| 摘要 |
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 the said HBV DNA strand(s), a promotor-primer, said promotor-primer having a 5' region comprising the sequence of a promotor 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 promotor-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, an enzyme having DNA dependent DNA polymerase activity, an enzyme having Rnase H activity, an enzyme having RNA 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. |
