CN112662795B - Positive reference substance for detecting infectious pathogens and preparation method and application thereof - Google Patents
Positive reference substance for detecting infectious pathogens and preparation method and application thereof Download PDFInfo
- Publication number
- CN112662795B CN112662795B CN202110102724.7A CN202110102724A CN112662795B CN 112662795 B CN112662795 B CN 112662795B CN 202110102724 A CN202110102724 A CN 202110102724A CN 112662795 B CN112662795 B CN 112662795B
- Authority
- CN
- China
- Prior art keywords
- positive control
- pathogen
- virus
- dna
- positive
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 244000052769 pathogen Species 0.000 title claims abstract description 82
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 16
- 230000002458 infectious effect Effects 0.000 title claims abstract description 9
- 239000013558 reference substance Substances 0.000 title claims description 5
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 61
- 239000013641 positive control Substances 0.000 claims abstract description 55
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 28
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 27
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 27
- 241000191963 Staphylococcus epidermidis Species 0.000 claims abstract description 12
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 11
- 241000702421 Dependoparvovirus Species 0.000 claims abstract description 10
- 241000711975 Vesicular stomatitis virus Species 0.000 claims abstract description 10
- 241000709744 Enterobacterio phage MS2 Species 0.000 claims abstract description 8
- 241000700584 Simplexvirus Species 0.000 claims abstract description 8
- 241000588724 Escherichia coli Species 0.000 claims abstract description 7
- 241000725643 Respiratory syncytial virus Species 0.000 claims abstract description 7
- 241000702670 Rotavirus Species 0.000 claims abstract description 7
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 5
- 241000588915 Klebsiella aerogenes Species 0.000 claims abstract description 5
- 241000186840 Lactobacillus fermentum Species 0.000 claims abstract description 5
- 241000193403 Clostridium Species 0.000 claims abstract description 4
- 241001288369 Escherichia virus T1 Species 0.000 claims abstract description 4
- 241000186781 Listeria Species 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims description 7
- 241000589516 Pseudomonas Species 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 206010036790 Productive cough Diseases 0.000 claims 1
- 210000003802 sputum Anatomy 0.000 claims 1
- 208000024794 sputum Diseases 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 32
- 208000035473 Communicable disease Diseases 0.000 abstract description 5
- 241000589540 Pseudomonas fluorescens Species 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 32
- 239000000523 sample Substances 0.000 description 27
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 19
- 238000012163 sequencing technique Methods 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 14
- 239000006166 lysate Substances 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- 239000012091 fetal bovine serum Substances 0.000 description 10
- 241000894007 species Species 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 238000012258 culturing Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 4
- 102000008579 Transposases Human genes 0.000 description 4
- 108010020764 Transposases Proteins 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 241000192125 Firmicutes Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 3
- 238000012165 high-throughput sequencing Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 238000003559 RNA-seq method Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 241000193470 Clostridium sporogenes Species 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 241000202221 Weissella Species 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229940012969 lactobacillus fermentum Drugs 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012473 microbial detection method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a positive control for detecting infectious pathogens, and a preparation method and application thereof, wherein the positive control comprises a first positive control and a second positive control, and pathogens in the first positive control comprise gram positive bacteria lactobacillus fermentum, clostridium, bacillus subtilis, staphylococcus epidermidis, listeria weiersensis, gram negative pseudomonas fluorescens, klebsiella aerogenes and escherichia coli; the pathogen nucleic acid in the second positive control comprises staphylococcus epidermidis, aspergillus niger, herpes simplex virus, bacteriophage T1, adeno-associated virus, rotavirus, respiratory syncytial virus, vesicular stomatitis virus and bacteriophage MS2. The invention provides a convenient, safe and directly available double-reference standard, and simultaneously carries out positive standard comparison from different angles, thereby ensuring the accuracy and stability of the metagenome detection flow of infectious diseases.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to a positive control for detecting infectious pathogens, and a preparation method and application thereof.
Background
Infectious diseases are major diseases in the world today that seriously threaten human health. Current infectious disease detection faces several pain points: 1. the pathogens causing infection are various, the pathogenic cause is unknown, and diagnosis is difficult; 2. the drug resistance information cannot be accurately obtained, so that the medication is difficult; 3. the traditional detection method has the limitations of long time consumption, difficulty in culturing part of pathogens, low sensitivity, window period, limited detection quantity and the like.
In recent years, the rapidly developed NGS (high throughput sequencing) technology can directly detect unknown pathogenic microorganisms without special probe design because the NGS technology does not depend on known nucleic acid sequences, the mNGS (metagenomic sequencing) technology does not depend on traditional microorganism culture, and can directly carry out high throughput sequencing on nucleic acids in clinical samples, so that the pathogenic microorganisms in the clinical samples can be rapidly and objectively detected, the technology breaks the limitation of traditional microorganism detection, and the technology has broad prospects in the field of clinical microorganisms.
However, infectious diseases are caused by various pathogenic pathogens, including bacteria, viruses, fungi, parasites and the like, and due to different pathogen structures, such as different cell wall thicknesses of gram positive bacteria and gram negative bacteria, different sizes and contents of pathogens and different pathogen nucleic acid types (single-stranded DNA, double-stranded DNA, single-stranded RNA and double-stranded RNA), difficulty is brought to pathogen detection, and the non-standardized treatment can cause deviation of pathogen detection results and even false negative and false positive results.
At present, the detection of infectious pathogens of an NGS platform requires strict quality control standards, and ensures the stability of a detection method. The quality control of the technology depends on the setting of standard substances, the preparation of pathogen standard substances is complex, the operation risk is high (generally more than BSL-2 level laboratory is required), and the pathogen detection technology has instability, so no mature and stable commercial standard substances aiming at clinical needs exist at present.
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 application thereof.
The aim of the invention is achieved by the following technical scheme:
the positive control for detecting infectious pathogens comprises a first positive control, wherein pathogens in the first positive control comprise gram positive bacteria lactobacillus fermentum, clostridium sporogenes, bacillus subtilis, staphylococcus epidermidis, listeria weiersensis, gram negative fluorescent pseudomonas, klebsiella aerogenes, escherichia coli, fungus aspergillus niger, saccharomyces cerevisiae and phage T1.
Preferably, the first positive control has the following pathogen copy number mixing proportion that gram positive bacteria are lactobacillus fermentum, clostridium, bacillus subtilis and staphylococcus epidermidis: weissella, gram-negative Pseudomonas fluorescens, klebsiella aerogenes, escherichia coli, aspergillus niger, saccharomyces cerevisiae, and phage T1, 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, the pathogen nucleic acid of which comprises: staphylococcus epidermidis; the fungus Aspergillus niger; herpes simplex virus; adeno-associated virus, rotavirus, respiratory syncytial virus, vesicular stomatitis virus, phage T1, phage MS2.
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 numbers of pathogen nucleic acids in the second positive control is staphylococcus epidermidis: the fungus aspergillus niger: herpes simplex virus: adeno-associated virus: rotavirus: respiratory syncytial virus: vesicular stomatitis virus: phage T1: phage MS2 is 2:1:2:2:2:2:2:2:2.
Preferably, the pathogen genomic structure ratio in the second positive control is DNA duplex: single strand of DNA: RNA double strand: 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 pathogen proportion, adding a bacterial lysate, and incubating for 20-40 minutes at 50-70 ℃; the composition of the bacterial lysate is a mixed solution of Tris-HCl, EDTA, guanidine isothiocyanate, liCl and SDS, wherein the pH value of the Tris-HCl is=9.
Preferably, the positive control is applied to samples for detecting and simulating microorganisms and simulating clinic.
Preferably, the positive control application mode includes, but is not limited to, high throughput sequencing, fluorescent quantitative PCR detection, multiplex PCR, pathogen probe capture detection, and the like.
Preferably, when the positive control is applied to microorganism detection, the first positive control is treated, wherein 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 an MP tissue crusher; the pathogens in the second positive reference substance are divided into two groups according to the types of nucleic acid genome, namely a first group consisting of staphylococcus epidermidis with DNA double chains, aspergillus niger with DNA double chains, herpes simplex virus with DNA double chains and bacteriophage T1 with DNA double chains, and a second group consisting of adeno-associated virus with DNA single chains, rotavirus with RNA double chains, respiratory syncytial virus with RNA single chains, vesicular stomatitis virus with RNA single chains and bacteriophage MS2 with RNA single chains.
The invention has the outstanding effects that: the invention provides a convenient, safe and directly available double-reference standard, and positive standard comparison is carried out from two angles at the same time, so that the accuracy and stability of the metagenome detection flow of infectious diseases are ensured.
The following detailed description of the embodiments of the present invention is provided with reference to the accompanying drawings, so that the technical scheme of the present invention can be understood and mastered more easily.
Drawings
FIG. 1 is a schematic diagram of a library yield stability analysis T1 standard curve setup.
FIG. 2 is a schematic diagram of a library yield stability analysis MS2 standard curve setup.
Detailed Description
The invention discloses a positive reference substance for detecting infectious pathogens, and a preparation method and application thereof.
Pathogen origin, culture and preservation
1) The desired pathogen is derived from a laboratory-studied strain or purchased from ATCC:
2) The required pathogen nucleic acid is extracted after pathogen culture, and can be directly purchased:
3) Pathogen culture
A bacteria, fungi
Culturing: according to the pathogen specification, the most suitable culture conditions (culture medium, temperature, culture time and the like) are selected for culturing the strain. The culture time should be grasped in the late growth stage because cells in the logarithmic growth phase are weak against freeze-drying. The bacteria are generally cultured for 24 to 48 hours, and during operation, the lawn or spores are gently scraped under the aseptic condition to prepare bacterial suspension.
Cell number calculation: resuscitating bacteria/fungi with specific culture medium, culturing to passable concentration, sucking 1mL of fully mixed bacteria/spore suspension with sterile pipetting gun head under aseptic operation, adding 9mL of physiological saline, and mixing to obtain 10 -1 Tube, dilute to 10 in turn by the same method -6 . Pouring the mixed solution into a culture dish containing bacterial solutions with different dilutions (0.2 mL), pouring about 10-15mL of the corresponding culture medium cooled to about 45 ℃ after being melted, placing the culture medium in a horizontal position, rapidly rotating and uniformly mixing, and culturing in a 37 ℃ incubator after solidification. After incubation for a corresponding period (e.g., 24 hours), the dishes are removed and the average number of colonies on three plates at the same dilution is calculated.
The calculation is performed according to the following formula:
colony forming units (cfu) =average colony count of triplicate of the same dilution x 5 per ml
Phage B (T1, MS 2)
Culturing: coli was used as phage host, cultured in liquid culture at appropriate temperature, and incubated with shaking until mid-log growth (OD 600-0.5). Dipping a small amount of phage into a liquid culture medium containing escherichia coli, shaking and culturing at 37 ℃ until bacterial suspension in a test tube turns clear from turbid, centrifuging (8000 rpm/30 min) the culture, taking supernatant, filtering with a 0.22um microporous filter membrane, and preserving at-20 ℃ for later use.
Phage titer assay: .
(1) Dilution of phages:
4 tubes containing 0.9ml of liquid medium were labeled 10-3, 10-4, 10-5 and 10-6, respectively.
0.1ml of 10-2 phage was pipetted into a 10-3 tube, the tube was swirled and mixed well.
0.1ml of the solution is sucked from the 10-3 tube by another sterile suction tube, added into the 10-4 tube, mixed uniformly, and the rest is analogically diluted to the 10-6 tube.
(2) Mixing bacteriophage and bacterial liquid
5 empty sterile tubes were labeled 10-4, 10-5, 10-6, 10-7 and control, respectively.
0.1ml was pipetted from the 10-3 phage dilution tube into a 10-4 empty tube, and 0.1ml was pipetted from the 10-4 dilution tube into a 10-5 empty tube with another pipette until 10-7 tubes.
Shaking up the host bacteria culture solution, taking 0.9ml of the bacteria culture solution by a suction pipe, adding into a control test tube, sucking 0.9ml of the bacteria culture solution into a 10-7 test tube, adding from the last tube until reaching 10-4 tubes, and adding 0.9ml of the escherichia coli culture solution into each tube.
Mixing the above test tubes.
(3) Adding the mixed solution into the upper layer culture medium
The 5-tube upper medium was thawed and labeled 10-4, 10-5, 10-6, 10-7 and controls. Cool to 48 ℃ and place into a 48 ℃ water bath.
The 4 tubes of the mixture and the control tube were each added to the upper medium tube. After each tube is added with the mixed solution, the mixed solution is immediately swirled and evenly mixed.
(4) Pouring the inoculated upper culture medium onto a bottom plate
And rapidly checking the number of the upper layer culture medium which is uniformly rotated and poured onto a bottom layer flat plate, and uniformly shaking the upper layer culture medium on a table top to make the upper layer culture medium spread on the flat plate.
After solidification, the cells were incubated at 37℃upside down.
(5) Plaques in the plates were observed and plates were selected at a pfu number of 30-300 to calculate the number of phages per ml of undiluted stock (titer). The titer-plaque forming units (pfu) per ml phage were 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:
(1) herpes simplex virus HSV-2:
DMEM complete medium of 10% Fetal Bovine Serum (FBS) Vero cells were cultured to 90% density in dishes;
b. the original medium was discarded, EMEM culture with 2% FBS was added, HSV virus (optimal MOI: 0.1) was added, and the mixture was incubated with 5% CO 2 Infection in incubator (37 ℃) for 1-2 hours, discarding supernatant again, replacing new medium (EMEM of 2% FBS);
c. after infection for 2-5 days, observing that most cells die and float, collecting cell supernatant, packaging, and freezing in a refrigerator at-80deg.C.
(2) Adeno-associated virus AAV-2:
HEK-293 cells were cultured in DMEM complete medium with 10% Fetal Bovine Serum (FBS) to a density of 80-90% on dishes;
b. the original medium was discarded, EMEM culture with 2% FBS was added, AAV virus and helper virus (optimal MOI: 1) were added, and the mixture was incubated with 5% CO 2 Infection in incubator (37 ℃) for 1-2 hours, discarding supernatant again, replacing new medium (EMEM of 2% FBS);
c. observing after infection for 2-3 days that most cells die and float, collecting cell supernatant, packaging, and freezing in-80deg.C refrigerator
(3) Vesicular stomatitis virus VSV:
a. VERO cells were cultured to a density of 80-90% using DMEM complete medium with 10% Fetal Bovine Serum (FBS);
b. changing culture medium to DMEM medium containing 5ml 2% serum, adding VSV virus (MOI about 1), infecting at 37deg.C in a box for 1 hr, discarding supernatant, washing with PBS, and changing to DMEM medium containing 5ml 2% serum;
C. and after 18-24 hours of infection, observing that most cells die and float, collecting cell supernatant, sub-packaging and freezing in a refrigerator at-80 ℃.
The first positive control provided in this example contained pathogens with different lysis difficulties, and in particular increased the proportion of gram positive bacteria, the specific composition and proportions are shown in table 1:
table 1: the first positive control contained a pathogen and cell proportion control table.
After culturing and counting each pathogen, each pathogen microorganism is diluted to the required cell number by PBS and split-packed into sterile centrifuge tubes for preservation.
When preparing the first positive control, a diluted pathogen was taken in each case according to the pathogen type in Table 1, and mixed by blowing, all pathogens were mixed into the same sterile centrifuge tube and the volume was measured.
Lysates were pipetted into a centrifuge tube of the first positive control mixed pathogen to give a total volume of 1ml for 30min at 60 ℃. This first positive control can be used 100 times in total.
The first positive control was aliquoted into 100. Mu.L/serving and stored for a long period at-80 ℃.
When in use, one part of the first positive control is divided into 10 mu L/part, 10 mu L of the first positive control is used each time, and the rest of the first positive control is stored at-80 ℃.
The selected formulation of the above lysates is shown in table 2:
table 2: lysate formulation table.
| Numbering device | Formula of lysate |
| L1 | 100mM Tris-HCl (pH 9), 40mM EDTA,4M guanidine isothiocyanate |
| L2 | 500mM NaCl，50mM Tris-HCl，50mM EDTA，4％SDS |
| L3 | 100Tris-HCl (pH 9), 40mM EDTA,4M guanidine isothiocyanate, 3.5M LiCl,3% SDS |
After the pathogen was mixed according to the amount of cells required for the first positive control, the mixture was blown and homogenized, 1/20 of the volume was removed before adding the lysate, and the mixture was aliquoted into 3 portions.
mu.L of L1, L2 and L3 lysates were added respectively.
20. Mu.g/. Mu.L proteinase K was added and digested for 30min at 55 ℃.
Physical disruption was performed using an MP tissue disruptor under conditions of 6m/s,20 s/time, 3 times/sample.
Pathogen DNA extraction was performed using the Kit AllPrep DNA/RNA Kit (Qiagen, cat No. 80204).
1ng of DNA was taken and a sequencing library was constructed using a transposase method, and sequenced using an Illumina Novaseq sequencer, yielding 6Gb of raw data per sample.
And performing quality control on the original Data by using Trimmomatic software to obtain effective Data (clear Data), comparing the effective Data with a host genome sequence by using Soap software, removing the host Data, further improving analysis efficiency, performing species annotation on the final effective Data by using MetaPhlAn2, and then counting species annotation results. Pathogen information was counted by species annotation.
The species annotation results are shown in table 3:
table 3: comparison of deviations from different lysate formulations versus the first positive control.
From the above, the lysate of the L3 formulation is preferably used as the cell preservation and nucleic acid extraction lysate of the first positive control.
To better embody the selection of pathogen treatment conditions, the following screening of corresponding experiments was performed:
table 4: different treatment patterns are shown for pathogens.
7 parts of the formulated first positive control were removed and 700. Mu.L of lysate was added.
Pathogen treatments were 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 No. 80204).
1ng of DNA was taken and a sequencing library was constructed using a transposase method, and sequenced using an Illumina Novaseq sequencer, yielding 6Gb of raw data per sample.
And performing quality control on the original Data by using Trimmomatic software to obtain effective Data (clear Data), comparing the effective Data with a host genome sequence by using Soap software, removing the host Data, further improving analysis efficiency, performing species annotation on the final effective Data by using MetaPhlAn2, and then counting species annotation results. Pathogen information was counted by species annotation.
Pathogen treatment results are shown in table 5:
table 5: comparison table of pathogen treatment results.
From the above results, it was determined that the pathogen treatment mode is preferably: lysozyme (3 mg/mL), proteinase K (20. Mu.g/. Mu.L), digestion at 55℃for 30min, MP tissue disruptor (6 m/s,30 s/times, 4 times/sample).
The second positive control provided by the invention is used as the supplement and perfection of the first positive control, 4 forms of pathogen genome structures are further supplemented under the condition of ensuring that high-difficulty pathogen nucleic acid is complete, and the nucleic acid complexity of a clinical sample is simulated by proportioning real pathogen nucleic acid.
Nucleic acids of staphylococcus epidermidis, aspergillus niger, phage T1, phage MS2 were extracted using the Kit AllPrep DNA/RNA Kit (Qiagen, cat No. 80204). Nucleic acids of herpes simplex virus, adeno-associated virus, vesicular stomatitis virus were extracted using kit QIAamp UltraSens Virus Kit (Qiagen, cat No. 53704). Both rotavirus and respiratory syncytial virus nucleic acids were purchased from ATCC. Pathogen nucleic acid copy number was detected using digital PCR. Wherein the ratio of the required copy numbers of pathogen nucleic acids is shown in Table 6:
table 6: pathogen nucleic acid information table.
For better selection of the grouping configuration for the second positive control, the genome structure in table 6 was grouped according to the difference in detection scheme, and the grouping scheme is shown in table 7.
Table 7: the nucleic acid genome groupings in the second positive control are shown schematically.
N1-1 and N2-1 library building: 1ng of DNA was taken and a sequencing library was constructed using a transposase method, and sequenced using an Illumina Novaseq sequencer, yielding 6Gb of raw data per sample.
N2-1 and N2-2 library building: 10pg RNA was used for library construction using the kit Ovation solo RNA-seq system (Tecan, cat. No. 0500-32), yielding 10Gb of raw data per sample.
The sequencing data was analyzed for species annotation. Pathogen nucleic acid sequencing data results are shown in table 8:
table 8: results table of pathogen nucleic acid sequencing data.
From the above results, it was determined that DNA pooling methods resulted in lower single-stranded DNA pathogen detection when DNA single-stranded is combined with DNA double-stranded; when DNA single strands and RNA are simultaneously pooled, detection of single strand DNA pathogens can be enhanced. The pathogen nucleic acid groupings from which the second positive control is determined are preferably: classification of N1 pathogen nucleic acids were mixed in groups according to the N1 format, which is the second positive control.
Clinical sample simulation processing
According to the grouping treatment of the second positive control, clinical specimens are particularly careful about the treatment of DNA single-stranded pathogens in order to ensure the integrity and accuracy of pathogen detection. The 9 pathogens in the table were mixed according to the requirement of the second positive control for pathogen nucleic acid copy number, mimicking the complexity of the clinical samples.
The simulation processing mode of the clinical samples is shown in table 9:
table 9: the clinical sample is simulated.
The mixed mock clinical samples were divided into 3 parts and DNA and RNA were simultaneously extracted using the Kit AllPrep DNA/RNA Kit (Qiagen, cat. No. 80204). Wherein the DNA and RNA of S1 are respectively preserved, and the DNA and RNA of S2 and S3 are mixed.
S2, sample processing: the S2 sample was divided into 2 parts and treated with DNase I and RNase A, respectively, to obtain S2-RNA and S2-DNA.
S3, sample processing: s3 samples were processed using HL-dsDNase to obtain S3-RNA.
S1-DNA, S2-DNA was library constructed using the transposase method, and the Illumina Novaseq sequencer was used to sequence, yielding 6Gb of raw data per sample.
S1-RNA, S2-RNA and S3-RNA were library constructed using the kit Ovation solo RNA-seq system (Tecan, cat. No. 0500-32), yielding 10Gb of raw data per sample.
The sequencing data was analyzed for species annotation. The clinical sample simulated sequencing data are shown in table 10:
table 10: the clinical samples simulate 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 mode can be selected pertinently according to the detection purpose of clinical samples: metagenome detection can select the processing methods of S1 and S2, and only double-stranded DNA is analyzed; detection of macrovirons requires binding to S1 and S3 or to S2 and S3, which can enhance detection of single stranded DNA viruses.
Stability detection
To accurately evaluate the stability of the microbial detection method, positive control, negative control and clinical samples were tested, each sample was subjected to 3 parallel experiments, and the experimental samples and descriptions are shown in table 11:
table 11: stability test sample for microbial detection.
(1) Stability analysis of sequencing data
Screening the sample sequencing data to obtain a processed sample sequence, comparing the processed sample sequence with a reference sequence, and drawing the pathogen ratio. The positive control pathogen ratio is compared with the theoretical value to determine the pathogen detection deviation. The positive control detection results are shown in tables 12-13:
table 12: stability the detection result of the first positive control was examined.
Table 13: stability the detection result of the second positive control was examined.
/>
The ratio of phage T1 reads (number of sequencing data) and phage MS2 reads to total reads of the sample was counted by species annotation results, as shown in tables 14-15:
table 14: t1 sequencing data strip number duty ratio results.
/>
Table 15: MS2 sequenced data bar duty cycle results.
/>
The ratio of T1 to MS2 in the sample is greatly different according to the difference of pathogen abundance in the sample, and the ratio of the number of data strips of T1 to MS2 in the positive control is more than 0.5 parts per million.
(2) Library yield stability analysis
Performing fluorescent quantitative PCR experiments on the initial nucleic acid of the sequenced sample and the sequencing library, respectively calculating absolute contents of phage T1 and MS2 in the initial sample and the sequencing library by absolute quantification of Taqman method, and calculating library yield stability (CV value), wherein the CV value is less than 20%.
CV value calculation formula:
single copy library yield = phage T1 (or MS 2) copy number in library +.starter nucleic acid phage T1 (or MS 2) copy number.
Library stability CV value = stdev.s (single copy library yield)/(AVERAGE (single copy library yield)).
The results of fluorescent quantitative PCR were as follows:
A. as shown in connection with fig. 1, the standard curve setting (T1):
library yield stability calculation (T1):
B. as shown in connection with fig. 2, standard curve setting (MS 2):
| average Ct | MS2 copy number | |
| std 1 | 18.20387268 | 531000 |
| std 2 | 21.8133405 | 53100 |
| std 3 | 25.69413567 | 5310 |
| std 4 | 29.82939021 | 531 |
| std 5 | 33.49228795 | 53.1 |
Library yield stability calculation (MS 2):
in conclusion, the positive control provided by the invention has stability when being applied to microorganism detection.
The invention has various embodiments, and all technical schemes formed by equivalent transformation or equivalent transformation fall within the protection scope of the invention.
Claims (1)
1. The double-control standard for detecting infectious pathogens in sputum disease samples is characterized in that: the kit comprises a first positive control, wherein pathogens in the first positive control comprise gram positive bacteria lactobacillus fermentum, clostridium, bacillus subtilis, staphylococcus epidermidis, listeria weiersonii, gram negative fluorescent pseudomonas, klebsiella aerogenes, escherichia coli, fungi aspergillus niger, saccharomyces cerevisiae and phage T1; the standard also comprises a second positive control, and the pathogen nucleic acid comprises: staphylococcus epidermidis; the fungus Aspergillus niger; herpes simplex virus; adeno-associated virus, rotavirus, respiratory syncytial virus, vesicular stomatitis virus, bacteriophage T1, bacteriophage MS2;
the second positive control has a pathogen genomic structure comprising: DNA double strand, DNA single strand, RNA double strand, and RNA single strand;
the first positive reference substance has the following pathogen copy number mixing proportion that gram positive bacteria lactobacillus fermentum, clostridium, bacillus subtilis and staphylococcus epidermidis: weiersion listeria gram-negative fluorescent pseudomonas, klebsiella aerogenes, escherichia coli, aspergillus niger and bacteriophage T1 are 16.44:16.44:16.44:16.44:16.44:5.48:5.48:5.48:0.55:0.55:0.27;
the ratio of the mixed copy numbers of pathogen nucleic acid in the second positive control is staphylococcus epidermidis: the fungus aspergillus niger: herpes simplex virus: adeno-associated virus: rotavirus: respiratory syncytial virus: vesicular stomatitis virus: phage T1: phage MS2 is 2:1:2:2:2:2:2:2:2;
the pathogen genome structure proportion in the second positive control is DNA double-strand: single strand of DNA: RNA double strand: the RNA single strand is 7:2:2:6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110102724.7A CN112662795B (en) | 2021-01-26 | 2021-01-26 | Positive reference substance for detecting infectious pathogens and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110102724.7A CN112662795B (en) | 2021-01-26 | 2021-01-26 | Positive reference substance for detecting infectious pathogens and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112662795A CN112662795A (en) | 2021-04-16 |
| CN112662795B true CN112662795B (en) | 2023-12-15 |
Family
ID=75414569
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110102724.7A Active CN112662795B (en) | 2021-01-26 | 2021-01-26 | Positive reference substance for detecting infectious pathogens and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112662795B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112569A (en) * | 2015-09-14 | 2015-12-02 | 中国医学科学院病原生物学研究所 | Virus infection detection and identification method based on metagenomics |
| WO2016094947A1 (en) * | 2014-12-16 | 2016-06-23 | Garvan Institute Of Medical Research | Sequencing controls |
| CN108239670A (en) * | 2016-12-26 | 2018-07-03 | Md保健株式会社 | The kit of the lung cancer of COPD patient and method are diagnosed by macro genome analysis |
| WO2018126036A1 (en) * | 2016-12-28 | 2018-07-05 | Ascus Biosciences, Inc. | Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities with tracer analytics, determination of functional relationships and interactions thereof, and synthesis of microbial ensembles including dosed microbial ensembles and inoculative microbial ensembles |
| CN110392738A (en) * | 2016-12-28 | 2019-10-29 | 埃斯库斯生物科技股份公司 | For being analyzed the microorganism strain in complex heterogeneous group, being determined its functional relationship and interaction and determine the method, apparatus and system of diagnosis and biological aspect management based on this |
| CN110938701A (en) * | 2019-11-26 | 2020-03-31 | 康美华大基因技术有限公司 | Standard substance for metagenome sequencing quantification |
| CN111471676A (en) * | 2020-03-13 | 2020-07-31 | 广州市达瑞生物技术股份有限公司 | Preparation method of database building sample for metagenome next generation sequencing |
| CN111607639A (en) * | 2020-05-08 | 2020-09-01 | 深圳华大因源医药科技有限公司 | Method and device for quantitative detection of metagenome pathogens based on internal reference |
| CN112111588A (en) * | 2020-09-22 | 2020-12-22 | 中国疾病预防控制中心传染病预防控制所 | Quantitative 16S metagenome sequencing method |
| CN112226488A (en) * | 2020-10-13 | 2021-01-15 | 上海宝藤生物医药科技股份有限公司 | Method and kit for constructing pathogenic microorganism sequencing library based on total nucleic acid and metagenome |
-
2021
- 2021-01-26 CN CN202110102724.7A patent/CN112662795B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016094947A1 (en) * | 2014-12-16 | 2016-06-23 | Garvan Institute Of Medical Research | Sequencing controls |
| CN105112569A (en) * | 2015-09-14 | 2015-12-02 | 中国医学科学院病原生物学研究所 | Virus infection detection and identification method based on metagenomics |
| CN108239670A (en) * | 2016-12-26 | 2018-07-03 | Md保健株式会社 | The kit of the lung cancer of COPD patient and method are diagnosed by macro genome analysis |
| WO2018124617A1 (en) * | 2016-12-26 | 2018-07-05 | 주식회사 엠디헬스케어 | Method for diagnosing lung cancer via bacterial metagenomic analysis |
| WO2018126036A1 (en) * | 2016-12-28 | 2018-07-05 | Ascus Biosciences, Inc. | Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities with tracer analytics, determination of functional relationships and interactions thereof, and synthesis of microbial ensembles including dosed microbial ensembles and inoculative microbial ensembles |
| CN110392738A (en) * | 2016-12-28 | 2019-10-29 | 埃斯库斯生物科技股份公司 | For being analyzed the microorganism strain in complex heterogeneous group, being determined its functional relationship and interaction and determine the method, apparatus and system of diagnosis and biological aspect management based on this |
| CN110938701A (en) * | 2019-11-26 | 2020-03-31 | 康美华大基因技术有限公司 | Standard substance for metagenome sequencing quantification |
| CN111471676A (en) * | 2020-03-13 | 2020-07-31 | 广州市达瑞生物技术股份有限公司 | Preparation method of database building sample for metagenome next generation sequencing |
| CN111607639A (en) * | 2020-05-08 | 2020-09-01 | 深圳华大因源医药科技有限公司 | Method and device for quantitative detection of metagenome pathogens based on internal reference |
| CN112111588A (en) * | 2020-09-22 | 2020-12-22 | 中国疾病预防控制中心传染病预防控制所 | Quantitative 16S metagenome sequencing method |
| CN112226488A (en) * | 2020-10-13 | 2021-01-15 | 上海宝藤生物医药科技股份有限公司 | Method and kit for constructing pathogenic microorganism sequencing library based on total nucleic acid and metagenome |
Non-Patent Citations (1)
| Title |
|---|
| 高通量宏基因组测序技术检测病原微生物的临床应用规范化专家共识;鲁辛辛等;《中华检验医学杂志》;20201231;第43卷(第12期);第1181-1195页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112662795A (en) | 2021-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112725287B (en) | Strong-lytic staphylococcus aureus phage RDP-SR-20001 and application thereof | |
| CN114521585A (en) | Method for preventing and treating postharvest diseases of jujube fruits by using antagonistic yeast to remold epiphytic microbial communities | |
| Ling et al. | Viral metagenomics reveals significant viruses in the genital tract of apparently healthy dairy cows | |
| CN114480731B (en) | Combined kit for phage bioamplification-real-time fluorescent quantitative PCR (polymerase chain reaction) for rapidly detecting staphylococcus aureus and method thereof | |
| CN101775443B (en) | LAMP kit for detecting PRV and preparation method thereof | |
| CN107034316A (en) | The system and its special LAMP primer of 6 boars virus are detected simultaneously | |
| CN114480679B (en) | Kit for rapidly detecting salmonella based on phage bioamplification combined with real-time fluorescence quantitative PCR and method thereof | |
| CN105039202B (en) | A kind of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious and preparation method thereof | |
| CN116042543A (en) | Salmonella phage with broad cleavage spectrum, phage composition and application | |
| CN101363056A (en) | High-flux microorganism identification method | |
| CN112662795B (en) | Positive reference substance for detecting infectious pathogens and preparation method and application thereof | |
| CN113462580B (en) | Trichoderma guizhouense Hz36 strain, biocontrol microbial inoculum and preparation method and application thereof | |
| CN114350738A (en) | Method for screening and identifying antibiotic resistant bacteria | |
| CN111549124A (en) | Molecular marker for detecting staphylococcus aureus mastitis of dairy cows and application thereof | |
| CN116891903A (en) | Cross primer isothermal amplification technology-based detection primer, kit and method for cheese bacillus paracasei | |
| AU2020103778A4 (en) | Primer Set for Detection of Streptococcus agalactiae, Detection Kit and Multiplex PCR Detection Method | |
| US20220098637A1 (en) | Methods of detecting microbial content in cannabis | |
| CN111676160A (en) | Application of beautiful millettia root endophyte RH5 in promoting strong growth of cows | |
| CN113215110A (en) | Method for improving multiplication capacity of African swine fever virus | |
| CN115558649B (en) | Solani fexofenadine phage and application thereof in low-temperature fresh-keeping | |
| CN117487664B (en) | Bifidobacterium separation culture method | |
| CN116606915A (en) | Simulation sample matrix, quality control product containing simulation sample matrix and application of simulation sample matrix | |
| CN118308310A (en) | Salmonella typhimurium phage ZK22 and application thereof | |
| CN106635901A (en) | Mycoplasma hyopneumoniae strain, vaccine and application thereof | |
| Faleh et al. | Molecular study of antimicrobial effect of bacteriocin isolated from Lactobacillus on Candida albicans |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |