Detection of Influenza A subtype H5N1 virus
using the LightCycler® Instruments

Roche Applied Science and TIB Molbiol announced the availability of a test system for the detection of H5N1 virus, using Roche Applied Science LightCycler® real-time PCR Systems, combined with parameter specific reagents (LightMix® reagent) from TIB Molbiol.next

Influenza A

Influenza viruses are about 100 nm in diameter. The InfA genome consists of eight single negative-stranded RNAs between 900 and 2400 nucleotides long.

Birds are carriers of a variety of InfA virus; in 2-5% of the wild bird population carry the InfA virus (Niesters, Virology Rotterdam). Most InfA viruses are not lethal for birds and can not be transferred to humans.

We differentiate the virus by its haemagglutinin (HA or H) and neuraminidase (N) glycoproteins, both of which are integrated in the virus membrane. Virus type H1N1 caused the fatal Spanish Flu pandemic in 1918-1920, types H2N2 and H3N2 are widely present in humans. The actual avian H5N1 virus caused several fatal humans cases in Asia and very recently in Turkey but does not seem to cause human to human infections.

RNA viruses are fast mutating and the detection must focus on conserved sequence regions. The preferred gene for broad InfA detection is the matrix protein (M) gene. Tests for the detection of the actual H5N1 virus must detect these two specific genes.

RNA virus tests based on FRET hybridization probes can be safer as they detect also new occurring variants since they will still generate a specific signal in the melting curve analysis even when a few bases at the binding site are exchanged.

LightMix® concept

LightMix products consist of premixed primers and hybridization probes for use with the LightCycler® Instruments series 1.x, 2.0 and 480 from Roche Diagnostics.The product is lyophilized in 6 individual vials, 16 reactions each, providing a total of 96 reactions. Also included, a lyophilized standard row for quantitative analysis ranging from 10 to 10E6 target equivalents per reaction. Products are tested with FastStart /FastStart Plus DNA Master Hybridization Probes on the LightCycler® Instruments versions 1.x and 2.0. LightMix® products do not contain polymerase, buffer or dNTPs.

LightMix® kits for Influenza H5N1 testing

The Influenza A virus is a RNA virus. Detection of the genomic RNA requires the preparation of cDNA prior to DNA amplification by PCR, which can optionally be performed in a One-Step RT-PCR reaction. LightMix® M2 targets the matrix protein gene to detect broad range Influenza A including the current Asian H5N1 isolates (2004/2005).

Order no.

Product

LightCycler®

Kit size

40-0219-16

LightMix H5

1.x, 2.0, 480

96 rxns

40-0230-16

LightMix N1

1.x, 2.0, 480

96 rxns

 

40-0234-16

LightMix M2

1.x, 2.0

96 rxns

 

40-0277-96

LightMix M2 HT

480

480 rxns

 

40-0242-16

LightMix H5N1

1.x, 2.0

96 rxns

 

 

The duplex PCR kits for the detection of M2 and H5N1 are designed for the LightCycler® Instruments 1.x and 2.0 only. The products for the detection of H5 and N1 can also be run on the LightCycler® 480. For an overview and instructions download next

For Reseach Use Only.

Detecting strategy – M2 screening and testing for the H5 and N1 genes

To screen samples for Influenza A virus cDNA/RNA (detected with probes labeled with LightCycler® Red 640) use the LightMix for the detection of Influenza virus A M2.

This LightMix also contains an internal PCR control (IPC, detected with probes labeled with LightCycler® Red 705) preventing false negative results due to PCR inhibition.

In case of positive results for Influenza A virus cDNA the sample can be further analyzed with the LightMix® for the detection of Influenza virus A H5 and if further specification is necessary with the LightMix® for the detection of Influenza virus A N1.

Alternatively the detection of the features H5 and N1 genes of Influenza virus A can be accomplished with the LightMix® for the detection of Influenza virus A H5N1 in a single capillary. This LightMix® is based on a duplex PCR reaction for the detection of Influenza virus A H5 cDNA with probes labeled with LightCycler® Red 640 and for Influenza virus A N1 cDNA with probes labeled with LightCycler® Red 705.

Background - Selection of Sequences

The primers and probes used for the detection of the H5 gene have been developed since January 2004 based on virus sequences isolated in Thailand. The amplified gene fragment is a component of the PCR product recently recommended (June 2005) by the WHO, Geneva. Our Blast analysis revealed 179 and 360 matches with 100% identity to the WHO primers; while the LightMix® H5 primers match 337 and 375 entries and 366 and 356/375 matches for the LightMix® H5N1 duplex PCR primers. The first version of the LightMix® H5 product has been tested on more than 50,000 samples in Asian countries and can be considered field-tested.

The German Reference Laboratory for Human Influenza certified a satisfactory sensitivity for the LightMix® H5 product; other validation studies are underway.

The primers and probes used for the detection of the N1 gene were selected based on alignments of Asian H5N1 virus sequences. Comparison among the primers, WHO show 114 and 124 matches in contrast to 357 and 258 matches with the LightMix® primers.

The German Reference Laboratory for Human Influenza found superior sensitivities using the LightMix®.

The LightMix® product for the detection of the Matrix gene is based on earlier designs published by Smith et. al., 2003. The detection probes were changed and the primers selected according to the publication from Schweiger et al, 2000 then modified to detect the actual H5N1virus isolates.

Specificity and Sensitivity

All LightMix® products have been tested using cDNA obtained from Asian H5N1 virus isolates. Sensitivity was measured by means of plasmid dilution rows containing the respective targets. The sensitivity of all PCR assays is at least 10 genome equivalents.

Validation

The single target assays for H5 and N1 and the matrix gene have been tested by the German AIV Reference laboratories for human influenza (Robert-Koch-Institut, RKI) and for veterinarian influenza (Friedrich-Löffler-Institut, FLI).

The statement from the RKI from november 2005 was positive. The sensitivity with the N1 assay was better than all other available assays.

The statement from FLI from january 2006 was also positive: 'suitable for the detection of the AIV genome' and 'the H5 assay and the N1 assay detected all current H5N1 isolates'.

References

Recommended laboratory tests to identify avian influenza A virus in specimens from humans, WHO Geneva, 06-2005

Rapid detection of influenza A and B viruses in clinical specimens by Light Cycler real time RT-PCR. Smith AB, Mock V, Melear R, Colarusso P, Willis DE. J Clin Virol. 2003 Sep 28(1):51-58

Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. Schweiger B, Zadow I, Heckler R, Timm H, Pauli G. JCM 38 (2000) 1552-1558

www.roche-applied-science.com/sis/avian

Notice to Purchaser

A license under U.S. Patents 4,683,202, 4,683,195 and 4,965,188 or their foreign counterparts, owned by Hoffmann-La Roche Inc. and F. Hoffmann-La Roche Ltd (“Roche”), has an up-front fee component and a running-royalty component. The purchase price of this product includes limited, nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction (“PCR”) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license. These rights under the up-front fee component may be purchased from Perkin-Elmer or obtained by purchasing an authorized thermal cycler. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR process for research applications may be obtained by contacting the Director of Licensing at The Perkin-Elmer Corporation, 850 Lincoln Center Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501. The purchase of this product does not convey any right for its use in clinical diagnostic applications. No rights for TaqManTM technology under U.S. Patents 5,210,015 and 5,487,972 are hereby conveyed.

These reagents were developed and manufactured by TIB MOLBIOL®, Berlin, Germany. LightCycler® hybridization probes produced under license from Roche Diagnostics. LightCycler® is a registered trademark of a member of the Roche group. For Research Use Only.