Accidental Amplification of the Avian Influenza N4 Gene Segment by N1-Specific Primers

Background: Avian influenza is a worldwide animal and public health risk. With increasing bird-to-human transmission, it has become a significant infectious agent. Objectives: This study investigated why N1-specific primers amplified the N4 gene segment. Materials and Methods: In the Central Veterinary Laboratory in the Kurdistan region, N4 was accidentally amplified by N1 primers. The polymerase chain reaction (PCR) product undergoes a confirmation process by designing specific primers for N4 and Sanger sequencing. Results: Out of thirteen samples amplified by N1-specific primers, one sample (KCVL-013) was N4 genotype. The results showed a 500bp PCR product as an N4 genotype amplified by N1 primers. The N4 PCR product was confirmed by DNA Sanger sequencing. For further confirmation, the sample (KCVL-013) was reamplified by specific primers designed for N4. Conclusion: Avian influenza A viral fragments are highly variable in their nucleotide sequences. Further confirmation of the PCR product is required to confirm the positive result. DNA sequencing for PCR products is a highly reliable method.