Abstract
For surveillance purposes real-time PCR assays for influenza viruses had to be adapted to the pandemic influenza A(H1N1)2009 strain. We combined published primers and probes for influenza A, influenza B and an internal amplification control with a detection system for influenza A(H1N1)2009 to set up a rapid, reliable, simple and cost-effective high-throughput multiplex one-step real-time RT-PCR. The workflow also includes automated sample preparation for high-throughput screening. The lower limit of detection of the multiplex assay was 3.5x10(2) RNA copies per PCR reaction. The diagnostic sensitivity of the multiplex assay was 87.7%, but increased to 99.4% for influenza-positive samples yielding C(t) values of less than 34 cycles in the respective diagnostic assay. High specificity was confirmed by sequencing and correct detection of 15 reference samples from two quality assurance studies. The multiplex PCR was introduced for surveillance of samples from a network of general practitioners and paediatricians in Bavaria, Germany during the influenza pandemic of 2009. Comparison with surveillance data from reported cases proved the reliability of the multiplex assay for influenza surveillance programmes.
Highlights
In April 2009, a novel influenza A(H1N1) virus emerged [1] that could not be detected by routine diagnostic assays for subtyping seasonal influenza A(H1N1) viruses
The optimised multiplex real-time RT-PCR assay in a 25 μl PCR reaction volume was composed as follows: 400 nM of all primers and 200 nM of each of the four TaqMan probes, 1x QuantiTect virus RT mix, 1x QuantiTect virus No Rox (NR) mastermix, 4U RNase inhibitor, 0.25 μl internal amplification control (IAC) RNA (2.5x104 copies) and 5 μl RNA extract
We report on a multiplex one-step real-time RT-PCR assay for the simultaneous detection of seasonal influenza A and B as well as influenza A(H1N1)2009 viruses
Summary
In April 2009, a novel influenza A(H1N1) virus emerged [1] that could not be detected by routine diagnostic assays for subtyping seasonal influenza A(H1N1) viruses. At that stage of the pandemic the World Health Organization (WHO), the European Centre for Disease Prevention and Control (ECDC) and the Robert Koch Institute in Germany (RKI) recommended strengthening the influenza surveillance. This surveillance should persist throughout the whole year and include the new influenza strain as well as seasonal influenza strains, because co-circulation was reported and expected in the future. Published diagnostic assays focused more on subtyping of influenza viruses using microarrays and sequencing [11,12,13,14] These tests are not suitable for high-throughput routine diagnostic screening
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