Eurasian H7 avian influenza real time RT-PCR
This protocol is a copy of the standard operating procedure used by the WOAH/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
OIE/FAO international reference laboratory for AI
SOP: VI.536 Edition 11 (02/10/2018)
1. Introduction
1.1 Purpose and scope of this protocol
1.1.1 This protocol provides the details required to (i) detect Eurasian isolates of H7 avian influenza virus (AIV) by RealTime reverse transcriptase (RRT) PCR and also to (ii) pathotype any H7 positive specimen by amplicon sequencing.
1.2 Background information
1.2.1 Two H7 RRT PCRs are presented in this document. Both are rapid, sensitive and specific methods for detection of Eurasian H7 AIV isolates (Slomka et al 2009). In brief, the two H7 RRT PCR methods differ as follows:
1.2.2 “HA2” H7 RRT PCR: Validation studies at APHA have shown this to be the more sensitive of the two H7 detection methods, amplifying a product within the HA2 region of the H7 gene. Detection utilises a H7-specific hydrolysis (“TaqMan”) probe with fluorescence emission in RealTime.
1.2.3 Cleavage Site (“CS”) H7 RRT PCR: While validation studies have shown that this may be the less-sensitive of the two H7 RRT PCRs, this method amplifies a product which spans the H7 gene cleavage site (CS) region. Sequencing of this H7 CS amplicon allows for successful pathotyping of the H7 AIV as either low pathogenic (LP) or highly pathogenic avian influenza (HPAI).
1.2.4 Validation at APHA has revealed both H7 RRT PCR approaches to be effective in detecting H7 AI virus directly from clinical specimens,(a minimum of 0.5g of clinical material, 0.2ml of amplified virus or a single swab) ie there is no need to first isolate the suspect H7 isolate in embryonated eggs by growth to a high titre (biological amplification, see SOP VI.444). H7 RRT PCR has been described previously (Spackman et al, 2002), but here primer and probe design was tailored for the detection of N American H7 AI virus isolates.
1.2.5 For the methods described in this protocol, haemaglutinnin (HA) gene sequences from Eurasian H7 isolates (mainly 1995-2005) were aligned. This served to optimise primer and probe design for the detection of Eurasian H7 AIV isolates. The HA2 H7 RRT PCR was designed at APHA, while the CS H7 RRT PCR was designed by Mr John Voermans and Dr Guus Koch at CIDC-Lelystad in the Netherlands.
1.2.6 In this protocol there is considerable commonality for both the HA2 and CS H7 RRT PCRs as regards Materials (section 3), Procedure / Method (section 4) and Results (section 5). However, it is important to note the differences between these two H7 RRT PCRs as regards (i) Chemicals and Reagents (3.2) for primers and probes, and (ii) the results interpretation guide for the two methods, ie sub-sections 5.2.5 and 5.2.6.
1.2.7 In addition, the CS H7 RRT PCR provides the additional opportunity to pathotype by amplicon sequencing as stated in Procedure / Method (section 4.7). Examination of this H7 CS amplicon sequence will determine whether the H7 specimen is low pathogenic (LP) or highly pathogenic avian influenza (HPAI).
2. Safety
2.1 It is your laboratory’s responsibility 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 Worksheet for RealTime plate layout.
Instruction manual for RealTime PCR platform and software for in-house use in your laboratory.
3.2 Chemicals and reagents
3.2.1 “HA2” H7 RRT PCR primers and probe
LH6H7 5'-GGC CAG TAT TAG AAA CAA CAC CTA TGA-3'
RH4H7 5'-GCC CCG AAG CTA AAC CAA AGT AT-3'
H7pro11 5'-FAM-CCG CTG CTT AGT TTG ACT GGG TCA ATC T-BHQ-3'
3.2.2 “CS” H7 RRT PCR primers and probe
H7F 5’-CGT GCA AGT TTT CTG AGA GG-3’
H7R 5’-GAC CTT CCC ATC CAT TTT CA-3’
H7-TM1 5’-FAM-AAC CCG CTA TGG CAC CAA ATA GGC CTC-BHQ-3’
3.2.3 HA2 H7 RRT PCR master mix
DEPC treated water (ie RNAse-free) Ambion or similar
(x5) Qiagen 1 step RT PCR buffer
Rox ref dye (pre-diluted 1 in 500 from Stratagene or similar Stratagene stock in DEPC water)
Qiagen dNTP mix
LH6H7 (50µM) Sigma or similar
RH4H7 (50µM) Sigma or similar
H7pro11 (30µM) Sigma or similar
25mM magnesium chloride Promega or similar
RNAsin (40U/µl) Promega or similar
Qiagen 1 stepRT PCR enzyme mix
3.2.4 CS H7 RRT PCR master mix
DEPC treated water (ie RNAse-free) Ambion or similar
(x5) Qiagen 1 step RT PCR buffer
Rox ref dye (pre-diluted 1 in 500 from Stratagene or similar Stratagene stock in DEPC water)
Qiagen dNTP mix
H7F (50µM) Sigma or similar
H7R (50µM) Sigma or similar
H7-TM1 (30µM) Sigma or similar
25mM magnesium chloride Promega or similar
RNAsin (40U/µl) Promega or similar
Qiagen 1 stepRT PCR enzyme mix
3.2.5 H7 controls:
1. Negatives:
Include at least one “no template control” (NTC) where 2µl RNase free water are added.
2. H7 extraction control (positive):
A Eurasian H7 AIV specimen eg aliquot of inactivated A/African Starling/Eng/983/79 H7N1 from the Avian Virology repository. This should serve as an RNA extraction control (manual or robotic). The should be within the Ct range 26-32. This control is included in the same RNA extraction procedure alongside the test samples. Higher titre (ie lower Ct) 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 H7 RRT PCR experiment, although for high sample volume work (eg during an outbreak) one H7 positive extraction control will suffice. Enter as “POS FAM” during experimental set-up using the software on the RealTime instrument. See Appendix one for the preparation of new batches.
3. There are situations where RNA extraction will be followed by up to three AIV RRT-PCR tests, namely M gene, H5 and H7, eg for Reference Lab or Report Case submissions, or possibly at the early stage of an outbreak to help fully characterise the index case. Here the RNA extraction control may be an H5 AIV isolate, and provided that this has given satisfactory results in the H5 and / or M gene RRT-PCR, then there is no need to include a H7 positive extraction control.
4. H7 RNA dilution series (“standards”):
A Eurasian H7 AIV RNA preparation from extracted egg fluid as x10 fold dilution series (at least 4 dilutions) eg A/African Starling/Eng/983/79 H7N1. 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 H7 RRT PCR efficiency. It is important to include low positive H7 controls which correspond to approximately 10 EID50/ml and 100 EID50/ml or approx. ct 36 of H7 AIV in the sample prior to RNA extraction – this will relate to defining the H7 RRT PCR positive cutoff, where the corresponding Ct value may differ in the HA2 and CS versions (sections 5.2.5 and 5.2.6, below). See Appendix 1 for preparation of new batches.
5. Quantification of H7 positive controls:
For testing scenarios where a qualitative result is required, ie specimens from Report Cases, Reference Laboratory submissions and outbreak / screening investigations), both the H7 AIV positive extraction control (paragraph 2 above) and the H7 specimen used to prepare the ten-fold RNA dilution series (standards, paragraph 4 above) do not require accurate quantification by virus titre determination. However, for certain research settings it may be necessary to quantify both these H7 AIV positive controls which will permit the H7 AIV RRT PCR results to be interpreted semiquantitatively. Here classical virus titre determination in embryonating chickens’ eggs (SOP VI.444) may be used. Alternatively, a dilution series of H7 RNA standards may be calibrated by M-gene RRT PCR, but this will require comparison to AIV RNA standards derived from an AIV specimen of known titre.
4. Procedure and method
4.1 Preparation of PCR master mix and loading
4.1.1 Preparation of PCR master mix and loading of RRT 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.
HA2 H7 RRT PCR master mix
This utilises the Qiagen OneStep RT-PCR kit (Cat No. 210212). Reagents not contained in the kit can be obtained from other suppliers. Volumes indicated below will be sufficient for 10 x 25µl reactions, ie divide the Master Mix into 10 x 23µl volumes & add 2µl extracted RNA to each.
DEPC treated water (ie RNAse-free) 139µl
(x5) Qiagen 1 step RT PCR buffer 50µl
Rox ref dye (pre-diluted 1 in 500 from Stratagene stock in DEPC water) 3.75µl
Qiagen dNTP mix 10µl
LH6H7 (50µM) 2µl
RH4H7 (50µM) 2µl
H7pro11 (30µM) 1.25µl
25mM magnesium chloride 12.5µl
RNAsin (40U/µl) 1µl
Qiagen 1 stepRT PCR enzyme mix 10µl
CS H7 RRT PCR master mix
This utilises the Qiagen OneStep RT-PCR kit as described above (3.2.3) for the HA2 H7 RRT PCR. The only differences are the choice of primers and probe. The below will be sufficient for 10 x 25µl reactions, ie divide the Master Mix into 10 x 23µl volumes & add 2µl extracted RNA to each.
DEPC treated water (ie RNAse-free) 139µl
(x5) Qiagen 1 step RT PCR buffer 50µl
Rox ref dye (pre-diluted 1 in 500 from Stratagene stock in DEPC water) 3.75µl
Qiagen dNTP mix 10µl
H7F (50µM) 2µl
H7R (50µM) 2µl
H7-TM1 (30µM) 1.25µl
25mM magnesium chloride 12.5µl
RNAsin (40U/µl) 1µl
Qiagen 1 stepRT PCR enzyme mix 10µl
4.1.3 Thoroughly mix the master mix and centrifuge for 30 secs to remove bubbles.
4.1.4 Aliquot 23µl of master mix per well of the RRT plate / strip(s).
NB: Ensure the plasticware (ie plates / strips and covers / caps) is appropriate for the chosen RealTime PCR platform.
4.1.5 Loosely place the plate caps on the portion of the plate being used.
4.1.6 Bring the plate / strip(s) out of the PCR clean room. If samples and controls are not to be added immediately, place the plate/strip(s) on ice.
4.2 Preparation of H7 RNA controls
4.2.1 H7 AIV positive and negative controls are as described above in 3.2.5.
4.2.2 Extract H7 RNA as per the manufacturer’s instructions.
4.3 Manual addition of samples and standards
4.3.1 Regardless of whether RNA extraction has been conducted manually or robotically, manually add 2µl of the sample RNA to the 23µl of master mix. A dedicated multichannel pipette is convenient for adding robotically extracted RNA.
NB: Take care to avoid cross contamination of samples at this stage. Change gloves frequently and do not hold tips containing RNA above the incorrect wells. It is advised to cover strips loosely which have not yet been filled with caps (strip covers) to further minimise the risk of cross contamination.
4.3.2 Add 2µl DEPC water as a no template control (NTC), replace cap on this well, add 2µl of the negative RNA extraction control, replace cap on this well, and then add the real time PCR RNA standards and positive RNA extraction control to appropriate wells based on your worksheet layout
4.3.3 Once RNA is added, fit caps (strip covers) to all wells.
4.3.4 It is important that the caps are fitted firmly and correctly onto the wells before insertion into the RRT instrument. In addition centrifuge PCR plate to ensure samples and mastermix are located in the bottom of the wells.
4.3.5 If the RRT 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 RRT plate / strip(s) in the appropriate RRT instrument.
4.4.2 The thermocycling profile is identical for both the HA2 and CS H7 RRT PCRs. These are also identical to that used for the H5 RRT PCR which enables more than one of these assays to be ran simultaneously in one plate:
RT step:
50C for 30 mins
95C for 15 mins
PCR step (x40 cycles)
95C for 10 secs
54C for 30 secs
72C for 10 secs
NB: Collect fluorescence data at end of 54C step using the ROX and FAM filters.
4.4.3 Open a new file using the instrument software to define all the necessary RealTime PCR experimental parameters. This is conducted according to the manufacturer’s software instructions. Provide the file an appropriate H5 RealTime PCR name
4.5 Molecular pathotyping by sequencing the CS H7 RRT PCR amplicon
4.5.1 Here the amplicon generated by the CS H7 RRT PCR can be directly purified from the reaction mix after completion of this RRT PCR. There is no need to electrophorese this H7 amplicon through an agarose gel prior to purification. Amplicon purification and sequencing is as described by protocol “OneStep RT PCR for detection of H5 & H7 avian influenza and cleavage site sequencing”, with the difference that primers H7F and H7R (3.2.2, above) are used to prime the BigDye sequencing reactions. The assembled sequence from the CS H7 RRT amplicon is then used to determine the H7 sample as being either low pathogenic (LP) or highly pathogenic avian influenza (HPAI) virus.
5. Results
5.1 Analysis and display of results by using MxPro software
5.1.1 Conduct this after familiarising yourself with your RealTime PCR software and instructions
5.2 Interpretation of results in both H7 RRT PCRs
5.2.1 Analyse the data by (i) first checking the fluorescence threshold cutoff and (ii) comparing the results obtained for the negative (NTC) and H7 positive controls.
5.2.2 Negative controls (NTCs):
All NTCs should give “No Ct” as their final result. High Cts in all NTC wells with a linear character (eg >38) and 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, eg 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 H7 probe for subsequent experiments & note result.
If the late Ct 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 signals may occasionally occur spuriously. Repeat the H7 RRT PCR experiment.
5.2.3 H7 positive controls
1. H7 extraction control
These should reproducibly amplify with a Ct value +/- 2 Cts either side of their predetermined Ct value. Greater increases above the predetermined Ct value suggest that:
RNA extraction from the specimens has been suboptimal. Repeat.
It is also possible that the H7 AIV positive control may have degraded if stored incorrectly eg 4C, excessive freeze-thawing etc….
Deviation (+/- 2 Cts) 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 value of the predetermined H7 AIV positive control. It may be possible to reset the software (eg by checking the fluorescence threshold) after the experiment to restore the expected Ct value provided the other controls are in order.
2. H7 AIV RNA dilution series “standards” (3.2.5, paragraph 4)
Ten-fold dilution series should yield a straight line with R2 value of >0.975 (ideally >0.985), slope in the range of approximately -3.0 to –3.9 which should correspond to H7 RRT PCR efficiency in the range 80 – 115%. These tolerances are acceptable for obtaining a qualitative RRT-PCR result in the context of testing submission cases, outbreak specimens and screening work, and do not require accurate quantification of the neat H7 RNA used to construct the ten-fold dilution series. 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 H7 RRT-PCR experiments. If conducting research work where a semi-quantitative result is required, then the use of more accurately quantified H7 RNA standards may be merited as regards the requirements of the research project.
5.2.4 If all controls are within acceptable limits, analyse the data for the test samples.
5.2.5 Criteria for assessing results and decision making steps for any repeat testing of samples are as follows in the HA2 H7 RRT PCR:
1. Examine the H7 RNA dilution series (standards): Note the Ct value of the control RNA extracted from a Eurasian H7 sample at a titre of approximately 10 EID50/ml (average Ct value if tested in duplicate) – refer to section 3.2.5 paragraph 4 (above). This should yield a Ct value in the range 34 – 37 by both H7 AIV RRT PCRs. In this example, where Ct 36 is obtained from the 10 EID50/ml RNA control, a Ct < 36 in the HA2 H7 RRT PCR is considered clearly H7 positive, while a specimen recording “No Ct” value is clearly negative.
2. Samples with Ct >36 in the HA2 H7 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 “H7 [very low] positive” or “H7 negative”:
3. Repeat testing of extracted RNA:
Simply repeat the same H7 AIV 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” 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 value for the (i) original and (ii) re-extracted RNA by the M gene RRT PCR: A Ct value of 34-37 by M gene RRT PCR suggests that this may be a very weak positive HA2 H7 RRT result. A reproducible result obtained after re-extraction of RNA from the same clinical specimen by HA2 H7 / M gene RRT PCRs would tend to confirm this. It is possible that such consistent inconclusive results may eg 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 risers” (particularly those from field submissions) would yield positive results by other tests eg virus isolation or conventional H7 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 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 AI RRT-PCR experiment.
5. However, if HA2 H7 / M gene RRT PCR testing of re-extracted RNA (each test conducted in duplicate) yields a “No Ct” result then such specimens are considered negative.
5.2.6 Criteria for assessing results and decision making steps for any repeat testing of samples are as follows in the CS H7 RRT PCR:
1. These criteria are similar in principle to those listed in the above four paragraphs of section 5.2.5. However, validation with clinical specimens has shown the CS H7 RRT PCR may be less sensitive (up to x10 fold) in comparison to the HA2 RRT PCR, and this discrepancy may be even greater for Ct values of >30. Examine the H7 RNA dilution series: The approximately 100 EID50/ml extracted RNA standard typically gives a Ct value of 32 (+/- 2) in the CS H7 RRT. In this example, a Ct <32 in the CS H7 RRT PCR is considered clearly H7 positive, while a specimen recording “No Ct” value is clearly negative.
2. It is possible that CS amplification plots (ie with a sigmoidal / logarithmic character) may be observed in the Ct range 32-38. Samples which give such inconclusive specimens should be re-tested, re-extracted and / or reconsidered with the results of another RRT PCR (eg M gene and /or HA2 H7) as outlined for the HA2 version, section 5.2.5, paragraphs 2 - 5 (above).
5.2.7 All H7 positive results from a potential new case 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 H7 PCR and sequencing before reporting is crucial in order to confirm the H7 subtype and pathotype.
5.2.8 Other considerations for the two H7 RRT PCRs:
Both H7 RRT PCRs described in this protocol were designed, optimised and validated to detect recent Eurasian H7 isolates. Because of clear sequence differences between American and Eurasian H7 AIVs, it was not considered likely the former H7s would be detected reliably by the two H7 RRT PCRs described in this protocol. Results from the validation work affirmed this supposition.
Primer and probe considerations for Eurasian H7 isolates:
As Eurasian H7 viruses continue to evolve, it is possible that new isolates will emerge which will be less efficiently detected by the two described H7 RRT PCR methods, or possibly not detected at all. Revision of H7 primer / probe sequences may be necessary and this will be reflected in any future updated versions of this protocol.
6. References
6.1.1 Slomka MJ, Pavlidis T, Coward VJ, Voermans J, Koch G, Hanna A, Banks J and Brown IH (2009). Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses. Influenza and Other Respiratory Viruses, 3: 151-164.
6.1.2 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.
Appendix 1
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 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 according to the SOP VIR.0165, 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 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 two 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 (eg. 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.