阅读说明：本技术 基于实时数字pcr检测人端粒绝对长度的方法及试剂盒 (Method and kit for detecting absolute length of human telomere based on real-time digital PCR ) 是由 沈燕龙 景奉香 吴东平 颜进取 于 2020-06-11 设计创作，主要内容包括：本申请提供了基于实时数字PCR检测人端粒绝对长度的方法,包括制备标准品、合成特异性扩增引物和探针引物、标准品实时数字PCR检测、待测样品实时数字PCR检测、计算端粒的绝对长度等步骤。本申请所提供的基于实时数字PCR检测人端粒绝对长度的方法对样本要求低,既能检测单个染色体端粒的绝对长度,又能提供端粒长度分布,专用于短端粒检测。并且,本申请通过加入特定染色体基因的扩增引物和探针,可以得到特定染色体端粒绝对长度,操作快捷、简便、经济、高效。(The application provides a method for detecting the absolute length of human telomeres based on real-time digital PCR, which comprises the steps of preparing a standard substance, synthesizing a specific amplification primer and a probe primer, detecting the real-time digital PCR of the standard substance, detecting a sample to be detected by the real-time digital PCR, calculating the absolute length of the telomeres and the like. The method for detecting the absolute length of the human telomere based on the real-time digital PCR has low requirements on samples, can detect the absolute length of the telomere of a single chromosome, can provide telomere length distribution, and is specially used for short telomere detection. In addition, the absolute length of the telomere of the specific chromosome can be obtained by adding the amplification primer and the probe of the specific chromosome gene, and the method is quick, simple, convenient, economic and efficient to operate.)

1. A method for detecting the absolute length of human telomeres based on real-time digital PCR is characterized by at least comprising the following steps:

(1) preparing a standard product:

synthesizing double-stranded DNA (deoxyribonucleic acid) which takes a single copy gene A of human origin and a telomere sequence B with known copy number m (TTAGGG) as a core sequence, connecting the double-stranded DNA to a PMD19-T vector through TA cloning, sequencing to verify the sequence, and determining the concentration of a plasmid solution to obtain a PMD19-T-A & B plasmid which is taken as a telomere detection nucleic acid standard substance;

(2) synthesis of specific amplification primers and probes:

designing and synthesizing a telomere detection specific amplification primer and a specific probe, and an A gene specific amplification primer and a specific probe according to the gene A and the gene B sequences;

wherein, the forward primer of the telomere detection specific amplification primer comprises a specific sequence of which the 5' end does not belong to a telomere sequence, a quenching group is marked, and a plurality of uniformly spaced sequences which are not matched with a telomere repetitive sequence;

the reverse primers of the telomere detection specific amplification primers comprise a plurality of uniformly spaced sequences which are not matched with telomere repetitive sequences and do not form dimers with the forward primers;

the telomere detection specific probe sequence is complementary with the specific sequence of the telomere detection forward primer or the specific sequence of the telomere detection reverse primer, a blocking group is added at the 3 'end of the telomere detection specific probe sequence to prevent the telomere detection specific probe sequence from extending, and a report group is marked at the 5' end;

(3) and (3) detecting a standard product by real-time digital PCR:

carrying out amplification process of A gene and B gene in the real-time digital PCR detection standard product, and recording: the fluorescence intensity and the amplification cycle number of the A gene in the standard when the amplification reaches a threshold line, and the fluorescence intensity and the amplification cycle number of the B gene in the standard when the amplification reaches the threshold line;

(4) real-time digital PCR detection of a sample to be detected:

collecting a sample to be detected, carrying out real-time digital PCR detection on the amplification process of the gene A and the gene B in the sample to be detected, and recording the fluorescence intensity and the amplification cycle number of the gene A in the sample to be detected when the amplification reaches a threshold line and the fluorescence intensity and the amplification cycle number of the gene B in the sample to be detected when the amplification reaches the threshold line;

(5) calculate the absolute length of telomeres:

I. absolute length L of A gene-containing chromosome telomere in sample to be detected_b：

Wherein the content of the first and second substances,

the amount of an amplification product when the amplification of the gene B in the double positive holes of the sample to be detected reaches a threshold line is the fluorescence intensity of the gene B in the double positive holes of the sample to be detected when the amplification reaches the threshold line;

X_B’the number of the B gene original template in the standard sample is m;

Ct_bthe amplification cycle number of the B gene in the double positive holes of the sample to be detected when the amplification reaches a threshold line;

Ct_B’the amplification cycle number of the B gene in the standard when the amplification reaches a threshold line;

Y_Ctathe quantity of an amplification product when the amplification of the gene A in the double positive holes of the sample to be detected reaches a threshold line is the fluorescence intensity of the gene A in the standard when the amplification reaches the threshold line;

Y_CtA’the quantity of an amplification product when the amplification of the A gene in the standard reaches a threshold line is the fluorescence intensity of the A gene in the standard when the amplification reaches the threshold line;

X_A’the number of A gene original templates in the standard sample is shown;

Ct_athe amplification cycle number of the gene A in the double positive holes of the sample to be detected when the amplification reaches a threshold line;

Ct_A’the amplification cycle number of the A gene in the standard when the amplification reaches a threshold line;

6 is the base number of 6 bases of human telomere gene (TTAGGG);

II. Absolute length L of chromosome telomere without gene A in sample to be detected_Bn：

Wherein the content of the first and second substances,

the amount of an amplification product of the B gene in the single positive hole of the sample to be detected when the amplification reaches a threshold line;

X_B’the number of the B gene original template in the standard sample is m;

Ct_B’the amplification cycle number of the B gene in the standard when the amplification reaches a threshold line;

Ct_Bnthe amplification cycle number of the B gene in the single positive hole of the sample to be detected when the amplification reaches a threshold line;

Y_Ctathe quantity of an amplification product when the amplification of the gene A in the double positive holes of the sample to be detected reaches a threshold line is the fluorescence intensity of the gene A in the standard when the amplification reaches the threshold line;

Y_CtA’the amount of the amplification product when the amplification of the gene A in the standard reaches the threshold line is taken as the fluorescence signalA value;

X_A’the number of A gene original templates in the standard sample is shown;

Ct_athe amplification cycle number of the gene A in the double positive holes of the sample to be detected when the amplification reaches a threshold line;

Ct_A’the amplification cycle number of the A gene in the standard when the amplification reaches the threshold line.

2. The method for detecting the absolute length of human telomeres based on real-time digital PCR according to claim 1,

the nucleotide sequence of the telomere detection forward primer is shown as SEQ ID NO: 1 is shown in the specification;

the nucleotide sequence of the telomere detection reverse primer is shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the telomere detection probe is shown as SEQ ID NO: 3, respectively.

3. The method for detecting the absolute length of human telomeres based on real-time digital PCR according to claim 2,

the 5' end of the forward primer of the telomere detection specific amplification primer is marked with a quenching group;

the 5 'end of the telomere detection specific probe sequence is marked with a report group, and the 3' end is added with a blocking group.

4. The method for detecting the absolute length of the human telomere based on real-time digital PCR of claim 1, wherein the A gene is ACTB gene.

5. The method for detecting the absolute length of human telomeres based on real-time digital PCR according to claim 4,

the nucleotide sequence of the ACTB gene forward primer is shown as SEQ ID NO: 4 is shown in the specification;

the nucleotide sequence of the ACTB gene reverse primer is shown as SEQ ID NO: 5 is shown in the specification;

the nucleotide sequence of the ACTB gene probe is shown as SEQ ID NO: and 6.

6. The method for detecting the absolute length of human telomeres based on real-time digital PCR according to claim 5,

the 5' end of the forward primer of the ACTB gene specific amplification primer is marked with a quenching group;

and a reporter group is marked at the 5 'end of the ACTB gene specific probe sequence, and a blocking group is added at the 3' end.

7. The method for detecting the absolute length of the human telomere based on the real-time digital PCR as claimed in claim 1, wherein the PCR reaction system for performing the real-time digital PCR detection on the standard substance and the sample to be detected comprises: 10 x dPCR BufferMix 3.5 u l, upstream and downstream primers 1 u l, probe primer 1 u l, genomic DNA1.5 u l, water to 35u l.

8. The method for detecting the absolute length of the human telomere based on the real-time digital PCR as claimed in claim 1, wherein the amplification reaction conditions for the real-time digital PCR detection of the standard substance and the sample to be detected are as follows: pre-denaturation at 90 ℃ for 10min, and PCR circulation at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles.

9. A kit for detecting the absolute length of human telomeres based on real-time digital PCR is characterized by comprising:

SEQ ID NO: 1, telomere detection forward primer;

SEQ ID NO: 2, telomere detection reverse primer;

SEQ ID NO: 3, a telomere detection probe;

SEQ ID NO: 4, an ACTB gene forward primer;

SEQ ID NO: 5, an ACTB gene reverse primer;

SEQ ID NO: ACTB gene probe shown in FIG. 6.

10. The kit for detecting the absolute length of human telomeres based on real-time digital PCR according to claim 9,

the 5' end of the forward primer of the telomere detection specific amplification primer is marked with a quenching group;

the 5 'end of the telomere detection specific probe sequence is marked with a report group, and the 3' end is added with a blocking group;

the 5' end of the forward primer of the ACTB gene specific amplification primer is marked with a quenching group;

and a reporter group is marked at the 5 'end of the ACTB gene specific probe sequence, and a blocking group is added at the 3' end.

Technical Field

The application relates to the technical field of digital PCR, in particular to a method and a kit for detecting the absolute length of human telomeres based on real-time digital PCR.

Background

Telomere lengths in human cells can vary from very short (e.g., less than 500bp) to very long (e.g., greater than 20 kbp). Kimura et al describe the telomeric length (Nature Protococ., 2010,5(9): 1596-. One measure of telomere length distribution is the percentage of short telomeres, e.g., the percentage of telomeres less than 1kb, less than 2kb, or less than 3kb in length. For example, samples can be tested to determine whether short telomeres account for more than 15%, more than 20%, more than 25%, or more than 30% of the total number of telomeres. Recent studies have shown that the score of short telomeres is likely to correlate better with health measures than the average telomere length. The measure of telomere abundance and the rate of change of these measures are indicative of health, risk or presence of a pathological state, and responsiveness to a drug, and the measure of telomere abundance of cells from the sample or the rate of change of these measures can be correlated with telomeric disease or drug responsiveness, etc.

Disclosure of Invention

In view of the problems in the background art, the invention provides a method and a kit for detecting the absolute length of human telomeres based on real-time digital PCR.

In order to achieve the above object, the method for detecting the absolute length of human telomeres based on real-time digital PCR provided by the first aspect of the present application at least comprises the following steps:

(1) preparing a standard product:

synthesizing double-stranded DNA (deoxyribonucleic acid) which takes a single copy gene A of human origin and a telomere sequence B with known copy number m (TTAGGG) as a core sequence, connecting the double-stranded DNA to a PMD19-T vector through TA cloning, sequencing to verify the sequence, and determining the concentration of a plasmid solution to obtain a PMD19-T-A & B plasmid which is taken as a telomere detection nucleic acid standard substance;

(2) synthesis of specific amplification primers and probes:

designing and synthesizing a telomere detection specific amplification primer and a specific probe, and an A gene specific amplification primer and a specific probe according to the gene A and the gene B sequences;

wherein, the forward primer of the telomere detection specific amplification primer comprises a specific sequence of which the 5' end does not belong to a telomere sequence, a quenching group is marked, and a plurality of uniformly spaced sequences which are not matched with a telomere repetitive sequence;

the reverse primers of the telomere detection specific amplification primers comprise a plurality of uniformly spaced sequences which are not matched with telomere repetitive sequences and do not form dimers with the forward primers;

the telomere detection specific probe sequence is complementary with the specific sequence of the telomere detection forward primer or the specific sequence of the telomere detection reverse primer, a blocking group is added at the 3 'end of the telomere detection specific probe sequence to prevent the telomere detection specific probe sequence from extending, and a report group is marked at the 5' end;

(3) and (3) detecting a standard product by real-time digital PCR:

carrying out amplification process of A gene and B gene in the real-time digital PCR detection standard product, and recording: the fluorescence intensity and the amplification cycle number of the A gene in the standard when the amplification reaches a threshold line, and the fluorescence intensity and the amplification cycle number of the B gene in the standard when the amplification reaches the threshold line;

(4) real-time digital PCR detection of a sample to be detected:

collecting a sample to be detected, carrying out real-time digital PCR detection on the amplification process of the gene A and the gene B in the sample to be detected, and recording the fluorescence intensity and the amplification cycle number of the gene A in the sample to be detected when the amplification reaches a threshold line and the fluorescence intensity and the amplification cycle number of the gene B in the sample to be detected when the amplification reaches the threshold line;

(5) calculate the absolute length of telomeres:

I. absolute length L of A gene-containing chromosome telomere in sample to be detected_b：

Wherein the content of the first and second substances,

the amount of an amplification product when the amplification of the gene B in the double positive holes of the sample to be detected reaches a threshold line is the fluorescence intensity of the gene B in the double positive holes of the sample to be detected when the amplification reaches the threshold line;

the amount of an amplification product when the amplification of the gene B in the standard reaches a threshold line is the fluorescence intensity of the gene B in the standard when the amplification reaches the threshold line;

X_B’the number of the B gene original template in the standard sample is m;

Ct_bthe amplification cycle number of the B gene in the double positive holes of the sample to be detected when the amplification reaches a threshold line;

Ct_B’the amplification cycle number of the B gene in the standard when the amplification reaches a threshold line;

Y_Ctathe quantity of an amplification product when the amplification of the gene A in the double positive holes of the sample to be detected reaches a threshold line is the fluorescence intensity of the gene A in the standard when the amplification reaches the threshold line;

Y_CtA’the quantity of an amplification product when the amplification of the A gene in the standard reaches a threshold line is the fluorescence intensity of the A gene in the standard when the amplification reaches the threshold line;

X_A’the number of A gene original templates in the standard sample is shown;

Ct_athe amplification cycle number of the gene A in the double positive holes of the sample to be detected when the amplification reaches a threshold line;

Ct_A’the amplification cycle number of the A gene in the standard when the amplification reaches a threshold line;

6 is the base number of 6 bases of human telomere gene (TTAGGG);

II. Absolute length L of chromosome telomere without gene A in sample to be detected_Bn：

Wherein the content of the first and second substances,

the amount of an amplification product of the B gene in the single positive hole of the sample to be detected when the amplification reaches a threshold line;

the average amount of the amplification products when the amplification of the B gene containing the A gene chromosome in the double positive holes of the sample to be detected reaches a threshold line is as follows:wherein n is the number of double positive holes of the sample to be detected;the average length of the B gene containing the A gene chromosome in the double positive holes of the sample to be detected is as follows:

wherein n is the number of double positive holes of the sample to be detected;

the amount of an amplification product when the amplification of the gene B in the standard reaches a threshold line is the fluorescence intensity of the gene B in the standard when the amplification reaches the threshold line;

X_B’the number of the B gene original template in the standard sample is m;

Ct_B’the amplification cycle number of the B gene in the standard when the amplification reaches a threshold line;

Ct_Bnthe amplification cycle number of the B gene in the single positive hole of the sample to be detected when the amplification reaches a threshold line;

the average amplification cycle number of the B gene containing the A gene chromosome in the double positive holes of the sample to be detected when the amplification reaches a threshold line is as follows:wherein n is the number of double positive holes of the sample to be detected;

Y_Ctathe quantity of an amplification product when the amplification of the gene A in the double positive holes of the sample to be detected reaches a threshold line is the fluorescence intensity of the gene A in the standard when the amplification reaches the threshold line;

Y_CtA’when the amplification of the gene A in the standard reaches a threshold line, the amount of an amplification product can be taken as a fluorescence signal value;

X_A’the number of A gene original templates in the standard sample is shown;

Ct_athe amplification cycle number of the gene A in the double positive holes of the sample to be detected when the amplification reaches a threshold line;

Ct_A’the amplification cycle number of the A gene in the standard when the amplification reaches the threshold line.

Preferably, in the method for detecting the absolute length of human telomeres based on real-time digital PCR provided by the invention, the nucleotide sequence of the telomere detection forward primer is as shown in SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the telomere detection reverse primer is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the telomere detection probe is shown as SEQ ID NO: 3, respectively.

Preferably, the 5' end of the forward primer of the telomere detection specific amplification primer is labeled with a quenching group; the 5 'end of the telomere detection specific probe sequence is marked with a report group, and the 3' end is added with a blocking group.

Preferably, the a gene is the ACTB gene.

Preferably, in the method for detecting the absolute length of human telomeres based on real-time digital PCR provided by the invention, when the gene a is the ACTB gene, the nucleotide sequence of the ACTB gene forward primer is as shown in SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the ACTB gene reverse primer is shown as SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of the synthesized ACTB gene probe primer is shown as SEQID NO: and 6.

Preferably, the 5' end of the forward primer of the ACTB gene-specific amplification primer is labeled with a quencher; and a reporter group is marked at the 5 'end of the ACTB gene specific probe sequence, and a blocking group is added at the 3' end.

Preferably, the PCR reaction system for real-time digital PCR detection of the standard and the sample to be detected comprises: 10 x dPCR Buffer Mix 3.5 u l, upstream and downstream primers 1 u l each, probe primer 1 u l, genomic DNA1.5 u l, water to 35u l.

Preferably, the amplification reaction conditions for performing real-time digital PCR detection on the standard substance and the sample to be detected are as follows: pre-denaturation at 90 ℃ for 10min, and PCR circulation at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles.

The invention also provides a kit for detecting the absolute length of human telomeres based on real-time digital PCR, which comprises:

SEQ ID NO: 1, telomere detection forward primer; SEQ ID NO: 2, telomere detection reverse primer; SEQ ID NO: 3, a telomere detection probe;

SEQ ID NO: 4, an ACTB gene forward primer; SEQ ID NO: 5, an ACTB gene reverse primer; SEQ ID NO: ACTB gene probe shown in FIG. 6.

Preferably, the 5' end of the forward primer of the telomere detection specific amplification primer is labeled with a quenching group; the 5 'end of the telomere detection specific probe sequence is marked with a report group, and the 3' end is added with a blocking group; the 5' end of the forward primer of the ACTB gene specific amplification primer is marked with a quenching group; and a reporter group is marked at the 5 'end of the ACTB gene specific probe sequence, and a blocking group is added at the 3' end.

Compared with the prior art, the technical scheme provided by the invention at least has the following beneficial effects:

conventional PCR, which typically employs two oligonucleotide primers each allowing polymerase extension in the direction of the other primer to anneal to the antisense complement of the target amplicon, presents a significant challenge for repeated DNA sequences (e.g., telomeric repeats) because the primers complementary to the reverse strands of the repeated sequences will bind to each other to form dimers.

The probe method can avoid repeated telomere sequences and reduce the dimer and mismatch phenomena of the probe, the template, the primer and the amplification product. In the PCR process of the present invention: at the melting temperature, the probe primer, the forward primer, the reverse primer and the DNA template are all in a free state; when the temperature is reduced, the probe primer is preferentially combined with the 5 'end of the forward primer in a specific manner to form a complex, and the 5' end of the forward primer contains a quenching group, so that a reporter group of the probe primer does not emit light; upon further temperature drop, the forward primer complex and the reverse primer bind to the DNA template strand; when the extension temperature is reached, the DNA polymerase starts to work, when the probe primer is contacted, the hydrolysis reporter group of the probe primer is separated, and the system starts to have a fluorescent signal which is detected and recorded by a machine. The next round of PCR is started, the unhydrolyzed probe from the previous round is recombined with the forward primer or with a new DNA strand, and when the probe primer is hydrolyzed again by the DNA polymerase, the fluorescence signal of the system is increased again. Until the probe primer is completely hydrolyzed or the cycle is finished.

Therefore, the method for detecting the absolute length of the human telomere based on the real-time digital PCR has low requirements on samples, can detect the absolute length of the telomere of a single chromosome, can provide telomere length distribution, and is specially used for short telomere detection. In addition, the absolute length of the telomere of the specific chromosome can be obtained by adding the amplification primer and the probe of the specific chromosome gene, and the method is quick, simple, convenient, economic and efficient to operate.

Drawings

FIG. 1 is a schematic representation of the standard PMD19-T-A & B plasmid in mutexample 2;

FIG. 2 is a schematic diagram of telomere amplification by real-time digital PCR of example 2.

Detailed Description

The present application is further described below in conjunction with the detailed description. It should be understood that these specific embodiments are merely illustrative of the present application and are not intended to limit the scope of the present application.