Human pandemics of flu, including the recent swine flu epidemic originally stemmed from the avian influenza virus, A IV, which is a species of the influenza A virus. Large surveillance programs have been implemented to collect information on the ecology of A IV and to detect highly pathogenic strains. In this video, a method is presented for isolating and identifying a IV samples from wild birds.
First ducks are trapped. Their fecal matter is collected and applied to an FTA card, which preserves the viral RNA for dry storage Samples are taken from the FDA cards and the fecal material is extracted. The RNA is extracted and magnetic beads are used to purify it.
Reverse transcriptase PCR is performed to generate CD NA, which is then sequenced. The resulting sequences are then compared to known a IV sequences to reveal the type of a IV present in the samples. The main advantage of this technique over existing methods like propagating infectious viruses is that no strict culture needs to be maintained and remote areas are sampled easily.
Moreover, biosafety regulations with FDA cards are easier to implement. This method can help answer key questions in disease ecology field, such as which influenza strains occur. Where Though this method can provide insights into the study of avian influenza.
It can also be applied to other systems such as different wildlife disease caused by viruses and other microorganisms. Generally, individuals new to this method will struggle because catching and sampling life ducks or other birds needs practice and lab work with RNA is difficult. We first had the idea for this method when we successfully sampled and isolated regular DNA from animal feces stored on what Watman FDA guards Trap dabbling ducks or other birds in a cage by attracting them with lu ducks or food.
Here, ducks of the genus onus are trapped. Place the traps ducks into individual cardboard boxes. Take the duck outta the box by holding its wings tight to its body.
In most cases, fresh fluid droppings will be present at the bottom of the box. Using a new sterile swab every time, pick up the droppings and apply them to a watman FTA card until the card target surface is soaked. If droppings are not present at the bottom of the box, perform cloacal swabbing.
By first turning the duck on, its back to permit access to the clo acre. The clo acre is a protruding structure situated below the abdomen close to the base of the tail. An experienced person can hold and sample the bird at the same time if needed.
A second person can assist taking the sample while the first person holds the bird. Carefully insert a sterile plastic rayon swab approximately one centimeter and make a gentle SW of the clo acre. Remove the swab, then roll this over the surface of a watman FTA card.
Dry the FTA cards at room temperature for at least one hour. Place each sample in a paper envelope. Then store the envelopes at room temperature for short term storage or freeze them for longer periods.
FTA cards can be sent by regular mail since the pathogens become inactive. Upon contact with the FDA card surface working under a fume hood. Begin the viral RNA isolation procedure by extracting the FDA card material, including the feces, using a Harris two millimeter puncher punch three discs and place all of them into a well of an RNAs free 96 well plate between each new sample.
Clean the puncher carefully with alcohol and a Kim precision wipe. To generate a positive control. Apply a laboratory strain to an FTA card as a negative control.
Punch out discs from an empty FTA card. Place these in the plate as before, be sure to leave another well free for a PCR control. Later in the experiment, add 70 microliters of RNA rapid extraction solution to the well.
Then using a thermo sealer heat seal the plate, place the plate on a shaker and set the speed, ensuring that it does not cause spillover. Incubate the plate shaking at room temperature for five minutes. Following the incubation, use ambience Mag Max 96 kit to isolate the viral RNA at 130 microliters of prepared lysis binding solution, which is provided in the kit to each well of the empty plate in the kit.
Then transfer 50 microliters of extracted FDA card material to each. Well place the plate on the shaker for one minute. Next, add 20 microliters of prepared bead mix from the kit to each sample and mix by pipetting up and down shake for five minutes.
During this incubation, the RNA molecules will bind into the magnetic beads. Next, move the plate to a magnetic 96. Well stand to capture the beads when the solution has turned completely clear.
Use a pipette to remove and discard the supinate. Then remove the plate from the magnetic stand. Wash the beads by adding 150 microliters of prepared wash solution one to each sample, shake for one minute, then capture the beads on the magnetic stand.
When the solution is completely clear, remove and discard the supernatant. Repeat this wash once. Then perform two washes using solution two.
After the last wash, use a pipette to remove as much of the solution as possible and place the dry plate on the shaker for two minutes at room temperature to air dry the beads. Once the beads are dry, add 50 microliters of elucian buffer from the kit and shake for four minutes to release the RNA from the beads. Capture the beads as before.
The supernatant now contains the isolated RNA, which is ready for downstream applications to generate and amplify. CDNA of the viral genes. Carry out reverse transcription PCR using the one step access R-T-P-C-R system in 25 microliter reactions as described in the accompanying written protocol.
Once the PCR is completed, the samples can be screened for a IV positivity and purified. To determine whether samples are a IV positive run two microliters of the PCR product mixed on a 1%aro gel stained with 1%atherium bromide for one hour at 120 volts. Visualize with a gel documentation system, then identify samples with amplified fragments in the expected size range.
Here the target fragment is 244 base pairs. So look for samples with bands between 200 base pairs and 300 base pairs, for instance, like those circled in red. Next, load the remainder of the PCR reaction.
About 23 microliters of the candidates on a 2%atherium bromide stained aros gel and run for two hours. Place the gel on a UV trans illuminator. Then using a scalpel, excise bands of the correct size and place them into individual 1.5 milliliter reaction tubes using A DNA recovery kit.
Purify the DNA from the gel fragments for sequencing and identification. Carry out Sanger sequencing of the target fragments here. The forward primer M 52 C is used to sequence the A IV matrix gene.
Once the sequencing results have been obtained. Compare the cDNA sequences against nucleotide databases such as GenBank using local or web-based tools such as blast at the National Center for Biotechnology information, gene fragment sequences can be matched to existing entries. Thus, the specific A IV strain can be characterized and valuable information on sequence features obtained.
84 mallards were sampled at Autumn B Bird Observatory in December, 2007 from each mallard a sample on an FTA card was taken as described in this protocol. After shipping the FTA cards were kept in a freezer at minus 20 degrees Celsius for two years. A gel picture of the PCR products from these 84 samples is shown here from natural samples.
A multitude of unspecific bands can be observed due to the presence of various microbial contaminations in the feces. The target fragment of the primer pair used here is 244 base pairs. Long bands of the expected size are circled in red.
Blue circles indicate an unspecific amplicon or a primer dimer artifact below 100 base pairs in size. The samples indicated by red arrows were isolated and sequenced. The green arrow indicates the positive control.
The samples chosen were confirmed to be a IV positive by comparing their cDNA sequences to the NCBI nucleotide database. Once mastered, this technique can be done in two hours from loaded FDA card to clean RNA template if it is performed properly. While attempting this procedure, it's important to remember to pay attention to clean work spaces, use of gloves, separation of pre and post PCR working environments, filter tips, and working under the safety hood Following this procedure.
Other downstream applications like cloning fragments into vectors can be used to answer additional questions like if there are multiple influenza strains infecting one host After its development. This procedure could pave the way for researchers in the field of disease ecology to explore AI infections in remote areas where AI sampling was not feasible.Previously. After watching this video, you should have a good understanding of how to sample birth for avian influenza from the cloaca isolate and purify RNA molecules from FDA cards and determine if and by which terrain they are infected.
Don't forget that working with wild birds bears the risk of handling individuals that are infected with avian influenza or other zoonotic diseases. And because of that, it is very important to always wear protective clothing while performing this procedure.
