Extraction of nucleic acids from swabs using the QIAGEN BioRobot Universal
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 procedure
1.1.1 | This procedure covers the extraction of nucleic acid from swabs and tissues collected in order to detect influenza A and Newcastle disease virus (NDV) using molecular tests. |
1.1.2 | The procedure not only covers the extraction but also the treatment of swabs prior to the extraction. Tissue preparation is covered in the procedures entitled “Procedure for the Isolation and Detection of Viruses from Avian Species using Embryonated Eggs” and “Swine influenza-Preparation of clinical specimens for swine Influenza “. Once tissues have been processed the extraction procedure is the same as for swabs. |
1.1.3 | The extraction of RNA is carried out in a QIAGEN BioRobot Universal robotic machine. |
1.2 Background information
1.2.1 | Realtime reverse transcription polymerase chain reaction (RT-PCR) assays are performed on RNA extracted from tissues, cloacal, tracheal or nasal swabs in order to assess the presence of influenza A viruses and AOAV-1. If a positive sample is detected it is essential that the HA/NA subtype (for influenza A) of the virus and pathotype for AOAV-1 is determined using further RT-PCR methods and ultimately DNA sequencing of the viral genome for these genes. |
1.2.2 | Dry swabs submitted for testing are introduced into 1ml of brain heart infusion broth with antibiotics (BHIB) and agitated briefly in order to release material into the medium. The BHIB is the starting material for the extraction. Virus transport media cannot be used. Wooden or charcoal swabs are not suitable for testing. |
1.2.3 | The extraction process involves lysis of virus under highly denaturing conditions using guanidine-HCL, labelled ‘AVL’ and 100% ethanol that inactivates any virus/bacteria present within and RNases introduced with the sample. Any nucleic acid released from material is then associated with filters, containing silica fibre, in 96 well vacuum plates (QIAGEN). Contaminating material (that may be inhibitory to the final PCR) is removed by washing the filters with two preparatory wash buffers (AW1, AW2) prior to elution of the purified nucleic acids (RNA and DNA) in nuclease free water or elution buffer (AVE). |
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. |
2.2 | 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. |
2.3 | Due to the nature of samples submitted for testing for avian influenza virus as part of the wild bird survey all material should be treated as potentially infected with influenza virus or other pathogens that might affect human beings. All manipulations prior to inactivation of material in AVL lysis buffer and 100% ethanol should be carried out in a class 1, 2 or 3 microbiological safety cabinet according to the BAAC RA 2006/06, BAAC RA 2011/01 and local microbiological safety cabinet class assignment tables. Please note: The minimum time required in order for virus to be inactivated in lysis buffer AVL, is 10 minutes. After the addition of 100% ethanol and surface decontamination following local procedures, it is safe to remove the samples from the microbiological safety cabinet (MSC) to a lower containment level. |
2.4 | AVL is chaotropic 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. |
2.5 | When decontaminating items for removal from an MSC the approved disinfectant – 1% Virkon S with a contact time of 10 minutes – must be used. |
3. Materials
3.1 Documentation and software
3.1.1 | The software (QIAsoft) to run the BioRobot Universal is loaded on the PC accompanying the extraction robot. All extraction methods are located in the QIAsoft software. |
3.2 Chemicals and reagents
3.2.1 | Brain heart infusion broth (BHIB) + antibiotics (1000IU penicillin G; 10µg/ml amphotericin B; 1mg/ml gentamicin) |
3.2.2 | AVL: Lysis buffer |
3.2.3 | Carrier RNA |
3.2.4 | AW1: Wash buffer 1 |
3.2.5 | AW2: Wash buffer 2 |
3.2.6 | AVE: Elution buffer |
3.2.7 | 1% Virkon S 1 x 5g Virkon S tablet dissolved in 500 ml of water produces a 1% solution. Once made, the solution has a shelf life of up to 5 days. When fresh, the solution has a strong pink colour which fades with age – a pale solution must not be used. Contact time is 10 minutes for decontamination. |
3.2.8 | Absolute Ethanol (molecular biology grade >99% Ethanol) hereafter referred to as 100% Ethanol |
3.2.9 | Distilled water |
3.3 Equipment
3.3.1 | QIAGEN BioRobot Universal |
3.3.2 | S block 96 deep well plasticware |
3.3.3 | Elution plate (96 well blue) |
3.3.4 | DNA extraction 96 well filter plate (QIAGEN) |
3.3.5 | 20ml Disposable plastic troughs (QIAGEN: #9232764) |
3.3.6 | 1ml BioRobot tips |
3.3.7 | Class 1, 2 or 3 microbiological safety cabinet |
4. Procedure and method
4.1 Test reliability
4.1.1 | Process of assuring ongoing test reliability: The Biorobot software keeps a record of all plate extractions using the barcodes on the elution plates. The Ct values obtained from the positive robot extraction controls after the RRT-PCR are recorded and monitored regularly to ensure the extraction process is working satisfactorily. |
4.2 Processing of swabs
4.2.1 | All procedures prior to inactivation of the sample material, using AVL lysis buffer and 100% ethanol, must be carried out in a class 1, 2 or 3 MSC according to locally employed risk assessments. |
4.2.2 | BHIB is dispensed in 1ml aliquots into 1.5ml Eppendorf tubes by RPU and stored at +2-8°C prior to use. |
4.2.3 | Label tubes containing 1 ml BHIB with sample identification. |
4.2.4 | Put the fibre end of swab into the appropriately labelled tube containing BHIB. Using a pair of scissors or bone cutters snip, or snap manually, the shaft of the swab near to the fibre end. Close the lid of the tube. |
4.2.5 | Immediately place the remainder of the swab in a sharps bin and dispose of the plastic sheath. |
4.2.6 | A new set of bone cutters should be used for each epidemiological unit. Alternatively, to reduce the possibility of cross-contamination among individual swabs - Dip the end of the bone crushers or scissors into a beaker of 100% ethanol. |
4.2.7 | Repeat this procedure for all swabs. |
4.2.8 | Mix each tube containing the swab for 5 seconds to elute material. Allow to stand for a minimum of 2 minutes to allow solid material to settle |
4.2.9 | The swab eluent can now be used for RNA extraction. |
4.3 S block preparation
4.3.1 | Care should be taken when handling AVL |
4.3.2 | Prepare the required volume of AVL by combining each 31ml bottle with310μg Carrier RNA. Dispense 420µl of AVL, with carrier RNA, into the appropriate number of wells of a 96 well S block plate (deep square well plate). |
4.3.3 | In an appropriate class of MSC dispense 140ml of tissue suspension or swab eluent into the AVL directly. |
4.3.4 | This process is repeated until a maximum of 88 samples (11 columns) have been introduced into the S block. |
4.3.5 | Incubate at room temperature for 10 minutes. |
4.3.6 | Dispense 420µl of 100% ethanol into each well of the 96 well S block plate containing samples. |
4.3.7 | At this point the S block plate can be removed from the MSC after following local decontamination procedures. |
4.3.8 | Residual BHIB tubes containing the swab head and tissue must then be frozen at -70°C or lower should they be required for further analysis, re-extraction or egg inoculation for isolation of viable virus. |
4.3.9 | Positive and negative extraction controls, appropriate for the downstream testing, must be added to AVL in the S block prior to extraction on the Qiagen Robot. Negative extraction Incubate at room temperature for 10 minutes. |
4.4 Running the BioRobot
4.4.1 | The BioRobot should already be switched on (turning it on and off frequently may interfere with the communication between the robot and the PC). Turn on the associated PC. |
4.4.2 | Double click on the QIAsoft icon on the desktop of the PC. |
4.4.3 | Select the extraction protocol from the menu. |
4.4.4 | Click the run arrow (Green). Select the columns you are extracting from on the map that is automatically displayed. The software will then lead you through the set up process including the positioning and volumes of the various components. This must be followed carefully |
4.4.5 | The ‘System Liquid Bottle’ (in carousel) should be filled with distilled water. The ‘System Liquid Container’ (below robot) should be filled with distilled water to between the MAX and MIN lines. The ‘Waste’ container (below robot) should not be allowed to reach MAX line (empty if necessary). The ‘Vacuum trap’ (below robot) should not be full (empty if necessary) (waste can be poured down a sink) |
4.4.6 | Follow all relevant menu instructions and finally run the extraction process. The extraction should take approximately 50 minutes. |
4.4.7 | Any errors will be indicated to the user with a warning. The software will allow you to run the program from the point of error or to start again from the beginning, which means preparing a new S block. |
4.4.8 | At the end of the run, make sure you have completed the entire protocol. Click ‘continue’ rather than ‘stop protocol’ and follow all the closing down instructions. |
4.4.9 | Remove your channelling blocks and holders and clean by:
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5. Results
5.1 What you should expect
5.1.1 | As a clarified liquid is used throughout there should be no “clogging” of the BioRobot and you should expect ~70µl of elution buffer containing the extracted nucleic acid. It should be clear and colourless. |
5.1.2 | When removing the S-block, filter and elution plates, check visually for any anomalies. Record any well/grid references affected. Report anomalies found to the test manager. |
5.2 What you should do next
5.2.1 | The elution plate can be stored for a limited duration (of up to a few hours) either on ice, a cool block or at +4°C prior to performing the required RRT-PCR. However, storage at -70°C is required in the event of a longer gap between the generation of the elution plate and the setting-up of the real- time PCR. The lid for the elution plate should be replaced on the plate by the BioRobot. |
5.2.4 | Extracted nucleic acids should be added to an RT-PCR TaqMan according to the appropriate realtime PCR procedure listed above |
5.2.5 | Once the PCR plate has been loaded, the blue elution plate containing nucleic acid should be capped and stored at -70°C or lower until use or for further analysis if necessary. |