An optimized molecular method for detection of influenza A virus using improved generic primers and concentration of the viral genomic RNA and nucleoprotein complex

Abstract

For reported primer sets used to detect influenza A viruses (IAVs), we verified the nucleotide identities with 9,103 complete sequences of matrix (M) genes. At best, only 93.2% and 85.3% of the sequences had a 100% match with reported forward and reverse primers, respectively. Therefore, we designed new degenerate forward and reverse primers with 100% identity to 94.4% and 96.2% of compared genes, respectively, and the primer set was used with SYBR-based reverse-transcription real-time PCR (SYBR-RT-rtPCR) for lower detection limits. The sensitivity of SYBR-RT-rtPCR with the new primers was 10-fold higher than that with a conventional method in ~2.37% of all M genes in the database used in our study. We successfully increased the sensitivity of SYBR-RT-rtPCR by concentrating the viral ribonucleoprotein (RNP) using immunomagnetic beads and Triton X-100. The improved generic primer set and RNP concentration method may be useful for sensitive detection of IAVs.

Keywords: Generic primer; influenza A virus; matrix gene; real-time PCR; ribonucleoprotein.

Figure 1.

Positive response to various subtypes of influenza A viruses and negative response to other avian RNA viruses. A. Detection of IAV panels [A/Puerto Rico/8/1934 (H1N1), A/Singapore/1/57 (H2N2), A/duck/Ukraine/1/63 (H3N8), A/duck/Hong Kong/820/80 (H5N3), A/duck/Hong Kong/301/78 (H7N1), A/turkey/Ontario/6118/68 (H8N4), A/turkey/Wisconsin/1/66 (H9N2), A/Chicken/Germany/N49 (H10N7), A/duck/Memphis/546/74 (H11N9), A/duck/Alberta/60/76 (H12N5)] by SYBR-RT-rtPCR. B. Verification of specificity to various avian RNA viruses [NDV (La Sota), IBV (SNU11045), IBDV (SNU16001), REV (SNU16008). Negative control (DEPC-treated distilled deionized water).

Figure 2.

Comparison of the detection limits of RNP concentration and conventional RNA extraction methods. rPR8 virus was diluted 10-fold, and RNA from A. 10^-6 and B 10^-7. diluted samples were extracted directly or after RNP concentration. SYBR-RT-rtPCR was performed, and melting curves were compared for the presence and signal intensity of the specific amplicon.