阅读说明：本技术 一种用于ffpe样本dna质控的试剂盒及检测方法 (Kit for FFPE sample DNA quality control and detection method ) 是由 任绪义 张锋 曹子豪 毕亭亭 于 2019-08-23 设计创作，主要内容包括：本发明公开了一种用于FFPE样本DNA质控的试剂盒及检测方法,属于生物检测技术领域。本试剂盒中包含的引物和探针序列如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示,实施步骤包括：采用生物信息学技术手段在人类基因组序列上(hg19)设计了3条PCR扩增引物,通过三条引物采用荧光定量PCR技术对待评估标本进行PCR扩增(包含108bp和212bp的扩增产物),根据两个扩增产物的CT值差异以及浓度,对标本质量进行评估,为PCR扩增或第二代测序(NGS)等下游分子实验提供重要的样本质量依据。(The invention discloses a kit and a detection method for FFPE sample DNA quality control, and belongs to the technical field of biological detection. The sequences of the primers and the probes contained in the kit are shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4, the implementation steps comprise: 3 PCR amplification primers are designed on a human genome sequence (hg19) by adopting a bioinformatics technical means, PCR amplification (including amplification products of 108bp and 212 bp) is carried out on a sample to be evaluated by adopting a fluorescent quantitative PCR technology through the three primers, the quality of the sample is evaluated according to the difference of CT values and the concentration of the two amplification products, and an important sample quality basis is provided for downstream molecular experiments such as PCR amplification or second generation sequencing (NGS).)

1. A kit for controlling the quality of DNA in an FFPE sample is characterized by comprising the following 3 primers and 1 probe which are used as reagents for amplification, wherein the 3 primers are respectively as follows:

SEQ ID NO.：1：CCAAGAGGTAGAGATCATCAAGAGC；

SEQ ID NO.：2：TCCATTAGACCAGAAAAGACAGCA；

SEQ ID NO.：3：CCAAGAATCAAGACAGACACAAA；

The 1 probe is: SEQ ID No.: 4: FAM-TTGATGGGACGTATT-MGB;

Wherein SEQ ID No.: 1 and SEQ ID No.: 2 primer to obtain amplicon 1, and SEQ ID No.: 1 and SEQ ID No.: 3 primer pair to obtain amplicon 2, wherein amplicon 1 is 108bp in length, amplicon 2 is 212bp in length, and amplicon 1 and amplicon 2 have multiple copies on human genome (hg19) and are distributed on different chromosomes.

2. The kit of claim 1, wherein the quality of the DNA in the FFPE sample can be effectively evaluated by only 3 primers and 1 probe.

3. the kit of claim 1, wherein the kit comprises the following amplification systems:

System 1 comprises the following components: 1 × PCR premix, upstream primer SEQ ID No.: 1, downstream primer SEQ ID No.: 2, probe SEQ ID No.: 4, template DNA.

System 2 comprises the following components: 1 × PCR premix, upstream primer SEQ ID No.: 1, downstream primer SEQ ID No.: 3, probe SEQ ID No.: 4, template DNA.

4. A detection method for FFPE sample DNA quality control, which is characterized in that the detection kit is used for implementing the detection kit of any one of claims 1-3, and comprises the following steps:

1) preparing PCR reaction solution of the system 1 and the system 2;

2) Preparing a standard curve reaction system containing a sample to be evaluated and based on a standard substance;

3) Quantitative PCR amplification reaction is carried out.

4) and calculating the CT values of the amplicon 1 and the amplicon 2 and the concentration of the sample to be evaluated, and evaluating the quality of the sample.

5. the detection method for FFPE sample DNA quality control as claimed in claim 4, wherein the standard for evaluating the quality of the sample is as follows:

1) the CT value difference is less than 2, and the DNA integrity is good;

2) The difference of CT value is between 2 and 4, and the DNA is degraded, but can still be used in standard experiment;

3) The CT difference is between 4 and 6, and the DNA is degraded, so that the risk is high when the DNA is used for downstream experiments;

4) CT difference is less than 6, DNA degradation is serious, and subsequent experiments are not recommended.

Technical Field

The invention belongs to the technical field of biotechnology, and particularly relates to a kit and a detection method for FFPE sample DNA quality control.

Background

Tissue samples are processed by Formalin-fixed Paraffin-embedded (FFPE) methods, which can preserve the samples for a long time, and thus the methods are widely used in clinical pathological examination, medical science research, biological sample preservation, and the like. At present, in the tissue sample library of medical or biological research institution, there are a huge number of FFPE samples, which provide valuable research materials for the work of elucidating the pathogenesis of diseases, exploring the therapeutic targets of new drugs, and comprehensive retrospective research.

With the development of scientific technology, the emergence of Next-generation sequencing technology (NGS) has greatly improved the ability of researchers to detect genes. The sequencing speed is high, the throughput is high, the sequencing cost is reduced continuously, and the like, so that the NGS method is successfully applied to the medical inspection work and becomes a powerful tool for inspection personnel. Meanwhile, the NGS is also possible to perform large-scale gene sequencing of numerous FFPE samples stored in tissue sample banks. However, due to a plurality of uncontrollable factors in the sample processing and storing process, the FFPE sample has the risk of DNA degradation and fragmentation, and the DNA degradation degree/integrity of different FFPE samples is greatly different. In the NGS detection process, the same sequencing strategy, if used, will severely affect the yield of the final valid sequencing data. Therefore, it is essential to accurately assess DNA concentration and integrity before NGS sequencing or other molecular detection of FFPE sample DNA.

the existing method for detecting the DNA concentration mainly comprises a Nanodrop micro spectrophotometer and a Qubit fluorescence quantification. The Nanodrop calculates the DNA concentration mainly by using the light absorption values of the DNA at 260nm and 280 nm; the Qubit uses fluorescent dyes that specifically bind dsDNA and that emit fluorescence under excitation from a light source of a specific wavelength to calculate the DNA concentration. The 2 methods only detect DNA concentration and do not reflect DNA integrity.

Common methods for testing the DNA integrity of FFPE samples are: agarose gel electrophoresis, nucleic acid fragment analyzer and qPCR method. Agarose gel electrophoresis is a method that when nucleic acid molecules are placed in an electric field, negatively charged nucleic acid molecules migrate towards a positive electrode, and the migration speed depends on the size and configuration of the nucleic acid molecules, i.e., DNA molecules with smaller molecular weight migrate faster than DNA molecules with larger molecular weight. The fragment size distribution of the FFPE sample DNA can be distinguished by using the characteristic agarose gel electrophoresis. The principle of action of the nucleic acid fragment analyzer is similar to that of agarose gel electrophoresis.

According to the invention, 3 PCR primers and 1 probe are designed by adopting a bioinformatics method, fluorescence quantitative PCR amplification (amplification of fragments of 108bp and 212 bp) is carried out on a to-be-detected sample through the 3 primers and the 1 probe, the quality of the sample is evaluated according to the difference of CT values of two amplicons and the obtained concentration of the sample to be detected, and an important sample quality basis is provided for downstream experiments such as PCR amplification or second generation sequencing (NGS).

Disclosure of Invention

The invention provides a kit for quantitatively detecting the concentration and integrity of FFPE sample DNA by using a multi-copy homologous sequence in a human genome and based on a Taqman-MGB probe technology.

a kit for controlling the quality of DNA in an FFPE sample comprises the following reagents for amplification, namely 3 primers and 1 probe, wherein the 3 primers are respectively as follows:

SEQ ID NO.：1：CCAAGAGGTAGAGATCATCAAGAGC；

SEQ ID NO.：2：TCCATTAGACCAGAAAAGACAGCA；

SEQ ID NO.：3：CCAAGAATCAAGACAGACACAAA；

the 1 probe is: SEQ ID No.: 4: FAM-TTGATGGGACGTATT-MGB;

Wherein SEQ ID No.: 1 and SEQ ID No.: 2 primer to obtain amplicon 1, and SEQ ID No.: 1 and SEQ ID No.: 3 primer pair to obtain amplicon 2, wherein amplicon 1 is 108bp in length, amplicon 2 is 212bp in length, and amplicon 1 and amplicon 2 have multiple copies on human genome (hg19) and are distributed on different chromosomes.

Furthermore, the kit can effectively evaluate the quality of the DNA in the FFPE sample only by 3 primers and 1 probe.

further, the kit comprises the following amplification systems:

System 1 comprises the following components: 1 × PCR premix, upstream primer SEQ ID No.: 1, downstream primer SEQ ID No.: 2, probe SEQ ID No.: 4, template DNA.

System 2 comprises the following components: 1 × PCR premix, upstream primer SEQ ID No.: 1, downstream primer SEQ ID No.: 3, probe SEQ ID No.: 4, template DNA.

The invention also discloses another technical scheme: a detection method for FFPE sample DNA quality control is implemented by the detection kit, and comprises the following steps:

1) Preparing PCR reaction solution of the system 1 and the system 2;

2) Preparing a standard curve reaction system containing a sample to be evaluated and based on a standard substance;

3) And carrying out fluorescent quantitative PCR amplification reaction.

4) and calculating the CT values of the amplicon 1 and the amplicon 2 and the concentration of the sample to be evaluated, and evaluating the quality of the sample.

further, the standard for evaluating the quality of the specimen is as follows:

1) The CT value difference is less than 2, and the DNA integrity is good;

2) The difference of CT value is between 2 and 4, and the DNA is degraded, but can still be used in standard experiment;

3) The CT difference is between 4 and 6, and the DNA is degraded, so that the risk is high when the DNA is used for downstream experiments;

4) CT difference is less than 6, DNA degradation is serious, and subsequent experiments are not recommended.

The kit and the detection method for FFPE sample DNA quality control provided by the invention have the advantages that:

(1) The specificity is strong: and only the conserved region sequence of the amplified homologous sequence is identified by using the optimal gene primer and the corresponding MGB labeled probe, and primer dimer cannot interfere detection, so that false positive and false negative are effectively avoided.

(2) Safe and simple: in the detection system, amplification and detection are carried out in one tube, and a cover is not required, so that pollution can be effectively prevented; when in use, the PCR reaction can be started only by adding the DNA of a sample to be detected, and the use is simple and convenient.

(3) The accuracy is high: the kit adopts a standard curve method, the linear relation among the standard substances with different concentration gradients is good, and the relative concentration of corresponding amplifiable fragments in the DNA of a sample can be accurately quantified.

(4) And (3) fast: the speed is high, the flux is high, and the process can be finished within 1 hour.

The kit provided by the invention can accurately and quickly detect the DNA concentration and integrity of the FFPE sample. In clinical detection, the FFPE sample DNA is accurately evaluated, and an effective sample quality control basis can be provided for downstream molecular detection experiments such as NGS and the like.

drawings

FIG. 1A is a standard curve of a fluorescence quantitative PCR system 1 using a standard substance for gradient dilution;

FIG. 1B is a standard curve of the fluorescence quantitative PCR system 2 using the standard substance for gradient dilution;

FIG. 2 is a schematic diagram of primer probe design.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

the following examples are included to further illustrate the beneficial effects of the present invention: