Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

The overall goal of this procedure is to test wild birds for avian influenza virus or a IV at the point of capture, which is often in a remote field setting away from laboratory facilities. This is accomplished by first capturing wild birds using the technique of mist netting, followed by timely collection of cloacal and oropharyngeal samples. After relocating to an enclosed space, RNA is extracted from the viral transport media using the RNZ Minikit preparation of positive and negative controls, as well as the test samples is then performed using lyophilized reagents followed by transfer of the samples to micro capillary tubes.

The final step of the procedure is to amplify viral RNA from positive samples using a portable real-time PCR machine called the Ruggedized Advanced Pathogen Identification Device or Rapid. Ultimately, results can be obtained that show the presence of a IV and wild birds within 24 hours of sample collection in the field. The main advantage of this technique over existing techniques done in a diagnostic laboratory is that in the field we can look for positive or negative samples of avian influenza virus at the site of capture.

This method can help with containing outbreak, preventing transmission to other bird populations, and even help to answer ecological questions such as how far can an infected bird fly? Demonstrating the procedure will be Annie Schultz, a biologist from the US Geological Survey. For Shorebird capture, choose a location over an active foraging site such as a marsh shoreline, mudflat, or salt evaporation pond.

To erect mist nets, locate the trammel line loops at one end of the mist net and slide each loop over one of the poles. Then insert the end of the pole vertically into the mud. Once the first pole is in, stretch out the mist net over the foraging area.

Slip the tramal loops at the other end of the mist net over the second pole vertically. Insert the pole into the mud, making sure that the trammel lines are tau. Check to make sure the mist net is tau enough so that the net does not sag excessively when birds are in the net, but not so taught that birds bounce out when they strike.

Once a bird is captured, extract the bird from the net and return to the banding station. Birds need to be processed as quickly as possible to minimize distress to the animal. Begin collection of cloacal swabs by unwrapping a sterile dacron or rayon tipped swab stemmed first.

Then moisten the tip with sterile viral transport media for greater ease in swabbing. Next, locate the C cloaca of the wild bird. Once the C cloaca is located, push back the surrounding feathers.

Gently insert the entire head of the moistened swab into the C cloaca. Swab the inside circumference of the cloaca by slowly twirling the swab. Then insert the swab tip directly into the viral transport media.

In the sample vial, rotate the swab shaft between the thumb and forefinger. While the swab is in the media, pull the tip of the swab back from the bottom of the vial approximately one centimeter. Cut the shaft of the swab with the pair of scissors so that the swab tip remains in the vial and the shaft does not interfere with cap closure.

Cap the vial tightly, always disinfect the scissors with alcohol in between cutting, swab shafts. Following collection, place the vials in a cooler containing either dry ice or ice packs. To collect the oropharyngeal swab samples, unwrap a clean swab from the package stemmed first.

Then gently open the bird's bill without touching the tip to any other surfaces. Insert the swab into the mouth, swab the choanal opening on the roof of the mouth, and continue back towards the oropharyngeal or throat region as performed with the cloacal swab. Place the oropharyngeal swab tip directly into the viral transport media vial.

Rotate the swab shaft between the thumb and forefinger while the swab is in the media. Once again, pull the tip of the swab back from the bottom of the vial and cut the shaft with scissors. Cap the vial tightly and place it in.

A cooler. Samples can be tested in any enclosed space, such as a research trailer or tent with a power supply to run the centrifuge Vortex PCR unit and attached computer. Ideally, sample preparation should occur in two locations, one for RNA extraction and another for amplification to more effectively avoid contamination.

To begin sample preparation, extract the RNA from the swabbed samples using the RN easy kit as described in the written protocol Accompanying this video, place samples on ice and transfer to the location designated for amplification. Next, the PCR assay samples are prepared from the lyophilized reagents contained in the Idaho technologies'freeze dried reagent detection kit To avoid contamination, the negative control should be prepared first, followed by the unknown samples and ending with the positive control for preparation of each reagent vial. First, centrifuge the vial for three seconds.

Following centrifugation, remove the cap and visually confirm the pellet is at the bottom of the vial. Add 20 microliters of reconstitution buffer, followed by 20 microliters of the purified RNA. Recap the vial and vortex the sample for five seconds at maximum speed.

Next, centrifuge the sample for three seconds to bring any liquid to the bottom of the vial. Visually confirm that the pellet has rehydrated. Then pipette 19 microliters of the hydrated mixture into a light cycler capillary tube.

Pipette another 19 microliters into a second capillary tube. To obtain a duplicate, finally cap both the tubes using tweezers. Be sure to keep track of the sample numbers.

Once all of the capillary tubes for the batch have been prepared, load them into a mini centrifuge fitted with capillary adapters. Centrifuge at the lowest setting by holding the lid down for one second. This transfers liquid to the bottom.

In preparation for testing, unpack the ruggedized advanced pathogen identification device or rapid. Prepare the light cycler carousel for processing. Load the capillary tubes in the order in which they were prepared, and close the top.

To begin the analysis, turn on the device and open the rapid light cycler software. Click on the advanced options button. After a new window opens, select the run button under run light cycler.

From the file menu, select open exp. Select the Avian Influenza program, which has been pre-programmed with the denaturation and amplification parameters found in the written protocol. The program consists of RNA sample preparation hold, RNA DENATURATION and RNA amplification.

Next, ensure that the display mode is set to channel two in the upper box and channel one in the lower box. Then check the channel. One of the fluorimeter gain is set to one channel.

Two is set to eight and channel three set to one. Finally select the run button in the lower right corner of the screen. When prompted, rename the data file, select save and click done on the lower right corner of the screen, the light cycler will take about 60 minutes To complete the program upon completion, select exit from the file menu.

In the small window, select the analysis tab and select the light cycler data analysis button.Here. The data file that was just created can be open to interpret the results. An example of a fluoro Graham generated from a successful analysis by the rapid 7, 200 is shown.

The positive control is amplified and generates detectable fluorescence at approximately 25 cycles, producing a fluorescent signal within the accepted number of cycles for a IV detection. In contrast, the negative control does not generate a fluorescent signal even after 45 cycles. Similarly, the 12 samples collected from Western sandpipers did not generate a fluorescent signal indicating that the birds were negative.

For a IV Once mastered. This technique can be done in two hours, making it possible to run three batches of 42 screenings per day. It can be used in a remote field situation where access to a laboratory is unavailable and help to identify even influenza outbreaks.