Method for the amplification and detection of HBV DNA using a transcription based amplification

• Main Authors: Dieman, Birgit Alberta Louisa Maria, Frantzen, Inge Marjolein, Van Strijp, Arnoldina Margaretha Wilhelmina
• Format: Patent
• Language: English
• Published: 25.05.2010

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.