Test ID: 800167    
Influenza Virus Type A and Type B, Molecular Detection, PCR

The LightCycler PCR has been optimized to detect common conserved sequences in the matrix genes of influenza virus type A and influenza virus type B. Viral nucleic acid is extracted by the MagNA Pure automated instrument (Roche Applied Science) from respiratory specimens. Primers directed to the matrix gene amplify a specific sequence of the virus. For the test, influenza virus type A and influenza virus type B genomic RNA is transcribed to cDNA. The LightCycler instrument (Roche Applied Science) amplifies and monitors the development of target nucleic acid sequences after the annealing step during PCR cycling by fluorescence assay. This automated PCR system can rapidly (about 1 hour) detect amplicon development through stringent air-controlled temperature cycling and capillary cuvettes. The detection of amplified products is based on the fluorescence resonance energy transfer (FRET) principle. For FRET product detection, a hybridization probe with a donor fluorophore, fluorescein, on the 3'-end is excited by an external light source and emits light that is absorbed by a second hybridization probe with an acceptor fluorophore, LC-Red 640, at the 5'-end. The acceptor fluorophore then emits a light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. Analysis of the PCR amplification and probe melting curves is accomplished through the use of LightCycler software. (Cockerill FR III, Uhl JR: Applications and challenges of real-time PCR for the clinical microbiology laboratory. In Rapid Cycle Real-Time PCR Methods and Applications. Edited by U Reischel, C Wittwer, F Cockerill. Berlin, Germany, Springer-Verlag; 2002, pp 3-30)

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