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

A method for the transcription based amplification of a double stranded target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, said method comprising:

• a) simultaneously incubating the sample, suspected to contain HBV, in an amplification buffer, in the absence of a restriction primer, with
• i) 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 strand(s),
• ii) 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 defined 3′ end of the DNA strand, and
• iii) a second or reverse primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of said target sequence,
• b) maintaining the thus created reaction mixture of (a) under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place;
• c) subjecting the reaction mixture of (b) 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;
• d) adding the following reagents to the reaction mixture of (c):
• i) an enzyme having RNA dependent DNA polymerase activity;
• ii) an enzyme having DNA dependent DNA polymerase activity;
• iii) an enzyme having Rnase H activity;
• and
• iv) an enzyme having RNA polymerase activity;
• and
• e) maintaining the reaction mixture of (d) under the appropriate conditions for a sufficient amount of time for the amplification to take place.