CN112852751A

CN112852751A - Preparation and quality control method of new coronavirus nucleic acid detection and quality control virus

Info

Publication number: CN112852751A
Application number: CN202110151581.9A
Authority: CN
Inventors: 翁炳焕
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-05-28

Abstract

A preparation and quality control method of new coronavirus nucleic acid detection quality control virus, according to the mechanism that new coronavirus is easy to propagate in host cells expressing ACE2 and SV40LT can immortalize human tissue cells, ACE2 and SV40LT genes are transfected into amniotic fluid stem cells left after prenatal diagnosis, immortalized new coronavirus hatching cells which are easy to propagate in cells due to over-expression of ACE2 and SV40LT are developed, so that the new coronavirus and the hatching cells can propagate in large quantities along with infinite amplification of the hatching cells when being cultured together, then the propagated viruses are used for preparing nucleic acid detection quality control virus in batches, then the quality control virus replaces analogue virus or phage virus-like particles which are inconsistent with the detected sample and are prepared by a nucleic acid detection kit or used by related units, and the quality control virus is used for establishing a nucleic acid detection indoor quality control method based on a Levey-Jennings quality control diagram, so that the quality control substance of real virus can be used for monitoring whether the indoor quality control is out of control or not in real time, and whether the detection results of the same batch of nucleic acid are accurate and reliable or not can be judged.

Description

Preparation and quality control method of new coronavirus nucleic acid detection and quality control virus

Technical Field

The invention relates to a preparation and quality control method of a new coronary pneumonia nucleic acid detection quality control virus, belonging to the infectious disease gene diagnosis technology in the field of biomedicine.

Background

The novel coronavirus (SARS-CoV-2) is composed of a single-stranded positive-strand nucleic acid (ssRNA), a spike protein (S), a membrane protein (M), an envelope protein (E), and a nucleocapsid protein (N). The detection rate of bronchial/alveolar lavage fluid is relatively high because the patient with new coronary pneumonia has higher virus load in the lower respiratory tract, and the nucleic acid detection of new coronary virus is usually performed by using a nasopharyngeal swab at present.

The literature reports that the detection of new coronavirus nucleic acid by adopting a real-time fluorescent RT-PCR method is the gold standard for diagnosing new coronary pneumonia at present, more than 20 new coronavirus nucleic acid detection kits adopting real-time fluorescent RT-PCR technologies have been approved by the national drug administration so far, and the technology is sensitive, simple, convenient and low-cost, can identify trace new coronavirus particles in early stage or latent stage of diseases, but usually causes the nucleic acid detection result to generate false negative or false positive due to the influences of disease development stage, sample collection quality, sample transportation and storage conditions, reagent and consumable quality, the level of experiment operation technicians and the like.

Therefore, the laboratory operation specifications and expert consensus of various new coronavirus nucleic acid detection suggest monitoring the accuracy of the nucleic acid detection result by indoor quality control, but the standardized indoor quality control needs to adopt the same quality control substance as the detected sample and detect the same quality control substance and the detected sample under the same condition, namely the qualified indoor quality control substance is the new coronavirus particles, but the indoor quality control is difficult to realize, so that only the phage virus sample particles called analog quality control substances or pseudo viruses can be used for replacing the new coronavirus particles to carry out the indoor quality control.

The literature reports that at present, indoor quality control products of the new coronavirus nucleic acid detection kit developed by multiple biological companies at home and abroad are not prepared from new coronavirus particles but pseudo viruses, the price of the quality control products is high, and the monitoring effect of the indoor quality control is still left to be fed back by users.

In addition, although genome sequencing can identify new unknown crown virus strains, and can also be used for detection of early low-virus-content samples or confirmation of suspicious results of real-time fluorescence RT-PCR detection, the genome sequencing is still not suitable for routine detection of clinical new crown samples due to the lack of positive and negative reference substances for quality control and the like.

According to the literature report, 1 part of weak positive quality control and 3 parts of negative quality control are set in each batch of detection in the standardized indoor quality control, the indoor quality control and a sample to be detected participate in the whole detection process in the same batch, only when all the 3 negative quality controls are negative and the weak positive quality control is positive, the indoor quality control can be regarded as being controlled and can send a report, otherwise, the indoor quality control is out of control, the report cannot be sent, the error reason can be immediately analyzed, and the indoor quality control is re-detected if necessary. The quality control among rooms is uniformly organized by a health management department, the quality control substances are distributed to all participating laboratories for detection after being subpackaged, and then whether the detection results of all laboratories are accurate or not is analyzed.

In view of the lack of quality control substances for detecting the new coronavirus nucleic acid, domestic literature reports suggest that the quality control substances are prepared by self to replace weak positive control prepared in a kit, namely, true new coronavirus is used for replacing pseudovirus. The preparation method comprises the steps of inactivating a new coronavirus nucleic acid detection positive specimen found in a laboratory at 56 ℃ in a dry bath for 30min, preparing a low-value quality control product with 2 or more than 2 target points, and suggesting that physiological saline/DEPC (diethyl phthalate) treated water is used as a negative quality control, wherein at least 1 part of the negative quality control is used for evaluating environmental pollution when in use, and the quality control product which is about to be uncapped is placed on an extractor/operation table top for PCR detection overnight and then is detected.

In summary, the quality control substance is poor because no ideal method for preparing the quality control substance for detecting the nucleic acid of the new coronavirus exists at present, and no matter the positive quality control substance is prepared in the kit or other quality control substances used in the experiment, the simulated quality control substance, the pseudovirus or the phage virus-like particle is usually used for replacing the new coronavirus particle for quality control, which is difficult to play the expected quality control role.

The literature reports that SARS-CoV replicates in 293T cells transfected with the angiotensin converting enzyme 2(ACE2) gene, but not in 293T cells mock-transfected (without ACE2), as does SARS-CoV-2, both infecting cells via the ACE2 receptor. The literature also reports that human mesenchymal stem cells undergo senescence or death when passaged in vitro to 15-30 passages, that cells of other tissues undergo shorter passage times, and that cell lines transfected with the simian virus 40 large T antigen gene (SV40LT) and/or human telomerase reverse transcriptase (hTERT) can be cultured in vitro for more than 350 passages.

However, no literature report that ACE2, SV40LT and/or hTERT transfects human mesenchymal cells to prepare immortalized hatching cells for propagating new coronavirus and uses the hatching cells as a vector to prepare the new coronavirus is found so far.

Disclosure of Invention

The invention provides the method for preparing the novel coronary pneumonia nucleic acid detection quality control substance.

The invention aims to disclose a preparation method of a new coronavirus nucleic acid detection quality control virus by taking an artificial new coronavirus hatching cell as a vector, and also aims to disclose a nucleic acid detection indoor quality control method based on a Levey-Jennings quality control diagram.

The purpose of the invention is realized as follows: (1) collecting remaining amniotic fluid cells after prenatal diagnosis, separating fusiform amniotic fluid cells or lung tissue cells, transfecting SV40LT and/or hTERT genes, screening an immortalized cell line by G418 and/or puromycin, and obtaining the immortalized amniotic fluid mesenchymal stem cell line or the lung stem cell line through cell line biological identification and lung stem cell marker detection, wherein the immortalized amniotic fluid mesenchymal stem cell line or the lung stem cell line is named as MSC-SV40 LT/hTERT. (2) Angiotensin converting enzyme II, namely ACE2 gene is connected to a lentivirus expression vector pHBLV-CMV-ACE2-EF1-ZsGreen-T2A-puro or pGC-FU, respectively constructing recombinant plasmids pHBLV-OE-ACE2 or pGC-FU-ACE2, co-transfecting 293FT Cells with the recombinant plasmids pHBLV-OE-ACE2 and packaging plasmids (psPAX2 and pMD2G) or the recombinant plasmids pGC-FU-ACE2 and packaging plasmids (pHelper1.0 and pHelper2.0), packaging recombinant lentiviruses carrying ACE2, transfecting the recombinant lentiviruses with MSC-SV40LT/hTERT, integrating the ACE2 into DNA of immortalized stem Cells, and screening and identifying to obtain immortalized new Coronavirus hatching Cells which are easy to infect and propagate new Coronavirus, wherein the immortalized new Coronavirus hatching Cells are named as matrix Cells for preparing new Coronavirus inactivated vaccines (Novel Coronavir Cells). (3) Subpackaging the hatching cells into ampoules, each ampoule being 10X 10⁶And (4) storing the cells in a liquid nitrogen cell bank at the temperature of-196 ℃. (4) Taking hatching cells in cell bank, performing subculture to desired cell amount, transferring into a bioreactor containing DMEM cell culture solution containing 3-5g/L microcarrier, and culturing at 37 deg.C with 5% CO₂Culturing at pH 7.2 and stirring speed of 50 + -20 rpm to form 60% confluence degree of cells on the surface of the microcarrier, replacing virus maintaining solution, inoculating 0.01-0.3 MOI amount of new corona virus solution, culturing at 33 deg.C and 5% CO₂pH 7.4, stirring speed 50 + -20 rpmCulturing for 48-96 hours under the condition, and harvesting virus liquid when cells are completely diseased. (5) Filtering the virus liquid by 75 mu m, 0.45 mu m and 0.22 mu m filters, ultrafiltering and concentrating by a 100KD ultrafiltration membrane, purifying by Sepharose CL-6B gel filtration chromatography, sterilizing and filtering by a 0.22 mu m filter, inactivating 92.5 mu g/ml formaldehyde, verifying the inactivation effect, and detecting the purity of the virus liquid to obtain the inactivated virus liquid. (6) Preparing the inactivated virus liquid into high-value, medium-value and low-value quality control virus liquid, detecting the content of virus nucleic acid by a reference laboratory, determining a target value, and performing conventional subpackage and preservation. (7) The method comprises the steps of taking a quality control virus as an indoor quality control object, detecting the indoor quality control object and a detected specimen under the same condition, drawing a Levey-Jennings quality control diagram, establishing a new coronavirus nucleic acid detection indoor quality control method based on the Levey-Jennings quality control diagram, observing whether a detection result is out of control, and further judging whether the detection result of the detected specimen is accurate and reliable and whether a detection report can be sent. (8) The quality control virus is used as a chamber quality assessment substance for checking the detection quality of each laboratory.

The invention has the beneficial effects that: the quality control substance is poor because an ideal method for preparing the new coronavirus nucleic acid detection quality control substance does not exist at present, and the simulated quality control substance, the pseudovirus or the phage virus-like particle is usually used for replacing the new coronavirus particle for quality control no matter the positive quality control substance is prepared in a kit or the positive quality control substance is used in an experiment, so that the expected quality control effect is difficult to achieve. Although the literature suggests that clinical samples positive in nucleic acid detection for various laboratories are prepared as quality control substances, the quality control substances are difficult to prepare by using accepted strains with unified standards, so that the ideal requirements of the quality control of the nucleic acid detection are difficult to achieve, and sufficient ideal quality control substances are difficult to provide for national or international unified organization indoor evaluation. According to the mechanism that the new coronavirus infects a host cell through an ACE2 receptor and is easy to propagate in the host cell expressing ACE2, stem cells are separated from residual fetal cells after prenatal diagnosis, ACE2 and SV40LT gene transfection is carried out, and immortalized new coronavirus hatching cells which are easy to propagate in the cells due to the fact that the ACE2 and SV40LT are expressed are developed, so that the new coronavirus and the hatching cells can propagate in large quantities along with infinite amplification of the hatching cells when being cultured together, a virus source is provided for industrial batch preparation of the new coronavirus nucleic acid detection quality control viruses of unified batches, so that the viruses can be used by laboratories for using the same batch of quality control substances in a longer time after accurate detection and target value obtaining in a reference laboratory, and a foundation is laid for eliminating the phenomenon that each unit self-uses or uses non-new coronavirus quality control substances which are not in line with clinical samples. The novel coronavirus nucleic acid detection indoor quality control method established based on the quality control virus and Levey-Jennings quality control diagram can clearly observe whether the indoor quality control is out of control or not, so that whether the nucleic acid detection result is accurate and reliable or not can be judged.

Drawings

FIG. 1 is a clone of immortalized stem cells obtained by G418 screening.

FIG. 2 is a subculture map of immortalized stem cells.

FIG. 3 is a clone of a new coronavirus hatching cell obtained by puromycin screening.

FIG. 4 is a subculture of hatching cells of the novel coronavirus.

FIG. 5 shows the growth of cells incubated with new coronavirus for 5 days.

FIG. 6 is a new indoor quality control method for coronavirus nucleic acid detection based on Levey-Jennings quality control diagram designed by the invention.

In FIG. 1, since the G418 resistance gene is integrated into the DNA of stem cells successfully transfected with the SV40LT recombinant vector, the stem cells do not survive being killed by G418, and the surviving individual cells grow to form cell clones.

In FIG. 2, since the SV40LT gene is integrated into the DNA of the stem cell successfully transfected by the SV40LT recombinant vector, the characteristics of permanent survival and unlimited amplification are obtained, and the immortalized stem cell grows in fusiform adherent growth during repeated passages and grows vigorously.

In FIG. 3, since the DNA of immortalized stem cells successfully transfected with the ACE2 recombinant vector has incorporated the puromycin resistance gene, it will not be killed by puromycin and will survive, and the surviving individual cells, i.e., the new coronavirus hatching cells, grow to form cell clones.

In FIG. 4, when the quantitative new coronavirus hatching cells were cultured for 5 days, the cells grew in fusiform adherent manner and grew vigorously.

In FIG. 5, when the same amount of the hatching cells of the new coronavirus as that in FIG. 4 were cultured together with a fixed amount of the new coronavirus for 5 days, the cells were in a round, floating and dead state, which indicates that the expression of ACE2 gene in the hatching cells is favorable for the virus to be taken into and propagated in the cells, and finally the hatching cells were dead, and a large amount of virus content was detected.

In FIG. 6, the present invention designs that the high-value quality control virus is subjected to new coronavirus ORF1ab gene detection under the same condition with the clinical sample every day for 20 days, and the Ct value is calculated

And

the values were 90.3. + -. 2.3, 90.3. + -. 4.6 and 90.3. + -. 6.9, respectively, and these data were used to plot the Levey-Jennings quality control chart. Then, the same batch of quality control viruses and clinical samples are detected under the same condition every day, for example, if the detection data of each day are all drawn on the corresponding positions of the established Levey-Jennings quality control diagram and connected, whether the detection result of the day is out of control can be obviously found. As shown in the figure (the horizontal axis 1-31 represents the detection date), the detection results of 1-18 days only show that the detection results of 9 th day exceed 97.2(3SD), which is out of control, and the detection results of the new crown nucleic acid on the day are unreliable, and the reason needs to be analyzed or the detection needs to be carried out again.

Detailed Description

The following detailed description of the embodiments of the present invention will be made with reference to the accompanying drawings, but these are exemplary descriptions for public attention and research for prevention and control, and do not limit the scope of the present invention as defined in the appended claims.

1. Collection of Mesenchymal Stem Cells (MSC)

1.1. Collection of amniotic fluid mesenchymal stem cells

Collecting amniotic fluid cells of the pregnant woman to be detected according to the prenatal diagnosis process, performing cell culture and prenatal diagnosis, and screening out the fusiform mesenchymal stem cells growing adherently from the remaining amniotic fluid cells after the prenatal diagnosis under an inverted microscope.

1.2. Collection of umbilical cord mesenchymal stem cells

Taking out the frozen umbilical cord from liquid nitrogen tank, thawing rapidly in 37 deg.C water bath, cleaning with sterile PBS, removing residual blood stain, and cutting into size of about 1mm³The tissue block (2) is placed in a culture dish paved with fetal calf serum, placed at 37 ℃ for 6 hours, added with 10% fetal calf serum DMEM medium, and 5% CO at 37 DEG C₂Culturing in an incubator, observing the growth condition of cells around adherent tissues, and digesting and passaging by pancreatin when the cells reach 80-90% fusion degree to obtain the mesenchymal stem cells.

1.3. Collection of mesenchymal Stem cells

Aseptically taking parturient fetal lung tissue, mechanically dispersing, digesting with 0.25% trypsin, filtering with gauze with pore size of 100 μm, centrifuging at 1000r/min for 5min, discarding supernatant, and adding DMEM culture solution (0.1umol beta-mercaptoethanol, 100UI/mL streptomycin, 10% fetal calf serum). At 37 deg.C, 5% CO₂Culturing under the condition. And changing the solution after 45min to remove the cells which are not attached to the wall, and changing the solution every 48 h. After 80% cell confluence, 0.25% trypsin digestion was used for passage.

2. Immortalization of mesenchymal Stem cells (construction of MSC-SV40LT)

Construction of SV40LT/pLXSN

Performing PCR amplification on SV40LT high-fidelity long fragments by using SV40DNA (strain 766) as a template, an upstream primer of 5'-GCCCAGGATCCTTAACAACAACAACAAT-3' and a downstream primer of 5'-ACGCTGAATTCCCTCTGAGCTAT-3'; the PCR product of SV40LT and the pLXSN retrovirus vector are subjected to EcoR I/BamH I enzyme digestion, connection, transformation, screening and sequencing verification to obtain the SV40 LT/pLXSN-containing recombinant retrovirus vector.

2.2. Transfection of SV40LT into mesenchymal Stem cells

Mesenchymal cells to be transfected were matched to approximately 8X 10⁵Inoculating in single cell concentration, and placing in 5% CO₂And cultured at 37 ℃, infected with recombinant retrovirus containing SV40LT gene (Polybrene concentration is 8ug/mL) after 24h, and screened with G418 at 500ug/mL for 4 weeks after 1 week to obtain immortalized mesenchymal stem cell (MSC-SV40LT) clone (see FIG. 1). Or refer toThe literature refers to the preparation of hTERT transfected mesenchymal stem cells by an hTERT transfection method.

Identification of MSC-SV40LT

Characterization of biological Properties of MSC-SV40LT

Includes that the cell is spindle-shaped and fibroid. ② white bands appear at the relative molecular mass of 120000 and 93000 respectively by Western detection. ③ the growth curve of the cell line is typical of the "S" growth characteristics. And fourthly, the chromosome karyotype of the cell line is diploid. The cell line can not grow in soft agar. Sixthly, the nude mouse has negative tumorigenicity test.

Surface molecular assay for MSC-SV40LT

Detecting cell membrane surface molecules by using a flow cytometer, wherein positive molecules comprise CD73-APC, CD90-FITC, CD44-PE and CDl 05-Cy5.5; negative molecules include CDllb-PE, CDl9-PE, CD34-PE, CD45-PE, HLA-DR-PE. Through identification, the immortalized mesenchymal stem cells which can be permanently passaged in vitro are obtained. FIG. 2 shows immortalized stem cells that passed through passage 35.

2.3.3. Identification of immortalized Lung Stem cells (Lung MSC-SV40LT)

The human lung stem cell line needs to meet the following requirements: observing with a phase contrast microscope: growing in shuttle shape and arranging in vortex or barrier shape. Detecting by a flow cytometer: cell surface markers CD45, CDl 1a, CDl4, CD90, CD34, CD71, CD25, CDl05, CDll7, CDl66 and CD44 were positive. And thirdly, confocal technology detection: keratin expression is negative, and dryness-related factors c-Myc, Oct4, Nanog and Nestin are positive. Fourthly, indirect immunofluorescence detection: vimentin, type III collagen, fibronectin were positive, while surfactant protein C precursor, von willebrand factor, and alpha smooth muscle actin were negative.

ACE2 Gene Assembly of MSC-SV40LT (construction of MSC-SV40LT/ACE2)

3.1. Construction and identification of lentiviral expression vector pHBLV-ACE2 or pGC-FU-ACE2

ACE2 in plasmid pc-DNA3.1-hygro (+) -mACE2 (or ACE2 was PCR-amplified after RNA was extracted from lung tissue cells and reverse-transcribed to cDNA). Designing a PCR primer sequence according to the mRNA sequence of human ACE2 in GenBank, wherein an outer end primer amplified by human ACE 2: f1(F out) 5'-GAT GGA GTA CCG ACT GGA GTC-3', R1(Rout) 5'-CTA ATA TCG ATG GAG GCA TAA-3', product 547 bp; an inner end primer: f2(F in) 5'-GAG GAG GAT GTG CGA GTG GCT A-3', R2(R in) 5'-CCA ACC ACT ATC ACT CCC ATC A-3', and a product of 269 bp. The amplification primer sequence of the human beta-actin is as follows: F5'-GCT CGT CGT CGA CAA CGG CTC-3', R5'-CAA ACA TGA TCT GGGTCATCTTCT-3', product 353 bp. PCR conditions were as follows: 94 deg.C for 5min, then 94 deg.C for 30s denaturation, 55 deg.C for 30s annealing, 68 deg.C for 5min, circulating for 30 times, and finally extending at 68 deg.C for 10 min.

The vector pHBLV-CMV-ACE2-EF1-ZsGreen-T2A-puro and the PCR product are respectively cut by BamHI and EcoRI, or pGC-F and the PCR product are respectively cut by AgeI and EcoRI, agarose electrophoresis is used for purification, linearized vector DNA, the PCR product recovered by enzyme cutting, T4 phage DNA ligase and buffer thereof, and ddH20 are placed at 16 ℃ for overnight connection, the connection liquid is transformed into competent cells, and positive clones are identified by PCR and sequencing. The transformed bacteria are amplified by LB culture solution, pHBLV-ACE2-OE or pGC-FU-ACE2 is extracted by a plasmid extraction kit, 293FT cells are transfected by Lipofectamine 2000, and expression of ACE2 genes is detected by Western-Blot after 72h of transfection.

3.2. Packages carrying ACE2 lentivirus and titer determination thereof

A lentivirus expression vector (pHBLV-ACE2), a packaging plasmid (psPAX2 vector and pMD2G) or pGC-FU-ACE2 and the packaging plasmid (pHelper1.0 and pHelper2.0) are respectively co-transfected into 293T cells to obtain a recombinant lentivirus (Lentiviral-ACE) carrying an ACE2 gene. At the same time, another set of 293T cells was co-transfected with pHBLV empty vector (containing GFP gene), packaging plasmids (psPAX2 vector and pMD2G), and the empty vector (NC-GFP) carrying only GFP gene was obtained as a control.

And 8h after transfection, replacing the medium with a complete medium, culturing for 48h, collecting cell supernatant rich in lentivirus, centrifuging for 10min at 4 ℃ at 4000g, filtering the supernatant by using a 0.45-micron filter, removing cell debris, and centrifuging to obtain the lentivirus with high titer. Infecting 293T cells with the lentivirus stock solution to be identified, extracting RNA after 4d, and determining the lentivirus titer by Real-Time PCR.

Lentiviral-ACE transfection of MSC-SV40LT

At a rate of 1X 10 per hole⁶MSC-SV40LT was inoculated into 6-well plates at individual cell densities, and divided into an objective gene overexpression (OE-ACE2) group, an empty vector (NC-GFP) control group, and a blank group, each group had 2 wells, and when the cells reached 30%, 5-fold diluted lentivirus stock solution (Lentiviral-ACE) was added to the lentivirus group, 5-fold diluted empty vector stock solution was added to the empty vector group, 10% DMEM was replaced with FBS after 24h of culture, and puromycin (2.50. mu.g.mL) at the optimum screening concentration was added^-1) And maintaining the puromycin concentration, changing the liquid every other day until the blank control group completely dies, and finishing screening. The MSC-SV40LT not killed by puromycin is a stem cell line MSC-SV40LT/ACE2 which is transfected by recombinant lentivirus and has integrated ACE2 gene on DNA, namely a prefabricated new coronavirus hatching cell.

ACE2 Gene detection of MSC-SV40LT/ACE2

RT-PCR detection of mRNA transcription of ACE2 Gene

At 1 × 10⁶And (3) inoculating MSC-AT2R/ACE2 into a 6-well plate AT each cell density, culturing for 4d, observing fluorescence by using a fluorescence microscope, detecting transcription of ACE2 by using RT-PCR, wherein upstream primers are CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3', respectively; the downstream primer is EF 1-Rn: 5'-GCCAGTACACGACATCACTT-3', respectively; the upstream and downstream of beta-actin are respectively: 5'-TGGACTTCGAGCAAGAGATGG-3', 5'-ATCTCCTTCTGCATCCTGTCG-3'. And comparing and statistically analyzing the ACE2/B-actin integrated optical density value (IOD), calculating the mRNA expression amount of the test group and the control group, and screening the MSC-SV40LT/ACE2 with high ACE2 expression efficiency.

Western-Blot detection of ACE2 protein expression

Extracting proteins of cell lysates of each group, quantifying the proteins by a BAC method, and arranging 3 random multiple holes in each group. The sample proteins were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes, primary antibody was anti-VDR diluted 1: 400, visualized using ECL chemiluminescence kit, and the relative gray values of the protein bands determined using a gel image analysis system. And observing the expression of ACE2 protein of the test group and the control group, and screening the MSC-SV40LT/ACE2 with high expression of ACE 2.

Functional testing of MSC-SV40LT/ACE2

4.1. Sample collection

The throat swab of the patient with COVID-19 confirmed diagnosis is taken, double antibodies (10000IU penicillin and 10000 mug streptomycin) are added according to the proportion of 100: 1, the final concentration of the penicillin and the streptomycin is 100IU and 100 mug respectively, and the mixture is kept at 4 ℃ for overnight use.

4.2. Virus culture and isolation

Vero-E6 was inoculated into 12.5cm of DMEM medium containing (10% fetal bovine serum)²Placing in a culture flask at 36 deg.C and 5% CO₂Culturing to 30% confluent monolayer cells in incubator, removing culture medium, washing cells with DMEM for 2 times, adding 0.5mL of double antibody-treated COVID-19 patient sample into culture flask, standing at 36 deg.C and 5% CO₂Adsorbing for 90min in an incubator, removing the sample, adding 3.5mL DMEM culture solution (10% fetal bovine serum), observing cytopathic effect (CPE) every day, culturing for 5-7 d, taking supernatant of pathological cells, performing sucrose gradient ultracentrifugation, separating new coronavirus, and preparing 10% culture solution into 10% of the new coronavirus³～10⁵TCID₅₀Viral fluid/ml.

Co-culture of MSC-SV40LT/ACE2 with viruses

Vero group, MSC-SV40LT group, and MSC-SV40LT/ACE2 group were set up, and 12-well plates were inoculated per group to contain 2X 10 per well⁵Individual cells, 2mL of DMEM medium (10% fetal bovine serum), at 36 ℃ with 5% CO₂When the mixture was cultured in an incubator to 30% confluency, 0.5mL of virus solution was added to each well, and the culture was continued. Then, after incubation for 1 hour, 6 hours, 24 hours and 72 hours, 3-well supernatants were collected from each group, mixed and diluted 1: 4, 1: 12, 1: 36, 1: 108, 1: 324, 1: 972, 1: 2916 and 1: 8748 times, and subjected to RT-PCR detection.

4.4. Real-time fluorescent RT-PCR detection of viral RNA

Nucleic acid extraction kit (batch No. 2019004), 2019 novel coronavirus (ORF1ab/N) nucleic acid detection kit (batch No. 20200123) and DA3200 nucleic acid extractor from Daan Gen-stocky Co., Ltd, Zhongshan university, and ABI 7500 type PCR instrument from Thermo Fisher Scientific, USA. According to the operation of the kit specification, the amplification reaction conditions are as follows: 15min at 50 ℃; 15min at 95 ℃; 15s at 94 ℃; 45s at 55 ℃; for a total of 45 cycles, fluorescence signals were collected at 55 ℃.

According to the kit specification, the result judgment criteria are as follows: if the detected sample has no amplification curve in ORF1ab and N gene channel or Ct value is greater than 38, it is judged as SARS-CoV-2 negative; if the Ct value of the detected sample in ORF1ab and N gene channel is less than or equal to 38 and there is obvious amplification curve, it is determined as SARS-CoV-2 positive; and thirdly, if the Ct value of the detected sample in ORF1ab or N gene channel is less than or equal to 38, the other channel has no amplification curve, the retest result is consistent with the original result, and the SARS-CoV-2 is judged to be positive.

4.5. Results of viral RNA detection

In Table 1, the dilution at which viral RNA was detected positively in each group of the culture broth after the cell line and the virus were cultured in a mixed state for 1 hour was 1: 4, which was considered as the result of detection of the virus added exogenously to the culture broth in the state where it was not substantially replicated. In tables 2-3, after the cell line and virus were cultured for 6 hours in a mixed manner, the titers of the culture solution of MSC-SV40LT/ACE2 group and Vero cell group and the intracellular RNA detection positivity were all 1: 36, which was increased by 3 times compared with MSC-SV40LT group. In Table 4, after the cell line and virus were cultured in mixture for 24 hours, the titers of RNA detection positivity in the culture solutions of the MSC-SV40LT/ACE2 group and the Vero cell group were both 1: 972, which was increased 27-fold compared with the MSC-SV40LT group. In tables 5-6, the titers of positive RNA detection in the culture medium and the cells of MSC-SV40LT/ACE2 were 1: 26244 or more, respectively, after the cell line and virus were mixed and cultured for 72 hours.

The MSC-SV40LT/ACE2 that is the new coronavirus hatching cell can make the new coronavirus largely replicate in the cell, can be used as the host cell for virus propagation, and can be applied to the preparation of the new coronavirus nucleic acid detection quality control virus.

TABLE 1 results of viral RNA detection in 1 hour coculture of cell lines with isolated New coronavirus in culture broth

TABLE 2 results of viral RNA detection in culture broth co-cultured with isolated new coronavirus for 6 hours

TABLE 3 intracellular viral RNA assay results of cell lines co-cultured with isolated New coronavirus for 6 hours

TABLE 4 results of viral RNA detection of cell lines co-cultured with isolated New coronavirus in 24-hour medium

TABLE 5 results of viral RNA detection of cell lines cocultured with isolated New coronavirus for 72h

TABLE 6 intracellular viral RNA assay results of cell lines cocultured with isolated New coronavirus for 72 hours

4.6. Storage of new coronavirus hatching cells

Subpackaging the hatching cells into ampoules, each ampoule being 10X 10⁶And (4) storing the cells in a liquid nitrogen cell bank at the temperature of-196 ℃.

5. Preparation method of new coronavirus hatching cell-based new coronavirus nucleic acid detection quality control virus

5.1. Equipment

A bioreactor: can be produced by Holland application company, including 7 liters, 75 liters and 550 liters, is automatically controlled by a computer, can be sterilized in place, and automatically controls the dissolved oxygen, the pH value, the temperature, the stirring speed and other fermentation parameters; a clarification and filtration device: the multi-stage filtration can be carried out by using a filter produced by PALL company, a filter column is put into a stainless steel filter cylinder, and the filter cylinder is sterilized for standby after being cleaned; and (3) ultrafiltration concentration equipment: the Millipore Pelicon 2 system, polyethersulfone ultrafiltration membrane, 100 million molecule cut-off, can be used; a chromatography purification device: using a GE AKTA Primer chromatographic purification system, BPGl00/950 and BPG200/450 chromatographic columns; DMEM culture medium, M199 culture medium and fetal bovine serum are selected from Gibco company; the microcarrier is selected from GE Healthcare company of America;

5.2. collection of novel coronavirus strains

Preparing inactivated vaccine with epidemic strain designated by authority organization, or taking throat swab of patient with COVID-19 confirmed diagnosis, adding double antibody (10000IU penicillin and 10000 ug streptomycin) at ratio of 100: 1 to make final concentration of penicillin and streptomycin 100IU and 100 ug respectively, and standing at 4 deg.C overnight.

5.3. Incubation cell culture

Taking 1 or more ampoule hatching cells in a cell bank, inoculating the cells into culture flasks for culture, carrying out subculture amplification, inoculating a new coronavirus strain after the required amount of the hatching cells is reached, carrying out mixed culture on the virus and the hatching cells, and then collecting virus liquid.

Or culturing the cells to be incubated to a monolayer, digesting the cells by pancreatin, and inoculating the cells to a bioreactor for cell culture. Performing perfusion culture in a bioreactor, performing recirculation culture in a 75L bioreactor, and performing batch culture in a 550L bioreactor. The culture conditions of each stage of bioreactor are as follows: 3-5g/L of Cytodexl microcarrier and DMEM cell culture solution, the culture temperature is 37 +/-0.5 ℃, the dissolved oxygen is 50 percent or 20 percent, the pH value is 7.2 or 0.2, the stirring speed is 50 +/-20 rpm, and the culture is carried out for 4-7 days. After growing into a monolayer on the microcarrier particles, digesting the cells by trypsin, dispersing the cells into uniform cell suspension, and inoculating the cell suspension into a next-stage fermentation tank for amplification culture; production cells (not less than 0.8X 10) expanded to an appropriate amount in 550 liters⁶Cells/ml) for virus inoculation.

5.4. Bioreactor culture of viruses

After the cells are cultured by 550 liters of microcarriers to reach a certain concentration, abandoning the cell culture solution, replacing with a virus maintenance solution, and inoculating single type virus working seeds with the inoculation amount of 0.01-0.3 MOI. The culture conditions were: the temperature is 33 ℃, the temperature is 0.5 ℃, the dissolved oxygen is 25% + -10%, the pH value is 7.4+0.2, and the stirring speed is 50 +/-20 rpm. And culturing the virus for 48-96 hours, and harvesting the virus suspension when the hatching cells are completely diseased.

5.5. Virus fluid harvesting and filtration

The virus liquid with complete lesion is clarified and filtered by filters with different pore size combinations of 75 μm, 75 μm and diatomite filter aid, 0.45 μm and 0.22 μm, and microcarriers and cell debris are removed.

5.6. Concentration of virus liquid

After clarification, the virus harvest liquid is ultrafiltered and concentrated by an ultrafiltration membrane with the molecular weight cutoff of 100KD by 400 times by using ultrafiltration concentration equipment.

5.7. Purification of virus liquid

Purifying the virus concentrated solution by Sepharose CL-6B gel filtration chromatography, wherein the elution buffer solution is PBS (pH7.0-4-0.2), the sample loading amount of each time should not exceed 10% of the column volume, the flow rate is 0.35 +/-0.2 cm/min, the detection wavelength is 280nm, and collecting the first peak. The collected virus liquid was subjected to gel filtration and purified by ion exchange chromatography using DEAE Sepharose f.f. The buffer solution is PBS (pH7.0 + -0.2), the eluent is 1mol/L NaCl, the flow rate is 0.5 + -0.15 cm/min, and the first peak is the purified virus peak.

5.8. Filtration and inactivation of virus fluids

And (3) sterilizing and filtering the virus purified solution by using a filter with the pore diameter of 0.22 mu m, and inactivating the filtered virus solution by storing the virus solution at 2-8 ℃ for not more than 72 hours. The purified solution after filtration is diluted by M199 and then inactivated by formaldehyde, and the purified solution of each type of virus is inactivated under the condition that the protein content is lower than 150 mu g/ml. Adding formaldehyde to a final concentration of 92.5. mu.g/ml, inactivating at 37 ℃. + -. 0.5 ℃ to day 6, refiltering the inactivation solution through a filter with a pore size of 0.22. mu.g, and continuing to inactivate at 37 ℃ and 0.5 ℃ until day 12 is terminated.

5.9. Virus inactivation verification test

Virus inactivation verification by adopting a cell culture method: taking two samples respectively at the 9 th day of inactivation and at the termination of inactivation, wherein the sampling amount is 1500 doses each time, performing virus inactivation verification detection on each sample, inoculating the samples to Vero monolayer cells, culturing at the temperature of 35.5 ℃ and 0.5 ℃ for 21 days (original culture), and taking original culture supernatant for 14 days and 21 days to perform subculture for 14 days respectively, wherein no CPE occurs in the original culture and the subculture, which indicates that the virus inactivation is thorough and no live virus exists.

5.10. Virus liquid purity detection

Detection is carried out using an HPLC water-soluble gel column or other suitable chromatographic column: taking 100 mu L of a purified solution sample of the new coronavirus inactivated vaccine for detection, wherein a mobile phase is 0.1mol/L phosphate-0.1 mol/L sodium chloride buffer solution (pH7.0 Shi 0.20), the flow rate is 0.3ml/min, the detection wavelength is 280nm, the running time is 40 minutes, and the purity of the virus solution is analyzed and is not lower than 95%.

5.11. Preparation of quality control virus stock solution

Respectively detecting the copy number of target nucleic acid of the quality control virus by 3-5 nucleic acid detection reference laboratories to prepare 10⁵～10⁶And copying/mL, and subpackaging in a cryopreservation tube, and performing cryopreservation at-80 ℃ for later use.

5.12. Preparation of quality control virus application liquid

According to the nucleic acid detection sensitivity of kits of different companies, according to reference documents, the quality control virus stock solution is subjected to gradient dilution by using 0.01mol/L PBS, oropharyngeal swab sample mixed liquor with new coronavirus nucleic acid detection negativity, mixed serum or mixed plasma, high-value, medium-value and low-value application quality control products with dilution degrees of 1: 25, 1: 125 and 1: 625 can be obtained, the nucleic acid copy number of the medium-value quality control product is the weak positive level indicated by the nucleic acid detection kit, each 210 mu L is packaged into 1.5mL centrifuge tubes, and the centrifuge tubes are frozen for standby.

5.13. Application of quality control virus in indoor quality control of nucleic acid detection

And (3) carrying out nucleic acid extraction, amplification, result analysis and indoor quality control diagram drawing on the quality control virus application liquid and the tested clinical specimen under the same condition, wherein the specific operation refers to different kit specifications.

5.13.1. Extraction of nucleic acid from novel coronavirus

And (3) taking out the nucleic acid extraction plate with 96 holes, adding 200 mu L of quality control virus into a biological safety cabinet, placing the biological safety cabinet in a BG-Flex-48 full-automatic nucleic acid extraction instrument, adding a magnetic sleeve, selecting an automatic extraction program to start extraction, and immediately detecting the extracted nucleic acid.

5.13.2. Nucleic acid amplification

Prepare 25 uL reaction system according to the 2019-nCoV nucleic acid detection kit instruction, 12 uL reaction solution containing nucleic acid amplification, 4 uL enzyme mixed solution, 4 uL ORF1ab/N reaction solution (mainly including primer and probe), and 5 uL nucleic acid sample. The real-time fluorescent RT-PCR amplification program comprises the following steps: 10min at 50 ℃; 5min at 95 ℃; 95 ℃ for 10s, 55 ℃ for 40s, 40 cycles. The new coronavirus ORF1ab and N target gene are detected by amplifying in an Anadas9850 full-automatic nucleic acid extraction and fluorescence PCR analysis system.

5.13.3. Analysis of results

Adjusting the start-stop value according to the actual amplification curve, analyzing, judging the detection result according to the result interpretation standard in the reagent specification, and determining the detection cycle threshold (Ct) value of the quality control virus target gene ORF1ab and N.

5.13.4. Statistical analysis

Performed with SPSS 13.0 software. Normal distribution data is expressed by mean + -standard deviation (x + -s), mean comparison between two groups is performed by independent sample t test, variation coefficient between two groups is performed by paired t test, and mean comparison between multiple groups is performed by single-factor variance analysis.

5.13.5. Method for establishing indoor quality control

And applying the Levey-Jennings quality control diagrams used in other detection projects to indoor quality control of new coronavirus nucleic acid detection, and establishing the new coronavirus nucleic acid detection indoor quality control method based on the Levey-Jennings quality control diagrams.

The Levey-Jennings quality control diagram is composed of a horizontal axis and a vertical axis, wherein the horizontal axis represents detection date or batch, and the vertical axis represents quantitative detection value. There are 3 balance lines on both sides of the horizontal axis, respectively representing quality control detection values

And

wherein the 3 balance lines above the horizontal axis represent the detected values respectively

And

the 3 balance lines below the horizontal axis respectively represent

And

when the indoor quality control result exceeds the Levey-Jennings quality control diagram

In time, the quality control is out of control, and the detection result of the clinical samples in the same batch is inaccurate.

When the indoor quality control of the new coronavirus nucleic acid detection based on the Levey-Jennings quality control diagram is established, the new coronavirus ORF1ab and N gene detection is carried out on the high-value, medium-value and low-value quality control viruses and clinical samples under the same condition every day (batch), the detection lasts for more than 20 days (batch), and the cycle threshold (Ct/delta Ct) of the detection result is calculated

And SD, and then calculate

And

and then drawing and establishing a Levey-Jennings quality control diagram for detecting the new coronavirus nucleic acid. In the application of the quality control chart, the same batch of quality control viruses and clinical samples are subjected to new crown disease under the same conditionsAnd (3) detecting toxic ORF1ab and N genes, drawing the Ct (delta Ct) of the detection result every day at the corresponding position of the quality control diagram, and then judging the quality control result according to a conventional method. For example, if the Ct of a certain quality control virus exceeds + -3 SD, this indicates that the detection is out of control, and the nucleic acid detection result of the same clinical sample may be inaccurate, and needs to analyze the cause or re-detect.

For example, the high-value quality control virus is subjected to new coronavirus ORF1ab gene detection under the same condition with a clinical sample every day for 20 days, and the Ct value is calculated

And SD to obtain corresponding

And

the values are 90.3 + -2.3, 90.3 + -4.6 and 90.3 + -6.9 respectively, and the Levey-Jennings quality control diagram is established by using the data. Then, the same batch of quality control viruses and clinical samples are detected under the same condition every day, for example, the detection data obtained from days 1 to 3 are respectively 88, 92 and 93, and then the detection data are plotted at the corresponding positions of the established Levey-Jennings quality control diagram (FIG. 6). As can be seen from FIG. 6 (the horizontal axis 1 to 31 represents the detection date), among the detection results for 1 to 18 days, only the detection result for the 9 th day exceeds 97.2(3SD), which means that the detection result is out of control, and this indicates that the detection result for the new crown nucleic acid is unreliable on that day and needs to be analyzed or re-detected.

5.13.6. Indoor quality control based indoor assessment for developing nucleic acid detection

The quality control supervisor sends the same batch of quality control viruses to each laboratory for nucleic acid detection and evaluates the conformity of the detection result fed back by each laboratory with a target value (true value) or the mean value of each laboratory so as to assess whether the nucleic acid detection result of each laboratory is accurate and reliable.

Claims

1. A method for preparing and controlling the quality control virus for detecting the nucleic acid of new coronavirus is characterized by that firstly, the method uses the new coronavirus as referenceInfecting host cells through ACE2 receptor and easily propagating in host cells expressing ACE2 and immortalizing human fibroblasts by SV40LT, separating stem cells from residual fetal cells after prenatal diagnosis, transfecting ACE2 and SV40LT genes, developing immortalized new coronavirus hatching cells which easily allow new coronavirus to enter cells to propagate due to overexpression of ACE2 and SV40LT, and allowing the new coronavirus and the hatching cells to be in a bioreactor containing DMEM cell culture solution containing 3-5g/L microcarrier at 37 ℃ and 5% CO₂The pH value is 7.2, and the stirring speed is 50 +/-20 rpm, and the cells can propagate in a large amount along with the infinite amplification of the hatching cells when being cultured together; further collecting a culture containing a large amount of propagated viruses, filtering by 75-micrometer, 0.45-micrometer and 0.22-micrometer filters, ultrafiltering and concentrating by a 100KD ultrafiltration membrane, purifying by Sepharose CL-6B gel filtration chromatography, sterilizing and filtering by a 0.22-micrometer filter, inactivating 92.5-mu g/ml formaldehyde, verifying the inactivation effect, and detecting the purity of the virus liquid to obtain an inactivated virus liquid; determining the target value of the target nucleic acid to be detected and preparing the inactivated virus liquid into new coronavirus detection quality control viruses with high value, medium value and low value according to the requirements of different nucleic acid detection kits; then the prepared quality control virus replaces the simulated quality control substances such as false virus or bacteriophage virus-like particles which are used by related units or are inconsistent with the tested sample and the like in the nucleic acid detection kit, or replaces the self-made quality control substances without uniform lot numbers of all units, so that all nucleic acid detection laboratories can use the uniform lot number quality control substances consistent with clinical real viruses to monitor the nucleic acid detection quality in real time; and finally, establishing a nucleic acid detection standardized indoor quality control method based on a Levey-Jennings quality control diagram by using the quality control virus, and judging whether the detection result of the same batch of new nucleic acid of the coronary pneumonia is accurate and reliable in real time.

2. The method for preparing and controlling the quality of the new coronavirus hatching cell as claimed in claim 1, wherein the preparation of the quality control virus by the new coronavirus hatching cell comprises preparing the quality control virus by a Vero cell, or preparing an inactivated virus by the Vero cell, thereby preparing the nucleic acid detection quality control virus.

3. The method for preparing and controlling the quality of the neocorolla pneumonia nucleic acid detection virus according to claim 1, wherein the SV40LT immortalizing human fibroblasts comprises preparing immortalized neocorolla virus hatching cells with hRERT.

4. The method for preparing and controlling the quality of the neocorolla pneumonia nucleic acid detection virus according to claim 1, wherein the method for establishing the indoor quality control of the neocorolla pneumonia nucleic acid detection comprises the steps of preparing the quality control virus into high-value, medium-value and low-value quality control viruses, carrying out the gene detection of the neocorolla virus ORFlab and N under the same condition with a clinical sample, detecting for more than 20 days in total, and calculating the cycle threshold Ct/delta Ct of the detection result

And SD value, and further calculating

And

and (3) values are drawn on a Levey-Jennings indoor quality control diagram, then new coronavirus ORFlab and N gene detection is continuously carried out on the same batch of quality control viruses and clinical samples under the same condition, Ct/delta Ct of a daily detection result is drawn at a corresponding position of the Levey-Jennings indoor quality control diagram, if the Ct/delta Ct of a certain day quality control virus exceeds +/-3 SD, the detection result is out of control, the nucleic acid detection result of the same batch of clinical samples is possibly inaccurate, and reasons need to be analyzed or the detection needs to be carried out again.

5. The method for preparing and controlling the quality of the new coronavirus used for nucleic acid detection and quality control of the coroneumoniae according to claim 1, which comprises the following steps:

(1) artificial neocoronavirus hatching cells:

collecting remaining amniotic fluid cells after prenatal diagnosis, separating fusiform amniotic fluid cells or lung tissue cells, transfecting SV40LT and/or hTERT genes, screening an immortalized cell line by G418 and/or puromycin, and obtaining the immortalized amniotic fluid mesenchymal stem cell line or the lung stem cell line through cell line biological identification and lung stem cell marker detection, wherein the immortalized amniotic fluid mesenchymal stem cell line or the lung stem cell line is named as MSC-SV40 LT/hTERT.

The ACE2 gene is connected to a lentivirus expression vector pHBLV-CMV-ACE2-EF1-ZsGreen-T2A-puro or pGC-FU, a recombinant plasmid pHBLV-OE-ACE2 or pGC-FU-ACE2 is respectively constructed, the recombinant plasmid pHBLV-OE-ACE2 and a packaging plasmid (psPAX2 and pMD2G) or the recombinant plasmid pGC-FU-ACE2 and a packaging plasmid (pHelper1.0 and pHelper2.0) are respectively co-transfected into 293FT Cells, recombinant lentiviruses carrying ACE2 are packaged, the recombinant lentiviruses are transfected into MSC SV40LT/hT, the ACE2 is integrated on DNA of the immortalized stem Cells, and immortalized new corona virus hatching Cells which are easy to infect and propagate the new corona virus are obtained through screening and identification and are used as matrix Cells which are named as Nolsonic virus vaccine Cells (Corronnic virus inactivated vaccine).

(2) Preparation of nucleic acid detection quality control virus based on hatching cell

Culturing the incubated cells to desired cell amount, transferring into DMEM bioreactor containing 3-5g/L microcarrier, and culturing at 37 deg.C with 5% CO₂Culturing at pH 7.2 and stirring speed of 50 + -20 rpm to form 60% confluence degree of cells on the surface of the microcarrier, replacing virus maintaining solution, inoculating virus solution with 0.01-0.3 MOI amount, culturing at 33 deg.C and 5% CO₂Culturing for 48-96 hours under the conditions of pH value of 7.4 and stirring speed of 50 +/-20 rpm until the cells are completely diseased, and harvesting virus liquid.

Filtering the virus liquid by 75 mu m, 0.45 mu m and 0.22 mu m filters, ultrafiltering and concentrating by a 100KD ultrafiltration membrane, purifying by Sepharose CL-6B gel filtration chromatography, sterilizing and filtering by a 0.22 mu m filter, inactivating 92.5 mu g/ml formaldehyde, verifying the inactivation effect, and detecting the purity of the virus liquid to obtain the inactivated virus liquid.

Preparing the inactivated virus solution into high-value, medium-value and low-value quality control virus solutions according to the detection requirements of the nucleic acid detection kit, detecting the content of virus nucleic acid by a reference laboratory, determining a target value, and conventionally subpackaging and storing in a quality control cell bank.

(3) Levey-Jennings indoor quality control diagram for establishing nucleic acid detection based on quality control virus

Performing nucleic acid detection on the quality control virus and clinical samples under the same condition, continuously detecting for more than 20 batches, and calculating the cycle threshold Ct or delta Ct of the detection result

And SD value, and calculating

And

the value is obtained.

By referring to Levey-Jennings quality control charts already used in other fields, the obtained results will be calculated

And

values are plotted on a Levey-Jennings quality control map, and a Levey-Jennings indoor quality control map suitable for detecting the new coronary pneumonia nucleic acid, which is called as an LJ new coronary quality control map for short, is created.

And then, in each nucleic acid detection of the clinical samples, detecting the same batch of quality control viruses under the same condition, drawing the detection results of the quality control viruses at corresponding positions of the LJ new corona quality control diagram, and judging whether the detection results of the same batch of clinical samples are accurate and reliable by judging whether the quality control is in control or out of control from the positions in the diagram.

(4) Indoor quality control based indoor assessment for developing nucleic acid detection

The quality control supervisor sends the same batch of quality control viruses to each laboratory for nucleic acid detection and evaluates the conformity degree of the detection results fed back by each laboratory and the target value so as to check whether the nucleic acid detection results of each laboratory are accurate and reliable.