Molecular detection of H1N1 virus by conventional reverse transcription PCR coupled with nested PCR

Abstract

Influenza A pdm09 virus has been spreading worldwide and creates a serious public health threat. Among different subtypes/lineages of swine influenza A viruses, the H1N1 subtype is more prevalent in all-over world infections followed by H3N2 which is present in the northern hemisphere. Influenza A pdm09 is dominant for centuries but the last pandemic was in 2009 and is responsible for 151,700–575,400 deaths within one year worldwide (Centres for Disease Control and Prevention). Rapid diagnosis for H1N1 is essential which must be highly sensitive and specific and can differentiate between influenza A subtypes and other respiratory pathogens. In this study, a conventional polymerase chain reaction assay and nested polymerase chain reaction assay were developed which can detect H1N1 pdm09. One outer set of primer is designed and one inner set of primer is designed specifically for hemagglutinin gene (H1). Specificity was checked using different pathogenic genomes for amplification of the H3N2 subtype, Neisseria meningitides, Salmonella typhi, etc. The sensitivity of the nested polymerase chain reaction is 0.001ng/6 ​μL of PCR amplified product of outer primer product of HA1 gene as compared to 50 ​ng/μL of two-step conventional polymerase chain reaction. The nested PCR and conventional PCR are performed on 80 samples which are consistent with the Real-Time PCR results. Real-Time is a standard test for H1N1 detection. But it is very expensive but current method is less expensive and provides comparable results with Real-Time PCR.

Keywords

• H1N1
• Nested PCR
• Conventional PCR
• Hemagglutinin gene
• Respiratory virus