Mutation analysis of Influenza RD8 from chicken egg

This project explored whether Influenza RD8 isolated from chicken eggs carried any genetic changes that could inform future vaccine development. The work began by collecting amniotic fluid from infected eggs and extracting viral RNA using a silica-column purification method. RNA quality was checked by Nanodrop, which confirmed that material was present but also highlighted contamination that could interfere with downstream reactions.

The RNA was converted into cDNA using influenza-specific, oligo(dT), and random hexamer primers. These cDNA samples were then amplified using primer sets targeting the HA and NA genes. The goal was straightforward: produce clean amplicons that could be cloned, sequenced, and compared against known influenza sequences to identify possible mutations.

Here’s where things changed direction. The PCR reactions produced primer dimers rather than clear HA or NA bands. Even so, the workflow continued. The reactions were ligated into a PCR 2.1 vector and transformed into E. coli. The transformation step worked well, confirmed by strong white-colony numbers during blue-white screening and a calculated transformation efficiency of 1 × 10⁸ cfu/ng.

Once plasmids were isolated and screened, digestion patterns again suggested that the expected viral insert was missing. Sequencing brought complete clarity. The NA sequence aligned with Corynebacterium pseudotuberculosis, and the HA sequence aligned with a cloning vector rather than influenza. No part of the recovered DNA showed similarity to influenza A, and therefore, no mutation analysis could be carried out.

Bottom line: the experimental workflow was completed successfully, but the viral genetic material never made it into the final clones. As a result, the study could not detect or characterize mutations in Influenza RD8.

Key Methods

Extracted viral RNA from amniotic fluid collected from infected chicken eggs.
Measured RNA concentration and purity using a Nanodrop reader.
Converted RNA into cDNA using influenza-specific and general primers.
Amplified target influenza genes (HA and NA) through PCR.
Checked PCR products on agarose gel to confirm amplification.
Cloned PCR products into a plasmid vector and transformed them into E. coli.
Screened bacterial colonies and isolated plasmid DNA.
Sequenced plasmid inserts and compared them with known sequences using BLAST.

Key Tools / Materials

QIAamp RNA extraction kit
Nanodrop spectrophotometer
Reverse transcription primers
PCR thermal cycler and HA/NA primer sets
Agarose gel electrophoresis
PCR 2.1 cloning vector and T4 DNA ligase
Competent E. coli and blue-white screening plates
Miniprep plasmid purification kit
DNA sequencing and BLAST analysis