Internal reference product for quantifying human nucleic acid in metagenome sample, quantifying method and application

Abstract

The invention discloses an internal reference product for quantifying human nucleic acid in a metagenome sample, a quantifying method and application, and provides a method for screening the concentration of an internal reference sequence for quantitative detection of the human nucleic acid in the metagenome and an internal reference product for quantitative detection of the human nucleic acid in the metagenome. The invention provides a construction method of a quantitative detection model for the content of human nucleic acid in a metagenome and the quantitative detection model for the content of the human nucleic acid in the metagenome. The invention provides a method for quantitatively detecting human nucleic acid in metagenome based on internal reference, a related system, equipment and a computer readable storage medium.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an internal reference product, a quantitative method and an application for quantifying human nucleic acid in a metagenomic sample. Background Art

[0002] Metagenomic Next-Generation Sequencing (mNGS) is a technical means to detect all nucleic acid sequences in a sample through unbiased sequencing. Based on its broad-spectrum detection characteristics, mNGS is increasingly used in the detection of pathogenic microorganisms in clinical samples, and has provided great help for clinical diagnosis, especially the identification of uncultivable pathogenic microorganisms. The content of human nucleic acid in the metagenomic sample directly determines the sensitivity of the metagenomic test. Excessive human nucleic acid content will lead to false negatives in the test. By quantifying the human nucleic acid in the sample, it is possible to assess whether the metagenomic test will have the risk of missing the microorganisms in the sample, so that corresponding remedial measures can be taken to address the risk.

[0003] The existing method for quantifying human nucleic acids in metagenomic test samples is mainly to add a certain amount of internal reference sequences to the sample to be tested. After nucleic acid extraction, library construction and sequencing, the human nucleic acid content in the sample is calculated based on the number of internal reference detection sequences, the total number of sequencing sequences, the amount of internal reference added, the length of the internal reference sequence, the size of the human genome and the internal reference characteristic coefficient. The calculation method is complicated, and this method is not suitable for nucleic acid extraction processes with host removal steps, because the internal reference sequence will be digested by nucleases together with human nucleic acids during the host removal process. Host removal can improve the sensitivity of pathogen detection and can greatly reduce the amount of sequencing data and sequencing costs. Therefore, host removal is widely used in metagenomic detection processes, which also limits the application of existing human nucleic acid quantification methods. In addition, the existing methods have not verified their accuracy in quantifying human nucleic acids in sample types other than cerebrospinal fluid. Summary of the invention

[0004] In order to solve the problem in the prior art that the detection sensitivity is affected by the human nucleic acid content in the sample (the higher the human nucleic acid content, the lower the detection sensitivity), and the great differences in the content of microorganisms and human nucleic acids between different clinical samples, the clinical application of mNGS still faces many challenges, mainly manifested in the lack of a method for quickly and accurately quantifying the human nucleic acid content in the sample (especially the human nucleic acid content in the nucleic acid sample after extraction), so it is impossible to determine the detection performance of mNGS for a specific sample, that is, it is impossible to judge whether the test result has a false negative risk based on the human nucleic acid content. The present invention provides the following technical solution.

[0005] The first aspect of the present invention provides a method for screening the concentration of an internal reference sequence for quantitative detection of human nucleic acid in a metagenome, the method comprising: The prepared internal reference sequences were mixed with human genomic DNA at different concentrations at a mixing volume ratio of 1:8, and then nuclease-free water was added at a volume ratio of 1. The samples were library built and sequenced, and the number of internal reference-specific detection sequences in all sequenced samples was analyzed. The amount of internal reference sequence and human genome to be added was determined by judging whether the internal reference could be stably detected and would not occupy the data volume.

[0006] Furthermore, the internal reference sequence is selected from the Arabidopsis thaliana genome sequence, and there is no crossover sequence between the internal reference sequence and the pathogenic nucleic acid sequence and the human nucleic acid sequence.

[0007] Furthermore, the internal reference sequence is shown in SEQ ID NO:1.

[0008] Furthermore, the concentration of the internal reference sequence is 0.001-0.1 ng / μl.

[0009] In some embodiments, the concentration of the internal reference sequence is 0.001 ng / μl, 0.002 ng / μl, 0.003 ng / μl, 0.004 ng / μl, 0.005 ng / μl, 0.006 ng / μl, 0.007 ng / μl, 0.008 ng / μl, 0.009 ng / μl, 0.01 ng / μl, 0.015 ng / μl, 0.02 ng / μl, 0.025 ng / μl l, 0.03ng / μl, 0.035ng / μl, 0.04ng / μl, 0.045ng / μl, 0.05ng / μl, 0.055ng / μl, 0.06ng / μl, 0.06 5ng / μl, 0.07ng / μl, 0.075ng / μl, 0.08ng / μl, 0.085ng / μl, 0.09ng / μl, 0.095ng / μl, 0.1ng / μl.

[0010] Furthermore, the concentration of the internal reference sequence is 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl.

[0011] Furthermore, the concentration of the human genome is 0.001-100 ng / μl.

[0012] In some embodiments, the concentration of the human genome is 0.001 ng / μl, 0.002 ng / μl, 0.003 ng / μl, 0.004 ng / μl, 0.005 ng / μl, 0.006 ng / μl, 0.007 ng / μl, 0.008 ng / μl, 0.009 ng / μl, 0.01 ng / μl, 0.015 ng / μl, 0.02 ng / μl, 0.025 ng / μl, 0.03 ng / μl, 0.035 ng / μl, 0.04 ng / μl, 0.045 ng / μl, 0.05 ng / μl, 0.055 ng / μl, 0.06 ng / μl, 0.065 ng / μl, 0.07 ng / μl, 0.08 ng / μl, 0.095 ng / μl, 0.10 .08ng / μl, 0.085ng / μl, 0.09ng / μl, 0.095ng / μl, 0.1ng / μl, 0.2ng / μl, 0.3ng / μl l, 0.4ng / μl, 0.5ng / μl, 0.6ng / μl, 0.7ng / μl, 0.8ng / μl, 0.9ng / μl, 1ng / μl, 2ng / μl, 3ng / μl, 4ng / μl, 5ng / μl, 6ng / μl, 7ng / μl, 8ng / μl, 9ng / μl, 10ng / μl, 20ng / μl, 30ng / μl, 40ng / μl, 50ng / μl, 60ng / μl, 70ng / μl, 80ng / μl, 90ng / μl, 100ng / μl.

[0013] Furthermore, the concentration of the human genome is 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl.

[0014] Furthermore, the total sequencing data volume of the human genome and internal reference sequences is 10M reads.

[0015] Further, the sample types include blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, pus, amniotic fluid, bone marrow, ascites, pelvic washing fluid, alveolar lavage fluid, pleural fluid, spinal fluid, lymph fluid, eye fluid, nasal swab extract, throat swab extract, genital swab extract, cell suspension of digested tissue, extract of fecal material, or tissue / organ sample.

[0016] In some embodiments, the sample is not further processed, for example, blood can be obtained from the peripheral circulatory system of the subject (e.g., by a blood collection needle). The sample can include, for example, blood, urine, feces, saliva, cerebrospinal fluid, and sweat. Non-limiting examples of such samples include blood (or components of blood - e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location of a subject (e.g., tissue, circulatory system, bone marrow), cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, tissue fluid from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluid, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, ocular fluids, sweat, mucus, earwax, oils, glandular secretions, cerebrospinal fluid, hair, nails, skin cells, plasma, nasal swab or nasopharyngeal wash, cerebrospinal fluid, umbilical cord blood, lymphatic fluid and / or other excreta or body tissues.

[0017] Furthermore, the source of the sample includes any one of the following: eukaryotes, prokaryotes, and viruses.

[0018] Furthermore, the eukaryotic organism includes humans or non-human mammals.

[0019] Furthermore, the prokaryotes include bacteria, actinomycetes, archaea, spirochetes, chlamydia, mycoplasmas, rickettsiae and cyanobacteria.

[0020] Furthermore, the viruses include novel coronavirus, influenza virus, hepatitis B virus, human immunodeficiency virus, herpes simplex virus, rubella virus, rabies virus, Ebola virus, norovirus, rotavirus, smallpox virus, vaccinia virus, human papillomavirus, varicella-zoster virus, cytomegalovirus, and parvovirus.

[0021] In some embodiments, the sample is derived from any subject in need of treatment or prevention, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys and apes, and including monkey species (e.g., genus Macaca (e.g., macaques, such as cynomolgus monkeys (Macaca fascicμLaris, and / or rhesus monkeys (Macaca mμLatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (Tamarins) (species from the genus Saguinus), and species of apes, such as chimpanzees (Pantroglodytes), rodents (e.g., mice, rats, guinea pigs), Leporidae (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgies, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish.

[0022] A second aspect of the present invention provides an internal reference product for quantitative detection of human nucleic acid in a metagenome, wherein the internal reference sequence is selected from the Arabidopsis thaliana genome sequence, and there is no crossover sequence between the internal reference sequence and the pathogenic nucleic acid sequence and the human nucleic acid sequence.

[0023] Furthermore, the internal reference product is the internal reference sequence shown in SEQ ID NO:1.

[0024] Furthermore, the concentration of the internal reference sequence is obtained by screening using the method described in the first aspect above.

[0025] Furthermore, the concentration ratio of the internal reference sequence to the genome to be detected is 0.01:0.008-80.

[0026] In some embodiments, the ratio of the concentration of the internal reference sequence to the genome to be detected is 0.01:0.001, 0.01:0.002, 0.01:0.003, 0.01:0.004, 0.01:0.005, 0.01:0.006, 0.01:0.007, 0.01:0.008, 0.01:0.009, 0.01:0.01, 0.01:0.02, 0.01:0.03, 0.01:0.04, 0.01:0.05, 0.01:0.06, 0.01:0.07, 0.01:0.08, 0.01:0 : 0.09, 0.01:0.1, 0.01:0.2, 0.01:0.3, 0.01:0.4, 0.01:0.5, 0.01:0.6, 0.01:0.7, 0.01:0.8, 0.01:0.9, 0.01:1, 0.01:2, 0.01:3, 0.01:4, 0.01:5, 0.01:6, 0.01:7, 0.01:8, 0.01:9, 0.01:10, 0.01:20, 0.01:30, 0.01:40, 0.01:50, 0.01:60, 0.01:70, 0.01:80.

[0027] Furthermore, the concentration of the internal reference sequence is 0.01 ng / μl.

[0028] Furthermore, the concentration of the genome to be detected is 80ng / μl-0.008ng / μl.

[0029] Furthermore, the number of cells with the genome to be detected is 1×10 7 cells / ml ~ 1×10 2 cells / ml.

[0030] In some embodiments, the number of cells containing the genome to be detected is 1×10 2 cells / ml、1×10 3 cells / ml、1×10 4 cells / ml、1×10 5 cells / ml、1×10 6 cells / ml、1×10 7 cells / ml.

[0031] The third aspect of the present invention provides a method for constructing a quantitative detection model for the content of human nucleic acid in a metagenome, the construction method comprising: The prepared internal reference sequences were mixed with human genomic DNA of different concentrations at different concentrations, with a mixing volume ratio of 1:8. Then, nuclease-free water was added at a volume ratio of 1. The samples were library built and sequenced respectively. The internal reference detection RPM in all sequenced samples was analyzed, and the relevant quantitative detection model was constructed using the internal reference detection RPM data and the human nucleic acid content in the corresponding samples.

[0032] Furthermore, the internal reference sequence is the internal reference sequence in the internal reference product of the second aspect.

[0033] Furthermore, the quantitative model formula for human nucleic acid content is human nucleic acid content (cells / ml) = 5E + 08 (internal reference RPM) -1.578 .

[0034] The fourth aspect of the present invention provides a quantitative detection model for the content of human nucleic acid in a metagenome, wherein the quantitative detection model includes a quantitative detection model obtained using the construction method described in the third aspect.

[0035] A fifth aspect of the present invention provides a method for quantitatively detecting human nucleic acid in a metagenome based on an internal reference, the method comprising: The nucleic acid of the sample to be tested is extracted, and an internal reference sequence is added to the extracted nucleic acid. Then, a sequencing library is constructed and sequenced to obtain sequencing data. The internal reference detection RPM in the sample is analyzed, and the internal reference detection RPM is brought into the quantitative detection model described in the fifth aspect of the present invention to calculate the human nucleic acid content.

[0036] There is no crossover sequence between the internal reference sequence and the nucleic acid sequence in the sample to be detected.

[0037] Furthermore, the nucleic acid extraction includes a step of removing host nucleic acid.

[0038] In some embodiments, the step of removing host nucleic acid includes the step of removing host nucleic acid using physical or chemical methods. In some embodiments, the step of removing host nucleic acid includes the step of processing the sample using immunoprecipitation, molecular biology methods or purification reagents.

[0039] Furthermore, the internal reference sequence is the internal reference sequence in the internal reference product described in the second aspect above.

[0040] Furthermore, the quantitative model formula for human nucleic acid content is human nucleic acid content (cells / ml) = 5E + 08 (internal reference RPM) -1.578 .

[0041] The sixth aspect of the present invention provides the application of the internal reference product described in the second aspect or the quantitative detection model described in the fourth aspect in the quantitative detection of human nucleic acids in the metagenome.

[0042] A seventh aspect of the present invention provides a system for quantitatively detecting human nucleic acid in a metagenome based on an internal reference, comprising: Data acquisition unit: acquires sequencing data, which is obtained by extracting nucleic acid from the sample to be tested, adding internal reference sequences to the extracted nucleic acid, and then constructing a sequencing library and sequencing the data on the machine.

[0043] Data processing unit: Analyze the internal reference in the sample based on the sequencing data and input it into the quantitative detection model for calculation.

[0044] Data output unit: outputs the results in the data processing unit to obtain the human nucleic acid content.

[0045] Furthermore, the quantitative detection model is the quantitative detection model described in the fourth aspect above.

[0046] In some embodiments, the above-mentioned units or steps can achieve the effect through a computing device. In some embodiments, the units or steps can be concentrated on a single computing device. In some embodiments, the units or steps can be distributed on multiple computing devices, and multiple computing devices are linked by a network or other linking methods. In some embodiments, the computing device stores a program code for executing the above-mentioned units or steps, and the program code is stored in a storage device. In some embodiments, the units or steps can be prepared into integrated circuits respectively, or combined into a single integrated circuit. In the specific embodiments of the present invention, no implementation method is limited, whether it is hardware or software.

[0047] An eighth aspect of the present invention provides a device for quantitatively detecting human nucleic acid in a metagenome based on an internal reference, comprising: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method described in the fifth aspect above is implemented.

[0048] In some embodiments, the memory includes read-only memory (ROM), phase-change random access memory (PRAM), static random access memory (SRAM), flash memory, random access memory (RAM), dynamic random access memory (DRAM) such as synchronous DRAM (SDRAM), electrically erasable programmable read-only memory (EEPROM), static memory (e.g., flash memory, flash disk, static random access memory), and other types of random access memory, cache memory, registers, compact disk read-only memory (CD-ROM), digital versatile disk (DVD) or other optical storage device, cassette, other magnetic storage device, or any other non-transitory medium that can be used to store information (e.g., stored in any format) that can be accessed by a processor or any other type of computer device, including images, data, or computer executable instructions. Computer program instructions can be accessed by a processor, read from ROM or any other suitable memory location, and loaded into RAM for execution by the processor.

[0049] In some embodiments, the device can be any device having one or more devices that can implement at least some parts of the present invention. The device is not limited to a specific configuration, type, or number of objects. In addition, the system is not limited to multiple components or specific embodiments. For example, a system can be implemented on one or more printed circuit boards or other substrates and can have movable or static components. Although the following description and examples use the system to describe various aspects of the present invention, the system is not limited to a specific configuration, type, or number of objects.

[0050] A ninth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the method described in the fifth aspect is implemented.

[0051] Advantages and beneficial effects of the present invention: The present invention designs a 1031 bp nucleic acid sequence that has no homology with humans and microorganisms as an internal reference. A certain amount of the internal reference is added to the extracted nucleic acid sample and library construction and sequencing are performed. The RPM (reads per million) detected based on the internal reference can accurately quantify the human origin in the sample.

[0052] 1. Determine the amount of internal reference sequence to be added to samples with different human nucleic acid contents: add different amounts (0.1 ng, 0.01 ng, 0.001 ng) of internal reference sequence to different amounts of human genomic DNA (800 ng to 0.008 ng). After library construction and sequencing, analyze the number of internal reference sequence specific detections in each sample. The internal reference can be stably detected in all samples and will not occupy too much sequencing data as the judgment standard. Finally, the amount of internal reference added is determined to be 0.01 ng.

[0053] 2. Construction method of human quantitative model: add a certain amount of internal reference sequence to nucleic acid samples with different human content, analyze the internal reference detection RPM in each sample, and then use the internal reference RPM and the sample human content to construct a correlation equation between the two, that is, the human quantitative model is obtained, human content (cells / ml) = 5E + 08 (internal reference RPM) -1.578 . BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is a correlation analysis chart between the internal reference detected RPM and the human content of the sample.

[0055] Figure 2 This is a comparison chart of the internal reference quantitative results and the digital PCR quantitative results. DETAILED DESCRIPTION

[0056] Specific embodiments of this specification have been described. Other embodiments are within the scope of the present invention. In some cases, the actions or steps recorded in the present invention can be performed in a different order than in the embodiments and still achieve the desired results. In addition, the processes depicted in the accompanying drawings do not necessarily require the specific order or continuous order shown to achieve the desired results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0057] Example 1

[0058] 1. Experimental materials.

[0059]

[0060] Human genomic DNA, Promega; library construction kit, Novigene; nuclease-free water, Thermo Fisher Scientific.

[0061] 2. Experimental method.

[0062] 1) Dilute the prepared internal reference sequence to 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl respectively.

[0063] 2) Dilute human genomic DNA in a gradient manner to 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl.

[0064] 3) Take 8 μl of human genomic DNA with concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl, respectively, and add 1 μl of internal reference sequence with a concentration of 0.1 ng / μl and 1 μl of nuclease-free water, respectively, to obtain sample a1, sample b1, sample c1, sample d1, sample e1, and sample f1.

[0065] 4) Take 8 μl of human genomic DNA with concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl, respectively, and add 1 μl of internal reference sequence with a concentration of 0.01 ng / μl and 1 μl of nuclease-free water, respectively, to obtain sample a2, sample b2, sample c2, sample d2, sample e2, and sample f2.

[0066] 5) Take 8 μl of human genomic DNA with concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl, respectively, and add 1 μl of internal reference sequence with a concentration of 0.001 ng / μl and 1 μl of nuclease-free water, respectively, to obtain sample a3, sample b3, sample c3, sample d3, sample e3, and sample f3.

[0067] 6) The 18 samples prepared in steps 3, 4, and 5 were separately constructed and sequenced, with three technical replicates.

[0068] 7) Analyze the number of specific internal reference sequences in all sequencing samples.

[0069] 3. Experimental results.

[0070] According to the sequencing results (as shown in Table 1), the human content is 80 ng / μl to 0.008 ng / μl (converted to 1×10 7 cells / ml ~ 1×10 2 When 0.01 ng of internal reference sequence is added to a sample with 10 cells / ml, the internal reference can be stably detected and does not occupy the data volume (the total sequencing data volume of each sample is about 10M reads on average), so the amount of internal reference added is determined to be 0.01 ng.

[0071] Table 1 Number of internal reference detection sequences corresponding to different human content and internal reference addition amount

[0072] Example 2 1. Experimental materials.

[0073] The internal reference sequence SEQ ID NO: 1 with a length of 1031 bp, human genomic DNA, Promega; library construction kit, Novigene; nuclease-free water, Thermo Fisher Scientific.

[0074] 2. Experimental method.

[0075] 1) Dilute the prepared internal reference sequence gradient to 0.01 ng / μl.

[0076] 2) Dilute human genomic DNA in a gradient manner to 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl.

[0077] 3) Take 8 μl of human genomic DNA with a concentration of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, and 0.001 ng / μl, and add 1 μl of internal reference sequence with a concentration of 0.01 ng / μl and 1 l of nuclease-free water to obtain a human content equivalent to 1×10 7 cells / ml, 1×10 6 cells / ml, 1×10 5 cells / ml, 1×10 4 cells / ml, 1×10 3 cells / ml, 1×10 2 cells / ml of sample a4, sample b4, sample c4, sample d4, sample e4, and sample f4.

[0078] 4) Prepare libraries and sequence the six samples prepared in step 3 separately, with three technical replicates.

[0079] 5) The experiment was repeated three times.

[0080] 6) Analyze the internal reference detection RPM in all sequencing samples.

[0081] 7) Use the internal reference detection RPM in each sample and the human content of the corresponding sample to construct a correlation equation between the two.

[0082] 3. Experimental results.

[0083] The internal reference detection RPM in samples with different human content is shown in Table 2. The correlation equation between the internal reference detection RPM and the human content of the corresponding samples (i.e., human quantitative model) was constructed. Human content (cells / ml) = 5E + 08 (internal reference RPM) -1.578 ,like Figure 1 As shown, the results showed that the RPM detected by the internal reference was exponentially negatively correlated with the human content of the sample, and the correlation coefficient between the two was >0.95.

[0084] Table 2 RPM of internal reference detection in samples with different human content

[0085] Example 3 1. Experimental materials.

[0086] The internal reference sequence SEQ ID NO:1 with a length of 1031 bp, different types of clinical samples (including bronchoalveolar lavage fluid, pus, cerebrospinal fluid, tissue, blood, sputum, ascites); host removal kit, Novigene; nucleic acid extraction kit, Novigene; library construction kit, Novigene; nuclease-free water, Thermo Fisher Scientific.

[0087] 2. Experimental method.

[0088] 1) Collect different types of clinical samples, including bronchoalveolar lavage fluid, pus, cerebrospinal fluid, tissue, blood, sputum, and ascites, remove the host from each sample, and then extract nucleic acid; 2) Take 10 μl of each sample nucleic acid and add 0.01 ng of internal reference sequence respectively, and then construct and sequence the library of each sample nucleic acid with internal reference sequence added; 3) Analyze the internal reference detection RPM in each sequencing sample; 4) Substitute the internal reference detection RPM into the human quantitative model constructed in Experiment 2, and the human content (cells / ml) = 5E +08 (internal reference RPM) -1.578 , calculate the human content of each sample; 5) Compare the results of internal reference quantitative human sources with the results of digital PCR quantitative human sources.

[0089] 3. Experimental results.

[0090] The RPM of internal reference detection and internal reference quantitative results in each sample are shown in Table 3. The internal reference quantitative results were compared with the digital PCR quantitative results (Table 3), and the fit between the two reached more than 0.98 ( Figure 2 ), indicating that the human-derived quantification method of the present invention has high accuracy, and the human-derived quantification method of the present invention is not limited by the sample type.

[0091] Table 3 Internal reference detection RPM, internal reference quantitative results and digital PCR quantitative results

Claims

1. A method for screening the concentration of an internal reference sequence for quantitative detection of human nucleic acid in a metagenome, the method comprising: The prepared internal reference sequences were mixed with human genomic DNA at different concentrations at a mixing volume ratio of 1:8, and then nuclease-free water was added at a volume ratio of 1. The samples were library built and sequenced, and the number of internal reference-specific detection sequences in all sequenced samples was analyzed. The amount of internal reference sequence and human genome to be added was determined by judging whether the internal reference could be stably detected and would not occupy the data volume.

2. The method according to claim 1, wherein the internal reference sequence is selected from the Arabidopsis thaliana genome sequence, and there is no crossover sequence between the internal reference sequence and the pathogenic nucleic acid sequence and the human nucleic acid sequence; Preferably, the internal reference sequence is as shown in SEQ ID NO: 1; Preferably, the concentration of the internal reference sequence is 0.001-0.1 ng / μl; Preferably, the concentration of the internal reference sequence is 0.1 ng / μl, 0.01 ng / μl, 0.001 ng / μl; Preferably, the concentration of the human genome is 0.001-100 ng / μl; Preferably, the concentration of the human genome is 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, 0.01 ng / μl, 0.001 ng / μl; Preferably, the total sequencing data volume of the human genome and the internal reference sequence is 10M reads; Preferably, the sample type includes blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, pus, amniotic fluid, bone marrow, ascites, pelvic washing fluid, alveolar lavage fluid, pleural fluid, spinal fluid, lymph fluid, eye fluid, nasal swab extract, throat swab extract, genital swab extract, cell suspension of digested tissue, extract of fecal material, or tissue / organ sample; Preferably, the source of the sample includes any one of the following: eukaryotes, prokaryotes, viruses; Preferably, the eukaryotic organism comprises a human or a non-human mammal; Preferably, the prokaryotes include bacteria, actinomycetes, archaea, spirochetes, chlamydia, mycoplasmas, rickettsiae and cyanobacteria; Preferably, the viruses include novel coronavirus, influenza virus, hepatitis B virus, human immunodeficiency virus, herpes simplex virus, rubella virus, rabies virus, Ebola virus, norovirus, rotavirus, smallpox virus, vaccinia virus, human papillomavirus, varicella-zoster virus, cytomegalovirus, and parvovirus.

3. An internal reference product for quantitative detection of human nucleic acid in a metagenome, wherein the internal reference sequence is selected from the Arabidopsis thaliana genome sequence, and there is no crossover sequence between the internal reference sequence and the pathogenic nucleic acid sequence and the human nucleic acid sequence; Preferably, the internal reference product is the internal reference sequence shown in SEQ ID NO: 1; Preferably, the concentration of the internal reference sequence is obtained by screening by the method of claim 1 or 2; Preferably, the ratio of the concentration of the internal reference sequence to the genome to be detected is 0.01:0.008-80; Preferably, the concentration of the internal reference sequence is 0.01 ng / μl; Preferably, the concentration of the genome to be detected is 80ng / μl-0.008ng / μl; Preferably, the number of cells with the genome to be detected is 1×10 7 cells / ml ~ 1×10 2 cells / ml.

4. A method for constructing a quantitative detection model for the content of human nucleic acid in a metagenome, the construction method comprising: The prepared internal reference sequences were mixed with different concentrations of human genomic DNA at different concentrations, with a mixing volume ratio of 1:8, and then nuclease-free water was added at a volume ratio of 1. The samples were library built and sequenced respectively, and the internal reference detection RPM in all sequenced samples was analyzed. The relevant quantitative detection model was constructed using the internal reference detection RPM data and the human nucleic acid content in the corresponding samples; Preferably, the internal reference sequence is the internal reference sequence in the internal reference product of claim 3; Preferably, the quantitative model formula for human nucleic acid content is human nucleic acid content (cells / ml) = 5E + 08 (internal reference RPM) -1.578 .

5. A quantitative detection model for the content of human nucleic acid in a metagenome, the quantitative detection model comprising a quantitative detection model obtained using the construction method described in claim 4.

6. A method for quantitative detection of human nucleic acid in a metagenome based on an internal reference, the method comprising: Extract nucleic acid from the sample to be tested, add an internal reference sequence to the extracted nucleic acid, then construct a sequencing library and perform sequencing on a machine to obtain sequencing data, analyze the internal reference detection RPM in the sample, and bring the internal reference detection RPM into the quantitative detection model described in claim 5 to calculate the human nucleic acid content; There is no crossover sequence between the internal reference sequence and the nucleic acid sequence in the sample to be tested; Preferably, the internal reference sequence is the internal reference sequence in the internal reference product of claim 3; Preferably, the quantitative model formula for human nucleic acid content is human nucleic acid content (cells / ml) = 5E + 08 (internal reference RPM) -1.578 .

7. Use of the internal reference product described in claim 3 or the quantitative detection model described in claim 5 in the quantitative detection of human nucleic acids in metagenomes.

8. A system for quantitative detection of human nucleic acid in a metagenome based on an internal reference, comprising: Data acquisition unit: acquires sequencing data, which is obtained by extracting nucleic acid from the sample to be tested, adding internal reference sequences to the extracted nucleic acid, and then constructing a sequencing library and sequencing the data on the machine; Data processing unit: Analyze the internal reference in the sample according to the sequencing data and input it into the quantitative detection model for calculation; Data output unit: outputs the results in the data processing unit to obtain the human nucleic acid content; Preferably, the quantitative detection model is the quantitative detection model described in claim 5.

9. A device for quantitative detection of human nucleic acid in a metagenome based on an internal reference, comprising: A memory and a processor, wherein the memory is used to store program instructions; The processor is used to call program instructions, and when the program instructions are executed, the method according to claim 6 is implemented.

10. A computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the method of claim 6 when executed by a processor.

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