Establishment of a new molecular method for influenza A virus detection using improved generic primers and concentration of viral RNP

Abstract

The mismatch rate and positions of primers and templates as well as the copy numbers of target genes in specimens may affect the sensitivity of polymerase chain reaction (PCR)-based diagnostics. To date, various generic primer sets to detect influenza A viruses (IAVs) have been reported

however, their mismatch rates have not been extensively evaluated. In this study, we verified the nucleotide identities of reported primer sets with 3,441 complete coding regions of matrix genes. According to the results, none of compared primers showed 100% identity to more than 86% of the compared genes. Therefore, we designed a new degenerate primer set with 100% identity to more than 98% of compared genes and compared the amplification efficiency of the primer set using SYBR-based real-time reverse transcription PCR (SYBR-RT-qPCR) with TaqMan probe-based RT-qPCR. The analytical sensitivity of each RT-qPCR method was assessed by determining the smallest amount that can be detected from the serial dilutions of the IAVs viral RNAs. According to our results, both RT-qPCR methods showed similar analytical sensitivity (1.37 EID50) when performed with viral RNAs of wild-type PR8 virus, whereas our new SYBR-RT-qPCR method showed about ten-fold higher analytical sensitivity (7.89 EID50) than that of Taqman probe-based RT-qPCR method (78.9 EID50) when performed with viral RNAs of recombinant PR8 virus which has a single nucleotide mismatch in the 5th position from the 3 end of Spackmans reverse primer. In addition, we compared the clinical sensitivity, specificity, and positive predictive value between our new SYBR-RRT PCR method and Taqman probe-based RT-qPCR in 293 fecal samples of migratory birds collected from the drainage basin of the the Geum-gang river. The clinical sensitivities of the two RT-qPCR methods were similar as 100%, while the clinical specificity and positive predictive value of SYBR RT-qPCR were 95.41%, and 43.48%, respectively, both of which are higher than 85.16%, and 19.23%, of Taqman probe-based RT-qPCR. Furthermore, we successfully increased the sensitivity of SYBR-RT-qPCR by concentrating the complex of viral RNA and nucleoprotein (RNP) in allantoic fluid and feces reacting with Triton X-100, anti-nucleoprotein mouse monoclonal antibodies, and anti-mouse immunoglobulin antibody-conjugated magnetic beads. The concentration of viral RNA in the eluate was significantly increased by 64-fold compared to the conventional RNA extraction method by using our RNP-concentrating method. In addition, we verified that our RNP concentration method could detect IAVs below the detection limit of the conventional RNA extraction method. Thus, the improved generic primer set and RNP concentration method together can be applied as useful tool for sensitive detection of IAVs.