O (Ambion [Thermofisher] or similar) 13.75 µl 137.5 µl
(x5) Qiagen 1-step RT PCR buffer 5 µl 50 µl
ROX ref dye, pre-diluted to 1:500 in DEPC-treated (RNase-
free)-H2O from neat stock (Agilent or similar): 0.375 µl 3.75 µl
Qiagen dNTP mix 1 µl 10µl
H5RH1 (50µM) 0.2 µl 2µl
H5LH1 (50µM) 0.2 µl 2 µl
H5Pro (30µM) 0.25 µl 2.5 µl
25mM MgCl2 (Promega or similar) 1.25 µl 12.5 µl
RNAsin (40U/µl, Promega or similar) 0.1 µl 1 µl
Qiagen 1-step RT-PCR enzyme mix 1 µl 10 µl
Total: 23 µl 230 µl
Thoroughly mix the master mix and centrifuge for 30 seconds to remove bubbles. And
aliquot 23 ul of each reaction mix, add 2ul of RNA to each reaction mix.
RRT-PCR Cycling conditions
HP H5 detection conditions*
Step Temp (⁰ C)
Time Cycles
RT 50 30min 1
Activation Taq 95 15min 1
Denaturation 95 10sec
40
Annealing 54 30sec
Elongation 72 10sec
Collect fluorescence data at end of 54ºC step using the ROX and FAM filters. Use Rox as the reference dye
Appendix 4
Real-time reverse transcription PCR (RRT-PCR) for the detection of
HPAIVs of H5 subtype
Primers and Probes
Oligo
Sequence (5- 3) Reaction conc. (µM)
2344b_H5_F3 CCT TGC GAC TGG GCT CAG 20
2344b_H5_R2 ATC AAC CAT TCC CTG CCA 20
2344b_DE_Sonde FAM-AGA AGA AAR AGA GGG CTG TTT GGG GCT-BHQ-1 5
RT-qPCR reaction mix
Ingredients – all except primers & probe from Qiagen OneStep RT-PCR kit (cat # 210212) & other stated suppliers Volumes for 1x reaction recipe Final concentration / strength
DEPC-treated (i.e. RNAse free)- H2O (Ambion [Thermofisher] or similar) 10.375 µl -
(x5) Qiagen 1-step RT PCR buffer 5 µl 1x
ROX ref dye, pre-diluted to 1:500 in DEPC-treated (RNase-
free)-H2O from neat stock (Agilent or similar): 0.375 µl 1x
Qiagen dNTP mix 1 µl 1x
2344b_H5_F3 (50µM) 0.4 µl 20 µM
2344b_H5_R2 (50µM) 0.4 µl 20 µM
2344b_DE_Sonde (50µM) 0.1 µl 5 µM
25mM MgCl2 (Promega or similar) 1.25 µl 3.75 mM
RNAsin (40U/µl, Promega or similar) 0.1 µl 0.16 U/µl
Qiagen 1-step RT-PCR enzyme mix 1 µl 1x
Total: 20 µl -
Thoroughly mix the master mix and centrifuge for 30 seconds to remove bubbles and
.aliquot 20 ul of each reaction mix, the add 5ul of RNA to each reaction mix.
RRT-PCR Cycling conditions
HP H5 detection conditions*
Step Temp (⁰C) Time Cycles
RT 50 30min 1
Activation Taq 95 15min 1
Denaturation 95 10sec
40
Annealing 54 30sec
Elongation 72 10sec
*Same conditions as the H5 HA2 RRT-PCR, collect fluorescence data at end of 54ºC step using the ROX and FAM filters.