阅读说明：本技术 一种在多重pcr体系内加入内参定量核酸拷贝数的方法 (Method for adding internal reference quantity of nucleic acid copy number in multiplex PCR system ) 是由 浦浩 黄文潘 张亚楠 李阳 于 2019-11-22 设计创作，主要内容包括：本发明提供了一种在多重PCR体系内加入内参定量核酸拷贝数的方法,属于核酸分子生物学检测技术领域；所述方法,包括以下步骤：1)构建内参和目标基因扩增效率系数训练集,获得内参与目标基因扩增系数之间的关系；2)在目标基因的多重PCR体系中添加确定拷贝数的内参以及扩增内参的引物后,进行两轮多重PCR扩增后获得测序文库；3)对所述测序文库进行测序,获得测序数据；4)分析所述测序数据获得内参和目标基因的reads数,利用步骤1)中获得的内参与目标基因扩增效率系数之间的关系,计算目标基因的拷贝数。本发明所述方法能够快速的定量样品中特定核酸基因的拷贝数,准确度高。(The invention provides a method for adding internal reference quantity of nucleic acid copy number in a multiplex PCR system, belonging to the technical field of nucleic acid molecular biology detection; the method comprises the following steps: 1) constructing an internal reference and target gene amplification efficiency coefficient training set to obtain the relationship between the internal reference and the target gene amplification coefficients; 2) adding an internal reference for determining copy number and a primer for amplifying the internal reference into a multiplex PCR system of a target gene, and performing two rounds of multiplex PCR amplification to obtain a sequencing library; 3) sequencing the sequencing library to obtain sequencing data; 4) analyzing the sequencing data to obtain the reads number of the internal reference and the target gene, and calculating the copy number of the target gene by utilizing the relation between the internal reference and the target gene amplification efficiency coefficient obtained in the step 1). The method can rapidly quantify the copy number of the specific nucleic acid gene in the sample, and has high accuracy.)

1. A method for adding an internal reference amount of nucleic acid copy number in a multiplex PCR system, comprising the steps of:

1) constructing an internal reference and target gene amplification efficiency coefficient training set to obtain the relationship between the internal reference and the target gene amplification coefficients;

2) adding an internal reference for determining copy number and a primer for amplifying the internal reference into a multiplex PCR system of a target gene, and performing two rounds of multiplex PCR amplification to obtain a sequencing library;

3) sequencing the sequencing library to obtain sequencing data;

4) analyzing the sequencing data to obtain the reads number of the internal reference and the target gene, and calculating the copy number of the target gene by utilizing the relation between the internal reference and the target gene amplification efficiency coefficient obtained in the step 1);

the nucleotide sequence of the internal reference is shown as SEQ ID No. 1.

2. The method of claim 1, wherein the construction of the training set of internal reference and target gene amplification efficiency coefficients comprises the following steps:

1.1) respectively diluting a target gene and an internal reference to different concentration gradients, and respectively mixing the target gene and the internal reference with the same concentration to obtain a plurality of template sets;

1.2) adding primers for amplifying a target gene and an internal reference into the template set, and performing two rounds of PCR amplification to obtain a sequencing library;

1.3) sequencing and analyzing the sequencing library to obtain reads of an internal reference gene and a target gene;

1.4) calculating and obtaining the amplification efficiency coefficients of the internal reference gene and the target gene according to the formula I;

wherein σ_iIs the amplification efficiency of the i-th target gene, d_inIs the number of reads of the ith target gene in the nth concentration gradient, a_nIs the average of reads in the nth concentration gradient test.

3. The method according to claim 2, wherein the different concentration gradients in step 1.1) comprise a total of 5 concentration gradients with copy numbers of 300, 1000, 3000, 10000 and 30000.

4. The method of claim 1, wherein the formula for calculating the copy number of the target gene in step 4) is shown in formula II,

wherein r is_iIs the copy number of the i-th target gene, d_iIs the reads number obtained by sequencing the ith target gene in the sequencing sample; sigma_iIs the amplification efficiency coefficient of the ith target gene and is obtained by calculation of a formula I; d_nIs the reads number of the internal reference; c is the copy number of the added internal reference.

5. The method according to claim 1 or 2, wherein the nucleotide sequence of the primers for the amplification of the internal reference in step 2) is shown as SEQ ID No.2 and SEQ ID No. 3.

6. The method of claim 1, wherein the length of the sequencing in step 3) is greater than PE 50.

7. The method of claim 1, wherein analyzing the sequencing data in step 4) comprises splitting Barcode, data quality filtering, removing low quality reads, and removing non-specific amplification reads.

Technical Field

The invention belongs to the technical field of nucleic acid molecular biology detection, and particularly relates to a method for adding internal reference quantity of nucleic acid copy number in a multiplex PCR system.

Background

In order to rapidly and accurately quantify the copy number of a particular gene in a nucleic acid sample, a number of techniques have been developed. These techniques have wide applications in detecting and quantifying nucleic acids in food, environmental, clinical, and other types of samples.

One common approach to accomplish such a task is nucleic acid hybridization, which is based on the principle that two nucleic acid strands containing complementary or substantially complementary sequences have the ability to form a double-stranded structure under appropriate conditions, thereby being able to bind specifically. In order to detect and quantify a particular nucleic acid sequence, a labeled probe complementary to the target sequence is prepared. At present, the Quantitative detection of DNA mostly adopts a real-time fluorescent Quantitative polymerase chain reaction (qPCR) technology, but the price is high, and certain technical limitations also exist. Although the quality of the domestic reagents is greatly improved, the sensitivity, accuracy, repeatability and the like of most reagents are far from the sensitivity, accuracy, repeatability and the like of imported high-quality reagents, and the time difference is more obvious when clinical samples with low nucleic acid amount or interfering substances are detected.

With the development of a new generation of high-throughput target sequence capture sequencing technology, represented by the IonPGMTM/ProtonTM series of Life Technologies and Hiseq and Miseq series sequencers of Illumina, the method has the advantages of high throughput, low cost, comprehensive detection variation type, high accuracy and the like which can not be replaced by other Technologies, and is widely applied to various biological researches and medical detection. Compared with the qPCR technology, the method for capturing the target sequence by using the multiple PCR amplification has the obvious advantages of simple operation, low cost, large enrichment of the target DNA sequence in a short time and the like, and greatly shortens the detection process and the detection time. For current nucleic acid detection and quantification methods based on target sequence amplification, it has been a well-known challenge to accurately detect and quantify genes with different genotypes or mutations (e.g., mutations that cause disease). Therefore, a method which is based on the multiple PCR capture technology and can rapidly, accurately and quantitatively detect and quantify the gene copy number in the sample at one time is developed, and the method has market application value.

Disclosure of Invention

In view of the above, the present invention provides a method for adding an internal reference amount of nucleic acid copy number into a multiplex PCR system, which can rapidly quantify the copy number of a specific nucleic acid gene in a sample, and has high accuracy and good universality.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a method for adding internal reference quantity of nucleic acid copy number in a multiplex PCR system, which comprises the following steps:

1) constructing an internal reference and target gene amplification efficiency coefficient training set to obtain the relationship between the internal reference and the target gene amplification coefficients;

2) adding an internal reference for determining copy number and a primer for amplifying the internal reference into a multiplex PCR system of a target gene, and performing two rounds of multiplex PCR amplification to obtain a sequencing library;

3) sequencing the sequencing library to obtain sequencing data;

4) analyzing the sequencing data to obtain the reads number of the internal reference and the target gene, and calculating the copy number of the target gene by utilizing the relation between the internal reference and the target gene amplification efficiency coefficient obtained in the step 1);

the nucleotide sequence of the internal reference is shown as SEQ ID No. 1.

Preferably, the construction of the training set of the internal reference and target gene amplification efficiency coefficients comprises the following steps:

1.1) respectively diluting a target gene and an internal reference to different concentration gradients, and respectively mixing the target gene and the internal reference with the same concentration to obtain a plurality of template sets;

1.2) adding primers for amplifying a target gene and an internal reference into the template set, and performing two rounds of PCR amplification to obtain a sequencing library;

1.3) sequencing and analyzing the sequencing library to obtain reads of an internal reference gene and a target gene;

1.4) calculating and obtaining the amplification efficiency coefficients of the internal reference gene and the target gene according to the formula I;

wherein σ_iIs the amplification efficiency of the i-th target gene，d_inIs the number of reads of the ith target gene in the nth concentration gradient, a_nIs the average of reads in the nth concentration gradient test.

Preferably, the different concentration gradients in step 1.1) comprise a total of 5 concentration gradients with copy numbers of 300, 1000, 3000, 10000 and 30000.

Preferably, the formula for calculating the copy number of the target gene in step 4) is shown in formula II,

wherein r is_iIs the copy number of the i-th target gene, d_iIs the reads number obtained by sequencing the ith target gene in the sequencing sample; sigma_iIs the amplification efficiency coefficient of the ith target gene and is obtained by calculation of a formula I; d_nIs the reads number of the internal reference; c is the copy number of the added internal reference.

Preferably, the nucleotide sequence of the primer for amplifying the internal reference in the step 2) is shown as SEQ ID No.2 and SEQ ID No. 3.

Preferably, the length of the sequencing in step 3) is greater than PE 50.

Preferably, analyzing the sequencing data in step 4) comprises splitting Barcode, data quality filtering, removing low quality reads and removing non-specific amplification reads.

The invention has the beneficial effects that: the invention provides a method for adding internal reference quantity nucleic acid copy number in a multiplex PCR system, wherein amplification internal reference is added in the multiplex PCR system, so that the phenomenon of false negative in PCR detection can be effectively solved; if the reaction system has no amplification internal reference, the negative result is detected probably because the reaction system has no target gene or because the reaction system has an inhibitor, the PCR instrument has a fault, the reaction system has an error, the polymerase is inactivated and the like. If the amplification internal reference is added into the reaction system, no matter whether a target gene appears in the reaction system or not, a signal of the amplification internal reference always appears, and if the amplification internal reference does not have a signal, the PCR reaction fails, so that the phenomenon of false negative can be effectively solved by adding the amplification internal reference. The internal reference is an artificially synthesized sequence, is very stable in each detection process, and quantifies the copy number of a target gene contained in a detection sample through the internal reference; the method is crucial to the stability and accuracy of the detection of specific genes in a sample; the method can also be combined with unique bioinformatics analysis, can detect the content of the specific genes contained in different samples to be detected at a molecular level with high sensitivity, and has high detection result accuracy.

Drawings

FIG. 1 is a schematic diagram of the main steps of the process of the present invention;

FIG. 2 is a schematic diagram of a template set according to the present invention;

FIG. 3 is a graph showing the amplification efficiency of each target gene involved in the examples.

Detailed Description

The invention provides a method for adding internal reference quantity of nucleic acid copy number in a multiplex PCR system, which comprises the following steps: 1) constructing an internal reference and target gene amplification efficiency coefficient training set to obtain the relationship between the internal reference and the target gene amplification coefficients; 2) adding an internal reference for determining copy number and a primer for amplifying the internal reference into a multiplex PCR system of a target gene, and performing two rounds of multiplex PCR amplification to obtain a sequencing library; 3) sequencing the sequencing library to obtain sequencing data; 4) analyzing the sequencing data to obtain the reads number of the internal reference and the target gene, and calculating the copy number of the target gene by utilizing the relation between the internal reference and the target gene amplification efficiency coefficient obtained in the step 1); the nucleotide sequence of the internal reference is shown as SEQ ID No. 1.

In the invention, a training set of the amplification efficiency coefficients of the internal reference and the target gene is constructed, and the relation between the amplification coefficients of the internal reference and the target gene is obtained. In the invention, the nucleotide sequence of the internal reference is shown as SEQ ID No. 1; the internal reference is an artificially synthesized sequence, and the nucleotide sequence of the internal reference does not match with the nucleotide sequence included in the genome of the currently known biological species; the internal reference is preferably synthesized by a biological synthesis company. The invention has no special limitation on the type and sequence of the target gene, and the target gene to be detected in any organism can be used as the target gene; the present invention is not limited to the number of target genes, and in the specific implementation process, the number, the type, and the like of specific target genes are set according to the detection requirements.

In the invention, the construction of the training set of the internal reference and target gene amplification efficiency coefficients comprises the following steps: 1.1) respectively diluting a target gene and an internal reference to different concentration gradients, and respectively mixing the target gene and the internal reference with the same concentration to obtain a plurality of template sets; 1.2) adding primers for amplifying a target gene and an internal reference into the template set, and performing two rounds of PCR amplification to obtain a sequencing library; 1.3) sequencing and analyzing the sequencing library to obtain reads of an internal reference gene and a target gene; 1.4) calculating and obtaining the amplification efficiency coefficients of the internal reference gene and the target gene according to the formula I;

wherein σ_iIs the amplification efficiency of the i-th target gene, d_inIs the number of reads of the ith target gene in the nth concentration gradient, a_nIs the average of reads in the nth concentration gradient test.

In the invention, after the target gene and the internal reference are respectively diluted to different concentration gradients, the target gene and the internal reference with the same concentration are respectively mixed to obtain a plurality of template sets. In the invention, the different concentration gradients preferably comprise 5 concentration gradients with copy numbers of 300, 1000, 3000, 10000 and 30000, the target genes and the internal references are respectively diluted to the copy numbers of 300, 1000, 3000, 10000 and 30000, and then the target genes and the internal references with the same concentration are mixed to obtain a plurality of template sets; in the present invention, two repetitive treatments are preferably provided for each concentration gradient. The invention sets different concentration gradients to obtain the ratios of the amplification efficiency coefficients of different template concentrations and internal references to each target gene.

After the template set is obtained, primers for amplifying a target gene and an internal reference are added into the template set, and a sequencing library is obtained after two rounds of PCR amplification. In the invention, the nucleotide sequences of the primers for amplifying the internal reference are shown as SEQ ID No.2 and SEQ ID No. 3.

In the invention, the first round of PCR amplification is used for enriching a target gene template and an internal reference template; the system of the first round PCR is calculated by 30 μ l, and preferably comprises the following components: primer Panel Mix 8. mu.l, PCR enzyme 10. mu.l, H₂O11. mu.l, internal amplification standard (5 gradient concentrations) 1. mu.l. The amplification procedure of the first round of PCR is preferably as follows: 3min at 95 ℃; (95 ℃ C., 20 s; 60 ℃ C., 4min) for 22cycles, 72 ℃ C., 4min, and 10 ℃ C. After the first round of PCR amplification is finished, preferably purifying PCR amplification products, wherein the purification is preferably carried out by adopting a PCR amplification product purification kit; the PCR amplification product purification kit preferably adopts an Ampure XP magnetic bead purification kit. In the invention, the purification can effectively enrich target region fragments and reduce the concentration of nonspecific amplification.

After obtaining a purified amplification product of the first round of PCR, performing second round PCR amplification by taking the purified amplification product of the first round of PCR as a template; the second round PCR amplification reaction system is calculated by 30 μ l, and preferably comprises the following components: h₂18. mu.l of O, 10. mu.l of PCR enzyme, 1. mu.l of 2PCRPrimer F, 1. mu.l of 2PCR BarcodeXX R. The procedure for the second round of PCR amplification reaction is preferably as follows: 3min at 95 ℃; (95 ℃, 15 s; 58 ℃, 15 s; 72 ℃, 1min) 6-8 cycles; keeping at 72 deg.C for 5min and 10 deg.C. The ligated sequencing adapters were as follows:

2PCR PrimerF: 5 '-CCTCTCTATGGGCAGTCGGTGAT-universal sequence-3'; (SEQ ID No.2)

2PCR BarcodeXX R：

5-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGAT-universal sequence-3' (SEQ ID No. 3);

n in the sequence represents any base of AGCT, and NNNNNN is used for identifying libraries constructed by different samples; the universal primer is a sequencing universal primer, and the nucleotide sequence is GTGACTGGAGTTCCTTGGCACCCGAGA (SEQ ID No. 4). In the invention, the second round of PCR amplification is used for connecting the sequence of the target gene template after enrichment and an internal reference template with a sequencing library joint. In the present invention, it is preferable that the method further comprises a step of purifying the second round PCR product after the second round PCR amplification, and the purification of the second round PCR product is the same as the purification method of the first round PCR product, and is not described herein again.

According to the invention, a sequencing library is obtained after the second round of PCR amplification, and the reads number of the internal reference and target genes is obtained after the sequencing library is sequenced and analyzed. In the invention, the sequencing library is preferably subjected to quality inspection, and the quality inspection of the sequencing library has no special requirement and can be realized by adopting the conventional library quality inspection steps and standards in the field; in the present invention, the length of the sequencing is preferably greater than that of PE50, and the depth of the sequencing is preferably such that more than 1000 reads can be obtained for each target gene. In the present invention, the analysis includes Barcode resolution, data quality filtering, removal of low quality reads, and removal of non-specific amplification reads. The method and parameter requirements for the analysis are not particularly limited in the present invention, and conventional analysis methods and parameter requirements in the art may be employed.

According to the invention, the amplification efficiency coefficients of the internal reference and the target gene are obtained by calculation according to the reads numbers of the internal reference and the target gene obtained by analysis and the formula I, so that the relation between the amplification coefficients of the internal reference and the target gene is obtained.

In the invention, after adding an internal reference for determining copy number and a primer for amplifying the internal reference into a multiplex PCR system of a target gene, two rounds of multiplex PCR amplification are carried out to obtain a sequencing library. The number of copies of the internal parameter to be added is not particularly limited, and a known and determined number of copies, for example, 300 copies, may be used. The amplification system and the amplification procedure of the two-round multiplex PCR amplification in the present invention are the same as those in step 1.2) above, and will not be described herein.

After the sequencing library is obtained, the sequencing library is sequenced to obtain sequencing data. The invention analyzes the sequencing data to obtain the reads number of the internal reference and the target gene, and calculates the copy number of the target gene by utilizing the relation between the internal reference and the target gene amplification efficiency coefficient obtained in the steps. In the present invention, analyzing the sequencing data includes resolving Barcode, data quality filtering, removing low quality reads, and removing non-specific amplification reads.

In the invention, the formula for calculating the copy number of the target gene is shown as a formula II,

wherein r is_iIs the copy number of the i-th target gene, d_iIs the reads number obtained by sequencing the ith target gene in the sequencing sample; sigma_iIs the amplification efficiency coefficient of the ith target gene and is obtained by calculation of a formula I; d_nIs the reads number of the internal reference; c is the copy number of the added internal reference.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.