This protocol can be used for the postmortem diagnosis of animal rabies from the initial view of the diction in the field to the molecular conformation in a centralized level of activity. Rapid immunochromatographic diagnosis testing is a useful alternative for rural and rural traits in Africa and Asia in which reference materials for rabies diagnosis are not available. Demonstrating the procedure will be Simon Bonas a technician from my laboratory.
Demonstrating the first part of the procedure will be Ibrahima Dicko, a technicion from the Laboratoire Central Veterinaire in Bamako, Mali. To access the foramen magnum, use a knife to remove the animal head before the first cervical vertebrae. And use an appropriate tool to collect a brain stem sample.
Such as a disposable pipette, drinking straw, clamp, or dropper as shown here. The brain stem is then clearly visible as demonstrated here with the tip of this pipette. Use an appropriate tool to collect a part of this brain stem such as a drinking straw.
Insert it carefully and turn gently to collect the brain tissue. Do not forget to plug the straw with your finger before removing it, in order to collect the biopsy. The use of a clamp is also possible.
In order to collect larger pieces of brain stem at the level of the foramen magnum. Alternatively, it is possible to use directly the dropper provided in the kit to collect the brain stem. Similarly to the drinking straw, insert the dropper carefully while pressing and turn gently to collect the brain tissue.
Then aspirate the brain tissue and place it in a container like the stopper of a tube. For an accurate diagnosis it is critical to collect the brainstem which contains the medulla ablongata, during the sample collection. Carefully crush the brain material directly in the tube containing the buffer with the swab or dropper for about 30 seconds until a homogeneous suspension is obtained.
Using the dropper, deposit four drops approximately 100 microliters of the suspension in the sample inlet on the test device. In case of delay due to high viscosity suspension or to accelerate the start of the migration, gently scratch the bottom of the deposit site of the device with the dropper. Wait for complete sample migration, one to five minutes before reading the test device.
A sample is considered positive for rabies when two lines are visible. Negative, if only the control line is present. And invalid if only the test line or no lines are visible.
To extract the RNA from a device sample, carefully open the device and remove the filter paper. Collect the deposit area of the sample and place the sample into one milliliter of trireagent LS.After one hour at room temperature with regular gentle manual agitation, extract the RNA from the tubes supernatant according to standard RNA extraction protocols. Adding two microliters of glycogen to facilitate the RNA precipitation according to the manufacturers recommendations.
Adjust the final volume of the RNA resuspension to 50 microliters with nuclease free water. And prepare the master mix reaction solution for the three different reverse transcriptase quantitative PCR assays according to the table. Using the primers and probes as indicated in the table.
At five microliters of the diluted RNA samples. And 15 microliters of master mix to each of the three different assays in duplicate in the appropriate 96 well reaction plates. Add the appropriate positive and negative controls for each assay to each plate in duplicate.
Then run the different assays following the thermal cycling conditions as indicated in the table. The results can be analyzed as outlined in the table. The collection of a brain biopsy is as simple as possible to guarantee the collection of high quality samples.
Compared to the reference technique the sensitivity and specificity of the rapid immunochromatographic diagnostic test was high in all of the laboratories using these protocols. Based on the cumulative number of tested samples, the overall sensitivity and specificity were over 95%The rapid immunochromatographic diagnostic test is suitable for detecting lisavirus in brain biopsies from infected animals. As the level of lisavirus antigens is important for rabies diagnosis, however, the limit of detection remains high when testing titrated virus suspensions.
In these representative results, obtained after RNA detection by dual combined panlisavirus reverse transcriptase quantitative PCR targeting of the viral polymerase of lisavirus, a panel of 51 positive rapid immunochromatographic diagnostic tests were performed. In addition, genotyping could be performed for fourteen of the samples using the hemi-nested PCR targeting of the partial nucleoprotein gene. Diagnostic and extracting for the test strip for genotyping based on after RT PCR, or by for additional long string analysis.
The rapid immunochromatographic diagnosis test can provide a quick and accurate rabies diagnosis. Which is crucial to maintaining wave function in continuous wave based surveillance and control systems. And minimizing humannnes.
