CN112662795A - Positive control for infectious pathogen detection and preparation method and application thereof - Google Patents
Positive control for infectious pathogen detection and preparation method and application thereof Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a positive control substance for detecting infectious pathogens and a preparation method and application thereof, and the positive control substance comprises a first positive control substance and a second positive control substance, wherein the pathogens in the first positive control substance comprise gram-positive bacteria such as lactobacillus fermentum, clostridium, bacillus subtilis, staphylococcus epidermidis, listeria welshimeri, gram-negative pseudomonas fluorescens, klebsiella aerogenes and escherichia coli; fungi aspergillus niger, saccharomyces cerevisiae and bacteriophage T1, and the pathogen nucleic acid in the second positive control comprises bacteria staphylococcus epidermidis, fungi aspergillus niger, virus herpes simplex virus, bacteriophage T1, virus adeno-associated virus, virus rotavirus, virus respiratory syncytial virus, virus vesicular stomatitis virus and bacteriophage MS 2. The invention provides a convenient, safe and directly usable dual reference standard, and simultaneously carries out positive reference from different angles, thereby ensuring the accuracy and stability of the detection process of infectious disease metagenome.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to a positive control substance for infectious pathogen detection, and a preparation method and application thereof.
Background
Infectious diseases are major diseases in today's world that seriously threaten human health. At present, infectious disease detection faces the following pain points: firstly, the infection is caused by a plurality of pathogens, the pathogenic cause is unknown, and the diagnosis is difficult; secondly, drug resistance information cannot be accurately acquired, so that the difficulty in medication is caused; thirdly, the traditional detection method has the limitations of long time consumption, difficult culture of part of pathogens, low sensitivity, window period, limited detection quantity and the like.
In recent years, the rapidly developed NGS (high throughput sequencing) technology does not depend on a known nucleic acid sequence, does not need special probe design, can directly detect unknown pathogenic microorganisms, and the mNGS (metagenomic sequencing) does not depend on traditional microorganism culture, directly carries out high throughput sequencing on nucleic acid in a clinical sample, can rapidly and objectively detect the pathogenic microorganisms in the clinical sample, breaks through the limitation of traditional microorganism detection, and shows wide prospects in the field of clinical microorganisms.
However, the pathogenic pathogens of infectious diseases are various, including bacteria, viruses, fungi, parasites, etc., and due to the different structures of pathogens, such as the different cell wall thicknesses of gram-positive and gram-negative bacteria, the difference in the size and content of pathogens, and the difference in the types of nucleic acids (single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA) of pathogens, the detection of pathogens is difficult, and the non-standardized treatment can cause the deviation of the detection result of pathogens and even the false negative and false positive results.
At present, the infectious pathogen detection of the NGS platform needs strict quality control standards to ensure the stability of the detection method. The quality control of the technology depends on the setting of a standard substance, the preparation of a pathogen standard substance is complex, the operation risk is high (generally requiring a laboratory with more than BSL-2 level), and the pathogen detection technology has instability, so that at present, no mature and stable commercialized standard substance aiming at clinical needs exists.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a positive control for detecting infectious pathogens, and a preparation method and applications thereof.
The purpose of the invention is realized by the following technical scheme:
a positive control for detecting infectious pathogens, comprising a first positive control, wherein the pathogens in the first positive control comprise gram-positive bacteria including lactobacillus fermentum, clostridium, bacillus subtilis, staphylococcus epidermidis, listeria welshimeri, pseudomonas fluorescens, klebsiella aerogenes, escherichia coli, fungi aspergillus niger, saccharomyces cerevisiae and bacteriophage T1.
Preferably, the copy number mixing ratio of the pathogen in the first positive control is as follows, gram-positive bacteria lactobacillus fermentum, clostridium sporogenes, bacillus subtilis and staphylococcus epidermidis: the Listeria welshii, gram negative Pseudomonas fluorescens, Klebsiella aerogenes, Escherichia coli, fungi Aspergillus niger Saccharomyces cerevisiae, and bacteriophage T1 were 16.44:16.44:16.44:16.44:5.48:5.48:5.48:0.55:0.55: 0.27.
Preferably, the control further comprises a second positive control whose pathogen nucleic acid comprises: staphylococcus epidermidis; fungus Aspergillus niger; herpes simplex virus; adeno-associated virus, rotavirus, respiratory syncytial virus, vesicular stomatitis virus, bacteriophage T1, bacteriophage MS 2.
Preferably, the second positive control has a pathogen genomic structure comprising: DNA double strand, DNA single strand, RNA double strand, and RNA single strand.
Preferably, the ratio of the mixed copy number of the pathogen nucleic acid in the second positive control is staphylococcus epidermidis: fungus Aspergillus niger: herpes simplex virus: adeno-associated virus: rotavirus: respiratory syncytial virus: vesicular stomatitis virus: bacteriophage T1: phage MS2 was 2:1:2:2:2:2:2:2: 2.
Preferably, the ratio of pathogen genome structure in the second positive control is DNA double strand: DNA single strand: RNA double-stranded: the RNA single strand is 7:2:2: 6.
Preferably, the preparation of the first positive control comprises the following steps of mixing according to the proportion of pathogens, adding a thallus lysate, and incubating for 20-40 minutes at 50-70 ℃; the composition of the thallus lysate is a mixed solution of Tris-HCl, EDTA, guanidinium isothiocyanate, LiCl and SDS, wherein the pH value of the Tris-HCl is 9.
Preferably, the positive control is applied to include, but is not limited to, a sample for microbial detection, simulated clinical use.
Preferably, the positive control is applied by methods including, but not limited to, high-throughput sequencing, fluorescent quantitative PCR detection, multiplex PCR and pathogen probe capture detection.
Preferably, when the positive control is applied to microbial detection, the first positive control is processed, and the steps comprise adding lysozyme, incubating at room temperature, and digesting after adding proteinase K; mechanically crushing by using an MP tissue crusher; the pathogens in the second positive control are divided into two groups according to the types of nucleic acid genomes, wherein the two groups are respectively a first group consisting of DNA double-stranded staphylococcus epidermidis, DNA double-stranded aspergillus niger, DNA double-stranded herpes simplex virus and DNA double-stranded bacteriophage T1, and a second group consisting of DNA single-stranded adeno-associated virus, RNA double-stranded rotavirus, RNA single-stranded respiratory syncytial virus, RNA single-stranded vesicular stomatitis virus and RNA single-stranded bacteriophage MS 2.
The invention has the following outstanding effects: the invention provides a convenient, safe and directly usable dual-control standard substance, and simultaneously carries out positive standard control from two angles, thereby ensuring the accuracy and stability of the detection process of infectious disease metagenome.
The following detailed description of the embodiments of the present invention is provided in connection with the accompanying drawings for the purpose of facilitating understanding and understanding of the technical solutions of the present invention.
Drawings
FIG. 1 is a schematic diagram of the standard curve set-up of library yield stability analysis T1.
FIG. 2 is a schematic diagram of a library yield stability analysis MS2 standard curve set-up.
Detailed Description
The invention discloses a positive control substance for infectious pathogen detection and a preparation method and application thereof.
Pathogen origin, culture and preservation
1) The desired pathogen is derived from a strain already studied in the laboratory or purchased from ATCC:
2) the required pathogen nucleic acid is extracted after the pathogen is cultured, and the pathogen nucleic acid can be directly purchased:
3) pathogen culture
A bacteria and fungi
Culturing: the culture is carried out under optimal culture conditions (medium, temperature, culture time, etc.) according to the pathogen specification. The culture time should be kept in the late growth phase because cells in the logarithmic growth phase are less resistant to freeze-drying. Culturing bacteria for 24-48 h, and scraping off lawn or spore under aseptic condition to obtain bacterial suspension.
Calculating the number of cells: recovering bacteria/fungi with specific culture medium, culturing to passable concentration, sucking 1mL of mixed bacteria/spore suspension with sterile pipette tip under aseptic condition, adding 9mL of normal saline, and mixing to obtain 10%-1Diluting to 10 degrees by the same method-6. Pouring 10-15mL of corresponding culture medium which is melted and cooled to about 45 ℃ into the culture dish containing bacterial liquid (0.2mL) with different dilutions, placing the culture dish in a horizontal position, quickly rotating and uniformly mixing the culture dish, and after solidification, placing the culture dish in an incubator at 37 ℃ for culture. After incubation for the corresponding time (e.g., 24h), the plates are removed and the average number of colonies on the three plates at the same dilution is calculated.
The following formula is used for calculation:
colony forming units (cfu) per ml-average number of colonies of triplicates at the same dilution x 5
B phage (T1, MS2)
Culturing: escherichia coli is used as a phage host, cultured at a proper temperature by a liquid culture method, and incubated with shaking until the middle logarithmic growth phase (OD 600-0.5). Dipping a small amount of phage into a liquid culture medium containing escherichia coli, placing the liquid culture medium at 37 ℃ for shaking culture until bacterial suspension in a test tube turns clear from turbidity, centrifuging the culture (8000rpm/30min), taking supernatant, filtering the supernatant by a 0.22um microporous membrane, and storing the supernatant at-20 ℃ for later use.
And (3) determining the titer of the phage: .
Firstly, phage dilution:
the 4 tubes containing 0.9ml of liquid medium are labeled 10-3, 10-4, 10-5 and 10-6, respectively.
0.1ml of 10-2 phage is absorbed by a 1ml sterile pipette, injected into a 10-3 test tube, and the test tube is shaken by rotation and mixed evenly.
Sucking 0.1ml from 10-3 tubes with another sterile pipette, adding into 10-4 tubes, mixing, and diluting to 10-6 tubes.
② mixing bacteriophage with bacterial solution
5 sterilized empty tubes are labeled 10-4, 10-5, 10-6, 10-7 and control, respectively.
0.1ml of the solution is sucked by a pipette from a 10-3 phage dilution tube and is added into a 10-4 empty test tube, and 0.1ml of the solution is sucked by another pipette from the 10-4 phage dilution tube and is added into a 10-5 empty test tube till reaching a 10-7 tube.
Shaking the host bacteria culture solution uniformly, taking 0.9ml of the bacteria solution by using a suction pipe, adding into a control test tube, sucking 0.9ml of the bacteria solution, adding into a 10-7 test tube, and adding from the last tube to 10-4 tubes, wherein 0.9ml of the escherichia coli culture solution is added into each tube.
The test tubes were shaken and mixed well.
③ the mixed liquid is added into the upper layer culture medium
The 5 tubes of upper medium were thawed and labeled 10-4, 10-5, 10-6, 10-7 and control. Cooling to 48 deg.C, and placing into 48 deg.C water bath box.
The 4 tubes of the mixture and the control tube were added in the upper medium test tube in duplicate, respectively. The mixture was immediately added to each tube and vortexed and mixed.
Fourthly, the inoculated upper layer culture medium is poured on a bottom layer flat plate
Quickly pouring the upper culture medium which is uniformly rotated in number onto the bottom flat plate, and uniformly shaking the upper culture medium on a table top to enable the upper culture medium to be fully paved on the flat plate.
After coagulation, the cells were inverted and cultured at 37 ℃.
Observing the plaques in the plate, and selecting the plate with 30-300 pfu numbers to calculate the number of the phage (titer) in each milliliter of undiluted stock solution. The titer of phage per ml, plaque forming units (pfu), was obtained by multiplying the number of plaques by the dilution.
C virus (herpes simplex virus HSV, adeno-associated virus AAV, vesicular stomatitis virus VSV) culture and preservation:
the herpes simplex virus HSV-2:
a.10% Fetal Bovine Serum (FBS) in DMEM complete medium to culture Vero cells to a plate density of 90%;
b. discarding original culture medium, adding 2% FBS EMEM for culture, adding HSV virus (optimum MOI: 0.1) in 5% CO2Infecting in an incubator (37 ℃) for 1-2h, discarding the supernatant again, and replacing with new culture medium (EMEM with 2% FBS);
c. observing that most of the cells die and float after 2-5 days of infection, collecting cell supernatant, and subpackaging and freezing at-80 deg.C.
② adeno-associated virus AAV-2:
a.10% Fetal Bovine Serum (FBS) in DMEM complete medium to culture HEK-293 cells to 80-90% density on dishes;
b. discarding original culture medium, adding 2% FBS EMEM culture, adding AAV virus and helper virus (optimum MOI: 1) in 5% CO2Infecting in an incubator (37 ℃) for 1-2h, discarding the supernatant again, and replacing with new culture medium (EMEM with 2% FBS);
c. observing cell death and floating 2-3 days after infection, collecting cell supernatant, and freezing at-80 deg.C
③ vesicular stomatitis virus VSV:
a. culturing VERO cells to reach the density of 80-90% by using a DMEM complete culture medium of 10% Fetal Bovine Serum (FBS);
b. changing the culture medium to DMEM medium containing 5ml of 2% serum, adding VSV virus (MOI of about 1), plating and infecting at 37 ℃ for 1h, discarding the supernatant after 1h, washing once with PBS, and changing to DMEM medium containing 5ml of 2% serum again;
C. observing that most of the cells die and float after 18-24 hours of infection, collecting cell supernatant, and subpackaging and freezing at-80 ℃ in a refrigerator.
The first positive control provided in this example contains pathogens with different lysis difficulties, particularly increasing the proportion of gram-positive bacteria, and the specific composition and proportion are shown in table 1:
table 1: the first positive control contains a control table of pathogen and cell ratios.
After each pathogen was cultured and counted, each pathogen microorganism was diluted to the desired number of cells using PBS and dispensed into sterile centrifuge tubes for storage.
When the first positive control is prepared, a diluted pathogen is taken according to the pathogen types in the table 1, respectively, blown and uniformly mixed, all the pathogens are mixed into the same sterile centrifuge tube, and the volume is measured.
The lysate was pipetted into the centrifuge tube containing the first positive control mixed pathogen to make the total volume of the first positive control 1mL, and incubated at 60 ℃ for 30 minutes. This first positive control can be used 100 times in total.
The first positive control was aliquoted at 100. mu.L/serving and stored at-80 ℃ for a long period of time.
When in use, one part of the first positive control is taken and equally divided into 10 mu L/part, 10 mu L of the first positive control is used each time, and the rest is stored at-80 ℃.
The selected formulation of the above lysates is shown in table 2:
table 2: and (4) a formula table of the lysis solution.
| Numbering | Lysate formula |
| L1 | 100mM Tris-HCl (pH 9),40mM EDTA,4M guanidinium isothiocyanate |
| L2 | 500mM NaCl，50mM Tris-HCl，50mM EDTA，4％SDS |
| L3 | 100Tris-HCl (pH 9),40mM EDTA,4M guanidinium isothiocyanate, 3.5M LiCl, 3% SDS |
After the pathogens were mixed according to the amount of cells required for the first positive control, they were blown up and mixed well, and 1/20 volumes were removed before adding the lysate, and aliquoted into 3 portions.
700. mu.L of lysate L1, lysate L2, and lysate L3 were added, respectively.
Add 20. mu.g/. mu.L proteinase K and digest for 30min at 55 ℃.
Physical disruption was performed using MP tissue disruptor set at 6m/s, 20 s/time, 3 times/sample.
Pathogen DNA extraction was performed using the Kit AllPrep DNA/RNA Kit (Qiagen, cat # 80204).
Taking 1ng of DNA, constructing a sequencing library by using a transposase method, sequencing by using an Illumina Novaseq sequencer, and generating 6Gb raw data of each sample.
Using Trimmomatic software to perform quality control on the original Data to obtain effective Data (clear Data), using Soap software to compare the effective Data with a host genome sequence, removing the host Data, further improving the analysis efficiency, performing species annotation on the final effective Data through MetaPhlAn2, and then counting species annotation results. And counting pathogen information through species annotation.
Species annotation results are shown in table 3:
table 3: the deviations of the different lysate formulations for the first positive control are compared to the table.
As described above, the lysate of the formula L3 is preferable as a lysate for cell preservation and nucleic acid extraction of the first positive control.
To better reflect the selection of pathogen treatment conditions, screening for the corresponding experiments was performed as follows:
table 4: different treatment regimens were performed on the pathogens.
The first positive control, which had been prepared, was removed in 7 portions and 700. mu.L of lysate was added.
Pathogen treatment was performed according to the different treatment conditions in table 4, respectively.
Pathogen DNA extraction was performed using the Kit AllPrep DNA/RNA Kit (Qiagen, cat # 80204).
Taking 1ng of DNA, constructing a sequencing library by using a transposase method, sequencing by using an Illumina Novaseq sequencer, and generating 6Gb raw data of each sample.
Using Trimmomatic software to perform quality control on the original Data to obtain effective Data (clear Data), using Soap software to compare the effective Data with a host genome sequence, removing the host Data, further improving the analysis efficiency, performing species annotation on the final effective Data through MetaPhlAn2, and then counting species annotation results. And counting pathogen information through species annotation.
Pathogen treatment results are shown in table 5:
table 5: the pathogen treatment results are tabulated.
From the above results it was determined that the pathogen treatment regime is preferably: lysozyme (3mg/mL), room temperature 10 minutes, proteinase K (20. mu.g/. mu.L), 55 ℃ digestion for 30 minutes, MP tissue disruptor (6m/s, 30 s/time, 4 times/sample).
The second positive control provided by the invention is used for supplementing and perfecting the first positive control, under the condition of ensuring that the high-difficulty pathogen nucleic acid is completely obtained, 4 forms of pathogen genome structures are further supplemented, and the real pathogen nucleic acid is matched to simulate the nucleic acid complexity of a clinical sample.
Nucleic acids of Staphylococcus epidermidis, Aspergillus niger, bacteriophage T1, bacteriophage MS2 were extracted using the Kit AllPrep DNA/RNA Kit (Qiagen, cat No. 80204). Nucleic acids of herpes simplex Virus, adeno-associated Virus and vesicular stomatitis Virus were extracted using the QIAamp UltraSens Virus Kit (Qiagen, cat # 53704). Both rotavirus and respiratory syncytial virus nucleic acids are purchased from ATCC. Pathogen nucleic acid copy number was detected using digital PCR. Wherein the desired copy number ratios of pathogen nucleic acids are, for example, as shown in Table 6:
table 6: pathogen nucleic acid information table.
To better select the grouping configuration for the second positive control, the genome structures in table 6 were grouped according to the detection method, as shown in table 7.
Table 7: nucleic acid gene component classes in the second positive control are shown schematically.
Creating libraries by N1-1 and N2-1: taking 1ng of DNA, constructing a sequencing library by using a transposase method, sequencing by using an Illumina Novaseq sequencer, and generating 6Gb raw data of each sample.
Creating libraries by N2-1 and N2-2: 10pg of RNA were taken and library construction was performed using the Ovation solo RNA-seq system kit (Tecan, cat No. 0500-32) yielding 10Gb of raw data per sample.
Sequencing data were analyzed for species annotation. Pathogen nucleic acid sequencing data results are shown in table 8:
table 8: pathogen nucleic acid sequencing data results table.
From the above results, it was determined that the DNA banking method resulted in low detection of single stranded DNA pathogens when DNA single strands were combined with DNA double strands; when the DNA single strand and RNA are simultaneously banked, the detection of single-stranded DNA pathogens can be improved. The pathogen nucleic acid grouping from which the second positive control is determined is preferably: classification of N1 pathogen nucleic acids were pooled in groups according to N1, which is a second positive control.
Clinical specimen simulation processing
According to the grouping treatment of the second positive control, in order to ensure the completeness and accuracy of pathogen detection, the clinical sample needs to pay attention to the treatment mode of DNA single-stranded pathogens. The 9 pathogens in the table were mixed to simulate the complexity of the clinical specimen, based on the pathogen nucleic acid copy number requirements of the second positive control.
The clinical sample simulation treatment regime is shown in table 9:
table 9: the clinical samples simulate the treatment mode.
The mixed mock clinical samples were divided into 3 aliquots and DNA and RNA were extracted simultaneously using the Kit AllPrep DNA/RNA Kit (Qiagen, cat # 80204). Wherein the DNA and RNA of S1 are preserved separately, and the DNA and RNA of S2 and S3 are mixed.
And S2 sample processing: the S2 sample was divided into 2 portions and treated with DNase I and RNAse A, respectively, to give S2-RNA and S2-DNA.
And S3 sample processing: s3 samples were treated with HL-dsDNase to obtain S3-RNA.
Library construction of S1-DNA, S2-DNA using the transposase method, sequencing by Illumina Novaseq sequencer, and 6Gb raw data per sample.
S1-RNA, S2-RNA and S3-RNA were library construction using the kit Ovation solo RNA-seq system (Tecan, cat No. 0500-32), yielding 10Gb of raw data per sample.
Sequencing data were analyzed for species annotation. The clinical sample mock sequencing data is shown in table 10:
table 10: clinical samples mimic sequencing data results.
From the above results, it was confirmed that the S3 treatment method can improve the detection of single-stranded DNA virus, and the S1 and S2 treatments can reduce the detection of single-stranded DNA virus, so that the treatment method can be selected specifically according to the purpose of detection of clinical specimens: the metagenome detection can select the processing methods of S1 and S2, and only double-stranded DNA is analyzed; detection of the macrovirus group requires binding to S1 and S3 or S2 and S3, which can improve detection of single-stranded DNA virus.
Stability detection
To accurately assess the stability of the microbiological assay, positive controls, negative controls and clinical samples were tested, each sample was run in 3 replicates, and the experimental samples and instructions are shown in table 11:
table 11: stability of microbial detection test samples.
Analysis of stability of sequencing data
And screening the sample sequencing data to obtain a processed sample sequence, comparing the processed sample sequence with a reference sequence, and drawing the proportion of each pathogen. And comparing the positive control pathogen ratio with a theoretical value to determine the pathogen detection deviation. The results of the positive control are shown in tables 12-13:
table 12: and (4) detecting the detection result of the first positive control in stability detection.
Table 13: and (4) detecting the detection result of the second positive control in stability detection.
From the species annotation results, the ratio of phage T1 reads number (number of sequencing data) to phage MS2 reads number in the total reads number of the sample was counted, as shown in tables 14-15:
table 14: t1 sequencing data number ratio result.
Table 15: MS2 sequencing data number ratio result.
The proportion of T1 and MS2 in the sample is greatly different according to the abundance of pathogens in the sample, and the proportion of T1 and MS2 data in the positive control is more than 0.5 ten-thousandth.
Analysis of library output stability
And performing a fluorescence quantitative PCR (polymerase chain reaction) experiment on the initial nucleic acid of the sequenced sample and the sequencing library, respectively calculating the absolute contents of bacteriophage T1 and MS2 in the initial sample and the sequencing library by absolute quantification of a Taqman method, and calculating the yield stability (CV value) of the library, wherein the CV value should be less than 20%.
CV value calculation formula:
single copy libraries yield the copy number of phage T1 (or MS2) in the library ÷ the copy number of the starting nucleic acid phage T1 (or MS 2).
Library stability CV value stdev.s (single copy library yield) ÷ AVERAGE (single copy library yield).
The results of the fluorescent quantitative PCR were as follows:
A. in connection with fig. 1, the standard curve setup (T1):
library yield stability calculation (T1):
B. in connection with fig. 2, standard curve set-up (MS 2):
| average Ct | MS2 | |
| std | ||
| 1 | 18.20387268 | 531000 |
| |
21.8133405 | 53100 |
| |
25.69413567 | 5310 |
| std 4 | 29.82939021 | 531 |
| |
33.49228795 | 53.1 |
Library yield stability calculation (MS 2):
in conclusion, the positive control provided by the invention has stability when applied to microbial detection.
The invention has various embodiments, and all technical solutions formed by adopting equivalent transformation or equivalent transformation are within the protection scope of the invention.
Claims (10)
1. A positive control for the detection of an infectious pathogen, characterized in that: the kit comprises a first positive control, wherein pathogens in the first positive control comprise gram-positive bacteria including lactobacillus fermentum, clostridium, bacillus subtilis, staphylococcus epidermidis, listeria welshimeri, gram-negative pseudomonas fluorescens, klebsiella aerogenes, escherichia coli, fungi including aspergillus niger, saccharomyces cerevisiae and bacteriophage T1.
2. The positive control for detection of an infectious pathogen of claim 1, wherein: the copy number mixing proportion of pathogens in the first positive control is as follows, gram-positive bacteria lactobacillus fermentum, clostridium, bacillus subtilis and staphylococcus epidermidis: the Listeria welshii, gram negative Pseudomonas fluorescens, Klebsiella aerogenes, Escherichia coli, fungi Aspergillus niger Saccharomyces cerevisiae, and bacteriophage T1 were 16.44:16.44:16.44:16.44:5.48:5.48:5.48:0.55:0.55: 0.27.
3. The positive control for detection of an infectious pathogen of claim 1, wherein: the control further comprises a second positive control whose pathogen nucleic acid comprises: staphylococcus epidermidis; fungus Aspergillus niger; herpes simplex virus; adeno-associated virus, rotavirus, respiratory syncytial virus, vesicular stomatitis virus, bacteriophage T1, bacteriophage MS 2.
4. The positive control for detection of an infectious pathogen of claim 1, wherein: the second positive control, whose pathogen genomic structure comprises: DNA double strand, DNA single strand, RNA double strand, and RNA single strand.
5. The positive control for detection of an infectious pathogen of claim 3, wherein: the ratio of the mixed copy number of the pathogen nucleic acid in the second positive control is staphylococcus epidermidis: fungus Aspergillus niger: herpes simplex virus: adeno-associated virus: rotavirus: respiratory syncytial virus: vesicular stomatitis virus: bacteriophage T1: phage MS2 was 2:1:2:2:2:2:2:2: 2.
6. The positive control for detection of an infectious pathogen of claim 4, wherein: the pathogen genome structure proportion in the second positive control is DNA double strand: DNA single strand: RNA double-stranded: the RNA single strand is 7:2:2: 6.
7. The method of claim 1 for preparing a positive control for the detection of an infectious pathogen, comprising: the preparation of the first positive control comprises the following steps of mixing according to the proportion of pathogens, adding thallus lysate, and incubating for 20-40 minutes at 50-70 ℃; the composition of the thallus lysate is mixed solution of Tris-HCl, EDTA, guanidinium isothiocyanate, LiCl and SDS, wherein the pH of the Tris-HCl = 9.
8. Use of a positive control for the detection of an infectious pathogen according to any one of claims 1 to 6, wherein: the positive control is applied to sample for microbe detection and clinical simulation.
9. Use of a positive control for the detection of an infectious pathogen according to any of claims 1 to 6, characterized in that: the application mode of the positive control product comprises and is not limited to methods such as high-throughput sequencing, fluorescent quantitative PCR detection, multiplex PCR, pathogen probe capture detection and the like.
10. Use of a positive control for the detection of an infectious pathogen according to claim 8 or 9, wherein: when the positive control is applied to microbial detection, the first positive control is processed, and the steps are that lysozyme is added, room-temperature incubation is carried out, and digestion is carried out after proteinase K is added; mechanically crushing by using an MP tissue crusher; the pathogens in the second positive control are divided into two groups according to the types of nucleic acid genomes, wherein the two groups are respectively a first group consisting of DNA double-stranded staphylococcus epidermidis, DNA double-stranded aspergillus niger, DNA double-stranded herpes simplex virus and DNA double-stranded bacteriophage T1, and a second group consisting of DNA single-stranded adeno-associated virus, RNA double-stranded rotavirus, RNA single-stranded respiratory syncytial virus, RNA single-stranded vesicular stomatitis virus and RNA single-stranded bacteriophage MS 2.
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