H6 (HA2) avian influenza virus real time PCR
This protocol is a copy of the standard operating procedure used by the WOAH and FAO International Reference laboratory for avian influenza at the Animal and Plant Health Agency. If you have any technical queries, please contact AIWRL@apha.gov.uk
1. Introduction
1.1 Purpose and scope of this SOP
1.1.1 | This SOP provides the details required to detect H6 avian influenza virus (AIV) by real-time reverse transcriptase (RRT)-PCR. |
1.2 Background information
1.2.1 | This is a rapid, sensitive and specific method for the detection of European H6 AIV isolates. Validation has revealed it to be effective in detecting H6 AIV directly from clinical specimens, i.e. there is no need to first isolate the suspect H6 isolate in embryonated eggs by growth to a high titre (biological amplification). |
1.2.2 | For the H6 (HA2) RRT-PCR described in this SOP, haemagglutinin gene sequences from European H6 virus isolates (mainly 2009-2020) were aligned. Primers and probe were designed from conserved regions within the haemagglutinin 2 (HA2) gene region for specific detection of currently-circulating AIV H6 subtypes. |
2. Safety
2.1 | It is the responsibility of the individual laboratory to ensure all work described in this protocol is conducted to a high safety standard. This includes an awareness of risks relating to e.g. dangerous or toxic chemicals, potentially hazardous procedures etc. Here local safety rules in your laboratory should be understood by all relevant members of staff. |
3. Materials
3.1 Documentation and software
3.1.1 | This procedure refers to the Mx3000 series and AriaMx real-time machines. Use relevant guidance for the machines to be used in the individual laboratory if different from above. |
3.2 Chemicals and reagents
3.2.1 | Primers & probe (note: degenerate nucleotides shown in bold). Forward primer H6-1569F AAC AGG CAG GAA ATA GAA T
Reverse primer H6-1642R ACC GTA CTA TAA ATA GCA AG |
| Probe H6-1602Pro AAT CTT GGT GTG TAT CAA AT NB – New batches of primers and probes should be tested as described in Appendix 1 |
3.2.2 | H6 (HA2) RRT-PCR master mix This utilises the Quantifast Probe RT-PCR +ROX Vial Kit (400) (Cat. No. 204554). Volumes indicated below will be sufficient for 10 x 25µl reactions, i.e. divide the master mix into 10 x 23µl volumes & add 2µl extracted RNA to each. (X2) QuantiFast Probe RT-PCR Master Mix (without ROX)
QuantFast RT mix (x50) ROX Dye Solution (supplied in kit) DEPC-treated water Ambion or similar |
3.2.3 | H6 (HA2) RRT-PCR controls: These fall into the following categories: Negatives ie “No template controls” (NTCs) H6 positive extraction controls Dilution series (ten-fold) of known H6 RNA ie RNA “Standards” See Appendix 2 for preparation of new batches. |
3.2.4 | 1. Negatives: Include at least one “no template control” (NTC) where 2µl RNase free water are added. |
3.2.5 | 2. H6 extraction control (positive): For the H6 (HA2) RRT-PCR, use eg an aliquot of a known UK/ European H6 isolate. This should serve as an RNA extraction control (manual or robotic). The Ct/Cq value of this extracted RNA has been predetermined earlier: This should 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 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 themselves. A positive control must be included for all clinical specimens where haemagglutination activity is unknown. Ideally use in duplicate for each H6 RRT-PCR experiment, although for high sample volume work (eg during an outbreak) one H6 positive extraction control will suffice. Enter as “POS FAM” during experimental set-up using the software on the RRT-PCR instrument. |
3.2.6 | 3. H6 RNA dilution series (“standards”): For the H6 (HA2) RRT-PCR, use an RNA preparation from extracted egg fluid as x10 fold dilution series (at least 4 dilutions) of a known H6 isolate. Ideally this should be a duplicate dilution series, although in outbreak investigations a high volume of samples may necessitate use of single replicates. This series (entered appropriately as “standards” with relative quantities indicated by using the software while setting up the RRT-PCR experiment) can be used to determine the H6 (HA2) RRT-PCR efficiency. It is important to include low positive H6 controls which correspond to approximately 10 EID50/ml and 100 EID50/ml i.e. approx. Ct/Cq 36 of H5 AIV in the sample prior to RNA extraction – this will relate to defining the RRT PCR positive cut-off, where the corresponding Ct value may differ in the HA2 and CS versions (sections 5.2.5 and 5.2.6, below). To prepare AIV RNA standards the AIV RNA is divided into aliquots for frozen storage. When a new batch of AIV RNA is to be used 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 H6 (HA2) RRT-PCR protocol alongside the previous AIV RNA batch, each as a 10-fold dilution series. |
4. Procedure and method
4.1 Preparation of PCR master mix and loading
4.1.1 | Preparation of PCR master mix and loading of real time plate/strip(s) to be carried out in the PCR clean room. |
4.1.2 | Make up master mix sufficient for the number of samples to be tested. |
4.1.3 | H6 (HA2) RRT PCR master mix This utilises the Quantifast Probe RT-PCR +ROX Vial Kit (400) (Cat. No. 204554). Volumes indicated below will be sufficient for 10 x 25µl reactions, i.e. divide the master mix into 10 x 23µl volumes and add 2µl extracted RNA to each. |
4.1.4
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4.1.5 Thoroughly mix the master mix and centrifuge for 30 seconds to remove bubbles.
4.1.6 Aliquot 23µl of master mix per well of the real time plate/strip(s).
4.1.7 Loosely place the plate caps on the portion of the plate to be used. All wells will need to be covered even though only a portion of the plate may be used.
4.1.8 Bring the plate/strip(s) out of the clean room and place on ice before addition of sample RNA and controls.
4.2 Preparation of H6 RNA controls
4.2.1 | H6 positive controls are as described above in 3.2.3. |
4.2.2 | Extract H6 RNA as per the manufacturer’s instructions. |
4.2.3 | Include H6 positive controls in each H6 RRT-PCR experiment. |
4.3 Manual addition of samples and standards
4.3.1 | In order to minimise the risk of contamination, the different types of controls and test specimens should be 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 2µl DEPC-water as a no template control (NTC), 2µl of the negative extraction control, Replace caps on tubes. Then add the sample RNA to the master mix. Replace caps on tubes. |
4.3.2 | Add the RRT-PCR RNA standards (from weakest to strongest) and positive RNA extraction control to appropriate wells based on your worksheet layout. |
4.3.3 | Once RNA is added, fit caps to all wells. It is important that the caps are fitted firmly and correctly onto the wells before insertion into the real-time instrument. |
4.3.4 | If the real-time plate/strip(s) are not to be loaded into the instrument immediately, keep the plate/strip(s) on ice until ready to test |
4.4 Reverse transcription (RT) and PCR
4.4.1 | Place the real time plate/strip(s) in the appropriate real-time machine. |
4.4.2 | Run the following thermocycling profile: RT step: 50°C for 10 mins Annealing step: 95°C for 5 mins PCR step (x40 cycles) : 95°C for 10 secs 50°C for 15 secs 72°C for 15 secs NB: Collect fluorescence data at end of 50°C step using the ROX and FAM filters. Use endpoint read option with 1 read. |
4.4.3 | Open a new file using the instrument software to define all the necessary RRT-PCR experimental parameters. This is conducted according to the manufacturer’s software instructions. Name the H6 RRT-PCR file appropriately. |
4.4.4 | The complete run takes approximately 1 hr 23 mins. 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 | Conduct this after familiarising yourself with your in-house RT-PCR software and instructions. |
5.2 Interpretation of results
5.2.1 | Analyse the data by comparing the results obtained for the negative (NTCs) and H6 positive controls. 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 H6-positive controls. |
5.2.2 | Negative controls (NTCs) All NTCs should give “No Ct/Cq” as their final result. High Ct/Cqs in all NTC wells with a linear character (eg >38) & giving a very low level final fluorescence (ie 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 discard the aliquot & thaw-out a fresh aliquot of the relevant H6 probe for subsequent experiments and note result. 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 H7 RNA may be considered. It is also possible that such very late Ct/Cq signals may occasionally occur spuriously. Repeat the H6 (HA2) RRT-PCR experiment. |
5.2.3 | Positive controls, which include reference to extraction controls (and the RNA dilution series (i.e. “standards”). H6 virus extraction control Quantified specimens should reproducibly amplify with a Ct/Cq value +/-2 Ct/Cqs either side of their predetermined Ct/Cq value. Greater increases above the predetermined Ct/Cq value suggest that: RNA extraction from the specimens has been suboptimal. Repeat. It is also possible that the H6 positive control may have degraded if stored incorrectly eg 4°C, excessive freeze-thawing etc. Deviation (+/- 2 Ct/Cq) may also occur where software settings on the instrument are inaccurate / inappropriate and the fluorescence cut-off level excessively high / low & is affecting the Ct/Cq value of the predetermined H6 positive control. It may be possible to reset the software after the experiment to restore the expected Ct/Cq value provided the other controls are in order. |
5.2.4 | Construct a standard curve with the positive standards Ct/Cq values. This should include a minimum of three standards on the straight line with respect to providing qualitative data e.g. flock level diagnostic results, namely infected / uninfected. If individual bird level results are required the minimum should be four standards on the straight line. The curve should comply with the following criteria: Efficiency: >80% and <120%=0’ 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 eg (i) inaccurate RNA addition (ii) degradation of the RNA standards by inappropriate storage or (iii) inefficient thermocycling / fluorescence reading due to an instrument problem. This indicates a need to address these matters for subsequent H6 (HA2) RRT-PCR experiments. If conducting research work where a semi-quantitative result is required, then the use of more accurately quantified H6 RNA standards may be merited as regards the requirements of the research project (see 3.2.5, paragraph 5). |
5.2.5 | Criteria for (i) assessing results and (ii) decision making steps for any repeat testing of samples are as follows in the H6 (HA2) RRT PCR: The H6 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 32-35 in the H5 RRT-PCR, i.e. this is the H6 positive cut-off. Hence any Ct/Cq below this value is clearly H6 positive, while “No Ct/Cq” results are clearly H6 RNA negative. The five paragraphs below outline how to resolve “indeterminate” results which amplify at a Ct/Cq value above that of the H6 positive cut-off Ct/Cq value. This involves (i) retesting of the original extracted RNA by H6 (HA2) RRT-PCR and may also include M-gene RRT-PCR, and (ii) re-extraction of the clinical specimen to obtain fresh RNA for testing. 1. Examine the H6 RNA dilution series (standards): Note the Ct/Cq value of the control RNA extracted from a H6 sample at a titre of approximately 10 EID50/ml (average Ct/Cq value if tested in duplicate) – refer to section 3.2.5 paragraph 4 (above). This should yield a Ct/Cq value in the range 34 – 37. In this example, where Ct/Cq value of 36 is obtained from the 10 EID50/ml RNA control, a Ct < 36 in the H6 (HA2) RRT-PCR is considered clearly H6 positive, while a specimen recording “No Ct/Cq” value is clearly negative. 2. Samples with Ct/Cq values >36 in the H6 (HA2) 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 the following three paragraphs present possible strategies for confirmation as either “H6 [very low] positive” or “H6 negative”: 3. Repeat testing of extracted RNA: Simply repeat the same H6 (HA2) RRT-PCR test with the same original RNA samples in duplicate and compare to the corresponding M-gene RRT-PCR result(s) (SOP VIR 0493). 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 again upon repeat testing of the original extracted RNA, then proceed to re-extraction of the clinical specimen for further testing: 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 H6 (HA2) RRT-PCR result. A reproducible result obtained after re-extraction of RNA from the same clinical specimen by H6 (HA2)/ M-gene RRT-PCRs would tend 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, and this inhibitor 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 eg virus isolation or conventional H6 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. However, if H6 (HA2) / 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. |
5.2.6 | All H6 positive results from a potential new cases of AI are repeated and for an index case should be confirmed by use of virus isolation in accord with current OIE/EU guidelines. For the latter, conventional H6 PCR and sequencing before reporting is crucial in order to confirm the H6 subtype of detected AIV. |
5.2.7 | Other considerations for the H6 (HA2) RRT-PCR: The H6 (HA2) RRT-PCR primers and probes were designed from conserved regions within the haemagglutinin (HA) 2 gene region for specific detection of currently-circulating AIV H6 subtypes. The performance of this assay was evaluated in parallel with the established screening RRT-PCR for generic detection of AIV (Nagy et al., 2010). Primer & probe design for European H6 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 H6 AIVs continue to evolve. |
6. References
6.1.1 | 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 trancriptase PCR for type A influenza virus and the avian H5 and H7 haemagglutination subtypes. J. Clin. Microbiol. 40: 3256-3260. |
6.1.2 | Hoffmann, B., Hoffmann, D., Henritzi, D., Beer, M., & Harder, T. C., (2016). Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses. Scientific reports, 6, 27211. |
6.1.3 | Nagy, A., Vostinakova, V.,Pirchanova, Z., Cernikova,L., Dirbakova, Z., Mojzis, M., Jirinkova, H., Havlikova, M., Dan, A., Ursu, K., Vilcek, S. and Hornickova, J., (2010). Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species. Arch. Virol. 155: 665-673. |
Appendix 1
Testing new batches of reagents
New batches of probe and primers should be tested when they are first re-suspended. Probe and primers should be 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 should 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 on the BioRobot according to procedure RNA extraction from biological samples or extraction of nucleic acids from swabs using the Qiagen BioRobot Universal.
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/Cq 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 RRT-PCR to confirm the Ct/Cq values from the dilution fall within the required range.
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.
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 RRT-PCR. Test the current ‘in-use’ RNA standard batch in parallel on the same PCR test run.
7. Directly compare the results of the 2 batches. The Ct/Cq values and test efficiencies should 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.