Preparation and processing of swab pools
Preparation and processing of swab pools for detection of Influenza A matrix gene and/or Avian Orthoavulavirus Type 1 by real time RT-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
1. Purpose/scope of this protocol
1.1.1.To rapidly detect avian influenza A or avian orthoavulavirus type-1 (AOAV-1)) viral RNA extracted from pools of up to five cloacal or five oro-pharyngeal swabs. This swab pool testing approach aims to provide an early warning for the presence of influenza A virus or AOAV-1 in a sample.
The protocols described are applicable for statutory testing for notifiable avian disease (NAD) including International Trade, cases where NAD is suspected (so-called Report cases), surveillance testing following the detection of NAD in poultry or wild birds and NAD exclusion testing. The swab pool testing approach is also applicable for the specific detection of avian influenza A virus subtypes H5 and H7.
1.1.2 The protocol covers the initial pooling and processing of swabs. Nucleic acid extraction and the subsequent PCR amplification of eluted pooled swab nucleic acid material using protocols for generic detection of influenza A virus, avian influenza A virus subtypes H5 and H7 or AOAV-1 are described in the protocols listed in section 3.1.2.
Background information
1.2.1 The protocols described in this SOP are applicable for statutory testing for Notifiable Avian Disease (NAD) including International Trade, cases where NAD is suspected (so-called Report cases), surveillance testing following the detection of NAD in poultry or wild birds and NAD exclusion testing. The swab pool testing approach is also applicable for the specific detection of avian influenza A virus subtypes H5 and H7.
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.
2.2 Due to the nature of samples submitted for testing for avian influenza A virus or AAvV-1, all material should be treated as possibly infected with influenza virus, AAvV-1 or other pathogens that might affect people. All manipulations prior to inactivation of material in AVL Lysis buffer and 100% ethanol should be carried out in a microbiological safety cabinet (MSC). The addition of both AVL and ethanol is necessary to inactivate the virus. This is therefore required before samples can be removed safely from the MSC.
2.3. AVL is a chaotropic agent and will dissolve proteins. Therefore, gloves should be worn at all times and care should be taken not to get this material on skin or in eyes. In this event, rinse the affected area immediately with tap water and consult with on site medical staff if burns have occurred.
3. Materials
3.1 Documentation and software
3.1.2 Procedures Document References:
RNA extraction from biological samples.
Extraction of nucleic acids from swabs using the Qiagen BioRobot Universal.
Influenza-Detection of influenza A matrix gene by real time Taqman® RT-PCR.
NDV screening real time PCR.
OneStep RT PCR for detection of H5 & H7 avian influenza & cleavage site sequencing.
Newcastle Disease Statutory Sequencing. Eurasian H5 avian influenza RealTimePCR
H7 Eurasian RealTime PCRs for the detection and pathotyping of Eurasian H7 avian influenza isolates
3.2 Chemicals and reagents
3.2.1 Brain-Heart Infusion Broth (BHIB) containing antibiotics (1000IU penicillin
G; 10µg/ml amphotericin B; 1mg/ml gentamicin).
3.2.2 If BHIB is not available - Antibiotic PBS can be used instead.
3.2.3 100% Ethanol (replenish once a month).
3.2.4 AVL Lysis Buffer.
3.3 Equipment
Microbiological Safety Cabinet (MSC). Microcentrifuge tubes (1.5 ml).
Microcentrifuge (with rotor for 2 ml tubes). Single-use round scissors or bone cutters.
-18 C or lower freezer for storage of chemicals and reagents.
-70 C or lower freezer for storage of extracted nucleic acid. Vortex mixer.
Calibrated pipettes: refer to document GE 183 – Pipettes – use maintenance.
Sterile, RNase-free pipette tips with aerosol barrier. Disposable gloves, latex-free.
Sharps bin.
4. Procedure
4.1 Test reliability
4.1.1 This protocol is a support process not a test.
The subsequent PCR assays performed include a positive control in eac hextraction. The Ct values obtained from the extraction control and standards (where used) must fall within the range stated in the relevant SOP. If not, the reason behind an out of range result being obtained must be investigated.
The Ct values obtained from the positive extraction control and standards
% efficiency values are compared over time to identify trends that may indicate poor performance of any component of the test. Once a trend is identified its cause should be investigated.
4.2 Swab sample preparation and pooling
4.2.1 Swabs from the same anatomical site (oropharyngeal or cloacal) are pooled for testing with one pool comprising a maximum of five swabs e.g. 20 swabs = 4 pools of 5 swabs.
Example, if 20 oropharyngeal swabs and 20 cloacal swabs are submitted from an epidemiological group for M-gene real-time PCR, there will be 8 PCR tests for that group (and/or 8 PCR tests for L-gene PCR depending on the test(s) requested).
A different pair of scissors or bone cutters must be used for the cloacal and oropharyngeal swabs from each epidemiological group. Therefore, for a 20/20 submission, two scissors/ bone cutters must be used.
4.2.2 In an MSC operating in Class I mode, pool a maximum of 5 swabs from the same anatomical site into an Eppendorf tube containing a total volume of 1ml BHIB or antibiotic PBS, as described below.
4.2.3 Put the cotton wool end of the swab into the appropriately labelled tube containing BHIB or antibiotic PBS..
4.2.4 Using a single-use round scissor or bone cutter, snip the shaft of the swab near to the cotton wool end. Discard the remaining un-usable swab applicator into a sharps bin. Repeat this process for the remaining swabs which form part of the pool.
4.2.5 Close the lid of the tube.
4.2.6 Mix the tube by gentle shaking for a minimum of five seconds to elute the material from the swabs.
4.2.7 Allow the tube to stand at room temperature for a minimum of two minutes for material to settle.
4.2.8 Repeat the process (4.3.3 to 4.3.8) for other swab pools
4.2.9 Depending on how many pools there are to perform RNA extraction on, choose either an S-block containing 420μl AVL buffer per well (automated extraction) or 1.5ml micro-centrifuge tubes containing 560μl AVL buffer (manual extraction).
Note: Manual extraction of RNA from each sample should be considered in the first instance. If 16 pooled samples or less (perform manual extraction). If more than 16 pooled samples (perform automated extraction).
4.2.10 Carefully pipette up and down twice before removing 140μl pooled swab eluent solution and directly add to AVL buffer.
Mix this solution again by gently pipetting up and down twice. Discard pipette tip.
Note: It may be necessary to ‘wring out’ the solution from each individual swab by pressing the swab against the inside of the tube in order to obtain 140μl solution for addition to AVL buffer.
4.2.11 Incubate at room temperature for 10 minutes.
4.2.12 Into each tube containing swab eluent and AVL buffer dispense 420l of 100% ethanol for (automated extractions) or 560l of 100% ethanol for (manual extractions).
4.2.13 At this point the reaction can be removed from the MSC according to local decontamination procedures
4.2.14 Tubes containing the five pooled swab tips must then be stored at -70 C
in the event that any of these should be required for further analysis.
Tubes testing negative should be retained at -70 C for a minimum of
three months then discarded.
4.2.15 The pooled swab eluent can now be used for manual or automated RNA
extraction described in (Section 4.4).
4.3. RNA extraction
4.3.1 As stated in section 4.2.1, when 16 pooled samples or less are to be tested, perform manual extraction of RNA using the reagents from the QIAamp Viral RNA Minikit (Qiagen) according to the protocol – “RNA extraction from biological samples”.
With more than 16 pooled samples, perform automated extraction of RNA according to the protocol – “Extraction of nucleic acids from swabs using the Qiagen Biorobot Universal”.
RNA extracted from positive swab pool eluates must be stored at -70 C
while extracts from negative eluates must be retained at -70 C for a minimum of three months before being discarded.
4.4 Real-time RT-PCR assays
4.4.1 Perform Real-Time RT-PCR assays on each nucleic acid extract
according to the customers testing requirements, following the appropriate procedure.