WO2021043139A1 - 获得cfDNA标准品的引物组、PCR扩增阳性标准品及其制备方法、试剂盒及应用 - Google Patents

获得cfDNA标准品的引物组、PCR扩增阳性标准品及其制备方法、试剂盒及应用 Download PDF

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WO2021043139A1
WO2021043139A1 PCT/CN2020/112866 CN2020112866W WO2021043139A1 WO 2021043139 A1 WO2021043139 A1 WO 2021043139A1 CN 2020112866 W CN2020112866 W CN 2020112866W WO 2021043139 A1 WO2021043139 A1 WO 2021043139A1
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sequence
plasma
cfdna
primer
standard
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徐旸
王敏
李剑
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深圳微伴生物有限公司
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to the field of biotechnology, in particular to a primer set for obtaining cfDNA standard products, PCR amplification positive standard products, and preparation methods, kits and applications thereof.
  • cfDNA cell-free DNA, cfDNA
  • Mandel and Metais in 1948. Tan and his colleagues first linked cfDNA to disease in 1966, because they observed high concentrations of cfDNA in the plasma of patients with systemic lupus erythematosus (SLE). Subsequent studies also made people realize that changes in the concentration of cfDNA in plasma are closely related to the individual's physiological state and clinical manifestations. For example, acute trauma, cerebral infarction, exercise, organ transplantation, infection, etc. can all cause the concentration of cfDNA in plasma to rise. The use of cfDNA as a potential cancer biomarker began in 1977.
  • Leon and his colleagues used radioimmunochemical methods to prove that the cfDNA content detected in the serum of cancer patients was significantly higher than that of healthy people. However, with the condition of cancer patients The improvement of the patient’s body cfDNA content will also be accompanied by a decline. Therefore, a technology that can quickly detect the content of cfDNA in plasma may provide a relatively convenient auxiliary dynamic monitoring index for evaluating individual health.
  • cfDNA mainly exists in double-stranded form in the blood. It is generally believed that the main source of cfDNA is released from apoptotic or necrotic cells. Its most concentrated fragment is about 166bp in length. cfDNA integrity (cfDNA integrity) measures the ratio of long fragments to short fragments (copy number) in the obtained cfDNA. Normally, apoptosis is the main form of normal cell death, releasing DNA fragments of about 200 bp. The death of tumor cells will increase the proportion of necrotic cells, and the size of DNA fragments released by necrotic cells is different from the size of apoptotic fragments.
  • the integrity of cfDNA in the blood will also change. Therefore, the integrity of cfDNA may also become a marker that can dynamically monitor the occurrence and changes of tumors and has auxiliary guiding significance.
  • the quantitative detection methods for measuring the concentration of cfDNA in plasma are roughly divided into two categories: one is to extract cfDNA from the plasma using a kit, and the extracted product can be directly used for quantitative detection using Qubit, qPCR or digital PCR; the other The class is to directly use plasma samples for quantitative detection, which can minimize the loss or contamination of the product during the extraction process, and retain the most primitive information of cfDNA in the plasma, and the demand for plasma is small, and the operation time is low. The cost is also relatively low.
  • qPCR primers For the second type of solution, usually targeting specific conserved regions (Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma, Sarah Breitbach, etc., PLos One), two pairs of qPCR primers that are fully complementary to the conserved regions are designed.
  • the product fragments amplified by the qPCR primers are of different lengths, and the long and short fragments (222bp, 90bp) of the conserved region in the plasma sample cfDNA can be amplified separately, and then the amount of cfDNA in the plasma sample (short fragment indicator) and integrity can be indicated (Long fragment/short fragment), the long fragment includes short fragments.
  • the PCR products were amplified with long/short fragment qPCR primers, and added to animal plasma (without primer amplification template) according to preset different concentrations, as long/short fragment cfDNA standard products of different concentrations ( Generally 6 concentration gradients).
  • the corresponding qPCR primers are used to amplify cfDNA standards with different lengths and concentrations to obtain cfDNA amplification standard curves of different fragment lengths.
  • the qPCR test results are compared to obtain the cfDNA content and integrity information of the plasma sample to be tested.
  • the first type of method mentioned above is relatively mature and is currently the most widely used cfDNA quantitative detection method, but its shortcomings are also obvious: there is a large demand for plasma (generally at least 0.5ml of plasma), and cfDNA may be lost during the extraction process. Or contamination, which cannot reflect the truest condition of the cfDNA concentration and integrity in the plasma. In addition, the experimental time cost and reagent cost are relatively high.
  • the main problems of the second type of methods are as follows: 1) When cfDNA quantitative detection is limited to the use of animal plasma in standard products, there may be component differences with human plasma, which will affect the accuracy of the detection results; 2) Simultaneous detection of cfDNA concentration and A set of standard products are required for completeness.
  • Treating the original plasma with DNAase can eliminate cfDNA in the original plasma; however, this method will significantly increase the preparation steps of plasma standards, reduce efficiency, and increase costs. It also needs to be added when DNAase is added. Corresponding buffer to make DNAase active to remove cfDNA in the original plasma, which will increase the difference between the composition of the obtained plasma standard and the plasma sample to be tested, thereby making the plasma standard and the plasma sample to be tested When performing qPCR direct amplification, the amplification efficiency is different, and because the newly added buffer may also affect the measurement of the absorbance value of the qPCR amplification product, the plasma standard obtained by this method can also be obtained during use. The standard curve is not accurate.
  • the present invention aims to provide a primer set for obtaining cfDNA standard products, PCR amplification positive standard products and preparation methods, kits and applications thereof, so as to improve the accuracy of cfDNA detection of plasma samples.
  • a primer set for amplifying and obtaining a cfDNA standard includes: a first upstream primer and a first downstream primer, the first upstream primer includes a first exogenous sequence and a first anchor sequence that are sequentially connected from 5'end to 3'end, and the first downstream primer includes The second exogenous sequence and the second anchor sequence connected sequentially from the 5'end to the 3'end; the first exogenous sequence and the second exogenous sequence are not complementary paired, and are compatible with any human chromosome
  • the nucleotide sequences are not complementary; the first anchor sequence and the second anchor sequence are conserved nucleotide sequences on the same chromosome of humans, and the combination of the first anchor sequence and the second anchor sequence can be used for amplification Increase the first target sequence; the first target sequence is the first anchor sequence and its complementary sequence on the human chromosome connected sequentially from the 5'end to the 3'end, the first anchor sequence and
  • the first target sequence is a conserved nucleotide sequence in the human chromosome.
  • the first target sequence has multiple copies on the human chromosome.
  • the first target sequence is located on L1PA2, Alu, hTERT, human APP, ⁇ -actin, EIF2C1 or RPPH1 gene.
  • the length of the first target sequence is between 50 bp and 150 bp.
  • the length of the first target sequence is between 60 bp and 120 bp.
  • a PCR amplification positive standard product is a single-stranded nucleotide molecule or a double-stranded nucleotide molecule.
  • nucleotide molecules it includes: the complementary sequence of the first exogenous sequence, the first intermediate sequence, and the second exogenous sequence from the 5'to the 3'end; or the second exogenous sequence from the 5'to the 3'end.
  • the complementary sequence of the source sequence, the second intermediate sequence and the first exogenous sequence; when the PCR-amplified positive standard product is a double-stranded nucleotide molecule, one of the strands includes: from the 5'to the 3'end, the first The complementary sequence of the exogenous sequence, the first intermediate sequence and the second exogenous sequence; or the complementary sequence of the second exogenous sequence, the second intermediate sequence and the first exogenous sequence from 5'to the 3'end; the first The intermediate sequence is complementary to the second intermediate sequence.
  • the double-stranded nucleotide molecule after the complementary pairing of the first intermediate sequence and the second intermediate sequence is the first target sequence;
  • the first target sequence is from the 5'end to the 3'end cis
  • the first anchor sequence and its complementary sequence on the secondary connected human chromosome, the double-stranded nucleotide sequence between the first anchor sequence and the second anchor sequence, and the second anchor sequence and its complementary sequence A double-stranded nucleotide molecule with a fixed length;
  • the first exogenous sequence and the second exogenous sequence are not complementary to each other, and are not complementary to any nucleotide sequence on any human chromosome;
  • the first anchor The sequence and the second anchor sequence are conserved nucleotide sequences on the same chromosome of humans, and the combination of the first anchor sequence and the second anchor sequence can be used to amplify the first target sequence.
  • a method for preparing a positive standard for PCR amplification includes using any of the above primer sets to perform PCR amplification on a standard amplification template to obtain a PCR amplification positive standard; wherein the standard amplification template is plasma, cfDNA extracted from plasma, or Artificially prepared gDNA-derived DNA fragments that mimic the distribution of cfDNA fragments.
  • the method for preparing a PCR-amplified positive standard product further includes a step of purifying the amplified product after performing PCR amplification on the standard product-amplified template.
  • a plasma positive standard for detecting cfDNA of a plasma sample.
  • the plasma positive standard includes the aforementioned PCR amplification positive standard and plasma.
  • the concentration of the PCR-amplified positive standard in the plasma positive standard is between 1 ng/mL and 1000 ng/mL.
  • the plasma is human plasma.
  • a positive quality control product for detecting cfDNA in plasma.
  • the positive quality control product includes the aforementioned PCR amplification positive standard product, a second target sequence, and standard plasma; the second target sequence is a nucleotide sequence with a fixed length in the human genome; the sequence length of the second target sequence is greater than that of the first target The sequence length of the sequence.
  • the second target sequence is a conserved nucleotide sequence in the human genome
  • the standard plasma is human plasma whose cfDNA content is lower than a preset threshold.
  • the second target sequence includes the first target sequence.
  • the preset threshold value is 30ng/mL, 20ng/mL, 15ng/mL, 10ng/mL or 5ng/mL.
  • the length of the first target sequence is between 50 bp and 150 bp, preferably between 60 bp and 120 bp.
  • the length of the second target sequence is between 150 bp and 300 bp, preferably between 180 bp and 300 bp.
  • the ratio of the copy number of the PCR amplification positive standard product and the second target sequence in the positive quality control product is (1 ⁇ 2): (1 ⁇ 2), preferably, the PCR amplification positive standard product in the positive quality control product,
  • the copy number ratio of the second target sequence is 1:1.
  • a kit for detecting cfDNA in a plasma sample includes a first primer set and/or the aforementioned PCR amplification positive standard, and the first primer set is the aforementioned primer set for amplifying and obtaining the cfDNA standard.
  • kit also includes the above-mentioned positive quality control products for detecting cfDNA in plasma.
  • the kit further includes a fourth primer set, the fourth primer set includes a fourth upstream primer and a fourth downstream primer; the fourth upstream primer includes the first exogenous sequence, and the fourth downstream primer includes the second exogenous sequence.
  • the fourth upstream primer is the first exogenous sequence
  • the fourth downstream primer is the second exogenous sequence
  • the kit further includes a second primer set and a third primer set; the second primer set includes: a second upstream primer and a second downstream primer; the second upstream primer includes the first anchor sequence, and the second downstream primer includes the first The second anchor sequence; the third primer set includes: the third upstream primer and the third downstream primer; the third upstream primer includes the third anchor sequence, the third downstream primer includes the fourth anchor sequence; the third anchor sequence and the third anchor sequence
  • the four anchor sequences are all conserved nucleotide sequences on the same chromosome of humans, and their combination can be used to amplify the second target sequence; the sequence length of the second target sequence is greater than the sequence length of the first target sequence.
  • the second upstream primer is the first anchor sequence
  • the second downstream primer is the second anchor sequence
  • the third upstream primer is the third anchor sequence
  • the third downstream primer is the fourth anchor sequence
  • the second target sequence includes the first target sequence; and/or the second primer set and the third primer set have the same upstream primer or downstream primer.
  • the length of the first target sequence is between 50 bp and 150 bp, preferably between 60 bp and 120 bp.
  • the length of the second target sequence is between 150 bp and 300 bp, preferably between 180 bp and 300 bp.
  • an application of the above primer set for amplifying and obtaining a cfDNA standard in a technique for detecting cfDNA in plasma is provided.
  • an application of the above-mentioned kit for detecting cfDNA in plasma samples in a technique for detecting cfDNA in plasma in a technique for detecting cfDNA in plasma.
  • a qPCR method for detecting cfDNA of a plasma sample includes: S1, using the fourth primer set to perform qPCR amplification on plasma positive standards containing different concentrations of positive standards to obtain qPCR amplification results of plasma positive standards containing different concentrations of positive standards; S2, based on The qPCR amplification results of plasma positive samples containing different concentrations of positive standards are fitted to a standard curve; the fourth primer set includes the fourth upstream primer and the fourth downstream primer; the fourth upstream primer includes the first exogenous sequence, and the fourth primer set includes the fourth upstream primer and the fourth downstream primer.
  • the downstream primer includes a second exogenous sequence, and the plasma positive standard is the plasma positive standard used for the detection of cfDNA in plasma samples.
  • S1 also includes: using a second primer set to perform qPCR amplification on the plasma sample to obtain the first qPCR amplification result of the plasma sample;
  • the second primer set includes: a second upstream primer and a second downstream primer;
  • the second upstream The primer includes a first anchor sequence
  • the second downstream primer includes a second anchor sequence.
  • the second upstream primer is the first anchor sequence
  • the second downstream primer is the second anchor sequence
  • the qPCR method further includes: S3, combining the first qPCR amplification result of the plasma sample and the standard curve to calculate the content of cfDNA in the plasma sample.
  • S1 also includes using a third primer set to perform qPCR amplification on the plasma sample to obtain a second qPCR amplification result of the plasma sample;
  • the third primer set includes: a third upstream primer and a third downstream primer; and a third upstream primer Including the third anchor sequence, the third downstream primer includes the fourth anchor sequence;
  • the third anchor sequence and the fourth anchor sequence are both conserved nucleotide sequences on the same human chromosome, and their combination can be used for amplification Increase the second target sequence; the sequence length of the second target sequence is greater than the sequence length of the first target sequence.
  • the fourth upstream primer is the first exogenous sequence
  • the fourth downstream primer is the second exogenous sequence
  • step S3 further includes: combining the first qPCR amplification result of the plasma sample, the second qPCR amplification result of the plasma sample, and the standard curve to calculate the integrity of the cfDNA in the plasma sample.
  • the third upstream primer is the third anchor sequence
  • the third downstream primer is the fourth anchor sequence
  • an application of the above primer set for amplifying and obtaining a cfDNA standard in a method for judging human health is provided.
  • the primer set for amplifying cfDNA standard products of the present invention can be applied to different sample types, and the corresponding cfDNA standard products can be obtained quickly and at low cost, that is, the PCR amplification positive standard products of the present invention; during cfDNA detection
  • the PCR amplification positive standard product of the present invention only one standard product can be used, which simplifies the experimental program and reduces the detection cost; compared with the prior art that uses plasma from a different species of the cfDNA sample to be tested It avoids the inaccurate standard curve caused by the inconsistent amplification efficiency caused by the different plasma components of different species or the difference in the fluorescence signal intensity measured by the qPCR instrument due to the different plasma components of different species, which will lead to the problem of inaccurate detection results.
  • Figure 1 shows a schematic diagram of the principle of n2 fragment amplification in an embodiment of the present invention
  • Figure 2 shows a schematic diagram of the principle of n1 fragment amplification in an embodiment of the present invention
  • Figure 3 shows a schematic diagram of the principle of n3 fragment amplification in an embodiment of the present invention
  • Figure 4 shows the standard curve of the pure nucleic acid amplification system in Example 1 (positive standard L1PA2-S1, amplification primers: L1PA2-P5&L1PA2-P6 primers);
  • Figure 5 shows the standard curve of the pure nucleic acid amplification system in Example 1 (positive standard L1PA2-S1, amplification primers: L1PA2-P1&L1PA2-P2 primers);
  • Figure 6 shows the standard curve of the pure nucleic acid system in Example 1 (positive standard L1PA2-S2, amplification primers: L1PA2-P7&L1PA2-P8 primers);
  • Figure 7 shows the standard curve of the PCR amplification system (positive standard L1PA2-S1, amplification primers: L1PA2-P5&L1PA2-P6 primers) using plasma as a template in Example 1;
  • Figure 8 shows the standard curve of the PCR amplification system (positive standard L1PA2-S1, amplification primers: L1PA2-P1&L1PA2-P2 primers) using plasma as a template in Example 1;
  • Figure 9 shows the standard curve of the PCR amplification system (positive standard L1PA2-S2, amplification primers: L1PA2-P7&L1PA2-P8 primers) using plasma as a template in Example 1;
  • FIG. 10 shows a schematic diagram of the principle of amplification by the L1PA2-P5 and L1PA2-P6 primers of Experiment 1 in Example 3;
  • Figure 11 shows the melting curve of the human plasma pretreatment of Experiment 1 in Example 3 without dilution
  • Figure 12 shows the standard curve of the pretreatment of human plasma in Experiment 1 in Example 3 without dilution
  • Figure 13 shows the melting curve of the human plasma pretreatment of Experiment 1 in Example 3 when diluted 2 times;
  • Figure 14 shows the standard curve in the case where the human plasma pretreatment of Experiment 1 in Example 3 is diluted 2 times;
  • Figure 15 shows the melting curve of the human plasma pretreatment of Experiment 2 in Example 3 without dilution
  • Figure 16 shows the standard curve of the human plasma pretreatment of Experiment 2 in Example 3 without dilution
  • Figure 17 shows the melting curve of the human plasma pretreatment of Experiment 2 in Example 3 when diluted 2 times
  • Figure 18 shows the standard curve in the case where the human plasma pretreatment of Experiment 2 in Example 3 is diluted 2 times.
  • FIG. 19 shows a histogram of the absolute quantitative value of each primer in Example 4.
  • cfDNA free DNA: Endogenous DNA released from cells in the body into the circulatory system that is free outside the cell. This type of DNA fragments are concentrated in the length of 160-170bp due to degradation in the body.
  • LINE Long interspersed nuclear elements
  • Plasma direct amplification PCR When performing PCR amplification, the amplification template in the PCR reaction system directly uses the separated plasma instead of the plasma DNA purified by nucleotide extraction.
  • Standard Curve Standard curve.
  • Temperature Temperature, in degrees Celsius.
  • a primer set for amplifying and obtaining cfDNA standards includes: a first upstream primer and a first downstream primer, the first upstream primer includes a first exogenous sequence and a first anchor that are sequentially connected from the 5'end to the 3'end.
  • the first downstream primer includes a second exogenous sequence and a second anchor sequence connected sequentially from the 5'end to the 3'end; there is no complementary pairing between the first exogenous sequence and the second exogenous sequence, and It is not complementary to any nucleotide sequence on any human chromosome; the first anchor sequence and the second anchor sequence are conserved nucleotide sequences on the same chromosome of human, and the first anchor sequence and the second anchor sequence
  • the combination of anchor sequences can be used to amplify the first target sequence; the first target sequence is the first anchor sequence and its complementary sequence on the human chromosome connected sequentially from the 5'end to the 3
  • first anchor sequence and the second anchor sequence can be conserved, and the sequence between them can be non-conserved or conserved, but when they are not conserved, the length of the first target sequence must be the same Yes, some mutations are allowed, of course, the preferred first target sequence is conservative.
  • the positive standard for PCR amplification includes: single-stranded nucleotide molecules or double-stranded nucleotide molecules, when the positive standard for PCR amplification is In the case of a single-stranded nucleotide molecule, it includes: the complementary sequence of the first exogenous sequence, the first intermediate sequence, and the second exogenous sequence from the 5'to the 3'end; or the first exogenous sequence from the 5'to the 3'end.
  • the first intermediate sequence is complementary to the second intermediate sequence.
  • the double-stranded nucleotide molecule after the complementary pairing of the first intermediate sequence and the second intermediate sequence is the first target sequence;
  • the first target sequence is from the 5'end to the 3'
  • a double-stranded nucleotide molecule with a fixed length is composed;
  • the first exogenous sequence and the second exogenous sequence are not complementary to each other, and are not complementary to any nucleotide sequence on any human chromosome;
  • first The anchor sequence and the second anchor sequence are conserved nucleotide sequences on the same chromosome of humans, and the combination of the first anchor sequence and the second anchor sequence can be used to amplify the first target sequence.
  • the primer set is used to amplify human plasma samples to obtain cfDNA standards
  • the first target sequence is located in L1PA2 (a member of the LINE family), Alu (a segment containing restriction endonucleases)
  • the short interspersed repetitive sequence of AluI recognition sequence belongs to the SINE (short interspersed element) family), hTERT (Telomerase Reverse Transcriptase, telomerase reverse transcriptase), human APP (Amyloid Precursor protein, human starch precursor) Protein), ⁇ -actin (actin), EIF2C1 (Eukaryotic Translation Initiation Factor 2C1, eukaryotic translation initiation factor 2C1, located on chromosome 1) or RPPH1 (Ribonuclease P RNA Component H1, ribonuclease P RNA component H1) Genes, etc.; preferably, the length of the first target sequence is between 50 bp and 150 b
  • a method for preparing a positive standard for PCR amplification includes using the above primer set for amplifying and obtaining cfDNA standard products, performing PCR amplification on the standard product amplification template to obtain PCR amplification positive standard product; wherein, the standard product amplification template is plasma, from plasma Extracted cfDNA or artificially prepared gDNA-derived DNA fragments that mimic the distribution of cfDNA fragments (refer to CN108913682A for specific schemes); preferably, after PCR amplification of the standard amplification template, the step of purifying the amplified product is also included .
  • a plasma positive standard for cfDNA detection of plasma samples includes the aforementioned PCR amplification positive standard product and plasma; preferably, the concentration of the PCR amplification positive standard product in the plasma positive standard product is between 1 ng/mL and 1000 ng/mL. More preferably, the plasma is human plasma.
  • the plasma positive standard is generally prepared by adding nucleic acid fragments of a certain concentration (the same fragments amplified during cfDNA concentration detection) into animal plasma (such as mouse plasma).
  • animal plasma such as mouse plasma
  • the reason why plasma from other species (such as mouse plasma) is used to replace the same type of plasma in the original sample is that the DNA in the replacement plasma cannot be amplified by primers during qPCR, which can eliminate the same type of original sample.
  • the influence of cfDNA in plasma on the preparation of plasma standards and standard curves.
  • plasma standards and standard curves there are significant differences in plasma components from different species. Therefore, when qPCR amplification is performed directly using plasma standard or test plasma samples as templates, the amplification efficiency will also vary, and differences in plasma components will also affect qPCR amplification.
  • the plasma positive standard obtained by this method will have the problem of inaccurate standard curve when used. Since the PCR amplification positive standard includes the first exogenous sequence and the second exogenous sequence, in the subsequent quantitative or qualitative detection of amplification, the primers are paired with the first exogenous sequence and the second exogenous sequence, so that the plasma of the present invention
  • the positive standard plasma can be the same type of plasma as the sample, thereby avoiding the inaccuracy caused by the difference in plasma composition that affects the measurement of the absorbance value of the qPCR amplification product. At the same time, it also avoids the influence of cfDNA in the same type of plasma on the preparation of the standard curve.
  • the primer set for amplifying cfDNA standard products of the present invention can be applied to different sample types, and the corresponding cfDNA standard products can be obtained quickly and at low cost, that is, the PCR amplification positive standard products of the present invention; during cfDNA detection At this time, only one standard product of the PCR amplification positive standard product of the present invention can be used, which simplifies the experimental scheme and reduces the detection cost. Compared with the prior art scheme that uses plasma of different species from the cfDNA sample to be tested, it avoids the inaccurate standard curve caused by the inconsistent amplification efficiency caused by the different plasma components of different species, or the inaccurate standard curve due to the plasma components of different species.
  • the difference causes the difference in the fluorescence signal intensity measured by the qPCR instrument, which leads to the problem of inaccurate detection results, which improves the accuracy of the detection results; compared with the prior art scheme that uses plasma of the same species as the cfDNA sample to be tested, It avoids the inaccuracy of the standard curve caused by the standard substance contained in the plasma itself in the plasma-positive standard, which leads to inaccurate test results, improves the accuracy of the test result, and reduces the source of plasma in the plasma-positive standard.
  • the limitation of the method improves the applicability of the method.
  • the first target sequence only needs to be a conservative nucleotide sequence in the human genome, and single-copy and multiple-copy genes are both feasible. However, for single-copy genes, the number of cycles can be appropriately increased during amplification to obtain more copies. Of course, there may be non-specific amplification, which will reduce the accuracy. Therefore, single-copy genes are used as
  • the sample may further preferably be cfDNA extracted from the plasma sample, rather than the plasma sample itself.
  • the first target sequence has multiple copies on the human chromosome, so that it is easier to obtain a positive standard for PCR amplification with higher accuracy.
  • a positive quality control product for detecting cfDNA in plasma includes the aforementioned PCR amplification positive standard product, a second target sequence and standard plasma; the second target sequence is a nucleotide sequence with a fixed length in the human genome; the sequence length of the second target sequence is greater than that of the first The sequence length of the target sequence.
  • the second target sequence is a conserved nucleotide sequence in the human genome; preferably, the standard plasma is human plasma whose cfDNA content is lower than a preset threshold; preferably, the second target sequence includes the first target sequence; preferably , The preset threshold is 30ng/mL, 20ng/mL, 15ng/mL, 10ng/mL or 5ng/mL; preferably, the length of the first target sequence is between 50bp and 150bp, more preferably between 60bp and 120bp , The length of the second target sequence is between 150bp and 300bp, more preferably between 180bp and 300bp; preferably, the ratio of the copy number of the PCR amplification positive standard product and the second target sequence in the positive quality control product is (1 ⁇ 2): (1 ⁇ 2), more preferably, the ratio of the copy number of the PCR-amplified positive standard product and the second target sequence in the positive quality control product is 1:1.
  • cfDNA standards short/long fragment standards + animal plasma
  • 6 concentrations used for standard curve preparation and quantification
  • 3 replicate holes for each concentration used for standard curve preparation and quantification
  • Use negative control (same plasma as the standard, no standard fragments added) 4-6 wells, 40 wells need to be used, and 56 wells; each plasma sample two pairs of primers respectively 3 duplicate wells (total 6 Well), that is, 9 samples/plate, the detection throughput is low, two standards are needed to make two standard curves.
  • the second target sequence includes the first target sequence
  • only one standard product is needed to make a standard curve, thereby reducing the workload, improving the detection efficiency, and improving each qPCR detection
  • the throughput of cfDNA samples when the second target sequence includes the first target sequence, only one standard product is needed to make a standard curve, thereby reducing the workload, improving the detection efficiency, and improving each qPCR detection The throughput of cfDNA samples.
  • a kit for detecting cfDNA of plasma samples includes a first primer set and/or the aforementioned PCR amplification positive standard, and the first primer set is the aforementioned primer set for amplifying and obtaining the cfDNA standard.
  • the kit further includes a fourth primer set; the fourth primer set includes a fourth upstream primer and a fourth downstream primer; the fourth upstream primer includes the first exogenous sequence, and the fourth downstream primer includes the second exogenous sequence.
  • the fourth upstream primer is the first exogenous sequence
  • the fourth downstream primer is the second exogenous sequence
  • the kit further includes the above-mentioned positive quality control product for detecting cfDNA in plasma.
  • the kit further includes a second primer set and a third primer set; the second primer set includes: a second upstream primer and a second downstream primer; the second upstream primer includes the first anchor sequence, and the second downstream primer includes the first The second anchor sequence; the third primer set includes: the third upstream primer and the third downstream primer; the third upstream primer includes the third anchor sequence, the third downstream primer includes the fourth anchor sequence; the third anchor sequence and the third anchor sequence
  • the four anchor sequences are all conserved nucleotide sequences on the same chromosome of humans, and their combination can be used to amplify the second target sequence; the sequence length of the second target sequence is greater than the sequence length of the first target sequence; preferably , The second upstream primer is the first anchor sequence, and the second downstream primer is the second anchor sequence; preferably, the third upstream primer is the third anchor sequence, and the third downstream primer is the fourth anchor sequence.
  • the first primer set is used to amplify and obtain PCR amplification positive standards; the second primer set is used to amplify short fragments to achieve cfDNA quantification; the third primer set is used to amplify The extended fragments are combined with the results of the second primer set to achieve cfDNA integrity detection; the fourth primer set is used to amplify PCR amplification positive standards of different concentrations to obtain a standard product curve. Therefore, the application of this kit can efficiently and accurately detect the concentration and integrity of cfDNA in plasma.
  • the second target sequence includes the first target sequence, which can further simplify the detection steps and improve the detection efficiency.
  • the second target sequence may not include the first target sequence, as long as it is longer than the first target sequence.
  • a fluorescent dye method can be used for detection.
  • the probe method can simultaneously detect two fragments based on the simultaneous amplification of the first target sequence and the second target sequence in one well.
  • This solution can increase detection throughput, reduce detection costs, and improve detection efficiency and accuracy.
  • there should be two types of probes which are located in the first target sequence and the second target sequence, and are not complementary to each other, and they are both conserved sequences on human chromosomes.
  • the 5'end of the probe has a quenching group that binds to the fluorescent group on the probe so that the probe does not emit fluorescence.
  • the two probes are respectively bound to itself
  • the targeted binding region (on the template) of the polymerase is based on the template strand.
  • the primer When the primer is extended, its 5'-3' exonuclease activity will act to quench the 5'end of the probe bound to the template
  • the group excision makes the fluorescent probe produce fluorescence, and then the amount/concentration of the first target sequence and the second target sequence in the sample are determined based on the two kinds of fluorescence detected, and then information such as the amount and completeness of the cfDNA in the sample can be obtained .
  • the second primer set and the third primer set have the same upstream primer or downstream primer, so that the components of the kit can be simplified and the production cost of the kit can be reduced.
  • the length of the first target sequence is between 50bp and 150bp, more preferably between 60bp and 120bp, and the length of the second target sequence is between 150bp and 300bp, and more It is preferably between 180bp and 300bp.
  • the above primer set for amplifying and obtaining a cfDNA standard product, the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the above primer set for amplifying and obtaining a cfDNA standard product, the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the application of quality control products or the aforementioned kits for detecting cfDNA in plasma samples in assessing individual health.
  • the applications in individual health include dynamic monitoring of exercise and fitness effects, individual aging assessment (reflected in the monitoring of aging markers) and so on. Studies have shown that the health of the body is closely related to changes in the concentration of cfDNA. Monitoring the changes in cfDNA concentration can reflect individual health, including changes in physical fitness; cfDNA contains fragments of telomeres, and the shortening of telomeres is closely related to cell aging Relationship.
  • the above primer set for amplifying and obtaining a cfDNA standard product the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the above primer set for amplifying and obtaining a cfDNA standard product the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the above primer set for amplifying and obtaining a cfDNA standard product the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the application in tumor treatment includes the application of treatment effect monitoring and prognosis evaluation.
  • the use of cfDNA as a potential cancer biomarker began in 1977. Leon and his colleagues used radioimmunochemical methods to prove that the cfDNA content detected in the serum of cancer patients was significantly higher than that of healthy people. However, with the condition of cancer patients The improvement of the patient’s body cfDNA content will also be accompanied by a decline. Therefore, the technology of detecting the content of cfDNA in plasma may provide a relatively convenient auxiliary dynamic monitoring index for evaluating individual health.
  • the above primer set for amplifying and obtaining a cfDNA standard product the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the above primer set for amplifying and obtaining a cfDNA standard product the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • the autoimmune disease can be systemic lupus erythematosus.
  • the above primer set for amplifying and obtaining a cfDNA standard product, the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • systemic organ damage includes glomerulonephritis, pancreatitis, inflammatory bowel disease, hepatitis and other inflammatory diseases.
  • the above primer set for amplifying and obtaining a cfDNA standard product, the above PCR amplification positive standard product, the above plasma positive standard product for detecting cfDNA of a plasma sample, and the above positive standard product are provided.
  • Application of quality control products or the above kits for detecting cfDNA in plasma samples in organ transplantation is an application in the monitoring of rejection in organ transplantation.
  • cfDNA comes from necrotic cells (released more than 10,000bp), apoptotic cells (180-200 mononucleosomal DNA) and some active release substances of the donor organ. Its half-life in plasma is 15-30min .
  • the test of therapeutic drugs is based on the toxicity of the test drugs.
  • cfDNA testing can be added in the early stage, the risk of early infection can be reduced.
  • cfDNA detection can not only detect the immune rejection of organs earlier, but also make the detection of kidney, heart, lung, and liver transplants easier and more economical. At the same time, it can also design transplant programs according to individual circumstances.
  • a qPCR method for detecting cfDNA of a plasma sample includes: S1, using the fourth primer set to perform qPCR amplification on plasma positive standards containing different concentrations of positive standards to obtain qPCR amplification results of plasma positive standards containing different concentrations of positive standards; S2, based on The qPCR amplification results of plasma positive samples containing different concentrations of positive standards were fitted to a standard curve; the fourth primer set included the fourth upstream primer and the fourth downstream primer; the fourth upstream primer included the first exogenous sequence, the fourth The fourth downstream primer includes the second exogenous sequence; the plasma positive standard is the above-mentioned plasma positive standard used for the detection of cfDNA in plasma samples.
  • S1 further includes using a second primer set to perform qPCR amplification on the plasma sample to obtain the first qPCR amplification result of the plasma sample;
  • the second primer set includes: a second upstream primer and a second downstream primer; a second upstream primer
  • the first anchor sequence is included, and the second downstream primer includes the second anchor sequence.
  • the second upstream primer is the first anchor sequence
  • the second downstream primer is the second anchor sequence
  • the fourth upstream primer is the first exogenous sequence
  • the fourth downstream primer is the second exogenous sequence
  • the qPCR method further includes: S3, combining the PCR detection result of the plasma sample and the standard curve to calculate the content of cfDNA in the plasma sample.
  • concentration of cfDNA ((number of copies of the first target sequence/number of copies of the first target sequence in the genome)*mass of the human haploid genome)/volume of the plasma sample.
  • S1 further includes using a third primer set to perform qPCR amplification on the plasma sample to obtain a second qPCR amplification result of the plasma sample;
  • the third primer set includes: a third upstream primer and a third downstream primer; and a third upstream
  • the primer includes a third anchor sequence
  • the third downstream primer includes a fourth anchor sequence; both the third anchor sequence and the fourth anchor sequence are conserved nucleotide sequences on the same chromosome of humans, and their combination can be used
  • the second target sequence is amplified; the sequence length of the second target sequence is greater than the sequence length of the first target sequence;
  • the step S3 also includes: combining the first qPCR amplification result of the plasma sample and the second qPCR amplification of the plasma sample As a result and the standard curve, the integrity of the cfDNA in the plasma sample is calculated.
  • the third upstream primer is the third anchor sequence
  • the third downstream primer is the fourth anchor sequence.
  • cfDNA integrity number of amplified copies of the second target sequence/number of amplified copies of the first target sequence.
  • a method for detecting the concentration and integrity of human plasma cfDNA by using L1PA2 as the measurement object is provided.
  • the specific steps are as follows: 1) Using L1PA2 as the internal reference target site; 2) preparing standard products 3) Preparation of quality control Yangshen; 4) Blood samples for plasma separation; 5) Preparation of qPCR mix suitable for amplification using plasma samples as a template for testing the samples to be tested; 6) QPCR data processing and analysis.
  • L1PA2-P5 SEQ ID NO: 5
  • L1PA2-P6 SEQ ID NO: 6
  • primers P5 and P6 cannot amplify the L1PA2 sequence in human plasma samples
  • primers L1PA2-P3 L1PA2-P5+L1PA2-P1
  • L1PA2-P4 L1PA2-P2+L1PA2 -P6;
  • the amplification principle is shown in Figure 1 (In Figure 1, the primer name is omitted
  • the gene names are L1PA2, L1PA2-P1, L1PA2-P2, L1PA2-P5 and L1PA2-P6 are abbreviated as P1, P2, P5 and P6, respectively.
  • the amplification primer of the standard product is the exogenous sequence of the primer (primers L1PA2-P5, L1PA2-P6), and the amplification of the sample to be tested is used (L1PA2-P1, L1PA2-P2 or L1PA2-P7).
  • L1PA2-P8 SEQ ID NO: 7
  • L1PA2-P1 L1PA2-P7
  • L1PA2-P1 ⁇ L1PA2-P2 amplification product is 90bp
  • L1PA2-P7 ⁇ L1PA2-P8 amplification product is 222bp).
  • the human plasma added during standard amplification can be the mixed human plasma (as long as it is a human plasma sample) obtained by mixing the remaining plasma samples of the test sample for each test, because the exogenous primers cannot amplify the cfDNA template in human plasma Therefore, the cfDNA contained in the mixed human plasma sample will not affect the quantification of the standard.
  • the background of Yangshen should also be human plasma, but the L1PA2-P1&L1PA2-P2 and L1PA2-P7&L1PA2-P8 primers used can amplify cfDNA in human plasma Therefore, it is necessary to eliminate cfDNA fragments in human plasma used in Yangshen or control the concentration of cfDNA fragments at a low level to ensure the stability of Yangshen quantification.
  • the L1PA2-P1&L1PA2-P2 primers can be used to determine the mass concentration of cfDNA in all existing plasma samples in the plasma direct amplification system, and then the concentration value is selected below 15ng/ml (below this concentration represents the plasma concentration
  • the concentration of cfDNA is low, and it has little effect on the artificially added template). Take a portion of the plasma and mix it. After testing its stability for sun ginseng, it is aliquoted and stored at -80°C. Take a tube and use.
  • a qPCR mix suitable for PCR amplification using plasma samples as a template The plasma components are complex and contain a variety of PCR inhibitors. One of the key factors for qPCR quantification is to ensure that the amplification efficiency of the template during the PCR process is close to 100%, so choose Appropriate DNA polymerase is very important.
  • a special polymerase (such as Phusion Polymerase) suitable for PCR amplification using plasma as a template is selected, and by adjusting Mg 2+ , primer concentration, fluorescent dye ratio, etc., Make the amplification efficiency of qPCR approach 100%.
  • the qPCR mix used for PCR amplification using plasma as a template is shown in Table 1:
  • Phusion Blood II DNA Polymerase is 0.6 ⁇ L (recommended in the manual), and the amount can be reduced according to the actual template amount.
  • MgCl 2 concentration 2 ⁇ Phusion Blood PCR buffer has provided MgCl2 with a final concentration of 3.0mM, and the MgCl2 concentration can be increased up to 4.5mM according to the actual situation.
  • Fluorescent dye SYBR (10 ⁇ )
  • the preferred final concentration range is about 1 ⁇ , for example: 0.5 ⁇ , 0.6 ⁇ , 0.7 ⁇ , 0.8 ⁇ , 0.9 ⁇ , 1.0 ⁇ , 1.1 ⁇ , 1.2 ⁇ , etc.
  • Primer The preferred range is 0.2-1 ⁇ M.
  • the amount of blood sample added preferably 1%-20% of the reaction system, and the ratio can reach 40% or even higher.
  • SYBR greenI is also available Replace fluorescent dyes such as Dy (Biotium).
  • Phusion Blood II DNA Polymerase can also be replaced by the following polymerases: VELOCITY DNA Polymerase (Bioline), Terra TM qPCR Direct Premix, qPCR ToughMix TM , ROX TM , MightyAmp TM for Real Time( Plus).
  • the absolute quantification method is used to calculate the concentration value and integrity value of the plasma sample to be tested through the standard product.
  • the specific amplified sequence and primer information are as follows:
  • the sequence of L1PA2 is SEQ ID NO :8.
  • Primer L1PA2-P1 is complementary to (SEQ ID NO: 9) in L1PA2, primer P2 is SEQ ID NO: 2, and primer P8 is SEQ ID NO: 7.
  • sequence of ALU is SEQ ID NO: 10.
  • ALU short primer binding region SEQ ID NO: 11 and SEQ ID NO: 12.
  • ALU long primer binding region SEQ ID NO: 13 and SEQ ID NO: 14.
  • genes, primers, and amplified fragments can also be shown in Table 2 below:
  • the treatment of plasma can be carried out by the following methods:
  • the blood sample is collected with EDTA or Streck anticoagulant tube, and after the blood collection is completed, gently invert 5 to 10 times to mix the sample and anticoagulant thoroughly.
  • Blood samples collected with EDTA blood collection tubes must be stored at 4°C, and plasma separation is generally required within 3 hours.
  • Blood samples collected using Streck blood collection tubes can be transported at room temperature, and plasma separation is generally required within 1 week.
  • Plasma separation uses a two-step method.
  • the blood collection tube with the whole blood sample is placed at 4°C and centrifuged at 1,600g for 10 minutes. After the low-speed centrifugation is completed, the plasma is collected. At the end of the suction, the height of the remaining plasma in the blood collection tube from the albuginea layer must be at least 0.5-1cm to prevent the nucleated cells in the albuginea layer from contaminating the absorbed plasma components.
  • the second step of centrifugation is high-speed centrifugation. Plasma is placed at 4°C and centrifuged at 1,6000g for 10 minutes. After the second step of centrifugation is completed, a small amount of cell debris will be found at the bottom of the tube. At this time, only the plasma supernatant is aspirated, not cell debris.
  • the plasma sample separated after two centrifugation is the subsequent experimental sample.
  • the method for preparing artificially prepared gDNA-derived DNA fragments that simulate the distribution of cfDNA fragments specifically includes the following steps:
  • the concentration range of the PCR-amplified positive standard in the plasma positive standard is as follows:
  • the 6-point copy number concentration (per reaction well) of the standard used in the present invention is: 1000 , 5000, 10000, 20000, 100000, 1000000.
  • the measurable cfDNA concentration range of plasma samples for this series of standards is: 1.44ng/mL-717.99ng/mL.
  • the principle of setting the concentration of the standard copy number is probably to reduce the range of the standard as much as possible while ensuring the coverage of the test sample concentration range to obtain more accurate quantitative results. For different test subjects, the standard copy number concentration range can be adjusted.
  • the plasma cfDNA concentration range of healthy people generally does not exceed 100ng/mL, and the standard concentration range can be narrowed; for example, the plasma concentration of tumor patients may be as high as possible. 1000ng/mL, the standard concentration range can be relaxed to improve the accuracy and applicability of preparing and obtaining the standard curve.
  • P1 ⁇ P8 can represent primers Take L1PA2 as an example, see Figure 1 and Figure 2.
  • the middle number of the primer name, such as the "1" in L1PA2-1-P7, can be understood as the group.
  • the primer of the same group is A pair of amplification primers, for example, the long primers L1PA2-1-P7 and L1PA2-1-P8 are a pair of amplification primers, and the short primers L1PA2-11-P1 and L1PA2-11-P2 are a pair of amplification primers) :
  • the amplification primers of the L1PA2 gene are shown in Table 3 to Table 7 below.
  • L1PA2-1-P5, L1PA2-2-P5, L1PA2-3-P5, L1PA2-4-P5, L1PA2-5-P5 in Tables 3 to 7 can be used with SEQ ID NO: 66 Replace the nucleotide sequence shown, and the corresponding primers L1PA2-1-P6, L1PA2-2-P6, L1PA2-3-P6, L1PA2-4-P6, L1PA2-5-P6 use SEQ ID NO: 166 Replace the nucleotide sequence shown.
  • the amplification primers of ALU gene are shown in Table 8 ⁇ Table 10.
  • the amplification primers of human APP gene are shown in Table 15 and Table 16.
  • the amplification primers of the EIF2C1 gene are shown in Table 17 and Table 18.
  • the primers and positive standards involved in this example are as follows:
  • Primer L1PA2-P1 SEQ ID NO:1.
  • Primer L1PA2-P8 SEQ ID NO: 7.
  • L1PA2-S1 Select L1PA2 as the target template, design primers (L1PA2-P3: SEQ ID NO: 3, L1PA2-P4: SEQ ID NO: 4) to amplify the target area to obtain the standard product, the standard product
  • the sequence length is 131bp, which is the positive standard L1PA2-S1.
  • L1PA2-S2 Select the sequence fragment of the L1PA2 gene as a template, and use L1PA2-P7 (SEQ ID NO: 1) and L1PA2-P8 (SEQ ID NO: 7) primers to amplify the target region to obtain amplicon L222 , That is, the positive standard L1PA2-S2.
  • L1PA2-P5 SEQ ID NO: 5
  • L1PA2-P6 SEQ ID NO: 6
  • One plate uses a pure nucleic acid qPCR system, and one plate uses plasma as a template.
  • the mix preparation of the two qPCR reaction systems is shown in Table 22:
  • Reagent Volume ( ⁇ L) The mix for PCR amplification using plasma as a template 15.2 Primer (including forward and reverse primers L1PA2-P5 and L1PA2-P6, each 10mM) 0.8 Positive standard 2 Original mixed plasma 2
  • Standard product 1 (P5&P6) Standard product 1 (P5&P6) Standard product 1 (P5&P6) B Standard product 2 (P5&P6) Standard product 2 (P5&P6) Standard product 2 (P5&P6) C Standard product 3 (P5&P6) Standard product 3 (P5&P6) Standard product 3 (P5&P6) D Standard product 4 (P5&P6) Standard product 4 (P5&P6) Standard product 4 (P5&P6) E Standard product 5 (P5&P6) Standard product 5 (P5&P6) Standard product 5 (P5&P6) Standard product 5 (P5&P6) F Standard 6 (P5&P6) Standard 6 (P5&P6) Standard 6 (P5&P6) G NTC(P5&P6) NTC(P5&P6) NTC(P5&P6) NTC(P5&P6) NTC(P5&P6)
  • Standard Number of copies in each well Standard product 1 1000000 Standard 2 100000 Standard 3 20000 Standard 4 10000 Standard 5 5000 Standard 6 1000
  • the intensity of the fluorescence value detected by qPCR is proportional to the length of the amplicon, so the quantitative value between 1), 2), and 3) can be determined by the ratio of the length of the amplicon. Conversion.
  • the preparation method and reaction procedure of the qPCR reaction mix for the pure nucleic acid system and the plasma template PCR amplification system are the same as above (comparison of the amplification efficiency of the positive standard L1PA2-S1 in the plasma template PCR amplification system and the pure nucleic acid system).
  • Observed copies refers to the number of copies measured by the qPCR instrument, and expected copies refers to the number of copies of the theoretical standard.
  • O/E Observed copies/Expected copies, Average(O/E) is the mean value of O/E of all points of the same standard product; CV(O/E) is the coefficient of variation of O/E of all points of the same standard product.
  • Observed copies refers to the number of copies measured by the qPCR instrument, and expected copies refers to the number of copies of the theoretical standard.
  • O/E Observed copies/Expected copies, Average(O/E) is the mean value of O/E of all points of the same standard product; CV(O/E) is the coefficient of variation of O/E of all points of the same standard product.
  • the above method to obtain the standard curve is to use the software that comes with the StepOne plus qPCR instrument for analysis. Enter the copy number corresponding to each point of the standard in the relevant setting page.
  • the software calculates the analysis result.
  • the analysis result includes the standard curve. Slope (slope), Y-intercept (y-intercept), regression coefficient (R 2 value), amplification efficiency (Eff%).
  • the key indicators are regression coefficient (R 2 value) and amplification efficiency (Eff%): the closer the R 2 value is to 1, the better the linear fit of the standard curve.
  • the preferred range is R 2 value ⁇ 0.99; Eff% is the most ideal
  • the status of 100%, and the preferred range is 95%-105% (in Figures 4-9, quantity is the number of copies).
  • the standard curve is to plot the log value of the copy number of the standard template against the Ct value of each diluted sample to obtain a decreasing linear graph.
  • the probe method can also be used.
  • the probe method can also be used.
  • several specific feasible implementation schemes will be listed.
  • the amplification product (long fragment/short fragment) of each set of primers in this scheme a shares a probe, so the concentration and integrity are detected in two separate tubes, that is, the short fragment detection is one tube (take scheme 1 as an example, This tube includes probe 1, primer 11-F, and primer 11-R), and long fragment detection is another tube (take scheme 1 as an example, this tube includes probe 1, primer 12-F, and primer 12-R).
  • the concentration detection qPCR reaction system is shown in Table 32 (Take Scheme 1 as an example):
  • the absolute quantification method is used to calculate the concentration value and integrity value of the plasma sample to be tested through the standard product.
  • the amplified products (long fragments/short fragments) of each set of primers are detected with different probes, so the concentration and integrity can be detected in the same PCR tube, that is, the same PCR tube contains long fragment Primer pair, short fragment primer pair, two probes (taking the simultaneous detection of short fragment 1 and long fragment 1 as an example, this PCR tube includes probe 31, probe 32, primer 31-F, primer 31- R, primer 32-F, primer 32-R).
  • the absolute quantification method is used to calculate the concentration value and integrity value of the plasma sample to be tested through the standard product.
  • the probe method uses plasma as a template, and PCR amplification polymerase can also be replaced by the following enzymes: Environmental Master Mix 2.0, VersaTaqTM Direct PCR Polymerase (71002), KAPA PROBE FORCE qPCR Master Mix (2 ⁇ ) Universal Kit.
  • Plan b was used to detect 10 plasma samples, and the results were consistent with the results obtained using dye-based QPCR (specific results are not shown), indicating that plan b is feasible.
  • L1PA2-P5 (SEQ ID NO: 5)
  • L1PA2-P6 (SEQ ID NO: 6) primers amplify the template shown in Figure 10, the template length is 363bp, used to test different reagents and plasma environment, standard The amplification efficiency of the product.
  • the melting curve of human plasma without dilution before treatment is shown in Figure 11 (where the ordinate derivative reporter is the derivative of the fluorescence value versus temperature, which can also be expressed as - ⁇ F/ ⁇ T(change in fluorescence/change in temperature) , which reflects the change of fluorescence value with temperature), the standard curve is shown in Figure 12.
  • Phusion reagents to amplify a 363bp target fragment with L1PA2-P5 and L1PA2-P6 primers.
  • the amplification efficiency of the standard amplicon can still be close to 100 when the plasma is not diluted or diluted 2 times in the pretreatment. %; From the melting curve, the amplification specificity is good, and the specificity is good.
  • the melting curve of the pretreatment of human plasma without dilution is shown in Fig. 15, and the standard curve is shown in Fig. 16.
  • the amplification efficiency of the standard amplicon can still be close to 100% without diluting or diluting the plasma by 2 times; from melting The curve shows the specificity of amplification, and the specificity is good.
  • D-g125 series standard products are another set of standard products prepared during the test. The difference from the aforementioned S1 and S2 standard products is that the preparation process of D-g125 has not undergone PCR amplification. The preparation process is as follows: Fragment gDNA, then screen out fragments close to the distribution of cfDNA fragments, and dilute the fragments at a certain concentration in equal proportions to obtain D-g125 series standard products. D-g125-1 to D-g125-6 are their specific numbers.
  • the numerical unit in the table is haploid human genome copies/ ⁇ L plasma, which is the equivalent of human haploid genome per microliter of plasma.
  • the amplification template of MSTN and EIF2C1 on the genome is a single copy on the genome, and the amplification template of L90 on the genome is multiple copies. Therefore, L90 has a relatively more relaxed requirement for the amount of genome template in the quantification process.
  • the separated plasma samples are divided into two parts for testing. One part takes 400 ⁇ L of plasma for cfDNA extraction, and quantifies the extracted cfDNA with a pure nucleic acid system, and the other part directly uses plasma for PCR amplification of cfDNA with plasma as a template. .
  • the extracted nucleic acid product quantification system is the same as the standard product, sun ginseng and primers used in the plasma template PCR amplification quantification system of the present invention.
  • the specific information of the standard product, sun ginseng and primer is in the specific implementation or the above examples Has been detailed in.
  • the quantification method of the nucleic acid product after extraction refers to the existing literature (Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma_PLos One, referred to as existing later), and the specific steps are not described in detail here.
  • Plasma separation uses a two-step method.
  • the blood collection tube with the whole blood sample is placed at 4°C and centrifuged at 1,600g for 10 minutes. After the low-speed centrifugation is completed, the plasma is collected. During the suction process, it is necessary to prevent the nucleated cells in the albuginea layer from polluting the absorbed plasma components.
  • the second step of centrifugation is high-speed centrifugation. Plasma is placed at 4°C and centrifuged at 16,000g for 10 minutes. The plasma sample separated after two centrifugation is the subsequent experimental sample and is stored at -80°C.
  • reaction mix (18 ⁇ L/well) and standard/yangshen/sample to be tested (2 ⁇ L//well) into a 96-well plate.
  • the ratio of PCR amplification using plasma as a template to extraction is relatively stable (mean value 1.95, CV value 11% ), the ratio of PCR amplification using plasma as a template to extraction in the existing literature is about 2.
  • Sample Complete PCR amplification using plasma as template Completeness after extraction Sample 1 25.87% 25.10% Sample 2 16.90% 23.85% Sample 3 34.02% 28.25% Sample 4 28.15% 29.72% Sample 5 22.79% 23.44% Sample 6 17.68% 18.37% Sample 7 23.30% 22.97% Sample 8 22.81% 25.50%
  • the PCR amplification results of each plasma sample to be tested using the method of the present invention using plasma as a template are compared with the quantitative integrity after extraction.
  • Extraction of plasma cfDNA use MAGEN free nucleic acid commercial extraction kit to extract 400 ⁇ L plasma sample, add 0.4mL EDTA-preserved plasma and 0.1mL PBS to a 2mL centrifuge tube, and refer to the extraction kit for the next steps The instructions for the operation. 80 ⁇ L EB was used to re-dissolve the extracted product in the last step.
  • Sample dilution Dilute the extracted pure nucleic acid sample by 4 times.
  • reaction mix (8 ⁇ L/well) and standard/yangshen/sample to be tested (2 ⁇ L/well) into a 96-well plate.
  • step one applying the technical solution of the present invention to directly use plasma for PCR amplification of cfDNA quantification
  • step two quantifying the extracted cfDNA with a pure nucleic acid system
  • the reproducibility results of the samples in the plasma-template PCR amplification system and the pure nucleic acid system are shown in Table 58:
  • the positive standard S1+P5&P6 primers are used to quantify the above samples.
  • the repeatability of cfDNA concentration and integrity is good (CV value ⁇ 10%) ;
  • the pure nucleic acid system has no significant difference in repeatability.

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Abstract

提供了获得cfDNA标准品的引物组、PCR扩增阳性标准品及其制备方法、试剂盒及应用。用于扩增获得cfDNA标准品的引物组包括：第一上游引物和第一下游引物，第一上游引物包括从5'端到3'端顺次连接的第一外源序列和第一锚定序列，第一下游引物包括从5'端到3'端顺次连接的第二外源序列和第二锚定序列；第一外源序列和第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；第一锚定序列和第二锚定序列为人类的同一染色体上的保守核苷酸序列。提供了血浆阳性标准品，其qPCR反应背景为与待测样本同类型的血浆，避免了因标准品与待测样本血浆成分差异造成的测量不准确问题。

Description

获得cfDNA标准品的引物组、PCR扩增阳性标准品及其制备方法、试剂盒及应用 技术领域
本发明涉及生物技术领域，具体而言，涉及一种获得cfDNA标准品的引物组、PCR扩增阳性标准品及其制备方法、试剂盒及应用。
背景技术
cfDNA(cell-free DNA,cfDNA)于1948年被Mandel和Metais在血浆中发现。1966年Tan和他的同事首次将cfDNA和疾病联系在一起，因为他们在系统性红斑狼疮(systemic lupus erythematosus，SLE)的病人血浆中观察到高浓度的cfDNA。之后的研究也让人们意识到血浆中cfDNA的浓度变化与个体生理状态及其临床表现息息相关，例如急性创伤，脑梗塞，运动，器官移植，感染等都能够引起cfDNA在血浆中的浓度上升。利用cfDNA作为一个潜在的癌症生物标记物始于1977年，Leon与其同事利用放射免疫化学的方法证明在癌症患者血清中检测出的cfDNA含量会比健康人的明显偏高，但随着肿瘤患者病情的好转，患者体内cfDNA的含量也会伴随着下降。因此，一种能够快速检测出血浆中cfDNA含量的技术将有可能为评判个体健康提供一种获取相对便捷的辅助性动态监控指标。
cfDNA在血液中主要是以双链形式存在的，通常人们认为cfDNA的主要来源是由凋亡或坏死的细胞释放而出，它最集中的片段长度约166bp。cfDNA完整性(cfDNA integrity)测量的是所获取的cfDNA中长片段与短片段(拷贝数)的比例。通常正常细胞死亡形式以细胞凋亡为主，释放出200bp左右的DNA片段。而肿瘤细胞的死亡会增加坏死细胞的比例，坏死细胞释放出的DNA片段大小与细胞凋亡的片段大小存在差异。所以当机体有肿瘤发生或肿瘤病情变化时，血液中cfDNA的完整性也将发生改变。所以，cfDNA完整性也可能成为一种能够动态监测肿瘤发生与变化的，具有辅助指导意义的标记物。
目前，测量血浆中cfDNA浓度的定量检测方法大致分为两类：一类是利用试剂盒先提取出血浆中的cfDNA，提取后的产物可直接使用Qubit，qPCR或者digital PCR做定量检测；另一类是直接利用血浆样本进行定量检测，这样可以最大限度的避免在提取过程中对产物造成的损失或污染，最大程度的保留血浆内cfDNA的最原始信息，且对血浆需求量少，操作时间和成本也都相对较低。针对第二类方案，通常是针对特定的保守区域(Direct Quantification of Cell-Free,Circulating DNA from Unpurified Plasma，Sarah Breitbach等，PLos One)，设计两对与保守区域完全互补配对的qPCR引物，该两对qPCR引物扩增的产物片段长度不同，可分别扩增出血浆样品cfDNA中的保守区域的长、短片段(222bp、90bp)，进而指示血浆样品中cfDNA的量(短片段指示)和完整性(长片段/短片段)，该长片段包括短片段。同时，另外用长/短片段qPCR引物扩增获得PCR产物，并按照预设的不同浓度分别加入至动物血浆(不含有引物扩增模板)中，作为不同浓度的长/短片段cfDNA标准品(一般为6个浓度梯度)。在对待测血浆样品进行qPCR检测时，利用相应的qPCR引物对不同长度浓度的cfDNA标准品进行扩增，从而获得不同片段长度的cfDNA扩增标准曲线，进而通过该标准曲线和待测血浆样品的qPCR检测结果进行比对，从而获得待测血浆样品cfDNA的含量和完整性信息。
上述的第一类方法比较成熟，是目前运用最广泛的cfDNA定量检测方法，但是其缺点也很明显：对血浆需求量较大(一般情况至少0.5ml血浆)，提取过程中可能伴随cfDNA的损失或污染，不能反映血浆内cfDNA浓度及完整性的最真实状况，此外实验时间成本和试剂成本相对较高。第二类方法现存的主要问题有以下几点：1)cfDNA定量检测时限于标准品中使用动物血浆，可能与人源血浆有成分差异，影响检测结果的准确性；2)同时检测cfDNA浓度和完整性时需各使用一套标准品，时间和试剂成本较高，单次检测的样本通量较低；3)cfDNA定量的标准品制备过程繁琐、稳定性较差；4)批间实验缺少有效的阳参质控。
现有技术中，为了解决上述问题，一般采用下述2种方法：
1)用DNAase处理原样血浆，即可消除原样血浆中的cfDNA；但是，这种方法除了会明 显增加血浆标准品的制备步骤，降低效率，增加成本外，还因为加入DNAase处理时，还需加入相应的缓冲液，以使DNAase发挥活性除去原样血浆中的cfDNA，这会使获得的血浆标准品与待测血浆样品的组成之间差异度加大，进而使得血浆标准品和待测血浆样品在进行qPCR直扩时，扩增效率存在差异，而且因为新加入的缓冲液还可能影响qPCR扩增产物吸光值的测量，所以，通过这种方法获得的血浆标准品，在使用时，同样存在获得的标准曲线不准确的问题。
2)直接使用原样血浆，使得标准品与待测血浆样品在进行qPCR直扩时，将因血浆环境引起的差异降到最低。但是，利用这种血浆标准品进行cfDNA检测时，制备血浆标准品的原样血浆中cfDNA的浓度未知，这些浓度未知的cfDNA片段在qPCR时，同样会被引物扩增出PCR产物，从而使得基于这种标准品扩增获得的标准曲线不准确，相应的使基于该标准曲线获得的待测血浆样品的cfDNA浓度偏低。
发明内容
本发明旨在提供一种获得cfDNA标准品的引物组、PCR扩增阳性标准品及其制备方法、试剂盒及应用，以提高对血浆样品的cfDNA检测的准确性。
为了实现上述目的，根据本发明的一个方面，提供了一种用于扩增获得cfDNA标准品的引物组。该引物组包括：第一上游引物和第一下游引物，第一上游引物包括从5’端到3’端顺次连接的第一外源序列和第一锚定序列，第一下游引物包括从5’端到3’端顺次连接的第二外源序列和第二锚定序列；第一外源序列和第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；第一锚定序列和第二锚定序列为人类的同一染色体上的保守核苷酸序列，且第一锚定序列和第二锚定序列的组合能够用于扩增第一靶序列；第一靶序列为由从5’端到3’端顺次连接的人类染色体上的第一锚定序列及其互补序列，第一锚定序列和第二锚定序列之间的双链核苷酸序列，和第二锚定序列及其互补序列组成的具有固定长度的双链核苷酸分子。
进一步地，第一靶序列是人类染色体中的保守核苷酸序列。
更进一步地，第一靶序列在人类的染色体上具有多个拷贝。
进一步地，第一靶序列位于L1PA2、Alu、hTERT、human APP、β-actin、EIF2C1或RPPH1基因上。
更进一步地，第一靶序列的长度在50bp～150bp之间。
更进一步地，第一靶序列的长度在60bp～120bp之间。
根据本发明的另一个方面，提供一种PCR扩增阳性标准品，该PCR扩增阳性标准品为单链核苷酸分子或双链核苷酸分子，当PCR扩增阳性标准品为单链核苷酸分子时，包括：从5’到3’端依次为第一外源序列、第一中间序列和第二外源序列的互补序列；或从5’到3’端依次为第二外源序列、第二中间序列和第一外源序列的互补序列；当PCR扩增阳性标准品为双链核苷酸分子时，其中的一条链包括：从5’到3’端依次为第一外源序列、第一中间序列和第二外源序列的互补序列；或从5’到3’端依次为第二外源序列、第二中间序列和第一外源序列的互补序列；第一中间序列和第二中间序列互补配对，第一中间序列和第二中间序列互补配对后的双链核苷酸分子为第一靶序列；第一靶序列为由从5’端到3’端顺次连接的人类染色体上的第一锚定序列及其互补序列，第一锚定序列和第二锚定序列之间的双链核苷酸序列，和第二锚定序列及其互补序列组成的具有固定长度的双链核苷酸分子；第一外源序列和第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；第一锚定序列和第二锚定序列为人类的同一染色体上的保守核苷酸序列，且第一锚定序列和第二锚定序列的组合能够用于扩增第一靶序列。
根据本发明的再一个方面，提供一种PCR扩增阳性标准品的制备方法。该制备方法包括采用上述任一种引物组，对标准品扩增模板进行PCR扩增，获得PCR扩增阳性标准品；其中，标准品扩增模板为血浆、从血浆中提取获得的cfDNA、或人工制备的gDNA来源的模拟cfDNA片段分布的DNA片段。
进一步地，PCR扩增阳性标准品的制备方法，在对标准品扩增模板进行PCR扩增后还包括对扩增产物进行纯化的步骤。
根据本发明的又一个方面，提供一种用于对血浆样本的cfDNA检测的血浆阳性标准品。该血浆阳性标准品包括上述PCR扩增阳性标准品和血浆。
进一步地，血浆阳性标准品中含PCR扩增阳性标准品的浓度在1ng/mL至1000ng/mL之间。
更进一步地，血浆为人类血浆。
根据本发明的再一个方面，提供一种用于血浆中cfDNA检测的阳性质控品。该阳性质控品包括上述PCR扩增阳性标准品、第二靶序列和标准血浆；第二靶序列为人类基因组中具有固定长度的核苷酸序列；第二靶序列的序列长度大于第一靶序列的序列长度。
进一步地，第二靶序列为人类基因组中的保守核苷酸序列
进一步地，标准血浆为cfDNA含量低于预设阈值的人类血浆。
进一步地，第二靶序列包括第一靶序列。
进一步地，预设阈值为30ng/mL、20ng/mL、15ng/mL、10ng/mL或5ng/mL。
进一步地，第一靶序列的长度在50bp～150bp之间，优选在60bp～120bp之间。
进一步地，第二靶序列的长度在150bp～300bp之间，优选在180bp～300bp之间。
进一步地阳性质控品中PCR扩增阳性标准品、第二靶序列的拷贝数比值为(1～2):(1～2)，优选的，阳性质控品中PCR扩增阳性标准品、第二靶序列的拷贝数比值为1:1。
根据本发明的又一个方面，提供一种用于对血浆样本的cfDNA进行检测的试剂盒。该试剂盒包括第一引物组、和/或上述PCR扩增阳性标准品，第一引物组为上述用于扩增获得cfDNA标准品的引物组。
进一步地，试剂盒还包括上述用于血浆中cfDNA检测的阳性质控品。
进一步地，试剂盒还包括第四引物组，第四引物组包括第四上游引物和第四下游引物；第四上游引物包括第一外源序列，第四下游引物包括第二外源序列。
更进一步地，第四上游引物为第一外源序列，第四下游引物为第二外源序列。
进一步地，试剂盒还包括第二引物组和第三引物组；第二引物组包括：第二上游引物和第二下游引物；第二上游引物包括第一锚定序列，第二下游引物包括第二锚定序列；第三引物组包括：第三上游引物和第三下游引物；第三上游引物包括第三锚定序列，第三下游引物包括第四锚定序列；第三锚定序列和第四锚定序列均为人类的同一染色体上的保守核苷酸序列，且它们的组合能够用于扩增第二靶序列；第二靶序列的序列长度大于第一靶序列的序列长度。
进一步地，第二上游引物为第一锚定序列，第二下游引物为第二锚定序列。
进一步地，第三上游引物为第三锚定序列，第三下游引物为第四锚定序列。
进一步地，第二靶序列包括第一靶序列；和/或第二引物组和第三引物组具有相同的上游引物或下游引物。
进一步地，第一靶序列的长度在50bp～150bp之间，优选在60bp～120bp之间。
进一步地，第二靶序列的长度在150bp～300bp之间，优选在180bp～300bp之间。
根据本发明的再一个方面，提供一种上述用于扩增获得cfDNA标准品的引物组在检测血浆中cfDNA的技术中的应用。
根据本发明的再一个方面，提供一种上述PCR扩增阳性标准品在检测血浆中cfDNA的技术中的应用。
根据本发明的再一个方面，提供一种上述用于对血浆样本的cfDNA检测的血浆阳性标准品在检测血浆中cfDNA的技术中的应用。
根据本发明的再一个方面，提供一种上述用于血浆中cfDNA检测的阳性质控品在检测血浆中cfDNA的技术中的应用。
根据本发明的再一个方面，提供一种上述用于对血浆样本的cfDNA检测的试剂盒在检测 血浆中cfDNA的技术中的应用。
根据本发明的又一个方面，提供一种对血浆样品的cfDNA检测的qPCR方法。该qPCR方法包括：S1，利用第四引物组对含有不同浓度阳性标准品的血浆阳性标准品进行qPCR扩增，得含有不同浓度阳性标准品的血浆阳性标准品的qPCR扩增结果；S2，基于含有不同浓度阳性标准品的血浆阳性样品的qPCR扩增结果，拟合出标准曲线；第四引物组包括第四上游引物和第四下游引物；第四上游引物包括第一外源序列，第四下游引物包括第二外源序列，血浆阳性标准品为上述用于对血浆样本的cfDNA检测的血浆阳性标准品。
进一步地，S1还包括：采用第二引物组对血浆样品进行qPCR扩增，得血浆样品的第一qPCR扩增结果；第二引物组包括：第二上游引物和第二下游引物；第二上游引物包括第一锚定序列，第二下游引物包括第二锚定序列。
更进一步地，第二上游引物为第一锚定序列，第二下游引物为第二锚定序列。
进一步地，qPCR方法还包括：S3，结合血浆样品的第一qPCR扩增结果和标准曲线，计算出血浆样品中cfDNA的含量。
进一步地，S1还包括采用第三引物组对血浆样品进行qPCR扩增，得血浆样品的第二qPCR扩增结果；第三引物组包括：第三上游引物和第三下游引物；第三上游引物包括第三锚定序列，第三下游引物包括第四锚定序列；第三锚定序列和第四锚定序列均为人类同一染色体上的保守核苷酸序列，且它们的组合能够用于扩增第二靶序列；第二靶序列的序列长度大于第一靶序列的序列长度。
进一步地，第四上游引物为第一外源序列，第四下游引物为第二外源序列。
进一步地，步骤S3还包括：结合血浆样品的第一qPCR扩增结果、血浆样品的第二qPCR扩增结果和标准曲线，计算出血浆样品中cfDNA的完整性。
更进一步地，第三上游引物为第三锚定序列，第三下游引物为第四锚定序列。
根据本发明的又一方面，提供了上述用于扩增获得cfDNA标准品的引物组在评判人体健康状况的方法中的应用。
根据本发明的又一方面，提供了上述PCR扩增阳性标准品在评判人体健康状况的方法中的应用。
根据本发明的又一方面，提供了上述用于对血浆样本的cfDNA检测的血浆阳性标准品在评判人体健康状况的方法中的应用。
根据本发明的又一方面，提供了上述用于血浆中cfDNA检测的阳性质控品在评判人体健康状况的方法中的应用。
根据本发明的又一方面，提供了上述用于对血浆样本的cfDNA检测的试剂盒在评判人体健康状况的方法中的应用。
根据本发明的又一方面，提供了上述对血浆样品的cfDNA检测的qPCR方法在评判人体健康状况的方法中的应用。
根据本发明的又一方面，提供了上述用于扩增获得cfDNA标准品的引物组在动态检测肿瘤发生和/或变化的方法中的应用。
根据本发明的又一方面，提供了上述PCR扩增阳性标准品在动态检测肿瘤发生和/或变化的方法中的应用。
根据本发明的又一方面，提供了上述用于对血浆样本的cfDNA检测的血浆阳性标准品在动态检测肿瘤发生和/或变化的方法中的应用。
根据本发明的又一方面，提供了上述用于血浆中cfDNA检测的阳性质控品在动态检测肿瘤发生和/或变化的方法中的应用。
根据本发明的又一方面，提供了上述用于对血浆样本的cfDNA检测的试剂盒在动态检测肿瘤发生和/或变化的方法中的应用。
根据本发明的又一方面，提供了上述对血浆样品的cfDNA检测的qPCR方法在动态检测肿瘤发生和/或变化的方法中的应用。
本发明的用于扩增cfDNA标准品的引物组，可适用于不同样本类型，并可快速、低成本的获得相应的cfDNA标准品，即本发明的PCR扩增阳性标准品；在进行cfDNA检测时，可仅使用本发明的PCR扩增阳性标准品这1个标准品，简化了实验方案，降低了检测成本；与现有技术中通过使用与待检测cfDNA样本不同物种的血浆的方案相比，避免了因为不同物种血浆成分不同而引起的扩增效率不一致而导致的标准曲线不准确或因为不同物种血浆成分不同造成qPCR仪测得的荧光信号强度的差异，进而导致检测结果不准确的问题，提高了检测结果的准确性；与现有技术中通过使用与待检测cfDNA样本相同物种的血浆的方案相比，避免了因为血浆阳性标准品中血浆自身含有的标准品相同序列而导致的标准曲线不准确，进而导致检测结果不准确的问题，提高了检测结果的准确性，同时还降低了血浆阳性标准品中血浆来源的限制，提高了方法的适用性。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明一实施方式中n2片段扩增原理示意；
图2示出了本发明一实施方式中n1片段扩增原理示意；
图3示出了本发明一实施方式中n3片段扩增原理示意；
图4示出了实施例1中纯核酸扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P5&L1PA2-P6引物)的标准曲线；
图5示出了实施例1中纯核酸扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P1&L1PA2-P2引物)的标准曲线；
图6示出了实施例1中纯核酸体系(阳性标准品L1PA2-S2，扩增引物为：L1PA2-P7&L1PA2-P8引物)的标准曲线；
图7示出了实施例1中以血浆为模板PCR扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P5&L1PA2-P6引物)的标准曲线；
图8示出了实施例1中以血浆为模板PCR扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P1&L1PA2-P2引物)的标准曲线；
图9示出了实施例1中以血浆为模板PCR扩增体系(阳性标准品L1PA2-S2，扩增引物为：L1PA2-P7&L1PA2-P8引物)的标准曲线；
图10示出了实施例3中实验1的L1PA2-P5，L1PA2-P6引物进行扩增的原理示意图；
图11示出了实施例3中实验1的人血浆前处理不稀释的情况下的熔解曲线图；
图12示出了实施例3中实验1的人血浆前处理不稀释的情况下的标准曲线；
图13示出了实施例3中实验1的人血浆前处理稀释2倍的情况下的熔解曲线图；
图14示出了实施例3中实验1的人血浆前处理稀释2倍的情况下的标准曲线；
图15示出了实施例3中实验2的人血浆前处理不稀释的情况下的熔解曲线图；
图16示出了实施例3中实验2的人血浆前处理不稀释的情况下的标准曲线；
图17示出了实施例3中实验2的人血浆前处理稀释2倍的情况下的熔解曲线图；
图18示出了实施例3中实验2的人血浆前处理稀释2倍的情况下的标准曲线；以及
图19示出了实施例4中各引物定量绝对值的柱状图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明中涉及的缩写、英文及术语解释如下：
cell-free DNA(cfDNA，游离DNA：机体内细胞释放到循环系统中的游离于细胞外的内源性DNA，此类DNA片段因为机体内的降解作用使得片段长度集中于160～170bp。
长散在重复序列(Long interspersed nuclear elements，LINE)：是一种序列较长的重复序列，分散在各个染色体中，约占人基因组的17％。
血浆直扩PCR：在进行PCR扩增时，PCR反应体系中的扩增模板直接使用分离出的血浆，而不是经过核苷酸提取纯化的血浆DNA。
Standard Curve：标准曲线。
Quanitity：数量。
Melt Curve：熔解曲线图。
Temperature：温度，单位为摄氏度。
根据本发明一种典型的实施方式，提供了一种用于扩增获得cfDNA标准品的引物组。该用于扩增获得cfDNA标准品的引物组包括：第一上游引物和第一下游引物，第一上游引物包括从5’端到3’端顺次连接的第一外源序列和第一锚定序列，第一下游引物包括从5’端到3’端顺次连接的第二外源序列和第二锚定序列；第一外源序列和第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；第一锚定序列和第二锚定序列为人类的同一染色体上的保守核苷酸序列，且第一锚定序列和第二锚定序列的组合能够用于扩增第一靶序列；所述第一靶序列为由从5’端到3’端顺次连接的人类染色体上的第一锚定序列及其互补序列，第一锚定序列和第二锚定序列之间的双链核苷酸序列，和第二锚定序列及其互补序列组成的具有固定长度的双链核苷酸分子。
也就是说，第一锚定序列和第二锚定序列是保守的即可，它们之间的序列可以是不保守的或保守的，但为不保守时，第一靶序列的长度必须是一致的，允许有点突变，当然优选的第一靶序列是保守的。
应用该引物组对样品进行扩增可以获得PCR扩增阳性标准品，该PCR扩增阳性标准品包括：为单链核苷酸分子或双链核苷酸分子，当PCR扩增阳性标准品为单链核苷酸分子时，包括：从5’到3’端依次为第一外源序列、第一中间序列和第二外源序列的互补序列；或从5’到3’端依次为第二外源序列、第二中间序列和第一外源序列的互补序列；当PCR扩增阳性标准品为双链核苷酸分子时，其中的一条链包括：从5’到3’端依次为第一外源序列、第一中间序列和第二外源序列的互补序列；或从5’到3’端依次为第二外源序列、第二中间序列和第一外源序列的互补序列；第一中间序列和第二中间序列互补配对，第一中间序列和第二中间序列互补配对后的双链核苷酸分子为第一靶序列；第一靶序列为由从5’端到3’端顺次连接的人类染色体上的第一锚定序列及其互补序列，第一锚定序列和第二锚定序列之间的双链核苷酸序列，和第二锚定序列及其互补序列组成的具有固定长度的双链核苷酸分子；第一外源序列和第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；第一锚定序列和第二锚定序列为人类的同一染色体上的保守核苷酸序列，且第一锚定序列和第二锚定序列的组合能够用于扩增第一靶序列。
在本发明一种典型的实施方式中，引物组用于扩增人血浆样品获得cfDNA标准品，第一靶序列位于L1PA2(属于LINE家族的一员)、Alu(一段含有限制性内切核酸酶AluI识别序列(AGCT)的短散在重复序列，属于SINE(short interspersed element)家族的一员)、hTERT(Telomerase Reverse Transcriptase，端粒酶逆转录酶)、human APP(Amyloid Precursor protein，人类淀粉前体蛋白)、β-actin(肌动蛋白)、EIF2C1(Eukaryotic Translation Initiation Factor 2C1，真核翻译起始因子2C1，位于1号染色体上)或RPPH1(Ribonuclease P RNA Component H1，核糖核酸酶P RNA组分H1)基因等上；优选的，第一靶序列的长度在50bp～150bp之间，更优选在60bp～120bp之间。
根据本发明一种典型的实施方式，提供了一种PCR扩增阳性标准品的制备方法。该制备方法包括采用上述用于扩增获得cfDNA标准品的引物组，对标准品扩增模板进行PCR扩增，获得PCR扩增阳性标准品；其中，标准品扩增模板为血浆、从血浆中提取获得的cfDNA或人工制备的gDNA来源的模拟cfDNA片段分布的DNA片段(具体方案可参考CN108913682A)；优选的，对标准品扩增模板进行PCR扩增后还包括对扩增产物进行纯化的步骤。
根据本发明一种典型的实施方式，提供了一种用于对血浆样本的cfDNA检测的血浆阳性标准品。该血浆阳性标准品包括上述PCR扩增阳性标准品和血浆；优选的，血浆阳性标准品 中含PCR扩增阳性标准品的浓度在1ng/mL至1000ng/mL之间。更优选的，血浆为人类血浆。
现有技术中，血浆阳性标准品一般是动物血浆(如小鼠血浆)中加入确定浓度核酸片段(与cfDNA浓度检测时扩增的片段相同)制备得到。之所以用其他物种来源的血浆(例如小鼠血浆)代替原来的样本同类型的血浆，是因为替代血浆中的DNA在qPCR时不能被引物扩增出产物，即可消除原来的样本同类型的血浆中的cfDNA对血浆标准品、标准曲线制作的影响。但是，不同物种来源的血浆成分差异明显，因此，直接以血浆标准品或待测血浆样品为模板进行qPCR扩增时，扩增效率也会存在差异，而且血浆成分的差异同样会影响qPCR扩增产物吸光值的测量，所以，通过这种方法获得的血浆阳性标准品，在使用时，会存在获得的标准曲线不准确的问题。由于PCR扩增阳性标准品包括第一外源序列和第二外源序列，在后续定量或定性检测扩增时，引物与第一外源序列和第二外源序列配对，这样本发明的血浆阳性标准品血浆可以为样本同类型的血浆，从而避免了血浆成分的差异造成影响qPCR扩增产物吸光值的测量带来的不准确问题。同时，也避免了样本同类型的血浆中的cfDNA对标准曲线制作的影响。
本发明的用于扩增cfDNA标准品的引物组，可适用于不同样本类型，并可快速、低成本的获得相应的cfDNA标准品，即本发明的PCR扩增阳性标准品；在进行cfDNA检测时，可仅使用本发明的PCR扩增阳性标准品这1个标准品，简化了实验方案，降低了检测成本。与现有技术中通过使用与待检测cfDNA样本不同物种的血浆的方案相比，避免了因为不同物种血浆成分不同而引起的扩增效率不一致而导致的标准曲线不准确，或因为不同物种血浆成分不同造成qPCR仪测得的荧光信号强度的差异进而导致检测结果不准确的问题，提高了检测结果的准确性；与现有技术中通过使用与待检测cfDNA样本相同物种的血浆的方案相比，避免了因为血浆阳性标准品中血浆自身含有的标准品而导致的标准曲线不准确，进而导致检测结果不准确的问题，提高了检测结果的准确性，同时还降低了血浆阳性标准品中血浆来源的限制，提高了方法的适用性。
第一靶序列是人类基因组中的保守核苷酸序列即可，单拷贝、多拷贝基因都是可行的。但是，对于单拷贝基因来讲，在扩增时可以适当的增加循环数来获得更多的拷贝，当然，这可能存在非特异性扩增，从而使准确性相对降低，因此，以单拷贝基因作为扩增对象，样本进一步优选的可以是从血浆样本中提取出的cfDNA，而不是血浆样本本身。在本申请中，优选的，第一靶序列在人类的染色体上具有多个拷贝，这样可以较为容易的获得准确性较高的PCR扩增阳性标准品。
根据本发明一种典型的实施方式，还提供了一种用于血浆中cfDNA检测的阳性质控品。该阳性质控品包括上述PCR扩增阳性标准品、第二靶序列和标准血浆；第二靶序列为人类的基因组中具有固定长度的核苷酸序列；第二靶序列的序列长度大于第一靶序列的序列长度。
优选的，第二靶序列为人类的基因组中的保守核苷酸序列；优选的，标准血浆为cfDNA含量低于预设阈值的人类血浆；优选的，第二靶序列包括第一靶序列；优选的，预设阈值为30ng/mL、20ng/mL、15ng/mL、10ng/mL或5ng/mL；优选的，第一靶序列的长度在50bp～150bp之间，更优选在60bp～120bp之间，第二靶序列的长度在150bp～300bp之间，更优选在180bp～300bp之间；优选的，阳性质控品中PCR扩增阳性标准品、第二靶序列的拷贝数比值为(1～2):(1～2)，更优选的，阳性质控品中PCR扩增阳性标准品、第二靶序列的拷贝数比值为1:1。
现有技术中，cfDNA浓度的定量检测时，一块96孔板，如仅检测血浆样品的cfDNA浓度，则cfDNA标准品(短片段标准品+动物血浆)6个浓度，每个浓度3个复孔(用于标准曲线的制作，定量用)，阴性对照(与标准品相同的血浆，未添加标准片段)2～3个孔，需用去20个孔，剩76个孔进行样品检测，每个样品3个复孔，约可检测25个样品/板。若同时检测血浆样品的cfDNA浓度和完整性，则需两个cfDNA标准品(短/长片段标准品+动物血浆)6个浓度，每个浓度3个复孔(用于标准曲线的制作，定量用)，阴性对照(与标准品相同的血浆，未添加标准片段)4～6个孔，需用去40个孔，剩余56孔；每个血浆样品两对引物分别3 个复孔(共计6个孔)，即9个样品/板，检测通量低，需要两个标准品，做两个标准曲线。
而采用本发明的技术方案，当第二靶序列包括第一靶序列时，可以只需要一个标准品，制作一个标准曲线，从而降低了工作量，提高了检测效率，且能提高每次qPCR检测cfDNA样品的通量。
为了方便应用，根据本发明一种典型的实施方式，提供了一种用于对血浆样本的cfDNA检测的试剂盒。该试剂盒包括第一引物组、和/或上述PCR扩增阳性标准品，第一引物组为上述用于扩增获得cfDNA标准品的引物组。
优选的，试剂盒还包括第四引物组；第四引物组包括第四上游引物和第四下游引物；第四上游引物包括第一外源序列，第四下游引物包括第二外源序列。
更优选的，第四上游引物为第一外源序列，第四下游引物为第二外源序列。
优选的，试剂盒还包括上述用于血浆中cfDNA检测的阳性质控品。
优选的，试剂盒还包括第二引物组和第三引物组；第二引物组包括：第二上游引物和第二下游引物；第二上游引物包括第一锚定序列，第二下游引物包括第二锚定序列；第三引物组包括：第三上游引物和第三下游引物；第三上游引物包括第三锚定序列，第三下游引物包括第四锚定序列；第三锚定序列和第四锚定序列均为人类的同一染色体上的保守核苷酸序列，且它们的组合能够用于扩增第二靶序列；第二靶序列的序列长度大于第一靶序列的序列长度；优选的，第二上游引物为第一锚定序列，第二下游引物为第二锚定序列；优选的，第三上游引物为第三锚定序列，第三下游引物为第四锚定序列。
通过上述描述可知，实际上，第一引物组是用于扩增获得PCR扩增阳性标准品的；第二引物组是用于扩增短片段实现cfDNA定量的；第三引物组是用于扩增长片段，与第二引物组结果配合实现cfDNA完整性检测的；第四引物组是用于扩增不同浓度的PCR扩增阳性标准品，进而获得标准品曲线的。所以，应用该试剂盒可以高效准确的对检测血浆中cfDNA的浓度和完整性进行检测。
优选的，第二靶序列包括第一靶序列，这样可以进一步简化检测步骤，提高检测效率。当然，第二靶序列可以不包括第一靶序列，只要比第一靶序列长即可。
当第二靶序列包括第一靶序列时，可以选用荧光染料法进行检测。
当第二靶序列不包括第一靶序列时，可以选用荧光染料法、探针法等进行检测。其中，探针法可基于在一个孔中同时进行第一靶序列和第二靶序列的扩增而同时检测两种片段，本方案能提高检测通量，降低检测成本，提高检测效率和准确率，避免相互干扰，减少误差；需要说明的是，探针应为两种，分别位于第一靶序列和第二靶序列，且相互之间不互补配对，且均为人类染色体上的保守序列，探针上的5’端具有猝灭基团，该猝灭基团与探针上的荧光基团结合，使得探针不发出荧光，当进行PCR扩增时，两种探针分别结合在自己的靶向结合区域(模板上)，聚合酶在以模板链为基础，延伸引物时，其5’-3’外切酶活性发生作用，将结合在模板上的探针的5’端的猝灭基团切除，使得荧光探针产生荧光，进而通过检测到的两种荧光的情况，确定样本中第一靶序列和第二靶序列量/浓度，进而获得样品中cfDNA的量以及完整性等信息。
优选的，第二引物组和第三引物组具有相同的上游引物或下游引物，从而可以简化试剂盒组分，降低试剂盒生产成本。
为了进一步提高检测的灵敏度和准确性，更优选的，第一靶序列的长度在50bp～150bp之间，更优选在60bp～120bp之间，第二靶序列的长度在150bp～300bp之间，更优选在180bp～300bp之间。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在检测血浆中cfDNA的技术中的应用。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上 述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在评估个体健康中的应用。其中，个体健康中的应用包括运动健身效果的动态监控、个体衰老评估(体现在衰老标志物的监控)等。研究表明，身体的健康状况与cfDNA的浓度变化息息相关，监控cfDNA浓度的变化可以反映个体健康状况，包括运动健身身体状况的变化；cfDNA包含端粒的片段，而端粒的缩短与细胞衰老有着密切的关系。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在中风诊断和/或治疗中的应用。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在肿瘤诊断中的应用。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在肿瘤治疗中的应用。
在肿瘤治疗中的应用包括治疗效果监控、预后评估中的应用。利用cfDNA作为一个潜在的癌症生物标记物始于1977年，Leon与其同事利用放射免疫化学的方法证明在癌症患者血清中检测出的cfDNA含量会比健康人的明显偏高，但随着肿瘤患者病情的好转，患者体内cfDNA的含量也会伴随着下降。因此，检测出血浆中cfDNA含量的技术将有可能为评判个体健康提供一种获取相对便捷的辅助性动态监控指标。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在自身免疫疾病的诊断中的应用。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在自身免疫疾病的治疗中的应用。
其中，自身免疫性疾病可以为系统性红斑狼疮，研究表明，系统性器官损伤的病人血浆中具有高浓度的cfDNA，而且cfDNA浓度变化与个体生理状态及其临床表现息息相关，cfDNA的浓度会在一定程度反映个体生理状态及其临床表现。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在系统性器官损伤的诊断和/或治疗中的应用。其中，系统性器官损伤包括肾小球肾炎、胰腺炎、炎症性肠病、肝炎等炎症疾病等。
根据本发明一种典型的实施方式，提供了上述用于扩增获得cfDNA标准品的引物组、上述PCR扩增阳性标准品、上述用于对血浆样本的cfDNA检测的血浆阳性标准品、上述阳性质控品或上述用于对血浆样本的cfDNA进行检测的试剂盒在器官移植中的应用。器官移植中应用为在器官移植排异性的监控中的应用。cfDNA来自于供体器官的坏死的细胞(释放量大于10，000bp)、凋亡的细胞(180-200个单核小体DNA)和一些活性释放物质，它在血浆中的半衰期为15-30min。治疗性药物的检测依据是根据检测药物的毒性，如果在早期可以加上cfDNA检测则可以降低早期感染的风险。与传统技术相比，cfDNA检测不仅能够更早的检测出器官的免疫排斥反应，而且使对肾脏、心脏、肺和肝脏的移植中的检测更简便经济，同时还可以根据个人情况设计移植方案。
根据本发明一种典型的实施方式，提供了一种对血浆样品的cfDNA检测的qPCR方法。该qPCR方法包括：S1，利用第四引物组对含有不同浓度阳性标准品的血浆阳性标准品进行qPCR扩增，得含有不同浓度阳性标准品的血浆阳性标准品的qPCR扩增结果；S2，基于对含 有不同浓度阳性标准品的血浆阳性样品的qPCR扩增结果，拟合出标准曲线；第四引物组包括第四上游引物和第四下游引物；第四上游引物包括第一外源序列，第四下游引物包括第二外源序列；血浆阳性标准品为上述用于对血浆样本的cfDNA检测的血浆阳性标准品。
优选的，S1还包括采用第二引物组对血浆样品进行qPCR扩增，得血浆样品的第一qPCR扩增结果；第二引物组包括：第二上游引物和第二下游引物；第二上游引物包括第一锚定序列，第二下游引物包括第二锚定序列。
优选的，第二上游引物为第一锚定序列，第二下游引物为第二锚定序列。
优选的，第四上游引物为第一外源序列，第四下游引物为第二外源序列。
优选的，qPCR方法还包括：S3，结合血浆样品PCR检测结果和标准曲线，计算出血浆样品中cfDNA的含量。其中，cfDNA的浓度＝((第一靶序列拷贝数/第一靶序列在基因组中的拷贝数)*人类单倍体基因组质量)/血浆样本的体积。
更优选的，S1还包括采用第三引物组对血浆样品进行qPCR扩增，得血浆样品的第二qPCR扩增结果；第三引物组包括：第三上游引物和第三下游引物；第三上游引物包括第三锚定序列，第三下游引物包括第四锚定序列；第三锚定序列和第四锚定序列均为人类的同一染色体上的保守核苷酸序列，且它们的组合能够用于扩增第二靶序列；第二靶序列的序列长度大于第一靶序列的序列长度；所述步骤S3还包括：结合血浆样品的第一qPCR扩增结果、血浆样品的第二qPCR扩增结果和所述标准曲线，计算出所述血浆样品中cfDNA的完整性。
优选的，第三上游引物为第三锚定序列，第三下游引物为第四锚定序列。cfDNA的完整性＝扩增出的第二靶序列拷贝数/扩增出的第一靶序列拷贝数。
在本发明一具体的实施方式中，提供一种以L1PA2为测定对象从而检测人血浆cfDNA浓度和完整性的方法，具体步骤如下：1)以L1PA2作为内参靶向位点；2)制备标准品；3)制备质控阳参；4)血液样本做血浆分离；5)配制适用于以血浆样本为模板进行扩增的qPCR mix对待测样本进行检测；6)对QPCR数据处理分析。
L1PA2内参引物(L1PA2-P1：SEQ ID NO：1；L1PA2-P2，SEQ ID NO：2)：选取人类基因组L1PA2作为靶向模板，该位点为多拷贝数位点，在单倍体人基因组中对应了3345个拷贝，扩增后的产物L90序列长度为n1＝90bp，扩增原理参加图2(图2中，引物名称中省略了基因名称L1PA2，L1PA2-P1、L1PA2-P2分别简称为P1、P2)。
标准品制作：选取L1PA2作为靶向模板，设计引物(引物L1PA2-P3：SEQ ID NO：3，L1PA2-P4：SEQ ID NO：4)对目的区域进行扩增得到标准品，标准品的序列长度为131bp(扩增子长度为n2＝131bp的cfDNA standard，即阳性标准品L1PA2-S1)。因为考虑到标准品与待测样本的扩增环境最好一致，即两者均需要添加等量的人血浆作为扩增环境。另一方面在标准品中添加人血浆作为反应背景势必会引入血浆中自带的L1PA2模板，因此上述标准品制作反应时我们利用了外源序列的引物(引物L1PA2-P5：SEQ ID NO：5，L1PA2-P6：SEQ ID NO：6)；引物P5和P6无法扩增人血浆样本中的L1PA2序列，而引物L1PA2-P3＝L1PA2-P5+L1PA2-P1，L1PA2-P4＝L1PA2-P2+L1PA2-P6；这样采用L1PA2-P3和L1PA2-P4扩增得到扩增子长度为n2＝131bp的cfDNA standard，即阳性标准品L1PA2-S1，扩增原理参加图1(图1中，引物名称中省略了基因名称L1PA2，L1PA2-P1、L1PA2-P2、L1PA2-P5和L1PA2-P6分别简称为P1、P2、P5和P6。
在对待测样本进行定量时，标准品的扩增引物为引物的外源序列(引物L1PA2-P5，L1PA2-P6)，待测样本的扩增使用(L1PA2-P1，L1PA2-P2或L1PA2-P7，L1PA2-P8(SEQ ID NO：7)，L1PA2-P1＝L1PA2-P7，L1PA2-P1\L1PA2-P2扩增产物为90bp，L1PA2-P7\L1PA2-P8扩增产物222bp)。
标准品扩增时添加的人血浆可为每次测试待测样本剩余的血浆样本混合得到的混合人血浆(只要是人血浆样本即可)，因为外源引物无法扩增人血浆中的cfDNA模板，所以混合的人血浆样本中自带的cfDNA不会对标准品的定量产生影响。
完整性阳参的制作：选取L1PA2基因的序列片段作为模板，序列长度为n3＝222bp，使 用L1PA2-P7与L1PA2-P8引物对目的区域进行扩增得到扩增子L222，即阳性标准品L1PA2-S2，然后将等拷贝数的标准品与L222混匀，制备成完整性阳参，其理论的完整性值I＝50％，扩增原理参考图3(图3中，引物名称中省略了基因名称L1PA2，L1PA2-P7、L1PA2-P8分别简称为P7、P8)。
鉴于本发明的体系为以血浆为模板进行PCR扩增的体系，所以阳参的背景也应该为人血浆，但是使用的L1PA2-P1&L1PA2-P2与L1PA2-P7&L1PA2-P8引物可以扩增人血浆中的cfDNA模板，所以需要消除阳参使用的人血浆中cfDNA片段或将cfDNA片段的浓度控制在较低的水平，以此来保证阳参定量的稳定性。
在本发明中，可首先在血浆直扩体系中用L1PA2-P1&L1PA2-P2引物测定现有的所有血浆样本的cfDNA质量浓度，再从中筛选浓度值在15ng/ml以下(此浓度之下代表血浆中cfDNA质量浓度低，对人为加入的模板影响较小)的几份血浆样本，各取部分血浆进行混合，测试了其用于阳参的稳定性后，分装后于-80℃保存，每次取一管使用。
配制适用于以血浆样品为模板进行PCR扩增的qPCR mix：血浆成分复杂含有多种PCR抑制剂，qPCR定量的关键因素之一就是需要保证PCR过程中模板的扩增效率接近100％，所以选择合适的DNA聚合酶至关重要，本发明中选用了适用于以血浆为模板进行PCR扩增的特殊聚合酶(例如：Phusion Polymerase)，并通过调节Mg 2+、引物浓度、荧光染料比例等，使得qPCR的扩增效率接近100％。在本发明一典型的实施方式中，用于以血浆为模板进行PCR扩增的qPCR mix如下表1：
表1
Figure PCTCN2020112866-appb-000001
需要进一步说明的是，聚合酶：Phusion Blood II DNA Polymerase使用量最大为0.6μL(说明书推荐)，可根据实际的模板量减少用量。
MgCl 2浓度：2×Phusion Blood PCR buffer已经提供了终浓度为3.0mM的MgCl2，可根据实际情况增加MgCl2浓度最高达4.5mM。
荧光染料：SYBR(10×)优选的终浓度范围为1×左右，例如：0.5×、0.6×、0.7×、0.8×、0.9×、1.0×、1.1×、1.2×等。
引物：优选范围为0.2～1μM。
血样样本加样量(每个反应孔)：优选的占反应体系的1％～20％，比例可达40％甚至更高。
SYBR greenⅠ还可用
Figure PCTCN2020112866-appb-000002
Dy(Biotium)等荧光染料替代。
Phusion Blood II DNA Polymerase还可用以下聚合酶替代:VELOCITY DNA Polymerase(Bioline)、Terra TM qPCR Direct
Figure PCTCN2020112866-appb-000003
Premix、
Figure PCTCN2020112866-appb-000004
qPCR ToughMix TM,ROX TM、MightyAmp TM for Real Time(
Figure PCTCN2020112866-appb-000005
Plus)。
采用绝对定量的方法，通过标准品计算出待测血浆样本浓度值和完整性值。在以阳性标 准品S1为标准品的情况下，标准品使用L1PA2-P5&L1PA2-P6引物进行扩增(其扩增子片段长度为131bp)，待测样本用L1PA2-P1&L1PA2-P2引物进行扩增(其扩增子片段长度为90bp)，则待测样本的真实拷贝数值A(L90)为qPCR软件根据标准曲线定量的拷贝数值O(L90)乘以系数Index1＝标准品扩增子长度/待测样本的扩增子长度＝131bp/90bp＝1.46，即A(L90)＝O(L90)*1.46。这样的计算可以消除由于标准品与待测样本扩增子长度差异而导致的定量结果间的偏差。同理，在以S1为标准品的情况下，如果待测样本用L1PA2-P7&L1PA2-P8引物进行扩增(其扩增子片段长度为222bp)，则待测样本的真实拷贝数值A(L222)为QPCR软件根据标准曲线定量的拷贝数值O(L222)乘以系数Index2＝标准品扩增子长度/待测样本的扩增子长度＝131bp/222bp＝0.59，即A(L222)＝O(L222)*0.59。计算得到待测样本真实拷贝数值A(L90)后，利用拷贝数与基因组质量的对应关系即3345个拷贝数的A(L90)对应一个单倍体基因组的质量(3.3pg)，待测样本的质量浓度C＝A(L90)/2/3345*3.3ng/ml。计算得到待测样本真实拷贝数值A(L90)与A(L222)后，可以得到完整性值I＝A(L222)/A(L90)。
在本发明的发明宗旨指导下，扩增的DNA及引物可以有多种选择，例如，在本发明一具体的实施方式中，具体的扩增序列及引物信息如下：L1PA2的序列为SEQ ID NO：8。
备注：引物L1PA2-P1与L1PA2中的(SEQ ID NO：9)互补，引物P2为SEQ ID NO：2，引物P8为SEQ ID NO：7。
ALU的序列为SEQ ID NO：10。
ALU短引物结合区域：SEQ ID NO：11和SEQ ID NO：12。
ALU长引物结合区域SEQ ID NO：13和SEQ ID NO：14。
在另外的实施方式中，基因、引物及扩增片段信息还可以如下表2中所示：
表2
Figure PCTCN2020112866-appb-000006
在本发明一具体的实施方式中，血浆的处理可以通过以下方法进行：
血液样本用EDTA或Streck抗凝管采集，采血完成后上下轻轻颠倒5～10次使样本和抗凝剂充分混匀。采用EDTA采血管收集的血液样本需保存于4℃环境，一般要求3h内需对样本进行血浆分离。采用Streck采血管收集的血液样本可常温运输，一般要求1周内需对样本进 行血浆分离。
血浆分离采用两步法，将带有全血样本的采血管置于4℃，1,600g离心10分钟，低速离心完成后吸取血浆部分。在吸取结束时，采血管中剩余血浆的液面距白膜层的高度需保证至少有0.5-1cm，以防止白膜层中的有核细胞污染吸取的血浆成分。第二步离心采用高速离心，将血浆置于4℃，1,6000g离心10分钟。第二步离心完成后，会发现管底存在少量细胞碎片，此时只吸取血浆上清，切勿吸到细胞碎片。经过两次离心并分离出的血浆样本即为后续的实验样本。
在本发明一具体的实施方式中，制备人工制备的gDNA来源的模拟cfDNA片段分布的DNA片段的方法具体包括如下步骤：
将4份以上的健康个体(男女各半)的血液gDNA等量混合在一起，DNA总量2～3μg。使用KAPA HyperPlus Kit提供的KAPA Frag Enzyme对混合后gDNA进行酶切打断(40min)，打断纯化后的DNA再用bluepippin 3％Agarose Gel对100bp-350bp之间的小片段gDNA进行富集。富集后的产物经过磁珠纯化，得到gDNA(100-350)的片段即为模拟cfDNA片段分布的DNA片段，使用Qubit对其进行浓度测定。
在本发明一具体的实施方式中，血浆阳性标准品中含PCR扩增阳性标准品的浓度范围如下：本发明使用的标准品6个点的拷贝数浓度(每个反应孔)依次为：1000、5000、10000、20000、100000、1000000。该系列标准品可测量的血浆样本cfDNA浓度范围为：1.44ng/mL-717.99ng/mL。标准品拷贝数浓度的设置原则大概是在保证覆盖测试样本浓度范围的同时尽可能缩小标准品的范围，以获取更精确的定量结果。针对于不同的测试对象，标准品的拷贝数浓度范围可以作调整，例如健康人的血浆cfDNA浓度范围一般不超过100ng/mL，标准品浓度范围可以缩窄；例如肿瘤病人的血浆浓度最高可能到1000ng/mL，标准品浓度范围可以放宽，以提高制备获得标准品曲线的准确性和适用性。
根据本发明一典型的实施例，提供几种在本发明宗旨指导下获得的可用于具体检测的目标序列及其对应的扩增引物，具体如下(注：在本发明中P1～P8可以代表引物的物质，以L1PA2为例，可参见图1和图2，引物命名的中间数字，例如L1PA2-1-P7中的“1”，可以理解为第几组，相同基因中，相同组的引物为一对扩增引物，例如，长片段引物L1PA2-1-P7和L1PA2-1-P8为一对扩增引物，短片段引物L1PA2-11-P1和L1PA2-11-P2为一对扩增引物)：
L1PA2基因的扩增引物如下表3～表7所示。
表3
Figure PCTCN2020112866-appb-000007
表4
Figure PCTCN2020112866-appb-000008
表5
Figure PCTCN2020112866-appb-000009
表6
Figure PCTCN2020112866-appb-000010
Figure PCTCN2020112866-appb-000011
表7
Figure PCTCN2020112866-appb-000012
在本发明中，表3～7中的L1PA2-1-P5、L1PA2-2-P5、L1PA2-3-P5、L1PA2-4-P5、L1PA2-5-P5均可采用具有SEQ ID NO：66所示的核苷酸序列替换，同时与之对应的引物L1PA2-1-P6、L1PA2-2-P6、L1PA2-3-P6、L1PA2-4-P6、L1PA2-5-P6采用具有SEQ ID NO：166所示的核苷酸序列替换。
ALU基因的扩增引物如下表8～表10所示
表8
Figure PCTCN2020112866-appb-000013
表9
Figure PCTCN2020112866-appb-000014
Figure PCTCN2020112866-appb-000015
表10
Figure PCTCN2020112866-appb-000016
hTERT基因的扩增引物如下表11和12所示
表11
Figure PCTCN2020112866-appb-000017
表12
Figure PCTCN2020112866-appb-000018
β-actin基因的扩增引物如下表13和表14所示
表13
Figure PCTCN2020112866-appb-000019
表14
Figure PCTCN2020112866-appb-000020
human APP基因的扩增引物如下表15和表16所示
表15
Figure PCTCN2020112866-appb-000021
表16
Figure PCTCN2020112866-appb-000022
EIF2C1基因的扩增引物如下表17和表18所示
表17
Figure PCTCN2020112866-appb-000023
表18
Figure PCTCN2020112866-appb-000024
RPPH1基因的扩增引物如下表19和表20所示
表19
Figure PCTCN2020112866-appb-000025
Figure PCTCN2020112866-appb-000026
表20
Figure PCTCN2020112866-appb-000027
下面将结合实验数据对本发明的有益效果进行进一步说明。
实施例1
下述实施例中所用的试剂信息如下表21所示。
表21
Figure PCTCN2020112866-appb-000028
本实施例中涉及的引物及阳性标准品信息如下：
引物L1PA2-P1：SEQ ID NO：1。引物L1PA2-P2，SEQ ID NO：2。引物L1PA2-P3：SEQ ID NO：3。引物L1PA2-P4：SEQ ID NO：4。引物L1PA2-P5：SEQ ID NO：5。引物L1PA2-P6：SEQ ID NO：6。引物L1PA2-P7：SEQ ID NO：1。引物L1PA2-P8：SEQ ID NO：7。
阳性标准品L1PA2-S1：选取L1PA2作为靶向模板，设计引物(L1PA2-P3：SEQ ID NO：3，L1PA2-P4：SEQ ID NO：4)对目的区域进行扩增得到标准品，标准品的序列长度为131bp，即阳性标准品L1PA2-S1。
阳性标准品L1PA2-S2：选取L1PA2基因的序列片段作为模板，使用L1PA2-P7(SEQ ID NO：1)与L1PA2-P8(SEQ ID NO：7)引物对目的区域进行扩增得到扩增子L222，即阳性标准品L1PA2-S2。
以L1PA2为例，“直接对血浆样本进行qPCR扩增从而实现cfDNA检测”(也称为直扩体系)与“纯cfDNA片段为模板进行扩增的扩增效率比对实验”如下：
一、以血浆为模板进行PCR扩增的PCR体系与纯核酸体系中阳性标准品L1PA2-S1扩增效率的比较
使用L1PA2-P5(SEQ ID NO：5)和L1PA2-P6(SEQ ID NO：6)引物，对阳性标准品 L1PA2-S1进行qPCR，一板使用纯核酸的qPCR体系，一板使用以血浆为模板进行PCR扩增的qPCR体系(阳性标准品中添加血浆成分)，两种qPCR反应体系的mix配制如下表22所示：
表22
试剂 体积(μL)
KAPA SYBR FAST qPCR Master Mix(2×)Universal 5
ROX(50×) 0.2
引物(含正反引物L1PA2-P5和L1PA2-P6)，各10mM) 0.4
阳性标准品 2
无核酸酶水 2.4
按照下表23所列程序进行反应：
表23
Figure PCTCN2020112866-appb-000029
按下表24准备相应的qPCR反应体系(也称血浆直扩mix，即以血浆为模板进行PCR扩增的mix)，并准备好96孔板。
表24
试剂 20-μL孔
无核酸酶水 2.97
2×Phusion blood PCR buffer 10
SYBR greenⅠ(10×) 1.1
ROX(50×) 0.43
Mg 2+(10mM) 0.3
Phusion Blood II DNA Polymerase 0.4
总计 15.2
阳性标准品的qPCR反应体系见表25：
表25
试剂 体积(μL)
以血浆为模板进行PCR扩增的mix 15.2
引物(含正反引物L1PA2-P5和L1PA2-P6，各10mM) 0.8
阳性标准品 2
原混合血浆 2
按照下表26所列程序进行反应：
表26
Figure PCTCN2020112866-appb-000030
Figure PCTCN2020112866-appb-000031
以上两板的阳性标准品上样排版均如下表27所示：
表27
  1 2 3
A 标准品1(P5&P6) 标准品1(P5&P6) 标准品1(P5&P6)
B 标准品2(P5&P6) 标准品2(P5&P6) 标准品2(P5&P6)
C 标准品3(P5&P6) 标准品3(P5&P6) 标准品3(P5&P6)
D 标准品4(P5&P6) 标准品4(P5&P6) 标准品4(P5&P6)
E 标准品5(P5&P6) 标准品5(P5&P6) 标准品5(P5&P6)
F 标准品6(P5&P6) 标准品6(P5&P6) 标准品6(P5&P6)
G NTC(P5&P6) NTC(P5&P6) NTC(P5&P6)
注，表27中的标准品均是L1PA2-S1稀释不同倍数得到的，具体稀释情况见表28。
在相关设置页面(例如，与QPCR仪器连接的电脑上QPCR软件里面的设置页面)填写标准品各点对应的模板拷贝数(copies/reaction)，标准品各点对应的模板拷贝数(copies/reaction)如下表28：
表28
标准品 每个反应孔中的拷贝数
标准品1 1000000
标准品2 100000
标准品3 20000
标准品4 10000
标准品5 5000
标准品6 1000
qPCR实验结束后，通过StepOne plus仪器自带的软件分析纯核酸体系和以血浆为模板PCR扩增体系标准品拟合的标准曲线及其扩增效率：纯核酸体系的回归系数R 2＝1.000，扩增效率Eff％＝98.577％；以血浆为模板PCR扩增体系的回归系数R 2＝0.999，扩增效率Eff％＝98.842％。
以上分析结果表明，制备的阳性标准品在纯核酸体系与以血浆为模板PCR扩增体系中扩增效率均正常，两种体系的扩增效率无明显差异。
二、以血浆为模板PCR扩增体系与纯核酸体系中不同标准品之间的偏差(可作为待测产物与标准品间比例关系换算的证明)
将2套标准品(阳性标准品L1PA2-S1和阳性标准品L1PA2-S2)用不同引物分别作标准曲线，共得到3条标准曲线，具体信息如下：
1)阳性标准品L1PA2-S1，引物用L1PA2-P5和L1PA2-P6做标准曲线；
2)阳性标准品L1PA2-S1，引物用L1PA2-P1和L1PA2-P2做标准曲线；
3)阳性标准品L1PA2-S2，引物用L1PA2-P7和L1PA2-P8做标准曲线。
在模板数相同的情况下，qPCR检测到的荧光值强度与扩增子长度成正比，所以说，1)、2)、3)标准品之间的定量值可以通过扩增子长度的比值进行换算。
纯核酸体系与以血浆为模板PCR扩增体系qPCR反应mix的配制方法及反应程序同上(以血浆为模板PCR扩增体系与纯核酸体系中阳性标准品L1PA2-S1扩增效率的比较)。
纯核酸体系中不同标准品之间的偏差，如下表29所示。
表29
Figure PCTCN2020112866-appb-000032
Figure PCTCN2020112866-appb-000033
备注：标准品1～6分别为对应的标准品不同的稀释产物。
Observed copies指qPCR仪实测的拷贝数，Expected copies指理论上标准品的拷贝数。
O/E＝Observed copies/Expected copies，Average(O/E)即同一标准品所有点O/E的均值；CV(O/E)即同一标准品所有点O/E的变异系数。
以血浆为模板PCR扩增体系中不同标准品之间的偏差，如下表30所示。
表30
Figure PCTCN2020112866-appb-000034
Figure PCTCN2020112866-appb-000035
备注：标准品1～6分别为对应的标准品不同的稀释产物。
Observed copies指qPCR仪实测的拷贝数，Expected copies指理论上标准品的拷贝数。
O/E＝Observed copies/Expected copies，Average(O/E)即同一标准品所有点O/E的均值；CV(O/E)即同一标准品所有点O/E的变异系数。
从结果来看，纯核酸体系与以血浆为模板PCR扩增体系中不同标准品之间的偏差均很小，其中以血浆为模板PCR扩增体系中不同标准品之间的偏差更小。基于这样的测试结果，我们认为只使用1)阳性标准品L1PA2-S1+L1PA2-P5&L1PA2-P6引物这1套标准品对待测样本进行浓度或完整性的测定，与同时使用2)阳性标准品L1PA2-S1+L1PA2-P1&L1PA2-P2引物和3)阳性标准品L1PA2-S2+L1PA2-P7&L1PA2-P8引物这2套标准品对待测样本进行浓度或完整性的测定，待测样本的测定结果基本等同。
三、标准曲线的获得及其适用性
上述获取标准曲线的方式为使用StepOne plus qPCR仪自带软件进行分析，在相关设置版面将标准品每个点对应的拷贝数按要求输入，软件经过计算后得到分析结果，分析结果包括标准曲线的斜率(slope)、Y-截距(y-intercept)、回归系数(R 2value)、扩增效率(Eff％)。关键指标为回归系数(R 2value)与扩增效率(Eff％)：R 2value越接近1，表示标准曲线的线性拟合程度越好，优选范围为R 2value≥0.99；Eff％最理想的状态为100％，优选范围为95％～105％(图4～9中，quantity为量，即拷贝数)。
1)纯核酸扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P5&L1PA2-P6引物，扩增子长度为131bp)的标准曲线如图4所示。
2)纯核酸体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P1&L1PA2-P2引物，扩增子长度90bp，)的标准曲线如图5所示。
3)纯核酸体系(阳性标准品L1PA2-S2，扩增引物为：L1PA2-P7&L1PA2-P8引物，扩增子长度222bp)的标准曲线如图6所示。
4)以血浆为模板PCR扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P5&L1PA2-P6引物，扩增子长度为131bp)的标准曲线如图7所示。
5)以血浆为模板PCR扩增体系(阳性标准品L1PA2-S1，扩增引物为：L1PA2-P1&L1PA2-P2引物，扩增子长度90bp)的标准曲线如图8所示。
6)以血浆为模板PCR扩增体系(阳性标准品L1PA2-S2，扩增引物为：L1PA2-P7&L1PA2-P8引物，扩增子长度222bp)的标准曲线如图9所示。
以上是StepOne plus软件自动分析结果，再同一扩增体系中，三种不同标准品的扩增效率接近。此处补充标准曲线分析的计算原理：标准曲线即将标准品模板拷贝数取log值对每个稀释样本的Ct值作图，得到的递减的线性图。R 2就是反映该递减线性关系的可信度，理想情况下R 2＝1。在对标准曲线进行线性拟合时，可以计算得到该直线的斜率slope，理想状态下slope＝-3.32。通过slope值计算即可得到扩增效率Eff％＝(10 (-1/slope)-1)*100％，理想状态下Eff％＝100％。
实施例2
除了染料法QPCR可以实现本发明，探针法同样也可以，在本实施例中，将列举几种具体的可行实施方案。
方案a：浓度和完整性分开检测
例举两组方案a中的引物及探针序列信息，如表31：
表31
Figure PCTCN2020112866-appb-000036
Figure PCTCN2020112866-appb-000037
注：本方案a中每组引物的扩增产物(长片段/短片段)检测共用一个探针，所以浓度与完整性分开两管检测，即短片段检测为一管(以方案1为例，此管包括探针1、引物11-F、引物11-R)，长片段检测为另一管(以方案1为例，此管包括探针1、引物12-F、引物12-R)。
1)配制qPCR反应体系如下。
浓度检测qPCR反应体系如表32所示(以方案1为例)：
表32
成分 20-μL/孔
无核酸酶水 4.8
KAPA PROBE FORCE qPCR Master Mix(2×)Universal 10
10μM正向引物1(引物11-F，SEQ ID NO:157) 0.4
10μM反向引物1(引物11-R，SEQ ID NO:158) 0.4
10μM探针1(探针1，SEQ ID NO:156) 0.4
模板DNA(血浆) 4
总计 20
完整性检测qPCR反应体系如表33所示(以引物组1为例)：
表33
成分 20-μL/孔
无核酸酶水 4.8
KAPA PROBE FORCE qPCR Master Mix(2×)Universal 10
10μM正向引物2(引物12-F，SEQ ID NO:157) 0.4
10μM反向引物2(引物12-R，SEQ ID NO:159) 0.4
10μM探针1(探针1，SEQ ID NO:156) 0.4
模板DNA(血浆) 4
总计 20
2)设置qPCR反应程序如表34：
表34
Figure PCTCN2020112866-appb-000038
3)分析定量数据：确认标准品扩增效率是否在正常范围(95～105％)；阳参的完整性值是否在正常范围(45％～55％)。
4)计算待测物浓度及完整性。
采用绝对定量的方法，通过标准品计算出待测血浆样本浓度值和完整性值。
对10个血浆样本采用方案a进行检测，结果与使用染料法QPCR得到的结果一致(具体结果未示出)，说明方案a切实可行。
方案b：浓度和完整性在同一管检测
例举两组方案b中的引物及探针序列信息，如表35：
表35
Figure PCTCN2020112866-appb-000039
注：本方案b中，每组引物的扩增的产物(长片段/短片段)检测分别用不同的探针，所以浓度与完整性可在同一PCR管检测，即同一PCR管中包含长片段引物对、短片段引物对、两种探针(以同时检测短片段1和长片段1的方案3为例，此PCR管中包括探针31、探针32、引物31-F、引物31-R、引物32-F、引物32-R)。
1)配制qPCR反应体系如表36(以引物组3为例)：
表36
成分 20-μL/孔
无核酸酶水 3.6
KAPA PROBE FORCE qPCR Master Mix(2×)Universal 10
10μM正向引物31(引物31-F，SEQ ID NO:157) 0.4
10μM反向引物31(引物31-R，SEQ ID NO:158) 0.4
10μM正向引物32(引物32-F，SEQ ID NO:161) 0.4
10μM反向引物32(引物32-R，SEQ ID NO:163) 0.4
10μM探针31(SEQ ID NO:156) 0.4
10μM探针32(SEQ ID NO:160) 0.4
模板DNA(血浆) 4
总计 20
2)设置qPCR反应程序如表37：
表37
Figure PCTCN2020112866-appb-000040
3)分析定量数据：确认标准品扩增效率是否在正常范围(95～105％)；阳参的完整性值是否在正常范围(45％～55％)。
4)计算待测物浓度及完整性：
采用绝对定量的方法，通过标准品计算出待测血浆样本浓度值和完整性值。
探针法以血浆为模板，PCR扩增聚合酶还可用以下酶替代：
Figure PCTCN2020112866-appb-000041
Environmental Master Mix 2.0、VersaTaqTM Direct PCR Polymerase(71002)、KAPA PROBE FORCE qPCR Master Mix(2×)Universal Kit。
对10个血浆样本采用方案b进行检测，结果与使用染料法QPCR得到的结果一致(具体结果未示出)，说明方案b切实可行。
实施例3
本实施例中给出了采用了Phusion Blood Direct PCR Kit(Thermo Scientific)，VELOCITY DNA Polymerase(Bioline)两款试剂的检测方法及测试结果。
实验1
样品名称：(phu h0f，h2f)
Phusion Blood Direct PCR Kit(Thermo Scientific)
实验方法：
L1PA2-P5(SEQ ID NO：5)，L1PA2-P6(SEQ ID NO：6)引物对如图10所示的模板进行扩增，模板长度为363bp，用于测试不同试剂与血浆环境下，标准品的扩增效率。
反应体系中加入对应稀释倍数下的人血浆(前处理稀释0倍或2倍)，标准品用L1PA2-P5，L1PA2-P6引物扩增。反应体系见表38。
表38
Figure PCTCN2020112866-appb-000042
具体的样本信息如下表39：
表39
Figure PCTCN2020112866-appb-000043
按照下表40所列程序进行反应：
表40
Figure PCTCN2020112866-appb-000044
Figure PCTCN2020112866-appb-000045
实验结果见表41：
表41
Figure PCTCN2020112866-appb-000046
人血浆前处理不稀释的情况下的熔解曲线如图11所示(其中，纵坐标derivative reporter为荧光值对温度的求导，也可以表述为–ΔF/ΔT(change in fluorescence/change in temperature)，反映的是荧光值随着温度的变化而变化的情况)，标准曲线如图12所示。
人血浆前处理稀释2倍的情况下的熔解曲线如图13所示，标准曲线如图14所示。
实验结论：
使用Phusion试剂，以L1PA2-P5,L1PA2-P6引物扩增长度为363bp的目标片段，血浆在前处理不稀释或稀释2倍的状态下，其标准品扩增子的扩增效率仍可以接近100％；从熔解曲线看扩增特异性，特异性良好。
实验2
实验方法：
同实验1，但使用VELOCITY DNA Polymerase(Bioline)。
实验结果如下表42：
表42
Figure PCTCN2020112866-appb-000047
人血浆前处理不稀释的情况下的熔解曲线如图15所示，标准曲线如图16所示。
人血浆前处理稀释2倍的情况下的熔解曲线如图17所示，标准曲线如图18所示。
实验结论：
使用velocity试剂，以L1PA2-P5，L1PA2-P6引物扩增长度为363bp的目标片段，血浆在不稀释或稀释2倍的情况下标准品扩增子的扩增效率仍可以接近100％；从熔解曲线看扩增特异性，特异性良好。
实施例4
单拷贝、多拷贝基因方法比较试验及结果
实验方法：
使用两种引物扩增定量提取后的cfDNA样本中单拷贝基因(MSTN，EIF2C1)，并与L90(L1PA2的引物L1PA2-P1&L1PA2-P2扩增子，90bp)的定量结果做比较，分析定量结果间的异同及不同样本间相对值的变化趋势。
引物具体信息见表43：
表43
Figure PCTCN2020112866-appb-000048
Figure PCTCN2020112866-appb-000049
使用L90，MSTN，EIF2C1引物定量时，标准品用D-g125-1至D-g125-6。
说明：D-g125系列标准品是在测试过程中制备的另一套标准品，与前述的S1、S2标准品的区别在于，D-g125的制备过程没有经过PCR扩增，制备流程如下：先将gDNA片段化，然后筛选出接近cfDNA片段分布的片段，将该片段按一定浓度等比稀释即得到D-g125系列标准品，D-g125-1至D-g125-6是其具体标号。
反应体系见表44，PCR程序见表45和46：
表44
Figure PCTCN2020112866-appb-000050
表45
Figure PCTCN2020112866-appb-000051
表46
Figure PCTCN2020112866-appb-000052
实验结果：
各引物定量的绝对值见表47：
表47
Figure PCTCN2020112866-appb-000053
Figure PCTCN2020112866-appb-000054
注：表中的数值单位为haploid human genome copies/μL plasma，即每微升血浆的人单倍体基因组当量。
各引物定量绝对值的柱状图如图19所示，其中MSTN结果为两次定量结果的平均值。
两两引物定量结果间的一致性见表48：
表48
  R 2 P value
L90 vs.MSTN 0.9781 0.0002
L90 vs.EIF2C1 0.9514 0.0009
MSTN vs.EIF2C1 0.9867 <0.0001
实验结论：
MSTN，EIF2C1在基因组上的扩增模板在基因组上为单拷贝，L90在基因组上的扩增模板为多拷贝。所以L90在定量过程中对基因组模板量的需求量相对更宽松。
从定量结果的绝对值角度分析，使用MSTN，EIF2C1两套引物分别定量，其结果差值最小。L90与EIF2C1间定量结果的差值也整体小于L90与MSTN间的定量差值。
从定量结果的一致性角度分析，三套引物定量结果的一致性都表现良好，R 2均能达到95％以上，P value<0.001。
实施例5
选取来自健康体检者8例，使用EDTA采血管收集志愿者全血，在短时间内完成血浆分离。并将分离好的血浆样本分两部分进行测试，一部分各取400μL血浆进行cfDNA提取，并对提取后的cfDNA用纯核酸体系进行定量，另一部分直接用血浆进行以血浆为模板PCR扩增cfDNA定量。提取后的核酸产物定量体系和本发明的以血浆为模板PCR扩增定量体系中使用的标准品、阳参及引物相同，标准品、阳参及引物的具体信息在具体实施方式或上述实施例中已作详述。提取后的核酸产物定量方法参照现有文献(Direct Quantification of Cell-Free,Circulating DNA from Unpurified Plasma_PLos One，后续称现有)，具体步骤不在此详述。
一、应用本发明的技术方案直接用血浆进行cfDNA扩增定量
1.样本制备：
血浆分离采用两步法，将带有全血样本的采血管置于4℃，1,600g离心10分钟，低速离心完成后吸取血浆部分。在吸取过程中，需要防止白膜层中的有核细胞污染吸取的血浆成分。第二步离心采用高速离心，将血浆置于4℃，16,000g离心10分钟。经两次离心后分离出的血浆样本即为后续的实验样本，保存于-80℃。
2.制备qPCR反应体系：
按下表49准备相应的qPCR反应体系(也称血浆直扩mix，即以血浆为模板PCR扩增mix，本实施例中所用引物的具体信息参见实施例1)，并准备好96孔板。
表49
成分 20-μL/孔
无核酸酶水 2.97
2×Phusion blood PCR buffer 10
SYBR greenⅠ(10×) 1.1
ROX(50×) 0.43
Mg 2+(10mM) 0.3
Phusion Blood II DNA Polymerase 0.4
总计 15.2
阳性标准品及阳性质控的qPCR反应体系见表50：
表50
Figure PCTCN2020112866-appb-000055
待测样本的反应体系见表51：
表51
试剂 体积(μL)
以血浆为模板PCR扩增mix 15.2
引物(含正反引物L1PA2-P1&L1PA2-P2或L1PA2-P7&L1PA2-P8，各10mM) 0.8
血浆样本 2
DdH 2O 2
将反应mix(18μL/孔)与标准品/阳参/待测样本(2μL//孔)加入96孔板。
3.设置反应程序；
按照下表52所列程序进行反应：
表52
Figure PCTCN2020112866-appb-000056
4.结果分析：
1)判断标准品扩增效率是否合格：正常情况下标准品扩增效率为95～105％，R 2范围为0.99<＝R 2<＝1，本次实验的扩增效率为99.132％，R 2值为1，判定为合格。
2)判断完整性阳参是否在正常范围：正常情况下完整性阳参的值为45～55％，本次实验中完整性阳参的值为48.85％，判定为合格。
3)各血浆样本的浓度值
用公式A(L90)＝O(L90)*1.46计算得到各待测样本的拷贝数A(L90)，再通过C＝A(L90)/2/3345*3.3ng/ml计算得到各待测样本的cfDNA质量浓度(ng/ml)，计算结果如下表53所示。
表53
Figure PCTCN2020112866-appb-000057
将各待测血浆样本用本发明的方法以血浆为模板PCR扩增结果与其提取后的定量浓度比较，以血浆为模板PCR扩增与提取后的比值相对稳定(均值1.95，CV值为11％)，现有文献的以血浆为模板PCR扩增与提取后的比值在2左右。
4)各血浆样本的完整性
使用公式计算A(L90)＝O(L90)*1.46，A(L222)＝O(L222)*0.59得到两对引物测定的真实拷贝数，再通过I＝A(L222)/A(L90)计算得到待测样本的完整性值，如下表54所示。
表54
样本 以血浆为模板PCR扩增完整性 提取后完整性
样本1 25.87％ 25.10％
样本2 16.90％ 23.85％
样本3 34.02％ 28.25％
样本4 28.15％ 29.72％
样本5 22.79％ 23.44％
样本6 17.68％ 18.37％
样本7 23.30％ 22.97％
样本8 22.81％ 25.50％
将各待测血浆样本用本发明的方法以血浆为模板PCR扩增结果与其提取后的定量完整性进行比较，统计学方法为Paired t test，检验结果P value＝0.5906(P value>0.05)，表明两种定量方法对同一份样本测定完整性无显著差异，即以血浆为模板PCR扩增与提取后定量的完整性接近(现有文献的以血浆为模板PCR扩增与提取后完整性接近)，说明本发明的方法可靠。二、对提取后的cfDNA用纯核酸体系进行定量
具体实施步骤：
1.血浆cfDNA的提取：使用MAGEN游离核酸的商业提取试剂盒对400μL血浆样本进行提取，在2mL离心管中，加入0.4mL EDTA保存的血浆和0.1mL PBS，接下来的步骤参照该提取试剂盒的说明书进行操作。最后一步提取产物回溶时使用80μL EB进行回溶。
2.样本稀释：将提取后的纯核酸样本进行4倍稀释。
3.制备qPCR反应体系(使用试剂KAPA
Figure PCTCN2020112866-appb-000058
FAST qPCR Master Mix(2×)Kit)：
按下表准备相应的qPCR反应体系(mix)，并准备好96孔板。
标准品的反应体系(mix/孔)如表55所示：
表55
试剂 体积(μL)
KAPA SYBR FAST qPCR Master Mix(2×)Universal 5
ROX(50×) 0.2
引物(含正反引物L1PA2-P5&L1PA2-P6，各10mM) 0.4
阳性标准品 2
ddH 2O 2.4
待测样本及完整性阳参的反应体系(mix/孔)如表56所示：
表56
Figure PCTCN2020112866-appb-000059
Figure PCTCN2020112866-appb-000060
将反应mix(8μL/孔)与标准品/阳参/待测样本(2μL/孔)加入96孔板。
4.设置反应程序；
按照下表57所列程序进行反应：
表57
Figure PCTCN2020112866-appb-000061
5.结果分析：
1)判断标准品扩增效率是否合格：正常情况下标准品扩增效率为95～105％，R 2范围为0.99<＝R 2<＝1，本次实验的扩增效率为99.132％，R 2值为1，判定为合格。
2)判断完整性阳参是否在正常范围：正常情况下完整性阳参的值为45～55％，本次实验中完整性阳参的值为48.85％，判定为合格。
3)各提取后纯核酸样本的浓度值
用公式A(L90)＝O(L90)*1.46计算得到各待测样本的拷贝数A(L90)，再通过C＝A(L90)/2.5/3345*3.3ng/ml计算得到各待测样本的cfDNA质量浓度(ng/ml)，计算结果在以血浆为模板PCR扩增部分已展示。
4)各提取后纯核酸样本的完整性
使用公式计算A(L90)＝O(L90)*1.46，A(L222)＝O(L222)*0.59得到两对引物测定的真实拷贝数，再通过I＝A(L222)/A(L90)计算得到待测样本的完整性值，计算结果在以血浆为模板PCR扩增部分已展示。
三、血浆样本在以血浆为模板PCR扩增体系与纯核酸体系中的重复性测试结果
按上述步骤一(应用本发明的技术方案直接用血浆进行PCR扩增cfDNA定量)和步骤二(对提取后的cfDNA用纯核酸体系进行定量)对5个样本进行三次重复测定，得到这5个样本在以血浆为模板PCR扩增体系与纯核酸体系中的重复性结果见表58：
表58
Figure PCTCN2020112866-appb-000062
Figure PCTCN2020112866-appb-000063
无论是纯核酸体系还是以血浆为模板PCR扩增体系，使用阳性标准品S1+P5&P6引物这套标准曲线对以上样本进行定量，cfDNA浓度与完整性的重复性均良好(CV值<10％)；纯核酸体系与以血浆为模板PCR扩增体系相比，重复性没有明显差异。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (59)

  1. 一种用于扩增获得cfDNA标准品的引物组，其特征在于，包括：第一上游引物和第一下游引物，所述第一上游引物包括从5’端到3’端顺次连接的第一外源序列和第一锚定序列，所述第一下游引物包括从5’端到3’端顺次连接的第二外源序列和第二锚定序列；所述第一外源序列和所述第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；所述第一锚定序列和所述第二锚定序列为人类的同一染色体上的保守核苷酸序列，且所述第一锚定序列和所述第二锚定序列的组合能够用于扩增第一靶序列；所述第一靶序列为由从5’端到3’端顺次连接的人类染色体上的所述第一锚定序列及其互补序列，所述第一锚定序列和所述第二锚定序列之间的双链核苷酸序列，和所述第二锚定序列及其互补序列组成的具有固定长度的双链核苷酸分子。
  2. 根据权利要求1所述的引物组，其特征在于，所述第一靶序列是人类染色体中的保守核苷酸序列；
    优选的，所述第一靶序列在人类的染色体上具有多个拷贝。
  3. 根据权利要求1所述的引物组，其特征在于，所述第一靶序列位于L1PA2、Alu、hTERT、human APP、β-actin、EIF2C1或RPPH1基因上；
    优选的，所述第一靶序列的长度在50bp～150bp之间，更优选在60bp～120bp之间。
  4. 一种PCR扩增阳性标准品，其特征在于，所述PCR扩增阳性标准品为单链核苷酸分子或双链核苷酸分子，
    当PCR扩增阳性标准品为单链核苷酸分子时，包括：从5’到3’端依次为第一外源序列、第一中间序列和第二外源序列的互补序列；或从5’到3’端依次为第二外源序列、第二中间序列和第一外源序列的互补序列；
    当PCR扩增阳性标准品为双链核苷酸分子时，其中的一条链包括：从5’到3’端依次为第一外源序列、第一中间序列和第二外源序列的互补序列；或从5’到3’端依次为第二外源序列、第二中间序列和第一外源序列的互补序列；
    所述第一中间序列和第二中间序列互补配对，所述第一中间序列和第二中间序列互补配对后的双链核苷酸分子为第一靶序列；所述第一靶序列为由从5’端到3’端顺次连接的人类染色体上的第一锚定序列及其互补序列，所述第一锚定序列和第二锚定序列之间的双链核苷酸序列，和所述第二锚定序列及其互补序列组成的具有固定长度的双链核苷酸分子；所述第一外源序列和所述第二外源序列之间不互补配对，且与人类的任一染色体上的任意核苷酸序列不互补配对；所述第一锚定序列和所述第二锚定序列为人类的同一染色体上的保守核苷酸序列，且所述第一锚定序列和所述第二锚定序列的组合能够用于扩增所述第一靶序列。
  5. 一种PCR扩增阳性标准品的制备方法，其特征在于，包括采用如权利要求1至3中任一项所述的引物组，对标准品扩增模板进行PCR扩增，获得PCR扩增阳性标准品；其中，所述标准品扩增模板为血浆、从血浆中提取获得的cfDNA、或人工制备的gDNA来源的模拟cfDNA片段分布的DNA片段；
    优选的，对所述标准品扩增模板进行PCR扩增后还包括对扩增产物进行纯化的步骤。
  6. 一种用于对血浆样本的cfDNA检测的血浆阳性标准品，其特征在于，包括如权利要求4所述的PCR扩增阳性标准品和血浆；
    优选的，所述血浆阳性标准品中含所述PCR扩增阳性标准品的浓度在1ng/mL至1000ng/mL之间；
    优选的，所述血浆为人类血浆。
  7. 一种用于血浆中cfDNA检测的阳性质控品，其特征在于，包括如权利要求4所述的PCR扩增阳性标准品、第二靶序列和标准血浆；所述第二靶序列为人类基因组中具有固定长度的核苷酸序列；所述第二靶序列的序列长度大于所述第一靶序列的序列长度；
    优选的，所述标准血浆为cfDNA含量低于预设阈值的人类血浆；
    优选的，所述第二靶序列包括第一靶序列；
    优选的，所述第二靶序列为人类基因组中的保守核苷酸序列；
    优选的，所述预设阈值为30ng/mL、20ng/mL、15ng/mL、10ng/mL或5ng/mL；
    优选的，所述第一靶序列的长度在50bp～150bp之间，更优选在60bp～120bp之间，所述第二靶序列的长度在150bp～300bp之间，更优选在180bp～300bp之间；
    优选的，所述阳性质控品中所述PCR扩增阳性标准品、所述第二靶序列的拷贝数比值为(1～2):(1～2)，更优选的，所述阳性质控品中所述PCR扩增阳性标准品、所述第二靶序列的拷贝数比值为1:1。
  8. 一种用于对血浆样本的cfDNA进行检测的试剂盒，其特征在于，包括第一引物组、和/或如权利要求4所述的PCR扩增阳性标准品，所述第一引物组为如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述试剂盒还包括：
    如权利要求7所述的用于血浆中cfDNA检测的阳性质控品；
    第四引物组；所述第四引物组包括第四上游引物和第四下游引物；所述第四上游引物包括第一外源序列，所述第四下游引物包括第二外源序列；
    如权利要求6所示的用于对血浆样本的cfDNA检测的血浆阳性标准品；
    优选的，所述第四上游引物为第一外源序列，所述第四下游引物为第二外源序列。
  10. 根据权利要求8所述的试剂盒，其特征在于，所述试剂盒还包括第二引物组和第三引物组；所述第二引物组包括：第二上游引物和第二下游引物；所述第二上游引物包括第一锚定序列，所述第二下游引物包括第二锚定序列；所述第三引物组包括：第三上游引物和第三下游引物；所述第三上游引物包括第三锚定序列，所述第三下游引物包括第四锚定序列；所述第三锚定序列和所述第四锚定序列均为人类的同一染色体上的保守核苷酸序列，且它们的组合能够用于扩增第二靶序列；所述第二靶序列的序列长度大于所述第一靶序列的序列长度；
    优选的，所述第二上游引物为第一锚定序列，所述第二下游引物为第二锚定序列；
    优选的，所述第三上游引物为第三锚定序列，所述第三下游引物为第四锚定序列。
  11. 根据权利要求10所述的试剂盒，其特征在于，所述第二靶序列包括第一靶序列；或所述第二引物组和所述第三引物组具有相同的上游引物或下游引物；
    优选的，所述第一靶序列的长度在50bp～150bp之间，更优选在60bp～120bp之间，所述第二靶序列的长度在150bp～300bp之间，更优选在180bp～300bp之间。
  12. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在检测血浆中cfDNA的技术中的应用。
  13. 如权利要求4所述的PCR扩增阳性标准品在检测血浆中cfDNA的技术中的应用。
  14. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在检测血浆中cfDNA的技术中的应用。
  15. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在检测血浆中cfDNA的技术中的应用。
  16. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在检测血浆中cfDNA的技术中的应用。
  17. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在评估个体健康中的应用。
  18. 如权利要求4所述的PCR扩增阳性标准品在评估个体健康中的应用。
  19. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在评估个体健康中的应用。
  20. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在评估个体健康中的应用。
  21. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在评估个体健康中的应用。
  22. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在中风诊断和/或治疗中的应用。
  23. 如权利要求4所述的PCR扩增阳性标准品在中风诊断和/或治疗中的应用。
  24. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在中风诊断和/或治疗中的应用。
  25. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在中风诊断和/或治疗中的应用。
  26. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在中风诊断和/或治疗中的应用。
  27. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在肿瘤诊断中的应用。
  28. 如权利要求4所述的PCR扩增阳性标准品在肿瘤诊断中的应用。
  29. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在肿瘤诊断中的应用。
  30. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在肿瘤诊断中的应用。
  31. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在肿瘤诊断中的应用。
  32. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在肿瘤治疗中的应用。
  33. 如权利要求4所述的PCR扩增阳性标准品在肿瘤治疗中的应用。
  34. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在肿瘤治疗中的应用。
  35. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在肿瘤治疗中的应用。
  36. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在肿瘤治疗中的应用。
  37. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在自身免疫疾病的诊断中的应用。
  38. 如权利要求4所述的PCR扩增阳性标准品在自身免疫疾病的诊断中的应用。
  39. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在自身免疫疾病的诊断中的应用。
  40. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在自身免疫疾病的诊断中的应用。
  41. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在自身免疫疾病的诊断中的应用。
  42. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在自身免疫疾病的治疗中的应用。
  43. 如权利要求4所述的PCR扩增阳性标准品在自身免疫疾病的治疗中的应用。
  44. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在自身免疫疾病的治疗中的应用。
  45. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在自身免疫疾病的治疗中的应用。
  46. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在自身免疫疾病的治疗中的应用。
  47. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在系统性器官损伤的诊断和/或治疗中的应用。
  48. 如权利要求4所述的PCR扩增阳性标准品在系统性器官损伤的诊断和/或治疗中的应用。
  49. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在系统性器官损伤的诊断和/或治疗中的应用。
  50. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在系统性器官损伤的诊断和/或治疗中的应用。
  51. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在系统性器官损伤的诊断和/或治疗中的应用。
  52. 如权利要求1至3中任一项所述的用于扩增获得cfDNA标准品的引物组在器官移植中的应用。
  53. 如权利要求4所述的PCR扩增阳性标准品在器官移植中的应用。
  54. 如权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品在器官移植中的应用。
  55. 如权利要求7所述的用于血浆中cfDNA检测的阳性质控品在器官移植中的应用。
  56. 如权利要求8至11中任一项所述的用于对血浆样本的cfDNA检测的试剂盒在器官移植中的应用。
  57. 一种对血浆样品的cfDNA检测的qPCR方法，其特征在于，包括：
    S1，利用第四引物组对含有不同浓度阳性标准品的血浆阳性标准品进行qPCR扩增，得含有不同浓度阳性标准品的血浆阳性标准品的qPCR扩增结果；
    S2，基于所述含有不同浓度阳性标准品的血浆阳性样品的qPCR扩增结果，拟合出标准曲线；
    所述第四引物组包括第四上游引物和第四下游引物；所述第四上游引物包括第一外源序列，所述第四下游引物包括第二外源序列；所述血浆阳性标准品为权利要求6所述的用于对血浆样本的cfDNA检测的血浆阳性标准品。
  58. 根据权利要求57所述的qPCR方法，其特征在于，所述S1还包括：采用第二引物组对血浆样品进行qPCR扩增，得血浆样品的第一qPCR扩增结果；所述第二引物组包括：第二上游引物和第二下游引物；所述第二上游引物包括第一锚定序列，所述第二下游引物包括第二锚定序列；
    优选的，所述第二上游引物为第一锚定序列，所述第二下游引物为第二锚定序列；
    优选的，所述第四上游引物为第一外源序列，所述第四下游引物为第二外源序列；
    优选的，所述qPCR方法还包括：
    S3，结合血浆样品的第一qPCR扩增结果和所述标准曲线，计算出所述血浆样品中cfDNA的含量。
  59. 根据权利要求58所述的qPCR方法，其特征在于，所述S1还包括采用第三引物组对所述血浆样品进行qPCR扩增，得血浆样品的第二qPCR扩增结果；所述第三引物组包括：第三上游引物和第三下游引物；所述第三上游引物包括第三锚定序列，所述第三下游引物包括第四锚定序列；所述第三锚定序列和所述第四锚定序列均为人类同一染色体上的保守核苷酸序列，且它们的组合能够用于扩增第二靶序列；所述第二靶序列的序列长度大于所述第一靶序列的序列长度；
    所述步骤S3还包括：结合血浆样品的第一qPCR扩增结果、血浆样品的第二qPCR扩增结果和所述标准曲线，计算出所述血浆样品中cfDNA的完整性；
    优选的，所述第三上游引物为第三锚定序列，所述第三下游引物为第四锚定序列。
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