Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures

Abstract

The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

FIG. 1.

Highly specific NA signature primers for the 2009 H1N1 pandemic influenza virus. (A) Amplification of pandemic 2009 H1N1 NA in a conventional RT-PCR assay. RNA extracted from 1 × 10⁷ to 1 × 10⁸ PFU/ml of the shown H1N1 or H3N2 influenza viruses was subjected to a one-step RT-PCR using either the 2009 H1N1 NA signature primers (upper panel) or M segment consensus primers (lower panel) and run on a 1.2% agarose gel for detection by ethidium bromide. (B) Performance of 2009 H1N1 NA signature primers in a SYBR green assay. Specific NA signal curves obtained in a SYBR green one-step RT-PCR assay for the reference strains are shown. (C) Melting curve confirmation of specific products. In panels B and C, each sample was run in triplicate in a 96-well plate (25-μl final volume) using a Bio-Rad CFX96 real-time PCR detection system. Data are representative of two independent experiments with identical results.

FIG. 2.

Performance of 2009 H1N1 NA signature primers. RT-PCR amplification curves for a SYBR green assay (A) and a probe-based assay (C) conducted with the 2009 H1N1 NA signature primers and RNA extracted from 10-fold serial dilutions of a negative nasal swab spiked with the indicated amounts of the Neth09 virus isolate. The results with respect to linearity of the reaction obtained by the 2009 H1N1 NA signatures SYBR green (B) and probe-based (D) RT-PCR assays are shown. Samples were run in triplicate in a 25-μl final volume as described for the experiment shown in Fig. 1, and data are representative of two independent experiments.

FIG. 3.

Human samples detected with a 2009 H1N1 NA signatures SYBR green-specific assay. Performance and accuracy of the 2009 H1N1 NA signatures SYBR green assay were assessed with 125 human clinical samples (nasopharyngeal swabs or tracheal aspirates) obtained from individuals presenting with influenza-like symptoms during the 2009 H1N1 influenza outbreak in Chile (May to August 2009). Results are plotted as the C_T values obtained with the CDC 2009 H1 assay versus the C_T values obtained with the 2009 H1N1 NA signatures SYBR green assay. The limits of detection for each assay were 37 and 35 cycles, respectively, and these are depicted as dotted lines on each axis. Regression curves made with 10-fold serial dilution standards were between 0.984 and 0.995 in two independent runs for each assay. Dots indicate the mean C_T signal obtained for triplicate reactions per sample for each assay performed, in a final volume of 10 μl, in a 384-well plate using a Roche LightCycler 480 real-time PCR system. Samples that were over the limit of detection were considered negative for each specific assay.