Patent ID: 9290801
Filing Date: 2016-03-22
CPC Classification: C12Q

Claim Text:
1. A method for detecting miRNA, the method comprising the following steps: (a) adding a polyA tail to the 3′ end of miRNA for a miRNA sample to be detected; (b) adding a primer which is complementary or substantially complementary to the polyA tail of miRNA to anneal the primer with miRNA containing the added polyA tail; (c) reverse-transcribing miRNA containing the added polyA tail into cDNA; (d) conducting PCR amplification to the cDNA, using forward and reverse primers complementary to the cDNA in a PCR reaction system containing a fluorescent dye, thereby forming double stranded DNA-fluorescent dye complex; and detecting the amount of the amplified products during or after the amplification to determine the presence and/or the amount of miRNA to be detected, wherein the forward primer has the following characteristics: (i) the sequence of 3′ end of the forward primer is the same or substantially the same as that of 5′ end of the miRNA to be detected; and the Tm value of the sequence of 3′ end with cDNA is 45-65° C.; (ii) the sequence of the 5′ end of the forward primer makes the Tm of the whole forward primer with cDNA between 45° C. and 75° C.; and (iii) the length of the forward primer is between 8 bp and 50 bp; and the reverse primer has the following characteristics: (i) 3′ end of the reverse primer contains a first complementary region that is complementary to the sequence of 3′ end of miRNA to be detected and consists of 1 to 15 bases; (ii) the middle sequence of the reverse primer is polyT having 8 to 30 bases in length; (iii) the sequence of 5′ end of the reverse primer makes the Tm value of the whole reverse primer with cDNA reach 45-75° C.; and (iv) the length of the reverse primer is 12-50 bp; wherein the forward primer and the reverse primer is selected from the group consisting of SEQ ID Nos.: 9-24.