Nucleic acid detection method

#The present application claims priority from Japanese application JP 2005-099179 filed on Mar. 30, 2005, the content of which is hereby incorporated by reference into this application.

#The present invention relates to a nucleic acid (DNA or RNA) detection method that is useful in genetic analysis. More particularly, the present invention relates to a method for detecting specific genes of viruses, prokaryotes, eukaryotes, and the like.

#In the field of biological sciences, amplification of DNA or RNA has been carried out for various purposes. For example, competitive PCR (A. Wang, et al., Proc. Natl. Acad. Sci. USA, 86, 9717-9721, 1989) and real-time PCR (S. H. Aliyu, et al., Journal of Antimicrobial, 54, 968, 2004) have been known as techniques for analyzing the expression of given genes and quantifying the expression levels thereof. These techniques involve the application of a common nucleic acid amplification technique, i.e., polymerase chain reaction (PCR) (R. K. Saiki, et al., Science, 239, 487-491, 1988), to determine expression levels based on the amplified genes.

#FIG. 1 shows the procedure according to the first embodiment of the present invention.

#FIG. 2 shows the procedure according to the second embodiment of the present invention.

#FIG. 3 shows the procedure according to the third embodiment of the present invention.

#In the present invention, a sequence complementary to a promoter sequence of RNA polymerase is used as a primer for isothermal amplification, and a tag sequence is added to the 5′ end thereof. Thus, the target nucleic acid is subjected to reverse transcription using the aforementioned primer, a promoter sequence is generated upon conversion of the sequence into double-strand DNA, and a sequence complementary to the tag sequence is obtained as a transcription product. According to the method of the present invention, a tag sequence unrelated to the target nucleic acid sequence is amplified instead of the target nucleic acid, and the tag sequence in the amplified product is detected. Thus, a gene can be detected rapidly with high sensitivity.

#FIG. 1 shows the procedure according to the first embodiment of the present invention. The present invention relates to a method for amplifying and detecting a nucleic acid sequence. This method comprises: step 1 of subjecting the template target gene 1 to reverse transcription using a P1 primer 5 comprising a base sequence 2 having a sequence specific to the base sequence of the target gene 1, a base sequence 3 complementary to a promoter sequence of RNA polymerase, and an arbitrary base sequence 4; step 2 of subjecting the template of the product obtained in step 1, i.e., the target gene 1, to digestion; step 3 of subjecting the P2 primer 8 comprising a base sequence 7 specific to the base sequence of the target gene to elongation with a reverse transcriptase using a reverse transcription product 6, which has been single-stranded via step 2, as a template; step 4 of subjecting double-strand DNA comprising the elongation product 9 obtained in step 3 to transcription as a template using RNA polymerase recognizing a promoter sequence 10 of RNA polymerase; and step 5 of detecting a transcription product 13 comprising a sequence 12 complementary to the base sequence 4.

#FIG. 2 shows the procedure according to the second embodiment of the present invention. The present invention relates to a method for amplifying a nucleic acid sequence. This method comprises: step 1 of subjecting the template target gene 1 to reverse transcription using the P1 primer 5, which further comprises a base sequence 10 that is a promoter sequence of RNA polymerase at the 5′ end of the primer comprising a base sequence 2 having a sequence specific to the base sequence of the target gene 1, a base sequence 3 complementary to a promoter sequence of RNA polymerase, and an arbitrary base sequence 4; step 2 of subjecting the template of the product obtained in step 1, i.e., the target gene 1, to digestion; step 3 of subjecting the P2 primer 8 comprising a base sequence 7 specific to the base sequence of the target gene to elongation with a reverse transcriptase using a template reverse transcription product 6, which has been single-stranded via step 2; step 4 of obtaining a reverse transcription product 15 comprising sequences 12 and 14 complementary to the base sequences 4 and 10 and a transcription product 18 comprising sequences complementary to the base sequences 7, 10, and 11 from the template double-strand DNA comprising the elongation product 9 obtained in step 3 using RNA polymerase; step 6 of subjecting the template transcription product 18 obtained in step 4 to reverse transcription using the P2 primer 8; step 7 of subjecting the transcription product 18 as a template of the product obtained in step 6 to digestion; step 8 of allowing the P1 primer 5 to hybridize to the reverse transcription product 19, which has been single-stranded via step 7, thereby elongating both the primer and the reverse transcription product using a reverse transcriptase; step 9 of subjecting the template double-strand DNA 20 obtained via step 8 to transcription with RNA polymerase; and step 10 of subjecting the template transcription product 18 obtained via step 9 to reverse transcription using the P2 primer 8 to resynthesize the reverse transcription product 19 generated via step 6. Also, the method detects a transcription product 15 amplified by repeating steps 6, 7, 8, 9, and 10.