Giuseppe
Emeritus
A multiplex one-step real-time RT-PCR assay for influenza surveillance (Euro Surveill., abstract, edited)
[Source: Eurosurveillance, full text: <cite cite="http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19798">Eurosurveillance - View Article</cite>. Abstract, edited.]
Eurosurveillance, Volume 16, Issue 7, 17 February 2011
Research articles
A multiplex one-step real-time RT-PCR assay for influenza surveillance
I Huber 1,2, H Campe 1,2, D Sebah 1, C Hartberger 1, R Konrad 1, M Bayer 1, U Busch 1, A Sing 1
1. Bavarian Health and Food Safety Authority (LGL), Oberschlei?heim, Germany
2. These authors contributed equally to this work
Citation style for this article: Huber I, Campe H, Sebah D, Hartberger C, Konrad R, Bayer M, Busch U, Sing A. A multiplex one-step real-time RT-PCR assay for influenza surveillance. Euro Surveill. 2011;16(7)
ii=19798. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19798
Date of submission: 22 July 2010
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 3.5x102 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 Ct 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.
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[Source: Eurosurveillance, full text: <cite cite="http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19798">Eurosurveillance - View Article</cite>. Abstract, edited.]
Eurosurveillance, Volume 16, Issue 7, 17 February 2011
Research articles
A multiplex one-step real-time RT-PCR assay for influenza surveillance
I Huber 1,2, H Campe 1,2, D Sebah 1, C Hartberger 1, R Konrad 1, M Bayer 1, U Busch 1, A Sing 1
1. Bavarian Health and Food Safety Authority (LGL), Oberschlei?heim, Germany
2. These authors contributed equally to this work
Citation style for this article: Huber I, Campe H, Sebah D, Hartberger C, Konrad R, Bayer M, Busch U, Sing A. A multiplex one-step real-time RT-PCR assay for influenza surveillance. Euro Surveill. 2011;16(7)
ii=19798. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19798Date of submission: 22 July 2010
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 3.5x102 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 Ct 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.
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