Development and Evaluation of a Real-time RT-PCR Technique for Detecting Matrix Gene of Influenza Virus Type A in Human Throat Swab Samples

Influenza A virus is now considered to be widespread in poultry and has demonstrated the ability to infect humans in Iran. For laboratory diagnosis of these respiratoryviruses, it is essential to have rapid methods, able to detect viruses in early stages of the infection in clinical specimens. The real-time reverse-transcription polymerase chain reaction n (rRT-PCR) assay has been established as a standard method for Influenza virus type A diagnosis inpatients. In this study, we evaluateda single-tube rRT-PCR assay, targeting to the highly conserved region of matrix(M) gene for detection of the virus. In this experimental study, after preparation of 100throat mucus samples, respective RNA was extracted from the virus by usingviral RNA extraction kit. Two specific primers were synthesized, based on the conserved region of Influenza type AM-gene and a home-brewedone-step SYBR Green based rRT-PCR was developed and evaluated for detection of Influenza type A infection in the viral samples, on the basis of melting curveanalysis. The presence of M-gene in RNAs, extracted from 53viralsamples, was confirmed by this single-tube rRT-PCR assay, and after 45 amplification cycles, themelting curve analysis revealed the melting temperature (Tm) of 83.2 ± 0.5°C for various viral samples, quite different from that of primer-dimers and the positive samples showed only a small variation in parameters. This study showed that thedeveloped one-step rRT-PCR assay is the proper molecular method for rapid and accurate diagnosis of Influenza A by detection of M-protein encoding gene.

Keywords:

Influenza A , Diagnosis , Real-Time RT-PCR , Matrix Gene