A non-assignable composite quality control product for DNA pathogen metagenomic high-throughput sequencing and a preparation method thereof

Abstract

The application discloses a non-fixed composite quality control product for high-throughput sequencing of DNA pathogenic macro genome and a preparation method thereof, and belongs to the technical field of DNA detection. The preparation method comprises the following steps: S1, preparing a DNA positive quality control product: (1) extracting bacterial genomic nucleic acid and fungal genomic nucleic acid; (2) obtaining viral genomic nucleic acid; (3) extracting human genomic nucleic acid; (4) mixing 1% target pathogenic genome and 99% human genome per milliliter of the DNA positive quality control product according to the following relative abundance: 1% target pathogenic genome, 99% human genome; the target pathogenic genome comprises bacterial genomic nucleic acid, fungal genomic nucleic acid and viral genomic nucleic acid; (5) mixing the target pathogenic genome according to a preset relative abundance p pD The application successfully constructs a complex background system similar to a clinical sample by adding human genomic DNA to the DNA positive quality control product and preparing a negative quality control product by using human cells, and provides a quality control effect closer to the actual situation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of DNA detection, and particularly relates to a non-fixed-value composite quality control product for DNA pathogen metagenomic high-throughput sequencing and a preparation method thereof. BACKGROUND

[0002] With the rapid development of next-generation sequencing technology (NGS), metagenomic sequencing has become an important means to study the composition and function of microbial communities in complex samples such as the environment and the human body. This technology can simultaneously detect and identify hundreds of pathogens through the overall sequencing and analysis of all microbial genomes in the sample, and has a wide range of applications in the fields of infectious disease diagnosis, microbiome research, environmental monitoring, etc. At present, metagenomic sequencing technology has the advantages of not requiring prior assumptions, being able to discover new pathogens, and covering multiple microorganisms in a single detection, but also faces technical challenges such as complex sample nucleic acid composition, large human background interference, low pathogen content, limited detection rate, and non-uniform data analysis standards. In terms of quality control, laboratories generally use commercial plasmids, synthetic fragments, or single microbial cultures as internal controls, and use ultrapure water or extraction reagents as negative controls, but lack uniform interlaboratory evaluation standards, resulting in poor comparability and repeatability of results among laboratories.

[0003] Existing quality control schemes mainly include two types: commercial quality control products and laboratory self-made quality control products. Commercial quality control products have the problems of limited target sequences, high prices, and insufficient stability. Laboratory self-made quality control products mainly use plasmid cloning and mixed strain culture methods, which are relatively simple to operate and have low cost, but cannot truly simulate the complexity of clinical samples, and the preparation process is tedious and has large batch-to-batch differences. With the continuous progress of sequencing technology, read length is increasing, throughput is improving, and cost is decreasing, and its application field has expanded to clinical diagnosis, public health, and microbiome research, etc., and the requirements for standardization, automation, and traceability of quality control products are increasing. The main problems of current quality control products include: lack of complex human background, single pathogen species, discontinuous abundance gradient, large batch-to-batch difference, insufficient stability, harsh storage conditions, etc. There are few comprehensive quality control products on the market that contain DNA pathogens such as bacteria, fungi, and viruses and truly simulate clinical backgrounds, which cannot meet the diversified needs of sequencing technology. Therefore, it is urgent to establish a standardized quality control system, develop a stable quality control product preparation method, and provide traceable fixed-value schemes to meet the needs of multi-platform compatibility, support full-process quality monitoring, and ensure the comparability of results.

[0004] The current technical solutions for metagenomic sequencing quality control mainly include three categories: artificially synthesized nucleic acid quality control products, pathogenic microorganism culture quality control products, and mixed matrix quality control products. The artificially synthesized nucleic acid quality control products are mainly prepared by chemical synthesis and plasmid cloning technology. The chemical synthesis can obtain 20-200 bp oligonucleotide fragments, and longer sequences can be constructed by PCR amplification and fragment ligation. The plasmid cloning technology can amplify 0.1-15 kb target gene fragments.

[0005] The pathogenic microorganism culture quality control products are mainly prepared by inactivating pathogens. The inactivation methods include heat inactivation (56-121℃), irradiation (γ-ray or electron beam), and chemical reagent (formaldehyde, β-propiolactone, etc.) treatment. The inactivated pathogens can be used as quality control products to evaluate the sensitivity and specificity of the detection method.

[0006] The mixed matrix quality control products mainly include cell line matrix and clinical sample simulation. The cell line matrix is prepared by using human immortal cell lines, primary cultured cells or genetically engineered cells, and obtained by cell culture amplification, counting and lysis extraction of nucleic acid. The clinical sample simulation is prepared by mixing multiple clinical samples and adding known concentration of pathogens.

[0007] There are many products in the existing technology for commercial application, including various quality control products from international and domestic manufacturers, which are applied to inter-laboratory quality evaluation, method validation and daily quality control.

[0008] The current metagenomic sequencing technology has the following problems in the field of laboratory internal quality control:

[0009] 1. The existing artificially synthesized nucleic acid quality control products have the advantage of controllable sequence, but their application is limited in many aspects. Due to the limitations of synthesis technology, the length of artificially synthesized DNA fragments is generally short, which is difficult to simulate the complete pathogen genome. At the same time, the DNA conformation of the quality control products obtained by plasmid cloning is significantly different from that of natural nucleic acid, which cannot truly reflect the characteristics of nucleic acid in clinical samples. In addition, the cost of chemical synthesis and plasmid cloning technology is high, and it is difficult to achieve economic benefit balance in large-scale production.

[0010] 2. The pathogenic microorganism culture quality control products face many technical bottlenecks in the preparation and application process. Many important pathogens are difficult to culture due to their special growth requirements or biological safety requirements, and require higher level of biological safety facilities. In the preparation process, no matter what inactivation method is used, it may cause different degrees of damage to the nucleic acid of the pathogen, reducing the reliability of the quality control product. At the same time, the growth state of different batches of cultures is significantly different, which leads to significant batch-to-batch differences in the final product, making it difficult to achieve standardized production.

[0011] 3. Mixed matrix quality control has unique advantages in simulating real clinical samples, but its preparation process faces multiple challenges. When using cell lines to prepare quality control, the growth state of cells is difficult to accurately control, leading to fluctuations in product quality. The preparation of clinical sample simulators is limited by sample sources, making it difficult to consistently and stably obtain sufficient raw materials. In addition, due to the complexity of the matrix components, various physicochemical factors can affect the performance of the quality control. These characteristics make it difficult to standardize the production and large-scale application of mixed matrix quality control.

[0012] 4. Current commercial quality control products have some common problems that seriously restrict their widespread application. First, the production cost of existing quality control is high, leading to expensive product prices, increasing the operating cost of the laboratory. Second, the stability of the quality control is poor, and the storage conditions and transportation environment are strict, bringing many inconveniences to the logistics and use of the product. In addition, the traceability of the quality control is insufficient, and it is difficult to establish a unified quality standard system. Finally, due to the limitations of the production process, the uniformity between different batches of products cannot be guaranteed, affecting the comparability of the test results.

[0013] 5. There is no composite quality control system on the market that can meet the needs of DNA pathogen detection. Most existing quality control products are designed for a single type of pathogen or a single detection process. Traditional quality control products are mostly developed for traditional molecular detection methods such as PCR, and their design concept and performance characteristics cannot meet the needs of metagenomic sequencing for multiple quality control. This singularity forces laboratories to purchase and use multiple quality control products, increasing detection costs and potentially affecting result interpretation due to performance differences between different quality control products. At the same time, due to the lack of unified quality control standards, the detection results between different types of quality control products cannot be effectively compared. This situation makes it difficult for laboratories to fully evaluate the detection performance of metagenomic sequencing and accurately control the quality of detection results for different types of pathogens.

[0014] 6. Lack of standardized quality control system: The existing indoor quality control of metagenomic sequencing lacks unified positive and negative quality control products, which cannot effectively evaluate the sensitivity, specificity and accuracy of the sequencing process.

[0015] 7. Insufficient simulation of real samples by quality control products: Some quality control products are prepared by synthesizing fragments or samples from a single source, which cannot simulate the complex sample background of humans and the actual proportion of pathogenic microorganisms.

[0016] 8. Difficulty in verifying technical performance: Existing quality control products are mostly limited to a single detection technology, which cannot adapt to the widespread application of next-generation sequencing (NGS) technology in DNA pathogen metagenomic analysis, lacking universality and flexibility. SUMMARY

[0017] In order to solve the above problems existing in the prior art, the present application provides a non-fixed-value composite quality control product for DNA pathogen metagenome high-throughput sequencing and a preparation method thereof.

[0018] The technical scheme of the present application is as follows:

[0019] A preparation method of a non-fixed-value composite quality control product for DNA pathogen metagenome high-throughput sequencing, comprising the following steps:

[0020] S1. Preparation of DNA positive quality control product:

[0021] (1) Extracting bacterial genomic nucleic acid and fungal genomic nucleic acid;

[0022] (2) Obtaining viral genomic nucleic acid;

[0023] (3) Extracting human genomic nucleic acid;

[0024] (4) Mixing 1% target pathogen genome and 99% human genome per milliliter of DNA positive quality control product according to the following relative abundance: the target pathogen genome includes bacterial genomic nucleic acid, fungal genomic nucleic acid, and viral genomic nucleic acid;

[0025] (5) The target pathogen genome is mixed according to the preset relative abundance p pD

[0026] In practice, the target pathogen can be selected from any pathogen, specifically, any bacteria, fungi, and viruses can be selected, which can be routinely selected and determined by a person skilled in the art according to practical needs and detection requirements. After determining the specific target pathogen, the relative abundance of each target pathogen can be preset according to the type and quantity of the target pathogen, and the sum of each target pathogen and its respective preset relative abundance in the quality control product is equal to 100%, which can be routinely determined and achieved by a person skilled in the art according to the specific pathogen type and quantity, combined with conventional technical knowledge reported in the prior art. The conventional technical knowledge reported in the prior art includes:

[0027] 1. Editorial Board of Chinese Journal of Infectious Diseases. Expert consensus on clinical application of second-generation sequencing technology for metagenomics in detection of infectious pathogens [with correction] [J]. Chinese Journal of Infectious Diseases, 2020, 38(11): 681-689. DOI: 10.3760 / cma.j.cn311365-20200731-00732.

[0028] ​2. Expert consensus points out that pathogen metagenomics second-generation sequencing detection can cover a wide range of pathogens, viruses, fungi, bacteria, and parasites can be detected at the same time, whether the clinical sample culture is successful or not, as long as it contains detectable DNA or RNA.

[0029] 3. Knight, R., Vrbanac, A., Taylor, B.C. et al. Best practices for analysing microbiomes. Nat Rev Microbiol 16, 410-422 (2018). https: / / doi.org / 10.1038 / s41579-018-0029-9; this article points out that relative abundance is essentially compositional data. Any valid statistical analysis must be normalized to a constant sum (such as 100%) to avoid spurious correlations.

[0030] In some embodiments, the target pathogen genome and its preset relative abundance p pD As follows: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, 0.5% Streptococcus pneumoniae; 10% Human Bocavirus type 1, 5% Adenovirus type 7; 4% Candida albicans, 4% Cryptococcus gattii, 4% Aspergillus fumigatus; innovative relative abundance gradient design: the DNA pathogens in the quality control product are designed according to the gradient ratio from high to low, forming a continuous relative abundance gradient, which can comprehensively evaluate the detection ability of the detection system for pathogens of different abundance.

[0031] The target pathogen genome is preset according to the relative abundance p pD The mixing includes the following steps:

[0032] 1) Calculate the initial input volume of each target pathogen using the formula ( a ) V pD1

[0033] ( a )

[0034] The formula ( a ) is: g p represents the genome size of the target pathogen; The mass volume concentration of the target pathogen nucleic acid;

[0035] 2) the initial input volume of nucleic acid of each target pathogen calculated according to step 1) V pD1 Mix, then add human cell line genomic DNA, and finally add TE buffer (1x, pH 8.0) to form a sample;

[0036] 3) take 20%-40% of the sample volume from the sample, construct a library using metagenomic sequencing technology, perform sequencing and bioinformatics analysis, and obtain the effective sequence number of each target pathogen r D1 ;

[0037] 4) calculate the actual input volume of each target pathogen according to formula ( b ) V pD2 :

[0038] ( b )

[0039] In formula ( b ), a and b are randomly assigned within 0-1000 and r D2 ≠0; r D2 ;

[0040] Two target pathogens: pathogen α and pathogen β Each of r D2 satisfies the conversion relationship of the following formula (c):

[0041] ( c )

[0042] In formula ( c ), a and b are randomly assigned within 0-1000 and r D2α represents the effective sequence number of pathogen α ; r D2β represents the effective sequence number of pathogen β ; p pD2α represents the relative abundance of pathogen α , p pD2β represents the relative abundance of pathogen β ;

[0043] 5) the adjusted input volume of nucleic acid of each target pathogen calculated according to step (4) V pD2Mix, add human cell line genomic DNA at 100 ng / mL, and add TE buffer to the final DNA positive quality control.

[0044] The target pathogen is selected from the group consisting of Pseudomonas aeruginosa, Mycobacterium avium, Klebsiella pneumoniae, Ochrobactrum anthropi, Bacteroides fragilis, Streptococcus pyogenes, Haemophilus influenzae, Streptococcus oralis, Staphylococcus aureus, Veillonella parvula, Peptostreptococcus anaerobius, Legionella pneumophila, Streptococcus pneumoniae; Human Bocavirus type 1, Adenovirus type 7; Candida albicans, Cryptococcus gattii, Aspergillus fumigatus;

[0045] Preferably, the genome size of the target pathogen is selected from the group consisting of Pseudomonas aeruginosa 6839777 bp, Mycobacterium avium 4956752 bp, Klebsiella pneumoniae 5548441 bp, Ochrobactrum anthropi 5226429 bp, Bacteroides fragilis 5234583 bp, Streptococcus pyogenes 1844942 bp, Haemophilus influenzae 1850809 bp, Streptococcus oralis 1931995 bp, Staphylococcus aureus 2806340 bp, Veillonella parvula 2132186 bp, Peptostreptococcus anaerobius 2192403 bp, Legionella pneumophila 3407565 bp, Streptococcus pneumoniae 2096425 bp; Human Bocavirus type 1 5099 bp, Adenovirus type 7 35197 bp; Candida albicans 14735515 bp, Cryptococcus gattii 17527853 bp, Aspergillus fumigatus 28825722 bp;

[0046] The target pathogen genome and its preset relative abundance p pD As follows: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, 0.5% Streptococcus pneumoniae; 10% Human Bocavirus type 1, 5% Adenovirus type 7; 4% Candida albicans, 4% Cryptococcus gattii, 4% Aspergillus fumigatus.

[0047] In S1, the extraction of bacterial genomic nucleic acid and fungal genomic nucleic acid comprises: selecting a suitable culture medium to culture bacteria or fungi respectively, collecting the bacteria in the logarithmic growth phase, extracting the genomic DNA, and performing quality detection and quantification;

[0048] The obtaining of the viral genomic nucleic acid comprises: obtaining the full-length sequence of the genome of the target virus by in vitro synthesis, recombining the full-length sequence of the genome of the target virus with an Ad5 adenovirus skeleton through a plasmid, amplifying in BJ5183-AD-1 and Stbl3 bacteria, and then transfecting 293T cells for packaging to obtain DNA pseudo-virus particles, and then extracting nucleic acid from the DNA pseudo-virus particles.

[0049] The nucleic acid final concentration in the DNA positive quality control is 100 ng / mL.

[0050] The preparation method of the non-constant-value composite quality control for DNA pathogen metagenomic high-throughput sequencing further comprises the following step: S2. Preparing a negative quality control: washing a human cell line 3-5 times to obtain a human cell line precipitate free of viruses or other exogenous microbial contamination.

[0051] A non-constant-value composite quality control for DNA pathogen metagenomic high-throughput sequencing is prepared by the preparation method; the non-constant-value composite quality control comprises: a DNA positive quality control.

[0052] The DNA positive quality control comprises: pathogenic genomic nucleic acid and human genomic nucleic acid.

[0053] The pathogenic genomic nucleic acid comprises: DNA viral genomic nucleic acid, bacterial genomic nucleic acid, and fungal genomic nucleic acid.

[0054] The bacterial genomic nucleic acid comprises the following components with the following relative abundances: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, and 0.5% Streptococcus pneumoniae.

[0055] The DNA viral genomic nucleic acid comprises the following components with the following relative abundances: 10% Human Bocavirus type 1 and 5% Adenovirus type 7.

[0056] The fungal genomic nucleic acid comprises the following components with the following relative abundances: 4% Candida albicans, 4% Cryptococcus gattii, and 4% Aspergillus fumigatus.

[0057] The non-constant-value composite quality control further comprises: a negative quality control; the negative quality control comprises: a human cell line precipitate washed 3-5 times and free of viruses or other exogenous microbial contamination.

[0058] The human cell line refers to a cell line obtained by immortalizing cells of human origin.

[0059] The human cell line precipitate contains 1x105 a cell.

[0060] The cell line is a virus-free or other exogenous microbial contamination-free cell line.

[0061] The innovation of the present application is:

[0062] 1. Provide a standardized quality control system: Through the innovative design of DNA positive (DPC) and negative (NC) quality control, a double indoor quality control system is constructed for evaluating the sensitivity, specificity and consistency of DNA pathogen metagenomic sequencing.

[0063] 2. Simulate the background of real samples: The quality control product contains nucleic acids extracted from pathogenic microorganisms such as bacteria, fungi and pseudovirus, as well as human cell nucleic acid background, with accurate proportion, which can highly simulate the complexity of real clinical samples.

[0064] 3. Support multiple detection technologies: The quality control product is suitable for next-generation sequencing (NGS) platform, covering the DNA pathogen detection process, and can be widely used in clinical, scientific research and laboratory standardized testing.

[0065] 4. Innovative relative abundance gradient design: The DNA pathogens in the quality control product are designed in a gradient proportion from high to low, forming a continuous relative abundance gradient, which can comprehensively evaluate the detection capability of the detection system for pathogens of different abundance.

[0066] The beneficial effects of the present application are as follows:

[0067] Firstly, the present application realizes the complete coverage of the whole process of DNA pathogen metagenomic sequencing by integrating various quality control forms. Among them, the DNA positive quality control product innovatively combines artificially synthesized nucleic acid, inactivated strain DNA and human genomic DNA, which maximizes the simulation of the real state of the coexistence of pathogens and host background in clinical samples; the negative quality control product provides a real human background to ensure the specificity of the detection. The present application first proposes and realizes the innovative design of DNA pathogen metagenomic sequencing quality control product, by integrating bacterial, fungal, viral and other DNA pathogen nucleic acids and human background nucleic acids into the same quality control system, a complete quality control scheme is constructed. Among them, the DNA positive quality control product adopts the composite design of artificially synthesized pseudovirus extraction nucleic acid, inactivated strain DNA and human genomic DNA, and the negative quality control product provides a real human background to ensure the specificity of the detection, which realizes the innovative breakthrough of the quality control system.

[0068] Secondly, the present application adopts a high-efficiency and reliable production process, successfully avoids the risk in the process of live virus culture and inactivation through pseudovirus technology, adopts a standardized preparation process to ensure the stability between batches, and through accurate quantitative design to ensure the reliability of the detection results, significantly improves the production efficiency and product quality of the quality control product.

[0069] Third, the present application realizes the precise quantification and batch uniformity of quality control products through standardized preparation process and proportion design. By adding human genomic DNA in the DNA positive quality control product and using human cell to prepare the negative quality control product, a complex background system similar to clinical samples is successfully constructed, providing a more realistic quality control effect. Full-process adaptability: the quality control product is suitable for the whole process of DNA extraction, library construction and sequencing analysis of metagenomic sequencing, covering a wide range. The present application has a significant advantage in simulating clinical samples. By innovatively adding human genomic DNA as host background nucleic acid in the DNA positive quality control product and using human cells to prepare the negative quality control product, a real complex nucleic acid background is provided, which maximizes the simulation of the actual situation of coexistence of pathogens and host nucleic acid in clinical samples, ensuring the authenticity and reliability of the quality control results.

[0070] Fourth, the present application first proposes the design concept of DNA pathogen metagenomic quality control product. By integrating DNA bacterial, fungal and viral nucleic acids and human background nucleic acids into one quality control system, the limitations of existing single quality control scheme are overcome, providing a complete quality control solution for clinical laboratories, which can meet the detection needs of different types of pathogens. Innovative simulation background: the complex nucleic acid background is provided by human cell precipitate, which accurately simulates the real environment in clinical samples. Stability and long-term preservation: the quality control product has been evaluated for uniformity and stability to ensure consistent performance in short-term and long-term preservation. The quality control product system of the present application realizes unprecedented comprehensive quality control coverage, which can meet the detection needs of DNA pathogens and support the whole process quality control from sample processing to bioinformatics analysis, especially suitable for high-throughput sequencing platforms such as NGS. Through the complementary advantages of different types of quality control products, multiple quality control functions are successfully realized, overcoming the limitations of existing single quality control scheme.

[0071] Fifth, the present application adopts an innovative abundance gradient design, which forms a continuous gradient distribution from high to low by accurately controlling the relative abundance of pathogens. In the DNA positive quality control product, the gradient design from 15% Pseudomonas aeruginosa to 0.5% Streptococcus pneumoniae not only can comprehensively evaluate the detection ability of the detection system for pathogens of different concentrations, but also provides a reliable basis for determining the lower limit of detection sensitivity. This gradient design is significantly superior to the simple high and low concentration setting in existing quality control products, providing a more comprehensive and accurate reference standard for clinical laboratories to evaluate the performance of the detection system. Innovative abundance gradient design: the relative abundance of pathogens is accurately prepared according to the gradient ratio from high to low, forming a continuous concentration gradient, which can effectively evaluate the detection sensitivity of the detection system for different concentrations of targets.

[0072] Sixth, the present application provides an innovative DNA pathogen quality control system for DNA pathogen metagenomic sequencing quality control, which not only realizes quality control of the whole detection process, but also significantly improves the accuracy and reliability of the quality control by simulating the nucleic acid composition of real clinical samples, and can effectively meet the requirements of clinical diagnosis for the stability, reliability and comprehensiveness of the quality control product. Through the above innovative design, the composite quality control product system of the present application significantly improves the quality control level of DNA pathogen metagenomic sequencing, and provides more reliable and comprehensive quality control guarantee for clinical diagnosis. The present application provides an economical and practical quality control solution, and a set of quality control product system can meet multiple quality control requirements, effectively reducing the procurement and use cost of the laboratory; at the same time, the product has excellent stability, reduces the storage and transportation requirements, and is easy to operate, which is convenient for popularization and application in clinical laboratories, and has significant practical value.

[0073] In addition, the present application has the following beneficial effects:

[0074] 1. The DNA positive quality control product of the present application is nucleic acid extracted from pseudo-virus and inactivated culture, covering the full length of the respective genome.

[0075] 2. The DNA positive quality control product of the present application is pathogen nucleic acid genome, which has lower requirement for laboratory biological safety, and can be used in P2 and lower level laboratories. The DNA positive quality control product can realize standardized production.

[0076] 3. The raw materials of the present application are stable in source and can be continuously obtained. The prepared quality control product is stored in TE buffer, and the matrix composition is simple, which avoids the situation that the detection result is unstable due to interference of other factors, realizes standardized production of the quality control product, and ensures the stability of the performance of the quality control product. Most of the pathogens contained in the quality control product are nucleic acid extracted from inactivated culture, and a small part of the virus is nucleic acid extracted from pseudo-virus. The plasmid is introduced into E. coli or cells to realize the coating of protein shell and form pseudo-virus particles, which can be theoretically reproduced infinitely, and is not directly used in large-scale production by chemical synthesis of plasmid, and the purpose of cost control can be realized in the process of large-scale production.

[0077] 4. The existing quality control products are mainly for single pathogens, and in order to cover multiple types of pathogens such as fungi, bacteria and viruses, multiple quality control products need to be prepared and produced at the same time, and the consumption of materials and the working hours of personnel are doubled, which greatly increases the production cost. The present application covers multiple types of pathogens in one tube, reduces the consumption of materials and the working hours of personnel, and reduces the production cost by several times. The raw materials for each pathogen in the present application are prepared in large quantities at a time, and when the raw materials are used to prepare quality control products, a single batch can produce sufficient quality control products, which can meet the use of users for a long time, greatly reducing the frequency of batch replacement; even if the batch is replaced, the concentration of the pathogen in the raw material is repeatedly set multiple times, and the concentration level of the previous batch is gradually adjusted, and then prepared according to the fixed input volume, which maximizes the uniformity between different batches, and the CV coefficient of variation in Experimental Example 1 can reflect the uniformity of the quality control products of the present application.

[0078] 5. Experimental Examples 1-2 of the present application include DNA positive quality control products and negative quality control products. They respectively contain multiple pathogens that are clinically focused on, covering bacteria, fungi and viruses, and covering the full length of the genome of the pathogen, which can meet the DNA pathogen metagenomic sequencing. In theory, DNA pathogen metagenomic sequencing mNGS can detect all DNA pathogens, unlike traditional molecular detection methods such as PCR, which only detect specific pathogens. The present application covers multiple types of pathogens, and users do not need to purchase multiple single pathogen quality control products, and do not need to construct libraries for sequencing and analysis respectively. The present application only needs to construct one DNA library, and 20 M reads of sequencing data can be analyzed, which effectively reduces the detection cost, eliminates the performance difference between different types of quality control products, realizes the purpose of quality control of multiple pathogens in one tube, and helps the laboratory to comprehensively evaluate the detection performance of DNA pathogen mNGS.

[0079] 6. Experimental Example 1 of the present application includes DNA positive quality control products and negative quality control products. In Experimental Example 1, the abundance of pathogens contained in the DNA positive quality control product is set in a gradient from high to low, including the concentration levels of medium positive and weak positive, which can effectively evaluate the sensitivity and accuracy of the sequencing process. Experimental Example 2 is the operation method of the negative quality control product, which can effectively evaluate the specificity and accuracy of the sequencing process. DPC is a positive quality control product, which focuses on sensitivity and accuracy, needs to detect the declared contained pathogens, and the relative abundance meets the expectation, and does not evaluate the specificity. The negative quality control product is used to evaluate the specificity.

[0080] 7. The pathogens contained in Experimental Example 1 of the present application cover the full length of their respective genomes, which can effectively simulate real samples. The human cell line genomic DNA is inserted in the DNA positive quality control product of Experimental Example 1, and the proportion reaches more than 99%, which effectively simulates the proportion of more than 99% of host nucleic acids in clinical samples.

[0081] 8. The existing quality control products are mostly single-pathogen containing quality control products, mostly prepared from pseudo-viruses or nucleic acids, covering part of the pathogen genome, and only suitable for PCR type in vitro diagnostic reagents. The DNA positive quality control prepared in the experimental example 1 of the present application contains a plurality of pathogens that are clinically focused on, and covers the full length of the pathogen genome, and can meet the DNA pathogen metagenomic sequencing. Some laboratories only carry out DNA mNGS detection, and only use DNA positive quality control and negative quality control. In the composite quality control of the present application, DPC and NC are separately separated and independent in the kit, and can also be used in combination. The quality control of the present application can meet the use requirements of such laboratories, and reflects the flexibility of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 The relative abundance box plot of pathogen detected by the DNA positive quality control (DPC) of the experimental example 1 of the present application.

[0083] Figure 2 The relative abundance line chart of pathogen detected by the DNA positive quality control (DPC) of the experimental example 1 of the present application. DETAILED DESCRIPTION

[0084] The detailed content of the present application will be further described in combination with the drawings, specific experimental examples and experimental examples, but the protection scope of the present application is not limited thereto.

[0085] Sources of biomaterials

[0086] Pseudomonas aeruginosa used in Experimental Example 1 was ATCC® 27853™ from American Type Culture Collection; Mycobacterium avium was ATCC® 25291™ from American Type Culture Collection; Klebsiella pneumoniae was ATCC® 13883™ from American Type Culture Collection; Ochrobactrum anthropi was ATCC® 49188™ from American Type Culture Collection; Bacteroides fragilis was ATCC® 25285™ from American Type Culture Collection; Streptococcus pyogenes was ATCC® 19615™ from American Type Culture Collection; Haemophilus influenzae was ATCC® 49766™ from American Type Culture Collection; Streptococcus oralis was ATCC® 35037™ from American Type Culture Collection; Veillonella parvula was ATCC® 10790™ from American Type Culture Collection; Staphylococcus aureus was ATCC® 25923™ from American Type Culture Collection; Peptostreptococcus anaerobius was ATCC® 27337™ from American Type Culture Collection; Legionella pneumophila was ATCC® 33152™ from American Type Culture Collection; Streptococcus pneumoniae was ATCC® 49619™ from American Type Culture Collection; Human Bocavirus type 1 and Adenovirus type 7 were both artificially prepared DNA pseudovirus particles covering the whole genome sequence of each virus, and the whole genome sequence of each virus can be downloaded from NCBI database (Human Bocavirus type 1 has accession number NC_007455.1 in NCBI RefSeq database, and Adenovirus type 7 has accession number AC_000018.1 in NCBI RefSeq database); Candida albicans was ATCC® 10231™ from American Type Culture Collection; Cryptococcus gattii was ATCC® 32609™ from American Type Culture Collection; Aspergillus fumigatus was ATCC® 204305™ from American Type Culture Collection; BJ5183-AD-1 competent cells, Stbl3 competent cells, 293T cells, LN229 cell line were commercially available.

[0087] II. The human cell line used in Experimental Example 2 is LN229 (ATCC® CRL-2611™) from the American Type Culture Collection. One skilled in the art can routinely replace the genomic DNA of LN229 cells used in the present application with nucleic acids from different sources (such as other human cell lines, for example: GM12878 (human lymphoblastoid cell line), HEK293 (human embryonic kidney cells, healthy human embryonic kidney cells immortalized by adenovirus 5 (Ad5) DNA fragments), MCF-10A (mammary epithelial cells, healthy female breast tissue, non-tumorigenic immortalized cells), HUVEC (human umbilical vein endothelial cells, primary cells from the umbilical vein of a healthy newborn, which can be passaged a limited number of times or immortalized), etc.). These cell lines can be purchased commercially.

[0088] Preparation method of the non-fixed-value composite quality control product of the first group of examples, the present application

[0089] The present group of examples provides a preparation method of a non-fixed-value composite quality control product for DNA pathogen metagenomic high-throughput sequencing. All examples in the present group of examples have the following common features: the preparation method of the non-fixed-value composite quality control product for DNA pathogen metagenomic high-throughput sequencing comprises the following steps:

[0090] S1. Preparation of DNA positive quality control product:

[0091] (1) Extracting bacterial genomic nucleic acids and fungal genomic nucleic acids;

[0092] (2) Obtaining viral genomic nucleic acids;

[0093] (3) Extracting human genomic nucleic acids;

[0094] (4) Mixing 1% target pathogen genome and 99% human genome per milliliter of DNA positive quality control product according to the following relative abundance: the target pathogen genome includes bacterial genomic nucleic acids, fungal genomic nucleic acids, and viral genomic nucleic acids;

[0095] (5) The target pathogen genome is mixed according to the pre-set relative abundance p pD

[0096] ​In practice, the target pathogen can be selected from any pathogen, specifically, any bacteria, fungi, virus can be selected, which can be routinely selected and determined by those skilled in the art according to the needs of practice and detection requirements. After determining the specific target pathogen, the relative abundance of each target pathogen can be preset according to the type and quantity of the target pathogen, and the sum of each target pathogen and its respective preset relative abundance in the quality control sample is equal to 100%, which can be routinely determined and achieved by those skilled in the art according to the specific pathogen type and quantity, combined with the conventional technical knowledge reported in the prior art. The conventional technical knowledge reported in the prior art includes:

[0097] 1. Editorial Board of Chinese Journal of Infectious Diseases. Expert consensus on clinical application of second-generation sequencing technology for metagenomics in detection of infectious pathogens [with correction] [J]. Chinese Journal of Infectious Diseases, 2020, 38(11): 681-689. DOI: 10.3760 / cma.j.cn311365-20200731-00732.

[0098] 2. The expert consensus points out that pathogenic metagenomics second-generation sequencing detection can cover a wide range of pathogens, viruses, fungi, bacteria, parasites can be detected at the same time, whether the clinical sample culture is successful or not, as long as it contains detectable DNA or RNA.

[0099] 3. Knight, R., Vrbanac, A., Taylor, B.C. et al. Best practices for analysing microbiomes. Nat Rev Microbiol 16, 410-422 (2018). https: / / doi.org / 10.1038 / s41579-018-0029-9; This article points out that relative abundance is essentially compositional data. Any valid statistical analysis must be normalized to a constant sum (such as 100%) to avoid spurious correlations.

[0100] In some embodiments, the target pathogen genome and its preset relative abundance p pDAs follows: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, 0.5% Streptococcus pneumoniae; 10% Human Bocavirus type 1, 5% Adenovirus type 7; 4% Candida albicans, 4% Cryptococcus gattii, 4% Aspergillus fumigatus; the relative abundance gradient design of the innovation: the DNA pathogens in the quality control product are designed in a gradient from high to low, forming a continuous relative abundance gradient, which can comprehensively evaluate the detection capability of the detection system for pathogens with different abundances.

[0101] The target pathogen genome is in a preset relative abundance p pD The mixing includes the following steps:

[0102] (1) The initial input volume of each target pathogen is calculated using formula ( a ) V pD1

[0103] ( a )

[0104] In formula ( a ) g p represents the genome size of the target pathogen; the mass-volume concentration of the nucleic acid of the target pathogen;

[0105] 3.0E+09 is a scientific notation, representing 3×10 9 ; The size of a human haploid genome is about 3 Gb (3 billion base pairs), and the DNA mass of a human haploid genome is about 3.3 pg. The preset mixed copy number concentration of the 18 pathogens is 1000 copies / mL. According to the genome size of the mixed 18 pathogens, the volume of each pathogen input can be calculated by substituting the above formula ( a ) V pD1 ; The genome size of each target pathogen can be queried through the official website of ATCC and the NCBI database; the mass-volume concentration of the pathogen nucleic acid This parameter is obtained by Qubit 4 fluorescence meter (invitrogen, USA) after extraction of each pathogen nucleic acid.

[0106] In this paper, the DNA pathogen refers to a pathogen with DNA as genetic material.

[0107] Specifically in the embodiments, the DNA pathogen includes bacteria, fungi, and DNA viruses.

[0108] In some specific embodiments, the bacteria include: Pseudomonas aeruginosa, Mycobacterium avium, Klebsiella pneumoniae, Ochrobactrum anthropi, Bacteroides fragilis, Streptococcus pyogenes, Haemophilus influenzae, Streptococcus oralis, Staphylococcus aureus, Veillonella parvula, Peptostreptococcus anaerobius, Legionella pneumophila, Streptococcus pneumoniae.

[0109] In some specific embodiments, the fungi include: Candida albicans, Cryptococcus gattii, Aspergillus fumigatus.

[0110] In some specific embodiments, the DNA viruses include: Human bocavirus 1, Adenovirus 7.

[0111] The target pathogen is selected from: Pseudomonas aeruginosa, Mycobacterium avium, Klebsiella pneumoniae, Ochrobactrum anthropi, Bacteroides fragilis, Streptococcus pyogenes, Haemophilus influenzae, Streptococcus oralis, Staphylococcus aureus, Veillonella parvula, Peptostreptococcus anaerobius, Legionella pneumophila, Streptococcus pneumoniae; Human bocavirus 1, Adenovirus 7; Candida albicans, Cryptococcus gattii, Aspergillus fumigatus.

[0112] Each of the above target pathogens is a common pathogen known to those skilled in the art and has a common technical meaning known to those skilled in the art, for example:

[0113] Pseudomonas aeruginosa can be the meaning of the term "Pseudomonas aeruginosa" as recorded in the article "Study on the antibacterial effect of rhizoma atractylodis based on bioinformatics analysis", or the meaning of the term "green pyocyanin" in the Baidu Encyclopedia entry;

[0114] Mycobacterium avium can be the meaning of the term "Mycobacterium avium" as recorded in the article "Evaluating the Effects of 60°C Heating for 90 Min on Bacterial Pathogen Viability and IgG Concentration in Bovine Colostrum", or the meaning of the term "Mycobacterium avium" in the Baidu Encyclopedia entry;

[0115] Klebsiella pneumoniae can be the meaning of the term "Klebsiella pneumoniae" as recorded in the article "Distribution and drug resistance of pathogenic bacteria in sterile body fluids", or the meaning of the term "Klebsiella pneumoniae" in the Baidu Encyclopedia entry;

[0116] Ochrobactrum anthropi can be the meaning of the term "Ochrobactrum anthropi" as recorded in the article "Microbial community structure and carbon sequestration capacity in oil-containing solid waste residues", or the meaning of the term "Ochrobactrum anthropi" in the Baidu Encyclopedia entry;

[0117] Bacteroides fragilis can be the meaning of the term "Bacteroides fragilis" recorded in the article "Exploratory study on intestinal and plaque flora characteristics of elderly patients with lower extremity arteriosclerosis obliterans"; it can also be the meaning of the term "Bacteroides fragilis" in the Baidu Baike entry;

[0118] Streptococcus pyogenes can be the meaning of the term "Streptococcus" recorded in the article "Rhamnose polysaccharide-decorated outer membrane vesicles as a vaccine candidate targeting Group A Streptococcus from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis"; it can also be the meaning of the term "Streptococcus pyogenes" in the Baidu Baike entry;

[0119] Haemophilus influenzae can be the meaning of the term "Haemophilus influenzae" recorded in the article "Analysis of clinical characteristics of 180 hospitalized children with pertussis"; it can also be the meaning of the term "Haemophilus influenzae" in the Baidu Baike entry;

[0120] Streptococcus oralis can be the meaning of the term "Streptococcus oralis" recorded in the article "Analysis of intestinal flora structure changes in neonatal hyperbilirubinemia caused by early-onset sepsis"; it can also be the meaning of the term "Streptococcus oralis" in the Baidu Baike entry;

[0121] Staphylococcus aureus can be the meaning of the term "Staphylococcus aureus" recorded in the article "Study on intelligent active film monitoring shrimp freshness based on sodium alginate with antibacterial and ammonia-sensitive functions"; it can also be the meaning of the term "Staphylococcus aureus" in the Baidu Baike entry;

[0122] Veillonella parvula can be the meaning of the term "Veillonella parvula" recorded in the article "Effect of Veillonella parvula and its acetate-producing gene-deficient engineered bacteria on rumen microbial fermentation";

[0123] Peptostreptococcus anaerobius can be the meaning of the term "Peptostreptococcus anaerobius" recorded in the article "Pharmaceutical practice of clinical pharmacists participating in the treatment of a parturient woman infected with Peptostreptococcus anaerobius";

[0124] Legionella pneumophila can be the meaning of the term "Legionella pneumophila" recorded in the article "Influence of herpes virus infection on clinical prognosis and respiratory tract microecology of patients with severe pneumonia"; it can also be the meaning of the term "Legionella pneumophila" in the Baidu Baike entry;

[0125] The S. pneumoniae can be the S. pneumoniae described in the article "Preparation of S. pneumoniae capsular refined polysaccharide and degraded polysaccharide", or the S. pneumoniae described in the article "S. pneumoniae" in the Baidu Encyclopedia;

[0126] The human bocavirus 1 can be the human bocavirus 1 described in the article "Progress in molecular biology of human bocavirus 1", or the human bocavirus 1 described in the article "Human bocavirus 1" in the Baidu Encyclopedia;

[0127] The adenovirus 7 can be the adenovirus 7 described in the article "Monocyte percentage combined with CRP and PCT to identify influenza A virus and adenovirus infections", or the adenovirus 7 described in the article "Adenovirus 7" in the Baidu Encyclopedia;

[0128] The C. albicans can be the C. albicans described in the article "Research progress of chromosomal aneuploidy in fungal drug resistance mechanisms", or the C. albicans described in the article "Candida albicans" in the Baidu Encyclopedia;

[0129] The C. gattii can be the C. gattii described in the article "Isolation, identification and antibacterial activity of secondary metabolites of plant endophytic fungus Stagonosporopsis sp. YZHH-J-1", or the C. gattii described in the article "C. gattii" in the Baidu Encyclopedia;

[0130] The A. fumigatus can be the A. fumigatus described in the article "Research progress of chromosomal aneuploidy in fungal drug resistance mechanisms", or the A. fumigatus described in the article "A. fumigatus" in the Baidu Encyclopedia.

[0131] The genome size of the target pathogen is selected from the group consisting of: P. aeruginosa 6839777 bp, M. avium 4956752 bp, K. pneumoniae 5548441 bp, P. hirii 5226429 bp, B. fragilis 5234583 bp, S. pyogenes 1844942 bp, H. influenzae 1850809 bp, S. oralis 1931995 bp, S. aureus 2806340 bp, W. cibonii 2132186 bp, P. anaerobius 2192403 bp, L. pneumophila 3407565 bp, S. pneumoniae 2096425 bp; human bocavirus 1 5099 bp, adenovirus 7 35197 bp; C. albicans 14735515 bp, C. gattii 17527853 bp, A. fumigatus 28825722 bp;

[0132] (2) The nucleic acid of each target pathogen is added to the initial input volume of each target pathogen calculated in step (1) V pD1mixing, then adding human cell line genomic DNA, finally adding TE buffer (1x, pH 8.0) to make the total volume 1 mL; in some embodiments, the adding human cell line genomic DNA refers to adding 100 ng human cell line genomic DNA.

[0133] (3) taking 20%-40% volume of the sample, constructing library using metagenomic sequencing technology, sequencing and bioinformatics analysis to obtain the effective sequence number of each target pathogen r D1 ;

[0134] In some embodiments, the 20%-40% volume of the sample taken from the above sample can be adjusted according to the total volume of the sample, for example, if the total volume of the sample is 1 mL, then 300 μL of the sample can be taken from the above 1 mL.

[0135] In specific embodiments, the effective sequence number r D1 has the conventional technical meaning well known to those skilled in the art, for example, it can be the meaning of the term "effective sequence number" described in the article "Study on the diversity and antibacterial activity of S. japonica endophytic bacteria".

[0136] constructing library using metagenomic sequencing technology, sequencing and bioinformatics analysis to obtain the effective sequence number of each target pathogen r D1 This operation is a conventional technical operation well known to those skilled in the art, and the specific operation method can be referred to the method described in the article "Metagenomic sequencing".

[0137] (4) calculating the actual input volume of each target pathogen according to formula ( b ) V pD2 :

[0138] ( b )

[0139] formula ( b ) r D2 randomly assigned within 0-1000 and r D2 ≠0;

[0140] Two target pathogens: pathogen α and pathogen β each of which satisfies the conversion relationship of formula (c): r D2

[0141] ​​c )

[0142] formula( c )middle, r D2α Representative pathogen α The number of valid sequences; r D2β Representative pathogen β The number of valid sequences; p pD2α Representative pathogen α The relative abundance, p pD2β Representative pathogen β The relative abundance;

[0143] Select a specific pathogen and set the pathogen's [value] within the range of 0 to 1000. r D2 Values, due to the relative abundance of each pathogen p pD Once preset and fixed, the other pathogens can be calculated. r D2 Value (Formula c).

[0144] (5) Adjust the volume of each target pathogen's nucleic acid as calculated in step (4) and add it to the appropriate volume. V pD2 Mix, add 100 ng of human cell line genomic DNA, and finally add TE buffer (1×, pH 8.0) to obtain a DNA positive control. In some specific embodiments, the total volume of the DNA positive control is 1 mL.

[0145] In specific embodiments, the target pathogens are selected from: Pseudomonas aeruginosa, Mycobacterium avium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacteroides fragilis, Streptococcus pyogenes, Haemophilus influenzae, Streptococcus stomatologicus, Staphylococcus aureus, Veillonella spp., anaerobic pepsinophilic streptococci, Legionella pneumophila, Streptococcus pneumoniae; human bocavirus type 1, adenovirus type 7; Candida albicans, Cryptococcus grease, Aspergillus fumigatus;

[0146] Preferably, the genome size of the target pathogen is selected from the group consisting of Pseudomonas aeruginosa 6839777 bp, Mycobacterium avium 4956752 bp, Klebsiella pneumoniae 5548441 bp, Ochrobactrum anthropi 5226429 bp, Bacteroides fragilis 5234583 bp, Streptococcus pyogenes 1844942 bp, Haemophilus influenzae 1850809 bp, Streptococcus oralis 1931995 bp, Staphylococcus aureus 2806340 bp, Veillonella parvula 2132186 bp, Peptostreptococcus anaerobius 2192403 bp, Legionella pneumophila 3407565 bp, Streptococcus pneumoniae 2096425 bp; Human bocavirus type 1 5099 bp, Adenovirus type 7 35197 bp; Candida albicans 14735515 bp, Cryptococcus gattii 17527853 bp, Aspergillus fumigatus 28825722 bp;

[0147] The target pathogen genome and its preset relative abundance p pD As follows: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, 0.5% Streptococcus pneumoniae; 10% Human bocavirus type 1, 5% Adenovirus type 7; 4% Candida albicans, 4% Cryptococcus gattii, 4% Aspergillus fumigatus.

[0148] In some embodiments, in S1, the extracting bacterial genome nucleic acid and fungal genome nucleic acid comprises: selecting a suitable culture medium to culture bacteria or fungi respectively, collecting the bacteria or fungi in the logarithmic growth phase, extracting the genome DNA, and performing quality detection and quantification.

[0149] The above DNA extraction operation is a conventional operation for extracting nucleic acid of bacteria known to those skilled in the art, for example, in some embodiments, bacterial nucleic acid extraction uses the bacterial genome DNA extraction kit (DP302) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and fungal nucleic acid extraction uses the fungal genome DNA extraction kit (D2300-100) of Beijing Solaybao Technology Co., Ltd., and the DNA extraction operation can be performed according to the product instruction manual of these kits.

[0150] The virus genomic nucleic acid is obtained by in vitro synthesis of the full-length sequence of the target virus genome, recombination of the full-length sequence of the target virus genome with the Ad5 adenovirus backbone through a plasmid, amplification in BJ5183-AD-1 and Stbl3 bacteria, and then transfection of 293T cells for packaging to obtain DNA pseudovirus particles. The collected virus supernatant is subjected to nucleic acid extraction using a kit, and the quality is confirmed by multiple detections such as digital PCR. The operation is a routine operation known to those skilled in the art, and the virus nucleic acid extraction and digital PCR operation can be performed according to the method described in the article "A Quality Control Product for HIV-1 Genotype Drug Resistance Detection Based on Lentiviral Vector System and Its Preparation Method and Application".

[0151] The above virus genomic nucleic acid acquisition step is a routine technical operation known to those skilled in the art, and specifically, the steps can be performed according to the method described in the article "Choi, V.W., Asokan, A., Haberman, R.A. and Samulski, R.J. (2007), Production of Recombinant Adeno-Associated Viral Vectors for In Vitro and In Vivo Use. Current Protocols in Molecular Biology, 78: 16.25.1-16.25.24. https: / / doi.org / 10.1002 / 0471142727.mb1625s78 " can be referred to.

[0152] Preferably, in the DNA positive quality control product, the final concentration of nucleic acid is 100 ng / mL. The final concentration of nucleic acid here refers to the total concentration of bacterial genomic nucleic acid, fungal genomic nucleic acid, viral genomic nucleic acid, and human genomic nucleic acid.

[0153] In a further embodiment, the method for preparing a non-assigned composite quality control product for DNA pathogen metagenomic high-throughput sequencing further comprises the following step: S2. Preparing a negative quality control product: obtaining a virus-free or other exogenous microbial contaminated human cell line precipitate from a human cell line by washing 3-5 times. The washing is a routine technical operation known to those skilled in the art, and specifically, the washing can be performed according to the steps described in the article "ATCC Animal Cell Culture Guide (Revised in 2023)" Chapter "Cell Washing". Contamination and Biosafety " can be referred to.

[0154] Example 2, non-assigned composite quality control product of the present application

[0155] The embodiments of the present application provide a non-constant composite quality control product for DNA pathogen metagenomic high-throughput sequencing. All the embodiments of the present application have the following common features: prepared by the preparation method of any one of the first group of embodiments; the non-constant composite quality control product comprises: a DNA positive quality control product;

[0156] The DNA positive quality control product comprises: pathogenic genomic nucleic acid and human genomic nucleic acid;

[0157] The pathogenic genomic nucleic acid comprises: DNA virus genomic nucleic acid, bacterial genomic nucleic acid, and fungal genomic nucleic acid;

[0158] The bacterial genomic nucleic acid comprises the following components with the following relative abundances: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, and 0.5% Streptococcus pneumoniae; the relative abundance refers to the percentage of the effective reads number of the genome of a specific bacterium in the effective reads number of the pathogenic genomic nucleic acid contained in the DNA positive quality control product;

[0159] The DNA virus genomic nucleic acid comprises the following components with the following relative abundances: 10% human bocavirus type 1 and 5% adenovirus type 7; the relative abundance refers to the percentage of the effective reads number of the genome of a specific virus (human bocavirus type 1 or adenovirus type 7) in the effective reads number of the pathogenic genome contained in the DNA positive quality control product;

[0160] The fungal genomic nucleic acid comprises the following components with the following relative abundances: 4% Candida albicans, 4% Cryptococcus gattii, and 4% Aspergillus fumigatus; the relative abundance refers to the percentage of the effective reads number of the genome of a specific fungus in the effective reads number of the pathogenic genome contained in the DNA positive quality control product.

[0161] The microorganisms are clinically most concerned, and are usually pathogenic microorganisms that cause respiratory tract, blood stream or reproductive system infections; the relative abundance of the human genome is more than 99%, the sum of the relative abundance of the target pathogens incorporated in the quality control product is within 1%, and the sum of the two is close to 100%. The reason why it is not 100% is that the sequencing data also contains sequences of non-target microorganisms, which may come from environmental pollution, reagent pollution (such as microbial DNA in reagents), sample collection or processing pollution (such as skin colonization bacteria, microorganisms introduced during laboratory operation); the percentage values involved in the present application are preset values for the user to refer to. The quality control product is a non-certified quality control product, a non-standard product, which needs to be used by the user to accumulate target values through detection in the user's own detection system for more than 20 consecutive days. The non-certified composite quality control product of the present application can be used as the final product to be marketed, and the core innovation of the present application lies in the preparation method of the non-certified composite quality control product. The non-certified quality control product is characterized in that the manufacturer does not provide specific numerical values, but only marks the expected range or concentration gradient. The term "non-certified" has the conventional technical meaning commonly understood by those skilled in the art, for example, it can have the meaning of the term "non-certified" in "According to whether the labeled value of the analyte is given, the quality control product can be divided into certified quality control and non-certified quality control" in "I. Scope of application" of "Guiding Principles for the Registration and Review of Quality Control Products - Quality Control Product Valuation Research (No. 36 of 2022)" issued by the National Medical Products Administration.

[0162] The genomic nucleic acids in the non-certified composite quality control product of the present application are all whole genome nucleic acid sequences.

[0163] The use of the non-certified composite quality control product of the present application for metagenomic sequencing can make the host rate, i.e., the percentage of the number of reads aligned to the human genome in the total number of effective sequencing reads, more than 99%, and the percentage of the number of reads aligned to the pathogen genome in the total number of effective sequencing reads within 1%; the proportion of DNA sequences derived from humans in the sequencing data, i.e., (the number of reads aligned to the human genome / the total number of effective sequencing reads) x 100%.

[0164] In further embodiments, the non-certified composite quality control product for DNA pathogen metagenomic high-throughput sequencing further comprises a negative quality control product; the negative quality control product comprises a human cell line precipitate that is free of viral or other exogenous microbial contamination after 3-5 washes.

[0165] In specific embodiments, the human cell line refers to a cell line obtained by immortalizing human-derived cells that can be passaged in vitro;

[0166] In more specific embodiments, the human cell line precipitate contains 1x10 5 cells;

[0167] In preferred embodiments, the cell line is a cell line that is free of viral or other exogenous microbial contamination.

[0168] The present application provides a composite quality control product for DNA pathogen metagenome high-throughput sequencing and a preparation method thereof. The quality control product comprises: a DNA positive quality control (DPC) and a negative quality control (NC), which can be used to evaluate the detection performance of the whole process of DNA pathogen metagenome sequencing.

[0169] The technical solution points of the present application are as follows

[0170] I. Quality control product specifications and storage conditions

[0171] Quality control product name Specification Storage condition

[0172] DNA metagenome positive quality control 0.5mL / branch -20℃±5℃

[0173] Metagenome negative cell quality control 1*10 5 cells / branch -20℃±5℃

[0174] II. Quality control product composition

[0175] 1. DNA positive quality control (DPC)

[0176] • The design of this quality control product covers various pathogenic microorganisms such as DNA viruses, bacteria and fungi, which can cause infectious diseases of multiple systems and organs such as the respiratory system, the digestive system, the urogenital system and the skin soft tissue, and has wide clinical application value.

[0177] • DPC is a nucleic acid quality control product, which contains genomic nucleic acids of DNA viruses, bacteria and fungal pathogens, and human genomic nucleic acids.

[0178] • Each component is mixed according to a predetermined ratio, and the proportion of human nucleic acid in the total nucleic acid should reach more than 99%.

[0179] • In each component of DPC, the relative abundance of bacterial pathogens is set as Pseudomonas aeruginosa 15%, Mycobacterium avium 10%, Klebsiella pneumoniae 10%, Ochrobactrum anthropi 8%, Bacteroides fragilis 5%, Streptococcus pyogenes 5%, Haemophilus influenzae 5%, Streptococcus oralis 4.5%, Staphylococcus aureus 4%, Veillonella parvula 3%, Peptostreptococcus anaerobius 2%, Legionella pneumophila 1%, and Streptococcus pneumoniae 0.5%. The relative abundance of DNA viruses is set as human bocavirus type 1 10% and adenovirus type 7 5%. The relative abundance of fungal pathogens is set as Candida albicans 4%, Cryptococcus gattii 4%, and Aspergillus fumigatus 4%.

[0180] • The nucleic acid content of each pathogen in the quality control sample is precisely designed to decrease in a gradient from 15% relative abundance of P. aeruginosa to 0.5% relative abundance of S. pneumoniae, forming an ordered abundance difference, which facilitates the evaluation of the detection capability of the detection system for different concentrations of targets.

[0181] 2. Negative quality control (NC)

[0182] • Consists of cell pellets prepared from human cell lines.

[0183] • Used to simulate the complex nucleic acid background in human samples.

[0184] • Preferably a cell line without respiratory infection virus background.

[0185] • Preferably 1.0 x 10 5 cells per vial.

[0186] III. Innovation points of the preparation method of the quality control sample

[0187] 1. The preparation of DPC components uses optimized extraction and purification processes to ensure nucleic acid integrity.

[0188] 2. The ratio of each component is accurately calculated and verified, and an innovative gradient decrease design is used, with the nucleic acid content of different pathogens showing regular differences (DNA pathogens from 15% to 0.5%), which not only ensures the reliability of the detection results, but also comprehensively evaluates the detection capability of the detection system for different concentrations of targets.

[0189] 4. The batch difference of component ratio is small, with a coefficient of variation (CV) controlled within a reasonable range, showing good uniformity.

[0190] IV. Technical effects

[0191] 1. Simultaneous evaluation of DNA pathogen detection performance

[0192] 2. Simulate the nucleic acid composition characteristics of real clinical samples

[0193] 3. Good batch uniformity, with CV value controlled within a reasonable range

[0194] 4. Simple storage conditions, convenient transportation

[0195] The present application will be further described in detail below with specific examples.

[0196] Experimental Example 1. Preparation and detection of DNA metagenomic positive quality control (DPC)

[0197] 1. Preparation of DPC:

[0198] (1) Acquisition of bacterial and fungal nucleic acids: select appropriate culture medium to culture various pathogenic bacteria, collect bacterial cells in the logarithmic growth phase, extract genomic DNA, and perform quality testing and quantification.

[0199] (2) Acquisition of viral nucleic acids: obtain the full genome sequence of the virus by in vitro segmental synthesis, recombine each segment of the full genome with the Ad5 adenovirus backbone through a plasmid, then amplify in BJ5183-AD-1 and Stbl3 bacteria, and finally transfect 293T cells for packaging to obtain DNA pseudovirus particles. The collected viral supernatant is subjected to nucleic acid extraction using a kit, and the quality is confirmed through multiple tests such as digital PCR.

[0200] (3) Cultivation of LN229 cell lines, extraction of human genomic DNA, quality testing and quantification.

[0201] (4) Nucleic acid concentration determination using digital PCR detection system (product catalog number: 23053, Xin Yi Manufacturing Technology (Beijing) Co., Ltd. The reaction system is 30 μL, containing 2x Probe dPCR SuperMix (no UNG) 15 μL, bidirectional primers (10 μM) 2.4 μL each, probe (10 μM) 0.75 μL, DNA template and ddH2O 9.45 μL. PCR reaction program: 95℃ 10min, 95℃ 30s, 60℃ 1min, 40 cycles, 12℃ 5min. Bidirectional primers and probes are designed and synthesized according to the genomic sequence of the target pathogen to be detected by the skilled person in the art and reference the following literature:

[0202] Edwards RL, Takach JE, McAndrew MJ, Menteer J, Lestz RM, Whitman D, Baxter-Lowe LA. Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design. Front Genet. 2023 Nov 10;14:1272964. doi: 10.3389 / fgene.2023.1272964. PMID: 38028620; PMCID: PMC10667681.

[0203] Wadle S, Lehnert M, Rubenwolf S, Zengerle R, von Stetten F. Real-time PCR probe optimization using design of experiments approach. Biomol Detect Quantif. 2015 Dec 30;7:1-8. doi: 10.1016 / j.bdq.2015.12.002. PMID: 27077046; PMCID: PMC4827641.

[0204] Qiagen digital PCR detection development guide (https: / / www.qiagen.com / zh-cn / applications / digital-pcr / beginners / dpcr-guide / setup-and-troubleshooting)

[0205] In practice, the entire operation of the above digital PCR can also be entrusted to commercial biological companies (for example, Xinyi Manufacturing Technology (Beijing) Co., Ltd.). The design and synthesis of bidirectional primers and probes, which can be routinely done by those skilled in the art, are not described in detail here due to space constraints.

[0206] (5) According to the quantitative results of digital PCR, mix each component according to the preset proportion, and the proportion of host human nucleic acid should reach more than 99%, and the final concentration of DPC nucleic acid is 100 ng / mL. The relative abundance of bacterial pathogens is set as Pseudomonas aeruginosa 15%, Mycobacterium avium 10%, Klebsiella pneumoniae 10%, Ochrobactrum anthropi 8%, Bacteroides fragilis 5%, Streptococcus pyogenes 5%, Haemophilus influenzae 5%, Streptococcus oralis 4.5%, Staphylococcus aureus 4%, Veillonella parvula 3%, Anaerobic peptococcus 2%, Legionella pneumophila 1%, Streptococcus pneumoniae 0.5%. The relative abundance of DNA viruses is set as human bocavirus type 1 10% and adenovirus type 7 5%. The relative abundance of fungal pathogens is set as Candida albicans 4%, Cryptococcus gattii 4%, and Aspergillus fumigatus 4%. According to the length of each pathogen genome, the normalization proportion in DPC is carried out, and the target sequence number after normalization is set to 100-1000.

[0207] (6) The prepared DPC is divided into aliquots and stored at -20℃±5℃.

[0208] 2. DPC operation procedure:

[0209] (1) DNA macro genome positive quality control was thawed at room temperature for 15 minutes, then vortexed and centrifuged;

[0210] (2) 300 μL was taken and added to a 1.5 mL centrifuge tube containing UMSI;

[0211] (3) The lysis reaction solution was prepared, and the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH, Beijing, China) was used for DNA extraction and purification;

[0212] (4) The Qubit 4.0 Fluorescence Quantifier (Invitrogen, USA) was used for DPC nucleic acid concentration determination;

[0213] (5) After concentration determination, nuclease-free water was added to homogenize the DPC concentration to 1.2 ng / μL;

[0214] (6) After homogenization, the genomic fragmentation kit (VM008-50, Guangzhou Microfar Medical Instrument Co., Ltd.) was used to break the DPC nucleic acid;

[0215] (7) The DPC broken nucleic acid was added with index and PCR reagent (provided by Guangzhou Microfar Medical Instrument Co., Ltd.) to complete the library construction;

[0216] (8) Magnetic bead screening fragments, using Qubit 4.0 Fluorescence Quantifier for DPC library concentration determination;

[0217] (9) Mixed with other libraries, diluted;

[0218] (10) Sequencing on NextSeq 550Dx sequencer (Illumina, USA).

[0219] Part 2 "DPC operation procedure" is the operation steps of quality control of laboratory daily detection with quality control, which is irrelevant to the preparation of quality control.

[0220] 3. DPC inter-bottle uniformity evaluation

[0221] (1) Randomly selected 6 DPCs, 1 per day, for 6 consecutive days.

[0222] (2) According to the results of continuous monitoring, the inter-bottle uniformity of DPC was evaluated. The data volume, host rate, sequence number, relative abundance of quality control were recorded, and the sequence number, relative abundance and mean and CV were calculated. Relative abundance is the number of single target sequences of the pathogen / total target sequences.

[0223] (3) All the quality control indicators of metagenomic sequencing are qualified. Table 1 shows that the detection results of the positive quality control show that all the pathogenic microorganisms expected to be contained are detected; Tables 2 and Figure 1 shows that the sequencing results (all measured values in Table 2) are basically similar to the preset relative abundance of the flora, the CV (coefficient of variation) values of 6 repeated experiments are within the acceptable range, the inter-bottle uniformity is qualified, and can be used as DNA positive quality control for metagenomic high-throughput sequencing. Figure 2 shows the mean value of the measured values of 6 repeated experiments and the preset value, and the fitting degree of the two lines is very high. The relative abundance of the quality control is as expected, which can prove the accuracy of the preparation of DPC.

[0224]

[0225]

[0226] Experimental Example 2. Preparation and detection of negative quality control (NC)

[0227] 1. Preparation of NC:

[0228] (1) The human cell line refers to a cell line that can be passaged in vitro after being obtained by immortalizing cells of human origin, preferably a cell line without respiratory tract infection virus background.

[0229] (2) Cell precipitate refers to a cell that is removed from the culture medium and is free of contamination and is cryopreserved. The cell precipitate is selected from a cell that is free of virus or other exogenous microbial contamination and is washed for multiple times (3-5 times) and is stored at -80 degrees Celsius or lower temperature for long-term storage.

[0230] (3) The number of cells contained in the cell precipitate is consistent with the number of cells obtained from human respiratory samples, preferably the sample contains 1x10 5 cells (counted using a cell counting plate, repeated counting 3 times, and the number of cells in the quality control can be controlled at this level).

[0231] 2. Metagenomic negative cell quality control (DNC) operation procedure:

[0232] (1) A tube of NC is centrifuged at low speed, 500 μL of physiological saline is added, and is ready for use;

[0233] (2) The above NC with physiological saline is taken 300 μL according to the DPC operation procedure to extract the library;

[0234] (3) DNC is obtained from the above (1) NC.

[0235] Part 2: Operation procedure of macro-genome negative cytoplasmic control (DNC) This is the operation procedure of using the control to control the daily detection of the laboratory, which is irrelevant to the preparation of the control.

[0236] 3. Inter-bottle uniformity evaluation of NC

[0237] The data volume, host rate, number of detected sequences, relative abundance, etc. of the control are registered, and the number of sequences, relative abundance, mean and CV are calculated. The detection results of the macro-genome sequencing quality control indicators are all qualified, the expected pathogenic microorganisms in the positive control are not detected in the negative control, which meets the use requirements of the negative control, and the specificity can be proved without detecting the pathogens. The NC does not need to evaluate the uniformity and accuracy.

Claims

1. A method for preparing a non-assigned composite quality control for DNA pathogen metagenomic high-throughput sequencing, characterized in that, The preparation method comprises the following steps: S1. Preparation of DNA positive quality control: (1) Extracting bacterial genomic nucleic acid and fungal genomic nucleic acid; (2) Obtaining viral genomic nucleic acid; (3) Extracting human genomic nucleic acid; (4) Mixing the following relative abundance per milliliter of DNA positive quality control: 1% target pathogen genome, 99% human genome; the target pathogen genome comprises: bacterial genomic nucleic acid, fungal genomic nucleic acid, viral genomic nucleic acid; (5) the target pathogen genome is in a preset relative abundance p pD mixing; The mixing comprises the following steps: 1) Use the formula ( a Calculate the initial input volume for each target pathogen. V pD1 （ a ） Formula (I) a ) wherein, g p represents the genome size of the target pathogen; mass volume concentration of the target pathogen nucleic acid; 2) Add the nucleic acid of each target pathogen to the initial input volume of each target pathogen calculated in step 1) V pD1 Mix, add human cell line genomic DNA, and finally add TE buffer to form the sample; 3) 20%-40% volume of the sample is taken from the above sample, a library is constructed using metagenomic sequencing technology, sequencing is performed and bioinformatics analysis is performed to obtain the effective sequence number of each target pathogen r D1 ; 4) Calculate the actual volume of input for each target pathogen according to the formula (Vactual = Vtarget x (1 + %error) b V pD2 :​ （ b ） In formula (I), b ) wherein, r D2 is randomly assigned within 0~1000 and r D2 ≠0; Two target pathogens: pathogenic α and pathogenic β each r D2 satisfy the conversion relationship of the following formula (c): （ c ） formula( c )middle, r D2α Representative pathogen α The number of valid sequences; r D2β Representative pathogen β The number of valid sequences; p pD2α Representative pathogen α The relative abundance, p pD2β Representative pathogen β The relative abundance; 5) Adjust the nucleic acid of each target pathogen to the volume calculated in step 4) for each target pathogen V pD2 Mix, add human cell line genomic DNA, and finally add TE buffer to obtain DNA positive quality control; The genome size of the target pathogen is selected from the group consisting of: Pseudomonas aeruginosa 6839777 bp, Mycobacterium avium 4956752 bp, Klebsiella pneumoniae 5548441 bp, Ochrobactrum anthropi 5226429 bp, Bacteroides fragilis 5234583 bp, Streptococcus pyogenes 1844942 bp, Haemophilus influenzae 1850809 bp, Streptococcus oralis 1931995 bp, Staphylococcus aureus 2806340 bp, Veillonella parvula 2132186 bp, Peptostreptococcus anaerobius 2192403 bp, Legionella pneumophila 3407565 bp, Streptococcus pneumoniae 2096425 bp; human bocavirus type 1 5099 bp, adenovirus type 7 35197 bp; Candida albicans 14735515 bp, Cryptococcus gattii 17527853 bp, Aspergillus fumigatus 28825722 bp; The target pathogenic genome and its preset relative abundance p pD As follows: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, 0.5% Streptococcus pneumoniae; 10% Human Bocavirus type 1, 5% Adenovirus type 7; 4% Candida albicans, 4% Cryptococcus gattii, 4% Aspergillus fumigatus; In S1, the extracting bacterial genomic nucleic acid and fungal genomic nucleic acid comprises: selecting a suitable culture medium to culture bacteria or fungi respectively, collecting the bacteria in the logarithmic growth phase, extracting genomic DNA, and performing quality detection and quantification; Obtaining viral genomic nucleic acid comprises: in vitro synthesis to obtain the full-length sequence of the genome of the target virus, recombining the full-length sequence of the genome of the target virus with an Ad5 adenovirus backbone through a plasmid, amplifying in BJ5183-AD-1 and Stbl3 bacteria, and then transfecting 293T cells to package DNA pseudo-virus particles and then extracting nucleic acid from the DNA pseudo-virus particles.

2. The method for preparing a non-fixed-value composite quality control for high-throughput metagenomic sequencing of DNA pathogens according to claim 1, characterized in that, The target pathogen is selected from the group consisting of: Pseudomonas aeruginosa, Mycobacterium avium, Klebsiella pneumoniae, Ochrobactrum anthropi, Bacteroides fragilis, Streptococcus pyogenes, Haemophilus influenzae, Streptococcus oralis, Staphylococcus aureus, Veillonella parvula, Peptostreptococcus anaerobius, Legionella pneumophila, Streptococcus pneumoniae; human bocavirus type 1, adenovirus type 7; Candida albicans, Cryptococcus gattii, Aspergillus fumigatus.

3. The method for preparing a non-fixed-value composite quality control for high-throughput DNA pathogen metagenomic sequencing according to claim 1, characterized in that, In the DNA positive quality control, the final concentration of nucleic acid is 100 ng / mL.

4. The method for preparing a non-assayed composite quality control for high-throughput sequencing of DNA pathogen metagenome according to any one of claims 1-3, characterized in that, Further comprising the following step: S2. Preparation of negative quality control: virus-free or other foreign microorganism-polluted human cell line precipitate of human cell line washed for 3-5 times.

5. A non-valued composite quality control for DNA pathogen metagenomic high-throughput sequencing, characterized in that, Prepared by the preparation method in any one of claims 1-4; the non-fixed value composite quality control comprises: DNA positive quality control; The DNA positive quality control comprises: pathogenic genomic nucleic acid and human genomic nucleic acid; The pathogenic genomic nucleic acid comprises: DNA viral genomic nucleic acid, bacterial genomic nucleic acid, and fungal genomic nucleic acid.

6. The non-assayed composite quality control for high-throughput sequencing of DNA pathogen metagenome according to claim 5, wherein, The bacterial genomic nucleic acid comprises each component with the following relative abundance: 15% Pseudomonas aeruginosa, 10% Mycobacterium avium, 10% Klebsiella pneumoniae, 8% Ochrobactrum anthropi, 5% Bacteroides fragilis, 5% Streptococcus pyogenes, 5% Haemophilus influenzae, 4.5% Streptococcus oralis, 4% Staphylococcus aureus, 3% Veillonella parvula, 2% Peptostreptococcus anaerobius, 1% Legionella pneumophila, 0.5% Streptococcus pneumoniae; The DNA viral genomic nucleic acid comprises each component with the following relative abundance: 10% Human Bocavirus type 1, 5% Adenovirus type 7; The fungal genomic nucleic acid comprises each component with the following relative abundance: 4% Candida albicans, 4% Cryptococcus gattii, 4% Aspergillus fumigatus.

7. The non-assayed composite quality control for high-throughput sequencing of DNA pathogen metagenome according to claim 5, characterized in that, The non-fixed composite quality control product further comprises a negative quality control product; the negative quality control product comprises a human cell line sediment which is free of viruses or other exogenous microbial contamination and which has been washed for 3-5 times.

8. The non-assayed composite quality control for high-throughput sequencing of DNA pathogen metagenome according to claim 7, characterized in that, The human cell line refers to a cell line which can be passaged in vitro and which is obtained by immortalizing cells of human origin.

9. The non-assayed composite quality control for high-throughput sequencing of DNA pathogen metagenome according to claim 7, characterized in that, Human cell line pellets contain 1 x 10 5 cells.

10. The non-assayed composite quality control for high-throughput sequencing of DNA pathogen metagenomes according to any one of claims 7 to 9, characterized in that, The cell line is a cell line which is free of viruses or other exogenous microbial contamination. The cell line is a cell line which is free of viruses or other exogenous microbial contamination.

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