Method for the Amplification and Detection of HBV DNA Using a Transcription Based Amplification

• Main Authors: FRANTZEN INGE MARJOLEIN, VAN STRIJP ARNOLDINA MARGARETHA WILHELMINA, DEIMAN BIRGIT ALBERTA LOUISA MARIA
• Format: Patent
• Language: English
• Published: 02.10.2008
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; TESTING; VINEGAR; WINE

The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of, -incubating the sample, suspected to contain HBV, in an amplification buffer with one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3' end of said HBV DNA stand(s), a promoter-primer, said promoter-primer having a 5' region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3' region complementary to the define 3' end of the DNA strand, a second or reverse primer, having the opposite polarity of the promoter-primer and comprising the 5' end of the said target sequence, and in case of HBV ssDNA as the target sequence, a restriction primer, maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place, subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded, adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity, and maintaining the thus created reaction mixture under the appropriate conditions to a sufficient amount of time for the amplification to take place.