Detection method of micro-rna with high specificity

Abstract

The invention provides a method for detecting miRNA based on polymerase chain reaction comprising EvaGreen dye, and the use of EvaGreen dye in elevating the binding specificity of primers to templates in PCR.

1 - 10 . (canceled) 
     
     
         11 . A method for detecting miRNA, wherein, the method comprises 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 EVAGREEN™ fluorescent dye, thereby forming double stranded DNA-EVAGREEN™ fluorescent dye complex; and detecting the presence and/or 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 5′ end of the forward primer makes the Tm value of the whole forward primer with cDNA reach 45-75° C.; and   (iii) the length of the forward primer is 8-50 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.   
     
     
         12 . The method according to  claim 11 , wherein, in step (d), the presence or the amount of the amplified products is determined by detecting the fluorescence value of EVAGREEN™ dye. 
     
     
         13 . The method according to  claim 11 , wherein the forward primer has the following characteristics:
 (i) the sequence of 3′ end of the forward primer is the same as that of 5′ end of the miRNA to be detected; and   (ii) the sequence of 5′ end of the forward primer makes the Tm value of the whole forward primer with cDNA reach 50-75° C.   
     
     
         14 . The method according to  claim 11 , wherein the sample to be detected is selected the following group: animal sample, food, fodder and medicament. 
     
     
         15 . A method for obtaining the information of non-coding RNA from a sample to be detected, wherein the method comprises the following steps:
 (a) extracting RNA sample containing the non-coding RNA from a sample to be detected;   (b) adding a poly A tail to 3′ end of the non-coding RNA for the extracted RNA in step (a);   (c) adding a primer which is complementary to the polyA tail of the non-coding RNA to anneal the primer with the non-coding RNA containing polyA;   (d) reverse-transcribing the noncoding RNA containing polyA tail into cDNA;   (e) conducting PCR amplification to the cDNA, using forward and reverse primers complementary to the cDNA in the PCR reaction system containing EVAGREEN™ fluorescent dye, thereby forming double stranded DNA-EVAGREEN™ fluorescent dye complex; and detecting the presence and/or the amount of the amplified products during or after the amplification to obtain information of the non-coding RNA including the presence and the amount of the non-coding RNA in the sample,   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 5′ end of the forward primer makes the Tm value of the whole forward primer with cDNA reach 45-75° C.; and   (iii) the length of the forward primer is 8-50 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.   
     
     
         16 . The method according to  claim 15 , wherein, the fluorescent dye is a DNA binding dye selected from the group consisting of: EVAGREEN™ dye, and SYBR™ green dye. 
     
     
         17 . A method for improving binding specificity between a primer and a template during PCR reaction, wherein the method comprises the step of adding the primer and a fluorescence dye in PCR reaction system,
 wherein the primer comprises a forward primer and a reverse primer;   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 5′ end of the forward primer makes the Tm value of the whole forward primer with cDNA reach 45-75° C.; and   (iii) the length of the forward primer is 8-50 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 0 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.   
     
     
         18 . The method according to  claim 17 , wherein the fluorescence dye is EVAGREEN™. 
     
     
         19 . A pair of primers for detecting miRNA and its cDNA, wherein the pair of primers comprise a forward primer and a reverse primer;
 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 5′ end of the forward primer makes the Tm value of the whole forward primer with cDNA reach 45-75° C.; and   (iii) the length of the forward primer is 8-50 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.   
     
     
         20 . A kit for detecting miRNA, wherein the kit comprises a forward primer, a reverse primer and a fluorescent dye,
 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 5′ end of the forward primer makes the Tm value of the whole forward primer with cDNA reach 45-75° C.;   (iii) the length of the forward primer is 8-50 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.   
     
     
         21 . A kit according to  claim 20 , wherein, the fluorescent dye is a DNA binding dye selected from the group consisting of: EVAGREEN™ dye, and SYBR™ green dye.