Utilization of a Mechanical Homogenization-Based Direct-to-PCR Method for Influenza A Virus Detection

Respiratory viruses, both novel and known, are a significant focus of public health efforts across the globe. With the mainstay of many public health efforts to combat these pathogens relying on accurate detection of the virus within human or animal populations, the need for clinical research to improve testing has driven innovation in the field of nucleic acid detection of these viruses. 

The current gold-standard method for detection of respiratory viruses, such as influenza, is polymerase chain reaction (PCR) amplification of a specific gene product. This method has two fundamental parts, the sample preparation step involving nucleic acid extraction and purification, and the PCR amplification of the targeted viral gene. While this method has been shown to have high specificity and reproducibility, the workflow is time intensive and costly. Hence, research is being done on improving diagnostic testing for respiratory viruses like Influenza A (IAV) and SARS-CoV-2 through a direct-to-PCR (dPCR) method to lower the time and resources needed for viral detection over traditional extraction-based PCR. 

Herein, we demonstrate the utility of a mechanical homogenization-based dPCR method for IAV detection utilizing a set of performance-verified universal IAV probes targeting the matrix protein (MP) gene to confirm presence of IAV on spiked swabs simulating nasopharyngeal swabs.

Table 1: Average Ct values following RT-PCR for M gene amplification at each given concentration.