Hello everyone, Currently, I am working on determining potential vector of West Nile virus among Culex sp. mosquitoes. 5 samples showed a band after carried out One-Step RT PCR and I sent the gel for sequencing service. From the sequencing result obtained, I compared the results of the sequencing with the NCBI database by using BLAST and it showed a higher percentage of Plasmodium relictum. Does anyone got like this same problem? is it because of sample contamination?
In brief, the methodology of my study, After species identification, 500uL of PBS was added and homogenized the pooled mosquitoes (10 mosquitoes per pool) by using mechanical homogenizer (Omni Tissue Homogenizer). RNA extraction was conducted by using Trizol followed by One Step RT-PCR (Bioline) and lastly, 1.5% of gel electrophoresis, were carried out.
Thank you for helping me.
What primers are you using? That is, what gene are you trying to amplify?
What are the BLAST statistics (score, % identify etc.)
Craig S Wilding May I know what it means by the first question? The primers was designed and the result of sequencing was checked with NCBI database using BLAST. The results shows 100% WNV. The maximum and minimum score is 38.2.The query coverage and percentage identification is 100%. The E value is 1.4.
An e-value of 1.4 is very high suggesting that this hit is unreliable and spurious. I would not believe it is a genuine strong hit to Plasmodium based on this score.
My question is asking what your PCR primers are and what gene they are targeting.
The most probable cause of the above finding could be contamination. I would advice that you repeat the whole process, carefully confirm the primer sequences that you are you using and ensure that there is no possible mix up of the template.
Craig S Wilding thank you for the information. Pertaining to the question, the PCR primer is highly conserved region targeting between capsid and pre-membrane protein of West Nile virus. I hope I am answering your question. Thank you for the suggestion, Sarah Nanyiti I will that revise the methodology of my study
The lower the e-value, the greater the chances that the result is due to chance. For your case, the e-value will be high as well. So review the whole methodology again
Thank you Nkemngo Francis Nongley for the answer. May I know what is the acceptable range of e-value? How low it should be so that the result is acceptable?
Thank you again for helping me...
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