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WO2025162424A1 - Procédé d'évaluation spécifique du degré de fragmentation d'acide nucléique cible - Google Patents

Procédé d'évaluation spécifique du degré de fragmentation d'acide nucléique cible

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Publication number
WO2025162424A1
WO2025162424A1 PCT/CN2025/075384 CN2025075384W WO2025162424A1 WO 2025162424 A1 WO2025162424 A1 WO 2025162424A1 CN 2025075384 W CN2025075384 W CN 2025075384W WO 2025162424 A1 WO2025162424 A1 WO 2025162424A1
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WIPO (PCT)
Prior art keywords
nucleic acid
pairs
target sequence
primer
target
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English (en)
Chinese (zh)
Inventor
马晓娟
赵雨航
何辉煌
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Maccura Biotechnology Co Ltd
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Maccura Biotechnology Co Ltd
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Publication of WO2025162424A1 publication Critical patent/WO2025162424A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present application relates to the field of molecular biology, and more particularly to a method and system for evaluating the fragmentation of nucleic acid samples, and more particularly to a method for specifically evaluating the degree of fragmentation of a target nucleic acid.
  • nucleic acid integrity which refers to the size and degree of fragmentation of nucleic acid fragments, is a crucial quality control indicator in molecular biological genetic testing.
  • Incomplete nucleic acids include both naturally fragmented and artificially generated fragments.
  • Naturally generated fragmented nucleic acids typically arise from several sources, including fragmented cfDNA (cell-free DNA) produced by apoptosis of normal cells or necrosis of tumor cells; nucleic acid degradation caused by improper sample storage or repeated freeze-thaw cycles; nucleic acid breakage caused by mechanical shear forces during nucleic acid extraction; genomic DNA damage caused by improper preparation or storage of FFPE samples; and highly fragmented ancient DNA samples.
  • Artificially generated fragmented nucleic acids include fragments generated by breaking genomic DNA during NGS library construction and simulated fragmentation samples prepared for certain scientific research purposes.
  • nucleic acid samples have high requirements for integrity.
  • DNA quality of FFPE samples such as poor nucleic acid integrity, will significantly affect the subsequent fluorescence quantification results or the success rate of second-generation sequencing library construction and the accuracy of sequencing data indicators. Therefore, before performing nucleic acid testing on FFPE samples, it is very useful to evaluate the nucleic acid fragmentation of the samples. This allows different treatments to be performed on samples with different degrees of fragmentation to avoid wasting time and money (Corces MR, Granja JM, Shams S, et al. The chromatin accessibility landscape of primary human cancers.
  • the degree of sample fragmentation should be evaluated to ensure the quality of downstream experiments, thereby better guiding subsequent genetic testing and the judgment of test results.
  • the main methods for detecting the degree of nucleic acid fragmentation are the electrophoresis methods described in GB/T 40974-2021 "Nucleic Acid Sample Quality Assessment Methods", including gel electrophoresis, capillary gel electrophoresis, and microfluidic analysis.
  • the electrophoresis method uses the "charge effect” and "molecular sieve effect” of electrophoresis to separate nucleic acid samples of different fragment sizes.
  • the gel electrophoresis method is time-consuming and labor-intensive, prone to aerosol contamination, and has low sensitivity resulting in poor accuracy. It is only suitable for laboratories with high safety levels to evaluate the quality of samples with high concentrations and low fragmentation levels.
  • Capillary gel electrophoresis is a new type of liquid phase separation technology that uses capillaries as separation channels and high-voltage DC electric fields as the driving force, such as Agilent's 4150 bioanalyzer and Bioptic's Qsep100 fully automatic nucleic acid protein analysis system.
  • Microfluidics analysis refers to the science and technology involved in systems that use microchannels with sizes ranging from tens to hundreds of microns to process or manipulate tiny fluids (with volumes ranging from nanoliters to microliters), such as Agilent's 2100 system and PerkinElmer's Labchip system.
  • capillary gel electrophoresis and microfluidics analysis have overcome many of the shortcomings of ordinary gel electrophoresis, are relatively simple to operate, and have greatly improved sensitivity, they can provide high-resolution DNA fragment peak distributions and are mainly used for second-generation and third-generation sequencing quality control, sample quality control, and analysis of residual DNA in host cells of biological products.
  • this type of method requires different detection reagents for different application scenarios, and each detection reagent analyzes a different range of fragments, and cannot analyze the overall fragmentation of the sample; some instruments claim to be able to achieve picogram-level nucleic acid detection sensitivity, but often when the instrument detects samples of this concentration, many fragments are below the detection threshold, resulting in missed detections and inaccurate fragment information. Indeed, scientific research or clinical practice often also has testing needs for clinical samples with lower concentrations, such as analyzing the fragmentation distribution of extremely low-concentration cfDNA. However, because the sample concentration is lower than the detection sensitivity, relevant fragment information cannot be obtained.
  • the learning model of Agilent's 2100 system is derived from the manual judgment and scoring of 1,300 RNA samples by experts.
  • the sample size is small, and the manual scoring itself has inaccuracies.
  • the cost of its related instruments and supporting reagents is high, the shelf life of the reagents is short, and the instruments can only be used for fragment information analysis. It cannot obtain more sample information from a single detection process, such as whether the nucleic acid sample to be tested contains the target to be tested, the specific content of the target, the fragmentation symptoms of the target, etc., and the cost-effectiveness is relatively low.
  • the electrophoresis step and the collection of DNA after electrophoresis have the potential for contamination.
  • the electrophoresis method lacks specificity and cannot evaluate the degree of fragmentation of specific target nucleic acids.
  • HPV ctDNA may serve as a residual tumor marker at the end of chemoradiotherapy or predict recurrence during follow-up (Jeannot E, Latouche A, Bonneau C, et al. Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer. Clin Cancer Res. 2021; 27(21): 5869-5877. doi: 10.1158/1078-0432. CCR-21-0625).
  • Mass spectrometry has also been used to analyze nucleic acid size because nucleic acid fragments of different sizes, such as those prepared by primer extension reactions, have different molecular weights (Ding and Cantor, 2003, Proc Natl Acad Sci USA, 100, 7449-7453).
  • this method also requires additional equipment and supporting reagents, making the detection costly.
  • Methods for detecting the degree of nucleic acid fragmentation also include Ellinger, J., et al. (Cell-Free Circulating DNA: Diagnostic Value in Patients With Testicular Germ Cell Cancer, Journal of Urology, Volume 181, Issue 1, January 2009, Pages 363-371), which disclosed quantification of 106 bp, 193 bp, and 384 bp actin- ⁇ DNA fragments by real-time quantitative PCR.
  • DNA integrity is expressed as the ratio of large fragments (193 or 384 bp) to short fragments (106 bp). Compared with healthy subjects, the level of free DNA fragments was found to be significantly increased in testicular cancer patients.
  • the qPCR method is a relative absolute quantitative method.
  • U.S. Patent Publication No. US20180105864A1 discloses a method for determining the integrity and/or quantity of cell-free DNA (cfDNA) using digital PCR (dPCR). This method uses primers designed for two target sequences: one smaller than 300 bp and the other larger than 300 bp. DNA integrity is determined based on the test results.
  • one of the purposes of this application is to provide a method for specifically evaluating the degree of fragmentation of a target nucleic acid, the method comprising:
  • the mixture After mixing the biological sample with the at least two pairs of primer sets, the detection marker, and the nucleic acid amplification reaction solution, the mixture is randomly distributed into a plurality of reaction units to perform a nucleic acid amplification reaction to obtain detection copy numbers of different target sequence lengths;
  • a calculation formula is fitted based on the length of the target sequence and the detected copy number of the corresponding length, in which the variable Y is the detected copy number of the target sequence and the variable X is the length of the target sequence;
  • the degree of fragmentation of the target nucleic acid is evaluated based on the intersection of the fitting curve of the calculation formula and the X-axis.
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 540 bp, preferably from 30 to 260 bp.
  • the at least two pairs of primer sets share one forward primer or one reverse primer.
  • the at least two primer pairs are 3 to 7 primer pairs, preferably 4 to 7 primer pairs.
  • the at least two pairs of primer sets are four pairs of primer sets.
  • the lengths of the target sequences of the four pairs of primer sets are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • the method further comprises the step of isolating nucleic acids in the biological sample before mixing the biological sample with the at least two pairs of primer sets.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted according to the length of the target sequence and the number of detected copies of the corresponding length.
  • the degree of fragmentation of the target nucleic acid is assessed based on the
  • Another object of the present application is to provide a reagent for detecting a target nucleic acid, comprising:
  • At least two pairs of primers are used to amplify the same target nucleic acid, wherein the target sequence lengths of the at least two pairs of primers are different from each other.
  • the coefficient of variation of the quantitative values between the at least two pairs of primer sets is ⁇ 10%.
  • the at least two pairs of primer sets share a forward primer or a reverse primer.
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 540 bp, preferably from 30 to 260 bp.
  • the primer set is preferably a set of 3 to 7 primer pairs, or a set of 4 to 7 primer pairs, and more preferably a set of 4 primer pairs.
  • the target sequence lengths of the four pairs of primer sets are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • kits for detecting a target nucleic acid comprising the reagents of the present application; preferably, the kit further comprises a detection marker and a nucleic acid amplification reaction solution;
  • the nucleic acid amplification reaction solution includes dNTPs, a buffer, salt ions, and a polymerase.
  • Another object of the present application is to provide a method for detecting a target nucleic acid, the method comprising the following steps:
  • the fragmentation degree of the target nucleic acid is estimated based on the intersection of the fitting curve of the first calculation formula and the X-axis and/or the copy number of the target nucleic acid is estimated based on the coefficient of the first calculation formula.
  • the method further comprises the step of isolating nucleic acids in the biological sample before mixing the biological sample with the at least two pairs of primer sets.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted according to the length of the target sequence and the detected copy number of the corresponding length.
  • the fragmentation degree of the target nucleic acid is assessed based on the
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 540 bp, preferably from 30 to 260 bp.
  • the at least two pairs of primer sets share one forward primer or one reverse primer.
  • the primer set is a set of 3 to 7 primer pairs, or a set of 4 to 7 primer pairs, preferably a set of 4 primer pairs.
  • the target sequence lengths of the four pairs of primer sets are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • the method further comprises:
  • control biological sample with the at least two pairs of primer sets, the detection marker, and the nucleic acid amplification reaction solution to obtain a second mixed solution;
  • intersection of the fitting curves of the first calculation formula and the second calculation formula with the X-axis is compared to evaluate the fragmentation degree of the target nucleic acid, and/or the coefficients of the first calculation formula and the second calculation formula are compared to evaluate the copy number of the target nucleic acid.
  • control biological sample comprises a biological sample from a normal subject or a biological sample from a subject before administration of a drug.
  • the method further comprises a step of isolating nucleic acids in the control biological sample before mixing the control biological sample with the at least two pairs of primer sets.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted according to the length of the target sequence and the detected copy number of the corresponding length.
  • value is compared to the
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 540 bp, preferably from 30 to 260 bp.
  • the at least two pairs of primer sets share one forward primer or one reverse primer.
  • the primer set is a set of 3 to 7 pairs or 4 to 7 pairs of primers.
  • the primer set is a set of 4 primer pairs.
  • the target sequence lengths of the four pairs of primers are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • Another object of the present application is to provide a method for accurately detecting cfDNA, the method comprising:
  • cfDNA Mixing the cfDNA with at least two pairs of target nucleic acid primers, a pair of gDNA primers, a detection marker, and a nucleic acid amplification reaction solution; wherein the at least two pairs of primers have target sequences of different lengths and amplify the same target nucleic acid;
  • the mixed solution is randomly distributed into multiple reaction units to perform nucleic acid amplification reaction;
  • the degree of cfDNA fragmentation is evaluated based on the intersection of the fitting curve of the calculation formula and the X-axis, and/or the copy number of cfDNA is evaluated based on the coefficient of the calculation formula.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted based on the copy number obtained after deducting the copy number of a pair of gDNA primer sets from the copy number of each corresponding target sequence and the target sequence length of the corresponding target nucleic acid primer set.
  • the degree of cfDNA fragmentation is assessed based on the
  • the target sequence length of the at least two primer pairs ranges from 30 to 420 bp, preferably from 30 to 260 bp.
  • the target sequence length of the pair of gDNA primer sets is ⁇ 466 bp.
  • the at least two pairs of primer sets share one forward primer or one reverse primer.
  • the primer set is a set of 3 to 7 pairs or 4 to 7 pairs of primers.
  • the primer set is a set of 4 primer pairs.
  • the target sequence lengths of the four primer pairs are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • the target nucleic acid fragmentation degree assessment method of the present application is not limited by the detection reagent to the detection fragment range, and can perform an overall assessment of the fragmentation degree of the nucleic acid sample.
  • the present application can simultaneously achieve the assessment of the fragmentation degree of the target nucleic acid and the detection of the concentration; compared with the existing electrophoresis analysis method, it has higher sensitivity, lower cost, and does not require additional equipment and instruments, and is simple to operate, saves time, and improves efficiency; in addition, the accuracy of the fragmentation assessment using the method of the present application is higher, and the calculated concentration is closer to the true value.
  • the contamination of gDNA is further avoided in the cfDNA fragmentation assessment process, and a more accurate cfDNA fragmentation degree and content are obtained.
  • FIG1 is a graph showing the fitting results of a linear model, a quadratic polynomial model, and a cubic polynomial model.
  • target sequences of different lengths can be obtained.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted to the test results of each sample.
  • the X-axis and Y-axis are interchangeable. In this case, the degree of fragmentation of the target nucleic acid needs to be evaluated based on the intersection of the target fitting curve and the Y-axis.
  • This step satisfies: the coefficient of variation (CV value) of the quantitative value obtained by repeating the test 10 times for each sample using each primer set is ⁇ 15%, and the R 2 of the linear equation is >0.90.
  • value is indeed close to the main peak of the ultrasonically purified simulated sample, indicating that this method can, to a certain extent, reflect the main fragment length of the fragmented sample, that is, indirectly reflect the degree of fragmentation of the sample.
  • Quantitative values include ct values or copy numbers.
  • qPCR is a relative absolute quantitative method that requires the use of standard products to prepare a standard curve, and then the mass of the sample to be tested is calculated based on the conversion relationship between the ct value obtained from the standard curve and the mass of the standard product.
  • the operation is relatively complicated and the reagent cost is high.
  • a large change in the fluorescence signal value is required to bring about a slight change in the ct value, so the use of dPCR for quantitative detection has better sensitivity.
  • the method of the present application uses dPCR for quantitative detection.
  • the present application provides a method for specifically assessing the degree of nucleic acid fragmentation of a target nucleic acid.
  • the proposed technical solution overcomes the defects of limited detection range of sample fragments, poor fragment analysis capability for low-concentration samples, low quantitative accuracy and precision, and single instrument and reagent functions; compared with existing PCR evaluation methods, it overcomes the problem of inaccuracy caused by a small number of detection targets and errors in determination when the detection target is a sequence such as a virus that is easily mutated.
  • a shorter target sequence is used to analyze the presence of very small fragments in the sample, which can more comprehensively and accurately reflect the degree of fragmentation of the entire sample and the true value of the concentration of the target nucleic acid.
  • This application discloses a method for quantifying the same sample using multiple target sequence primers of different lengths based on a nucleic acid amplification platform.
  • the method establishes a relationship between the sample quantification values obtained for different target sequences and the target sequence lengths, thereby assessing sample quality and the degree of nucleic acid fragmentation.
  • Nucleic acid amplification for example, is PCR, such as qPCR or dPCR.
  • gene refers to a DNA segment involved in producing a polypeptide chain or transcribed RNA product. It may include regions before and after the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • target nucleic acid refers to a nucleic acid that is the target of amplification, detection, or both.
  • target sequence refers to a segment of a target nucleic acid to be amplified by an agent that can anneal to a primer under annealing or amplification conditions.
  • unfragmented target nucleic acid refers to the full-length gene that has not been interrupted.
  • the unfragmented GAPDH gene refers to the full-length GAPDH gene consisting of 12 introns and 13 exons, with a gene length of approximately 38 kb.
  • At least 2 pairs of primer sets can be 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs, 10 pairs, 11 pairs, 12 pairs, 13 pairs, 14 pairs of primer sets, etc.
  • the detection label can be a probe that is complementary to the target nucleic acid; or it can be a probe that is not complementary to or identical to the target nucleic acid. In a preferred embodiment, the detection label is a probe that is not complementary to or identical to the target nucleic acid.
  • probe refers to a labeled oligonucleotide used to detect the presence of a target.
  • biological sample refers to a composition in which one or more target nucleic acids may be present, including patient samples, plant or animal materials, waste materials, materials used for forensic analysis, environmental samples, circulating tumor cell (CTC) free DNA (cfDNA), liquid biopsy samples, biological products, etc.
  • Samples can be blood samples, tissue samples, urine samples, etc. Samples include any tissue, cell, or extract derived from a living or dead organism that may contain target nucleic acids, such as peripheral blood, bone marrow, plasma, serum, biopsy tissue samples including lymph nodes, respiratory tissue or exudates, gastrointestinal tissue, urine, feces, sperm, or other body fluids.
  • Specific target samples are tissue samples (including body fluids) from humans or animals with or suspected of having a disease or condition (particularly viral infection).
  • Other target samples include biological products, such as samples of vaccines and pharmaceuticals.
  • Sample components may include target and non-target nucleic acids, as well as other materials (e.g., salts, acids, bases, detergents, proteins, carbohydrates, lipids, and other organic or inorganic materials).
  • Samples may or may not be treated to purify target nucleic acids prior to amplification. Further processing may be treatment with detergents or denaturants to release nucleic acids from cells or viruses, remove or inert non-nucleic acid components, and concentrate nucleic acids.
  • the method of the present application does not include the step of obtaining a biological sample from a subject, i.e., the biological sample is an isolated sample.
  • the biological sample is plasma from a pregnant woman, in which case, for example, the plasma can be analyzed for the presence of fetal circulating DNA.
  • the biological sample is a biological product produced by a host.
  • the method of the present application can assess the toxicity of the biological product by detecting the residual amount of host nucleic acid in the biological product.
  • nucleic acid refers to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and include naturally occurring (e.g., adenosine, guanosine (guanidine), cytosine, uracil, and thymidine) and non-naturally occurring (artificially modified) nucleic acids.
  • naturally occurring e.g., adenosine, guanosine (guanidine), cytosine, uracil, and thymidine
  • non-naturally occurring nucleic acids e.g., the length of the polymer (e.g., the number of monomers).
  • Nucleic acids can be single-stranded or double-stranded and typically contain a 5'-3' phosphodiester bond, although in some cases, nucleotide analogs may have other bonds.
  • the monomers are typically referred to as nucleotides.
  • the four traditional nucleotide bases are A, T/U, C, and G, of which T is found in DNA and U is found in RNA.
  • the nucleotides found in the target are usually natural nucleotides (deoxyribonucleotides or ribonucleotides), which is also the case for the nucleotides that form the primer.
  • amplicon refers to the product of an amplification reaction, specifically the amplification product obtained by performing a nucleic acid amplification reaction using primers in the presence of a target nucleic acid.
  • Amplicon refers to the amplification product. The 5' and 3' boundaries of the amplicon are defined by the forward and reverse primers.
  • primer set refers to a combination of a forward primer and a reverse primer that can amplify a target nucleic acid to produce an amplicon.
  • the term "forward primer,” also known as the upstream primer, is an oligonucleotide that extends uninterruptedly along the negative strand
  • the term “reverse primer,” also known as the downstream primer is an oligonucleotide that extends uninterruptedly along the positive strand.
  • the positive strand also known as the sense strand or the positive strand, is generally located at the top of the double-stranded DNA, oriented 5'-3' from left to right, and has a base sequence that is essentially the same as the mRNA of the gene in which the oligonucleotide is located.
  • the primer that binds to this strand is the reverse primer; the negative strand, also known as the nonsense strand or the non-coding strand, is complementary to the positive strand, and the primer that binds to this strand is the forward primer. It should be understood that when the designations of the sense strand and the antisense strand are interchanged, the corresponding forward and reverse primer nomenclature can also be interchanged.
  • fragmentation degree refers to the fragmentation pattern exhibited by nucleic acid molecules, including the locations where breaks and fragmentation occur or the distribution of fragments.
  • nucleases in cells can break DNA into fragments of varying lengths, resulting in different fragmentation patterns.
  • genomic regions with open chromatin arrangement are more susceptible to binding nucleases and being broken than tight regions.
  • naked DNA that is not bound to any protein is more easily broken by nucleases, while regions protected by nucleosomes, transcription factors, etc. are less likely to be broken. By analyzing this fragmentation pattern information, it can be used to indicate different nucleic acid molecules.
  • calculation formula includes formulas obtained by fitting different fitting models.
  • the fitting models include a linear model, a quadratic polynomial model, and a cubic polynomial model.
  • the fitted model comprises a linear model.
  • coefficients of a calculation formula includes parameters used to describe the relationship between different variables. In some cases, the “coefficients of a calculation formula” further include parameters obtained after operation.
  • the target sequences of the at least two pairs of primer sets range in length from 30 to 550 bp.
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 260 bp, such as 30 to 220 bp, and further such as 30 to 200 bp.
  • the at least two pairs of primer sets share a forward primer or a downstream primer.
  • Using a shared primer approach can reduce development costs.
  • the primer set is a set of 3 to 7 pairs or 4 to 7 pairs of primers
  • the primer set is a set of 4 primer pairs.
  • the target sequence length of the primer set is selected from 30-50bp, 50-60bp, 60-70bp, 70-80bp, 80-90bp, 90-100bp, 100-110bp, 110-120bp, 120-130bp, 130-140bp, 140-160bp, 160bp-180bp, 180-200bp, 200-220bp, 220-240bp any two or more of 1-80 bp, 240-260 bp, 260-280 bp, 280-300 bp, 300-320 bp, 320-340 bp, 340-360 bp, 360-380 bp, 380-400 bp, 400-420 bp, 420-440 bp, 440-460 bp, 460-480 bp, 480-500 bp, 500-520 bp, and 520-540 bp.
  • the target sequence lengths of the four pairs of primer sets are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • the method of the present application further comprises a step of isolating the nucleic acid in the biological sample before mixing the biological sample with the at least two pairs of primer sets.
  • a reagent for detecting a target nucleic acid comprising:
  • At least two pairs of primers are used to amplify the same target nucleic acid, and the target sequence lengths of the at least two pairs of primers are different from each other.
  • the coefficient of variation (CV value) of the quantitative values between primer sets of the present application is ⁇ 10%.
  • a coefficient of variation of quantitative values between groups of primers of ⁇ 10% means that, using an unfragmented target nucleic acid as a template, the amplification efficiencies of the primers used in each primer set are substantially consistent, i.e., the amplification efficiencies of each primer set are between 90% and 110%.
  • the coefficient of variation of quantitative values between the three primer sets is ⁇ 10%.
  • reaction unit refers to a basic unit that can react independently in a chemical reaction or a biological reaction. It can be a container, a tiny space, or a system that can accommodate reactants and react.
  • the reaction unit can be a droplet, a reaction chamber of a microfluidic chip, etc.
  • the mixed solution can be distributed to 5,000 to 100,000 reaction units, such as 10,000, 20,000, 50,000, or 80,000.
  • the at least two pairs of primer sets share a forward primer or a reverse primer.
  • the primer set is preferably a set of 3 to 7 primer pairs, or a set of 4 to 7 primer pairs, and more preferably a set of 4 primer pairs.
  • the target sequence lengths of the four pairs of primer sets are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • the reagent further comprises a detection label.
  • the detection marker is labeled with a detection group.
  • kits for detecting a target nucleic acid comprising the reagent of the present application.
  • the kit further comprises a detection marker and a nucleic acid amplification reaction solution.
  • the nucleic acid amplification reaction solution includes dNTPs, a buffer, salt ions, and a polymerase.
  • kit refers to any article of manufacture (eg, a package or container) that includes at least one reagent (such as a primer pair), eg, a reagent described herein for specifically amplifying a target nucleic acid.
  • reagent such as a primer pair
  • Deoxynucleoside triphosphates i.e., dNTPs
  • dNTPs can be used, for example, dATP, dCTP, dGTP, dTTP, dITP, dUTP, ⁇ -thio-dNTP, biotin-dUTP, fluorescein-dUTP, digoxigenin-dUTP, 7-deaza-dGTP.
  • dNTPs are well known in the art and are commercially available.
  • the buffers and salts used in the present application provide suitable stable pH and ionic conditions for nucleic acid synthesis, such as reverse transcriptase and DNA polymerase activity.
  • a variety of buffers and salt solutions and modified buffers that can be used in the present application are known in the art, including reagents not specifically disclosed herein.
  • Preferred buffers include, but are not limited to, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, and CAPS.
  • the kits provided include TRIS.
  • the buffer used in the methods of the present application comprises TRIS at a pH of about 8 to about 9.
  • TRIS is TRIS-HCl (pH 8.0-9.0).
  • Salt solutions corresponding to salt ions include, but are not limited to, ammonium sulfate, magnesium chloride, potassium acetate, potassium sulfate, potassium chloride, ammonium chloride, ammonium acetate, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium solution, chloride, sodium acetate, lithium chloride, and lithium acetate.
  • the compositions provided include ammonium sulfate and magnesium chloride.
  • the compositions provided include TRIS-HCl, ammonium sulfate, and magnesium chloride.
  • polymerase there is no particular limitation on the polymerase in this application.
  • Common polymerases in the field of diagnostic reagents can be used, and exemplary polymerases can be polymerases derived from thermostable bacteria that are known in the art. Specific examples include DNA polymerases derived from Thermus aquaticus (U.S.
  • Patents 4,889,818 and 5,079,352) (trade name Taq polymerase), DNA polymerases derived from Thermus thermophilus (WO 91/09950) (rTth DNA polymerase), DNA polymerases derived from Pyrococcus furiosus (WO 92/9689) (Pfu DNA polymerase, manufactured by Stratagenes), and DNA polymerases derived from Thermococcus litoralis (EP-A455430 (trademark Vent): manufactured by New England Biolabs), which are commercially available. Among them, heat-resistant polymerases derived from Thermus aquaticus are preferred.
  • Exemplary fluorescent groups can be selected from various fluorescent labels, such as one or more of ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, and Quasar 705; exemplary quenching groups can be selected from various quenchers, such as one or more of DABCYL, BHQs (such as BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA.
  • An exemplary modifying group is, for example, 3'MGB.
  • the evaluation of the degree of fragmentation of the target nucleic acid can be used to evaluate the quality of biological samples. For example, when testing FFPE samples, it is often necessary to perform second-generation sequencing on the samples, but second-generation sequencing has extremely high requirements for the degree of fragmentation of the samples.
  • the degree of fragmentation of biological samples can be effectively, accurately and sensitively evaluated, which is conducive to guiding subsequent experiments.
  • the degree of fragmentation can be evaluated by the
  • the degree of fragmentation size of the host cells when conducting a safety assessment of biological products, such as when preparing a vaccine, it is often necessary to evaluate the host cell residues in the vaccine, and there are strict requirements on the DNA fragmentation size of the host cells.
  • the size of the DNA fragments of the host cells in the vaccine can be evaluated with high sensitivity by the
  • value means that the
  • some samples not only need to understand the degree of nucleic acid fragmentation, but also need to consider relevant information such as the quality and concentration of the nucleic acid from multiple dimensions.
  • relevant information such as the quality and concentration of the nucleic acid from multiple dimensions.
  • characterizing the degree of fragmentation and content of tumor genes can often guide disease diagnosis.
  • drug efficacy it may be necessary to comprehensively consider the degree of viral nucleic acid fragmentation and content before and after medication. Based on this, the inventors found that after using primers that amplify target sequences of different lengths to detect specific target nucleic acids, the formula obtained by fitting the target sequence length and the detection copy number of the corresponding length can effectively solve the difficulties of the prior art.
  • value can evaluate the degree of fragmentation of the target nucleic acid, and the b value can obtain data that is extremely close to the true content value, and this method can obtain two data at the same time, which can effectively ensure the correlation between the data.
  • a method for detecting a target nucleic acid comprising:
  • the fragmentation degree of the target nucleic acid is estimated based on the intersection of the fitting curve of the first calculation formula and the X-axis, and/or the copy number of the target nucleic acid is estimated based on the coefficient of the first calculation formula.
  • the method further comprises a step of isolating nucleic acids in the biological sample before mixing the biological sample with the at least two pairs of primer sets.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted according to the length of the target sequence and the detected copy number of the corresponding length.
  • the fragmentation degree of the target nucleic acid is assessed based on the
  • the coefficient of variation of the quantitative values between the at least two pairs of primer sets is ⁇ 10%.
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 540 bp, preferably from 30 to 260 bp.
  • the at least two pairs of primer sets share a forward primer or a downstream primer.
  • the primer set is 3 to 7 pairs, 4 to 7 pairs of primers, preferably 4 pairs of primers; more preferably, the target sequence length of the 4 pairs of primers is 30 to 50 bp, 80 to 100 bp, 130 to 150 bp, or 160 to 180 bp.
  • the method further comprises:
  • intersection of the fitting curves of the first calculation formula and the second calculation formula with the X-axis is compared to evaluate the fragmentation degree of the target nucleic acid, and/or the coefficients of the first calculation formula and the second calculation formula are compared to evaluate the copy number of the target nucleic acid.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted according to the length of the target sequence and the detected copy number of the corresponding length.
  • value is compared to the
  • value reflects the length of the main peak of the major fragment in the biological sample
  • value reflects the length of the main peak of the major fragment in the control biological sample.
  • value indicates that the main peak of the main fragment of the biological sample is smaller relative to the control biological sample; a
  • value indicates that the main peak of the main fragment of the biological sample is larger relative to the control biological sample; or a
  • value indicates that the main peak of the main fragment of the biological sample has not changed relative to the control biological sample.
  • a b value less than a b1 value indicates that the number of copies of the target nucleic acid in the biological sample is less than that in the control biological sample; a b value greater than a b1 value indicates that the number of copies of the target nucleic acid in the biological sample is greater than that in the control biological sample; or a b value equal to a b1 value indicates that the number of copies of the target nucleic acid in the biological sample is equal to that in the control biological sample.
  • the control biological sample includes a biological sample from a normal subject or a biological sample of a subject before administration of a drug.
  • the method further comprises the step of isolating nucleic acid in the control biological sample before mixing the control biological sample with the at least two pairs of primer sets.
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 540 bp, preferably from 30 to 260 bp.
  • the at least two pairs of primer sets share a forward primer or a downstream primer.
  • the primer set is a set of 3 to 7 pairs or 4 to 7 pairs of primers.
  • the primer set is a set of 4 primer pairs.
  • the target sequence lengths of the four pairs of primers are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • Another object of the present application is to provide a method for accurately detecting cfDNA, the method comprising the following steps:
  • cfDNA Mixing the cfDNA with at least two pairs of target nucleic acid primers, a pair of gDNA primers, a detection marker, and a nucleic acid amplification reaction solution; wherein the at least two pairs of primers have target sequences of different lengths and amplify the same target nucleic acid;
  • the mixed solution is randomly distributed into a plurality of reaction units to perform a nucleic acid amplification reaction; the detection copy number of the target sequence corresponding to at least two pairs of target nucleic acid primer sets and the detection copy number of a pair of gDNA primer sets are obtained;
  • a calculation formula is provided for fitting the copy number obtained by deducting the copy number detected by a pair of gDNA primer sets from the detected copy number of the target sequence of each pair of at least two pairs of target nucleic acid primer sets and the target sequence length of the corresponding target nucleic acid primer set, wherein the variable Y is the copy number of the target sequence after deduction, and the variable X is the length of the target sequence;
  • the degree of cfDNA fragmentation is evaluated based on the intersection of the fitting curve of the calculation formula and the X-axis, and/or the copy number of cfDNA is evaluated based on the coefficient of the calculation formula.
  • a linear model, a quadratic polynomial model, or a cubic polynomial model is fitted based on the copy number obtained after deducting the copy number of a pair of gDNA primer sets from the copy number of each corresponding target sequence and the target sequence length of the corresponding target nucleic acid primer set.
  • the degree of fragmentation of cfDNA is assessed based on the
  • the coefficient of variation of the quantitative values between the at least two pairs of primer sets is ⁇ 10%.
  • the target sequence lengths of the at least two pairs of primer sets range from 30 to 420 bp, preferably from 30 to 260 bp.
  • the target sequence length of the pair of gDNA primers is ⁇ 466 bp; preferably, the target sequence length of the pair of gDNA primers is ⁇ 498 bp.
  • the at least two pairs of primer sets share a forward primer or a downstream primer.
  • the primer set is a set of 3 to 7 pairs or 4 to 7 pairs of primers.
  • the primer set is a set of 4 primer pairs.
  • the target sequence lengths of the four pairs of primer sets are 30-50 bp, 80-100 bp, 130-150 bp, and 160-180 bp.
  • the target sequences of at least two primer pairs can be named, in order of length from short to long, as a first target sequence, a second target sequence, ..., an Nth target sequence.
  • the first target sequence is the shortest
  • the Nth target sequence is the longest
  • the intermediate target sequences are arranged in order of length.
  • the length difference between adjacent target sequences is 8 to 90 bp, such as 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, or 90 bp.
  • the second target sequence and the first target sequence are adjacent target sequences
  • the second target sequence and the third target sequence are adjacent target sequences
  • the third target sequence and the fourth target sequence are adjacent target sequences.
  • two or more of the at least two pairs of primer sets share a forward primer or a downstream primer.
  • the method is ex vivo or in vitro. In some embodiments of the various methods of the present application, the method is for non-diagnostic purposes.
  • an electronic device comprising:
  • a memory for storing executable instructions
  • the processor is used to execute the executable instructions stored in the memory to implement the method of the present application.
  • a computer-readable storage medium storing executable instructions for causing a processor to execute the method of the present application.
  • a computer program product comprising a computer program or computer executable instructions, which implement the method of the present application when the computer program or computer instructions are executed by a processor.
  • the plasmids, primers and probe sets used were synthesized and provided by General Biotech (Anhui) Co., Ltd. Digital PCR detection was performed using a MacBio D600 digital PCR instrument.
  • the human GAPDH gene was selected as the detection target, and four pairs of specific primers were designed.
  • the forward primer was fixed and the reverse primer was mobile, thereby obtaining target sequences of different lengths (39, 86, 137, and 166 bp).
  • the 5' end of GAPDH-P1 is modified with FAM and the 3' end is modified with BHQ1;
  • the 5' end of GAPDH-P2 is modified with CY5 and the 3' end is modified with BHQ2;
  • the 5' end of P is modified with ROX and the 3' end is modified with BHQ2.
  • Genomic DNA from 293T cells was sheared for 200 seconds using a Covaris M220 ultrasonic disruptor, following the sonication protocol in Table 2.
  • the sheared samples were purified using Beckman Coulter magnetic beads (Agencourt AMPure XP Nucleic Acid Purification Kit, 60 mL Kit) . Four fragments of varying sizes were obtained, each exhibiting a normal distribution, with major peaks of 171, 274, 364, and 466 bp, respectively.
  • the purified nucleic acid samples were analyzed using an Agilent 4150 Fragment Analyzer.
  • Example 2 Evaluating the effect of the number of target sequences on the
  • the GAPDH gene was selected as the detection target, and seven specific primer sets were designed.
  • the forward primer was fixed, and the reverse primer was mobile, resulting in target sequences of 39, 50, 67, 86, 104, 137, and 166 bp in length.
  • These seven specific primer pairs were used for digital PCR detection of genomic DNA from 293T cell lines. Each primer pair was tested 10 times.
  • the repeatability CV values for each primer pair were 10.62%, 2.70%, 3.18%, 7.36%, 6.23%, 12.33%, and 1.58%, respectively.
  • the inter-group CV for the quantitative values of the seven primer pairs was 6.58%, indicating good consistency in the quantitative values of the seven primer pairs.
  • the sequences of the seven primer pairs are shown in Table 7.
  • the 5' end of GAPDH-P1 is modified with FAM and the 3' end is modified with BHQ1;
  • the 5' end of GAPDH-P2 is modified with CY5 and the 3' end is modified with BHQ2;
  • the 5' end of P is modified with ROX and the 3' end is modified with BHQ2;
  • the 5' end of GAPDH-P4 is modified with VIC and the 3' end is modified with BHQ1.
  • Target sequence length Relationships between target sequence length and the corresponding mean quantitative value were established for four primer pairs (target sequence lengths of 39, 86, 137, and 166 bp) and seven primer pairs (target sequence lengths of 39, 50, 67, 86, 104, 137, and 166 bp).
  • the GAPDH gene was selected as the detection target, and four pairs of specific primers were used to amplify target sequences of 39, 86, 137, and 166 bp, respectively.
  • the primer sets and detection probes were the same as those in Example 1.
  • values for the four sample concentrations were 288, 299, 289, and 288, respectively, with a calculated CV of 2.07%.
  • values for the four sample concentrations were 222, 221, 217, and 222, respectively, with a calculated CV of 1.11%.
  • the GAPDH gene was selected as the detection target.
  • Primer design scheme 1 the primer in one direction was fixed and the primer in the other direction was moved to obtain target sequences of different lengths (39, 86, 137, and 166 bp). The specific experimental process and results were the same as those in Example 1.
  • Primer Design Option 2 No fixed primers were used. Target sequences of varying lengths (50, 88, 145, and 167 bp) were obtained through primer design. Primer sequences and probes are shown in Table 11. Digital PCR was performed on genomic DNA from 293T cells using the four specific primer pairs from Primer Design Option 2 for the GAPDH gene. The repeatability coefficients (CVs) for each primer pair, after 10 replicates, were 3.56%, 3.51%, 1.10%, and 2.65%, respectively.
  • CVs repeatability coefficients
  • the 5' end of P was modified with ROX, and the 3' end was modified with BHQ2.
  • digital PCR detection and formula fitting were performed on four ultrasonically purified fragmented samples (prepared in Example 1, with main peaks of 171, 274, 364, and 466 bp, respectively). The detection results are shown in Table 12.
  • values obtained after testing and formula fitting of ultrasonically purified fragment samples with main peaks of 171, 274, 364, and 466 bp were 174, 301, 377, and 414, respectively.
  • the CV values for each main peak were 1.74%, 2.75%, 0.72%, and 2.39%, respectively.
  • values obtained after testing and formula fitting of ultrasonically purified fragment samples with main peaks of 171, 274, 364, and 466 bp were 178, 290, 381, and 400, respectively.
  • a Tiangen extraction kit (magnetic bead-based large-volume free nucleic acid extraction kit, catalog number: DP710-02) was selected. Following the instructions, a human plasma sample was divided into five equal portions, and free DNA was extracted in parallel. Sample concentration was determined using Qubit 3.0. The digital PCR detection system described in Example 1 was used to detect free DNA. Each sample was tested 10 times using four primer pairs. A linear fit was established using the target sequence lengths of the four primer pairs and the corresponding quantitative values.
  • the HBV-S region was selected as the detection target, and four pairs of specific primers were designed from conserved regions to amplify target sequences of 48, 87, 131, and 175 bp, respectively.
  • the primer sequences and detection probes are shown in Table 14. These four pairs of specific primers were used for digital PCR detection of HBV-B plasmids. Each primer pair was tested 10 times, and the repeatability CVs of the quantitative values for each primer pair were calculated to be 4.1%, 4.3%, 3.5%, and 0.2%, respectively. The total CV of all quantitative values for the four primer pairs was 5.32%. This result demonstrates good consistency in the quantitative values of the four primer pairs.
  • the 5' end of P was modified with ROX, and the 3' end was modified with BHQ2.
  • the HBV-B plasmid was sonicated for 420 s using a Covaris M220 ultrasonic disruptor according to the sonication program in Table 2.
  • Ultrasonic fragmentation samples were purified using Beckman Coulter magnetic beads (Agencourt AMPure XP Nucleic Acid Purification Kit, 60 mL Kit). The purified nucleic acid samples were analyzed using an Agilent 4150 Fragment Analyzer, yielding fragments with main peaks of 238, 320, 460, and 718 bp, respectively. All fragments showed a normal distribution. Digital PCR was performed on these four fragmented samples using four pairs of specific primers. Each primer pair was tested 10 times, and the repeatability (CV) of the 10 quantitative values for each primer pair was calculated. The specific test results are shown in Table 15.
  • a viral extraction kit Magnetic Bead-Based Large-Volume Free Nucleic Acid Extraction Kit, MacBio, Catalog No. DP710-02
  • Sample concentrations were determined using Qubit 3.0, and all concentrations were 0.
  • An Agilent 4150 Fragment Analyzer was used to analyze these samples; no fragment information was obtained for any of them.
  • Digital PCR was performed on these samples, with each primer pair (the same primer-probe pair as in Example 6) being used without duplication. A relationship between the target sequence length and the corresponding quantitative value for each of the four primer pairs was established, and a linear fit was performed. The
  • the degree of fragmentation of Escherichia coli nucleic acid samples was assessed.
  • the E. coli ydiJ gene was selected as the detection target, and five pairs of specific primers were designed from conserved regions to amplify target sequences of 65, 74, 91, 100, and 109 bp, respectively .
  • the primer sequences and detection probes are shown in Table 17.
  • Digital PCR detection of E. coli nucleic acid was performed using these five pairs of specific primers.
  • the quantitative values (CVs) for each primer pair were ⁇ 15% after 10 replicates.
  • the intergroup CVs for the five primer pairs were 3.39%, indicating good consistency in the quantitative values.
  • EC-P’s 5’ end is modified with T6 and FAM, and its 3’ end is modified with 3’MGB.
  • the Escherichia coli nucleic acid sample was ultrasonically fragmented for 420 seconds according to the ultrasonic program in Table 2.
  • the ultrasonically fragmented sample was purified using Beckman Coulter magnetic beads (Agencourt AMPure XP Nucleic Acid Purification Kit 60mL Kit).
  • the purified nucleic acid sample was analyzed using an Agilent 4150 fragment analyzer to obtain a fragmented sample with a main peak of 96bp, and the fragments were normally distributed. Five pairs of specific primers were used to perform digital PCR detection on the digital PCR instruments of Qiagen, Bio-Rad, and Michael, respectively.
  • Literature indicates that 70% of cfDNA fragments are haploid, approximately 166 bp in size, with a small number of diploid and triploid fragments also present. Therefore, fragments > 498 bp can be considered gDNA contamination .
  • This application designed the following experimental protocol to detect and subtract gDNA contamination from cfDNA.
  • cfDNA was extracted from fresh plasma from 10 healthy individuals using the Tiangen Extraction Kit (Magnetic Bead-Based Large-Volume Free Nucleic Acid Extraction Kit, Cat. No. DP710-02) according to the manufacturer's instructions.
  • the cfDNA extracted from these 10 samples was then mixed. Different ratios of gDNA were added to these samples (simulating gDNA contamination with increasing gDNA content, with cfDNA:gDNA ratios of 1:0.1, 1:0.5, 1:1, 1:3, and 1:10, respectively) to simulate cfDNA samples with varying degrees of gDNA contamination.
  • a detection system of four pairs of short target sequence primers for GAPDH 39, 86, 137, and 166 bp was used, as in Example 1;
  • a gDNA primer pair detection system consisting of long target sequence primers selected from a GAPDH gene primer set with a target sequence length >498 bp.
  • the intergroup CV of quantitative values for this long target sequence primer and four pairs of short target sequence primers for the same sample was ⁇ 10%.
  • the reproducibility CVs for each primer pair were 8.7%, 3.8%, 8.0%, 3.3%, and 1.7%, respectively, for a total intergroup CV of 5.42%.
  • the gDNA primer probe sequences are shown in Table 19.
  • GAPDH-PC is modified with ROX at its 5' end and BHQ2 at its 3' end. Simulated contaminated cfDNA was mixed with four pairs of short target sequence primers and one gDNA primer combination detection system and then tested by digital PCR. The results are shown in Table 20.
  • the method described above in this embodiment can also be used to detect the degree of nucleic acid sample fragmentation before and after sample storage, thereby further evaluating the sample quality. Specifically:
  • the residual fragment size distribution of residual host nucleic acids in intermediates, semi-finished products, and finished products of biological products derived from the HEK293 host cell line was evaluated.
  • the risk gene E1A of the HEK293 host cell was selected as the detection target, and four pairs of specific primers were designed to simultaneously amplify target sequences of 83, 132, 217, and 571 bp in length in one tube.
  • the primer sequences and detection probes are shown in Table 24, and the corresponding target sequences are shown in Table 25.
  • digital PCR detection was performed on 0.1, 1, and 10 ng/ul of HEK293 host cell line genomic DNA.
  • the 5' end of E1A-P2 is modified with ROX and the 3' end is modified with BHQ2; the 5' end of E1A-P8-FAM is modified with 6-FAM and the 3' end is modified with BHQ1; the 5' end of E1A-P7-cy5 is modified with Cy5 and the 3' end is modified with BHQ2; the 5' end of E1A-P10-VIC-MGB is modified with VIC and the 3' end is modified with MGB.
  • HEK293 host cell line genomic DNA of the same concentration was sonicated for fragmentation periods of 25s, 50s, 100s, 200s, and 400s, following the sonication schedule in Table 2, to simulate residual host nucleic acids from biological products at varying degrees of digestion. Unsonicated HEK293 host cell line genomic DNA of the same concentration was used as a control. Digital PCR was performed on the sonicated fragmented samples using this one-tube, four-plex system. Each sample was run in triplicate, and the mean of the three quantitative values for each primer pair was calculated. Detailed results are shown in Table 27.
  • the human GAPDH gene was selected as the detection target, and 4 pairs of specific primers were designed to amplify target sequences of 50, 88, 111, and 145 bp, respectively.
  • the primer sequences and detection probes are shown in Table 28, and the corresponding target sequences are shown in Table 29.
  • the 5' end of P is modified with ROX, and the 3' end is modified with BHQ2.
  • Genomic DNA from 293T cells was diluted to 4E4 copies/ ⁇ l and sonicated using a Covaris M220 ultrasonic disruptor for 50, 100, and 200 s, according to the sonication schedule in Table 2, to simulate nucleic acid samples with varying degrees of fragmentation.
  • a non-sonicated sample of the same concentration served as a full-length control.
  • Digital PCR was performed on both full-length and fragmented samples using four pairs of specific primers. Each primer pair was tested in triplicate, and the mean of the three quantitative values for each primer pair was calculated. Detailed results are shown in Table 30.
  • value can be used to assess the degree of sample fragmentation and, based on the b value, to obtain a quantitative value that is as close to the true value as possible for the sample being tested. This method can be applied to trace the quantitative values of nucleic acid standards, quality control products, and other materials.
  • a model for assessing the degree of nucleic acid fragmentation was established using Escherichia coli nucleic acid samples.
  • the E. coli ydiJ gene was selected as the detection target, and 22 pairs of specific primers were designed from conserved regions. Target sequence lengths ranged from 56 to 176 bp. Primer sequences are shown in Table 32.
  • the detection probe is SEQ ID NO. 31.
  • Escherichia coli genomic DNA was ultrasonically fragmented for 420 s using a Covaris M220 ultrasonic disruptor according to the ultrasonication program in Table 2.
  • the ultrasonically fragmented samples were purified using Beckman Coulter magnetic beads (Agencourt AMPure XP Nucleic Acid Purification Kit 60 mL Kit).
  • the purified nucleic acid samples were analyzed using an Agilent 4150 Fragment Analyzer. This method yielded six fragmented samples with a normal distribution, with the main peaks at 96, 150, 288, 347, 414, and 538 bp, respectively.
  • Digital PCR was performed on these fragmented samples using 22 pairs of specific primers, with each sample tested once per primer pair. Specific test data are shown in Table 33.
  • the test data in Table 33 provide quantitative values corresponding to target sequence lengths (bp) for each target sequence length, which helps select the most appropriate model to describe the relationship between them.
  • the relationship between target sequence length (bp) and the quantitative values corresponding to the target sequence was fit using linear, quadratic, and cubic polynomial models.
  • the mean squared error (MSE) and coefficient of determination (R 2 ) for each model were calculated.
  • the different fitting models were evaluated based on their ability to explain the biological significance of the relationship. This analysis was performed for each sample, resulting in the data in Table 34 and Figure 1 below.
  • the cubic polynomial model provides the lowest MSE and highest R2 value when modeling the relationship between target sequence length and corresponding quantitative values, demonstrating the best fit. Compared to other models, it more closely follows the data point trend and should be the optimal model for describing the relationship between target sequence length and corresponding quantitative values.
  • the cubic polynomial has relatively many parameters and is prone to overfitting when data is limited. This means that the model fits the training data well but generalizes poorly to new data.
  • the mean square error (MSE) and coefficient of determination ( R2 ) of the quadratic polynomial are slightly lower than those of the cubic polynomial, and the model's ability to explain the biological significance of this relationship is average.
  • the linear model Compared to cubic and quadratic polynomials, the linear model has a slightly higher mean squared error (MSE) and a lower R2 , but its biological significance in explaining the relationship between target sequence length and corresponding quantitative values is clear.
  • MSE mean squared error
  • the function graph is a straight line, with k being the slope of the line, which determines its inclination, and b being the intercept of the line on the y-axis. The coordinates of the intersection of the line and the y-axis are (0, b).
  • Y 0,
  • value is indeed close to the main peak of the ultrasonically purified simulated sample.
  • the specific data are shown in Table 35. This shows that this method can reflect the main fragment length of the fragmented sample to a certain extent, that is, it indirectly reflects the degree of fragmentation of the sample.
  • the b value obtained by this method can reflect the true concentration (copy number) of the sample being tested to a certain extent, which has another practical significance.

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Abstract

L'invention concerne un procédé d'évaluation spécifique du degré de fragmentation d'un acide nucléique cible. Le procédé comporte : le mélange d'un échantillon biologique avec au moins deux paires d'amorces d'acide nucléique cible, un marqueur de détection et une solution de réaction d'amplification d'acide nucléique, et l'exécution d'une réaction d'amplification d'acide nucléique pour obtenir des nombres de copies de détection de différentes longueurs de séquence cible ; l'ajustement d'une formule de calcul basée sur les longueurs de séquence cible et les nombres de copies de détection de la longueur correspondante ; et l'évaluation du degré de fragmentation de l'acide nucléique cible en déterminant l'intersection de la courbe d'ajustement obtenue avec l'axe des X.
PCT/CN2025/075384 2024-02-01 2025-01-27 Procédé d'évaluation spécifique du degré de fragmentation d'acide nucléique cible Pending WO2025162424A1 (fr)

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