阅读说明：本技术 用于定量pcr扩增的组合物及其应用 (Composition for quantitative PCR amplification and application thereof ) 是由 杨林 张薇婷 高雅 张艳艳 陈芳 夏军 蒋慧 徐讯 于 2017-06-20 设计创作，主要内容包括：提供了用于定量PCR扩增的组合物,其包括：一对PCR引物对和一条特异性探针,所述PCR引物对包括第一引物和第二引物,其中,所述第一引物包含第一特异性序列和第一随机序列,所述第一特异性序列位于所述第一引物的3’端,所述第一随机序列位于所述第一引物的5’端,所述第二引物包含第二特异性序列和第二随机序列,所述第二特异性序列位于所述第二引物的3’端,所述第二随机序列位于所述第二引物的5’端,并且,所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物,所述第一随机序列和所述第二随机序列反向互补,所述特异性探针的序列与所述靶序列的上游引物和下游引物之间的序列互补配对。(Provided is a composition for quantitative PCR amplification, comprising: a pair of PCR primer pairs and a specific probe, wherein the PCR primer pair comprises a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is located at the 3' end of the first primer, the first random sequence is located 5' to the first primer, the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is located at the 3 'end of the second primer, the second random sequence is located at the 5' end of the second primer, and the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at the target sequence, the first random sequence and the second random sequence are reverse complementary, and the sequence of the specific probe is complementary to the sequence complementary pairing between the upstream primer and the downstream primer of the target sequence.)

A composition for quantitative PCR amplification comprising: a pair of PCR primer pairs and a specific probe,

the PCR primer pair comprises a first primer and a second primer,

wherein the content of the first and second substances,

the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is positioned at the 3 'end of the first primer, and the first random sequence is positioned at the 5' end of the first primer,

the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is positioned at the 3 'end of the second primer, and the second random sequence is positioned at the 5' end of the second primer,

and the number of the first and second electrodes,

the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at a target sequence, and the first random sequence and the second random sequence are reversely complementary,

the 5 'end of the specific probe is connected with a fluorescent group, the 3' end of the specific probe is connected with a quenching group, and the sequence of the specific probe is complementarily paired with the sequence between the upstream primer and the downstream primer of the target sequence.

The composition of claim 1, wherein the TM values of the first specific sequence and the second specific sequence are 55-65 degrees celsius and the TM values of the first primer and the second primer are 65-75 degrees celsius.

The composition of claim 1, wherein the first random sequence and the second random sequence are 16-30bp in length, and the first specific sequence and the second specific sequence are 16-30bp in length.

The composition of claim 1, wherein the 1 st to 5 th bases at the 5 'end and the 3' end of the first primer and the second primer are thio modified.

The composition of claim 4, wherein the thio modification is any one selected from the group consisting of a thiophosphoric acid type modification, a methylsulfate type modification and a peptide nucleic acid modification.

The composition of claim 1, wherein the 5' end of at least one of the first primer and the second primer is modified by phosphorylation.

The composition of claim 1, wherein the sequence length of the specific probe is 18-30bp, and the TM value is 70-80 degrees celsius.

A quantitative PCR amplification kit comprising the composition for quantitative PCR amplification according to any one of claims 1 to 7.

A quantitative PCR amplification method, wherein the quantitative PCR amplification is performed using the composition for quantitative PCR amplification according to any one of claims 1 to 7 or the quantitative PCR amplification kit according to claim 8.

The method of claim 9, wherein the method comprises two rounds of amplification comprising:

carrying out first round linear amplification on the PCR primer pair and the template in the presence of the specific probe at the annealing temperature of 55-65 ℃; and

and carrying out second round of circular amplification on the products of the first round of linear amplification at the annealing temperature of 65-72 ℃.

The method of claim 10, wherein the amplification reaction sequence of the method is as follows:

a method for quantitative analysis of a DNA sample to be tested, comprising:

the method according to any one of claims 9 to 11, wherein the DNA sample to be tested is subjected to fluorescent quantitative PCR amplification and the quantitative analysis is carried out based on the collected fluorescent signal.

A method for performing gene expression differential analysis of a specific gene in a plurality of test DNA samples, wherein each of the plurality of test DNA samples comprises a cDNA sequence of the specific gene, the method comprising:

the method according to any one of claims 9 to 11, wherein the plurality of DNA samples to be tested are subjected to fluorescent quantitative PCR amplification respectively, and quantitative analysis is realized based on the collected fluorescent signals; and

comparing the quantitative analysis results of the plurality of DNA samples to be tested so as to determine the gene expression difference of the specific genes of the plurality of DNA samples to be tested.