I have read about RT-PCR as a molecular amplification assay for detecting the presence of flaviviruses. However, when used on the extracted RNA of mosquitoes carrying Japanese Encephalitis Virus, there is a risk of obtaining a false negative result due to the inhibitory effects of the mosquitoes, as detailed in the following journal attached. As mentioned in the publication, it was necessary for them to use dilution in order to detect the presence of JEV in their mosquito pools. Though effective, there were no other detailed methods to use with RT-PCR that would help with detection, in both the publication as well as others I have encountered in trying to answer this question. I would really like to find out if there are other methods you have read about or have done yourself that were effective in JEV detection.
http://journals.plos.org/plosntds/article?id=10.1371%2Fjournal.pntd.0002153#pntd.0002153-Nimmo1
Hi,
You may see this article "Detection of Japanese encephalitis virus in samples of JE patients by RT-PCR" at https://www.ncbi.nlm.nih.gov/pubmed/11503059
Good luck!
Hi Rose,
Dilution is the simpliest way to overcome the potential of inhibition in a PCR assay. A 1:10 dilution should be enough to dilute an inhibitor to a level where amplification will occur without negative effects, and will only result in a 3 cycle shift if using real-time technology which is a negligable effect for virtually all samples (only mosquito pool with levels of virus close to the limit of detection for the assay will become a 'false-negative' and at these levels they are difficult to confim as a true positive even if undiluted). If using conventional PCR, again, strong positives (which are the predominant target when using this techology) will still produce visible bands
If you want an alternative method, you can spike your mosquito homogenates with an extraction control of known concentration, or alternatively use a housekeeping gene that is known to be present in your sample such as beta-acton, and then monitor the amplification. If this deviates from the norm, then you can suspect inhibiton and dilute this individual sample. This method offers great confidence in your results, by is significantly more expensive. If money is an issue, I would recommend dilution as it is far cheaper and easier to implement. There is a small inherent risk of false-negatives, but only in samples that would be of questionable relevance
All the best
Thank you very much Ms. Rana and Mr. Barry, your inputs have been very helpful!
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