Multi-linked quality control product for quality control of novel coronavirus nucleic acid detection laboratory and preparation method of multi-linked quality control product

Abstract

The invention relates to the technical field of genetic engineering, and particularly provides a multi-linked quality control product for quality control of a novel coronavirus nucleic acid detection laboratory and a preparation method of the multi-linked quality control product. The multi-linked quality control product is a multi-component composite system and is formed by mixing three substances, namely pseudovirus, target gene RNA (Ribonucleic Acid) molecules and internal standard plasmid DNA (Deoxyribose Nucleic Acid). Wherein the pseudovirus is pseudovirus particles of novel coronavirus ORF1ab gene RNA (Ribonucleic Acid) wrapped by a vesicular stomatitis virus vector and is used for monitoring virus nucleic acid extraction; the target gene RNA molecule is a novel coronavirus N gene RNA molecule and can monitor the reverse transcriptase activity; the internal standard plasmid is plasmid DNA of a human housekeeping gene RPP30, and whether inhibition or failure exists in an amplification link is monitored. According to the method, the whole process of nucleic acid extraction, reverse transcription and amplification is synchronously and hierarchically monitored, the monitoring blind area of a traditional single-component quality control product is solved, and the accuracy and efficiency of laboratory detection quality control are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and relates to a multiple quality control product for laboratory quality control of novel coronavirus nucleic acid detection and a preparation method thereof. Background Art

[0002] During the nucleic acid testing process, errors can be introduced at multiple stages, from sample collection, nucleic acid extraction, reverse transcription, and amplification, leading to false positive or false negative test results. To ensure the accuracy and reliability of novel coronavirus nucleic acid testing, comprehensive quality control of the entire testing process is necessary. Current quality control products on the market often only monitor the quality of a single step and are unable to achieve comprehensive quality control of the entire process, including nucleic acid extraction, reverse transcription, and amplification. This makes it difficult to meet the comprehensive quality control needs of novel coronavirus nucleic acid testing laboratories.

[0003] In our previous research, the quality control products involved in the invention patent CN113943832A "A novel coronavirus nucleic acid diagnostic pseudovirus quality control product based on a VSV vector and its preparation method" have promoted the quality control work of the novel coronavirus nucleic acid detection laboratory. However, there are still deficiencies in monitoring the entire process of nucleic acid extraction, reverse transcription, and amplification, and it is impossible to achieve simultaneous monitoring of all aspects of nucleic acid detection. After investigating domestic hospital laboratories, nucleic acid detection bases, and disease control centers, and collecting extensive feedback from doctors and experts, it can be seen that laboratory quality control issues are becoming increasingly prominent. Moreover, since the operating steps of novel coronavirus nucleic acid detection involve many complex factors, once the laboratory test results are abnormal, the existing types of quality control products are difficult to assist staff in quickly and accurately identifying the specific link where the abnormality comes from. This is very unfavorable for timely problem solving and ensuring the smooth progress of detection work.

[0004] It is worth noting that there are currently no pseudovirus-RNA-DNA multiplex quality control products for the detection of novel coronavirus nucleic acid on the market, in relevant literature, or in patents. The development of such multiplex quality control products faces many difficult challenges. Taking pseudoviruses as an example, many pseudoviruses generally have residual DNA during the preparation and production process. DNA residues will seriously interfere with the effective monitoring of nucleic acid extraction, reverse transcription, and amplification processes by multiplex quality control products, resulting in false positive results. During the nucleic acid extraction process, residual DNA may be extracted together, resulting in deviations in subsequent test results and an inability to truly reflect the actual extraction of viral nucleic acid in the sample; during the reverse transcription and amplification stages, residual DNA may participate in the reaction and compete with the target RNA for reaction resources, affecting the amplification efficiency and the accuracy of the results, thereby making it impossible for the multiplex quality control products to accurately control the quality of the entire nucleic acid detection process.

[0005] Therefore, based on the actual needs of nucleic acid testing laboratories, it is urgent to develop a multi-component quality control product that can simultaneously monitor the entire process of novel coronavirus nucleic acid testing to ensure test accuracy. This quality control product will help laboratory testing personnel quickly and accurately identify laboratory quality control issues and ensure laboratory testing quality and efficiency. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a composite quality control product composed of three substances: pseudovirus, RNA and DNA, and a preparation method thereof. The composite quality control product is composed of three different types of nucleic acid substances, all of which are prepared by gene recombination technology: first, a carrier DNA (plasmid DNA) containing the target gene is constructed, and the gene plasmid DNA is used as an internal control for amplification to specifically monitor the nucleic acid amplification link; then, pseudovirus packaging technology is used to convert the carrier DNA into RNA, and then the RNA is packaged into pseudovirus particles to simulate real virus particles and monitor the nucleic acid extraction link; finally, naked RNA molecules are obtained from the pseudovirus through nucleic acid extraction and purification, and the purified target gene RNA molecules independently monitor the reverse transcription process. The composite quality control product is designed with multiple components to synchronously monitor the entire process of nucleic acid extraction, reverse transcription and amplification in all directions, and has the advantages of low cost, high stability and easy storage and transportation, meeting the large-scale quality control needs of the laboratory.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] The present invention provides a multi-component quality control product for laboratory quality control of novel coronavirus nucleic acid detection. The multi-component quality control product is a multi-component composite system, including a novel coronavirus ORF1ab gene pseudovirus, a purified target gene RNA molecule, a human internal standard RPP30 gene plasmid DNA, and a protective agent. It helps laboratory testing personnel to quickly and accurately monitor the laboratory testing process and significantly improve the efficiency of laboratory testing quality control.

[0009] The novel coronavirus ORF1ab gene pseudovirus uses a modified vesicular stomatitis virus (VSV) as a vector to encapsulate the novel coronavirus ORF1ab gene RNA. The nucleic acid extraction efficiency is monitored by simulating the structure of real virus particles. The target sequence of the ORF1ab gene RNA is NC_045512.2: 6266-9565.

[0010] The target gene RNA molecule is a novel coronavirus N gene RNA molecule, which is independent of the pseudovirus system and is specifically used to monitor enzyme activity and reverse transcription reaction efficiency during the reverse transcription process. Its target gene sequence is NC_045512.2: 28274-29533.

[0011] The human internal marker of the multiplex quality control product is a plasmid DNA of the human housekeeping gene Ribonuclease P / MRP subunit p30 (RPP30), and its target gene sequence is No. NC_006413: 4-820, which is used to monitor amplification failure and reaction system inhibition in the amplification link.

[0012] Furthermore, the concentrations of the components in the multiple quality control products are as follows: novel coronavirus ORF1ab gene pseudovirus 1×10 7 copies / mL, novel coronavirus N gene RNA molecules 1×10 7 copies / mL, human internal standard RPP30 gene plasmid DNA 1×10 7 copies / mL.

[0013] The protective agent includes: 10-20mM phosphate buffer saline (pH 7.2), 2% to 5% (w / v) trehalose, 2% to 10% (w / v) sucrose, 1% to 5% (w / v) gelatin, 0.1% to 1% (w / v) bovine serum albumin, 5-10mM EDTA (DNA nuclease inhibitor), and 1-5U / mL RNasin (RNA nuclease inhibitor).

[0014] The present invention also provides a method for preparing the above-mentioned multiple quality control product, comprising the following steps:

[0015] Step 1: Prepare the novel coronavirus ORF1ab gene pseudovirus. The specific steps are as follows:

[0016] (1) Gene construction: Based on the ORF1ab gene target sequence NC_045512.2: 6266-9565, an ORF1ab gene fragment with an MluI restriction site was designed and synthesized. The recognition sequence of the MluI restriction site was 5'-ACGCGT-3'. The fragment was ligated with the pVSV-RNA-Amplicon or pVSV-RNA-G-Amplicon vector by in-fusion recombination to obtain a recombinant plasmid containing the ORF1ab gene. The nucleotide sequence of the pVSV-RNA-Amplicon is shown in SEQ ID NO. 1, and the nucleotide sequence of the pVSV-RNA-G-Amplicon is shown in SEQ ID NO. 2.

[0017] (2) Pseudovirus packaging: The recombinant plasmid of step (1) was co-transfected into HEK293T cells with the packaging auxiliary vectors pP, pN, pL, and VSVG. The cell culture supernatant was collected 48 hours after transfection, centrifuged at 2000 g for 10 minutes, and filtered through a 0.22 μm filter to obtain the pseudovirus packaging rescue stock solution;

[0018] (3) Pseudovirus propagation: VSVG plasmid was transfected into HEK293T cells, and the pseudovirus stock solution from step (2) was added 6 to 8 hours later. After 48 hours, the supernatant was harvested and the propagation process was repeated until the pseudovirus titer reached 10 8 TCID50 / mL.

[0019] Step 2: Prepare a novel coronavirus N gene RNA molecule: The novel coronavirus N gene RNA molecule is a novel coronavirus N gene RNA transcribed in vitro or extracted from nucleic acid;

[0020] The preparation steps of the novel coronavirus N gene pseudovirus are as follows:

[0021] (1) Gene construction: Based on the novel coronavirus N gene target sequence NC_045512.2: 28274-29533, an N gene fragment with an MluI restriction site was designed and synthesized; the fragment was ligated with the pVSV-RNA-Amplicon or pVSV-RNA-G-Amplicon vector by in-fusion recombination to obtain a recombinant plasmid containing the N gene; the nucleotide sequence of the pVSV-RNA-Amplicon is shown in SEQ ID NO. 1, and the nucleotide sequence of the pVSV-RNA-G-Amplicon is shown in SEQ ID NO. 2;

[0022] (2) Pseudovirus packaging: The recombinant plasmid of step (1) was co-transfected into HEK293T cells with the packaging auxiliary vectors pP, pN, pL, and VSVG. The cell culture supernatant was collected 48 hours after transfection, centrifuged at 2000 g for 10 minutes, and filtered through a 0.22 μm filter to obtain the pseudovirus packaging rescue stock solution;

[0023] (3) Pseudovirus expansion: VSVG plasmid was transfected into HEK293T cells. After 6 to 8 hours, the pseudovirus packaging and rescue stock solution of step (2) was added. After 48 hours, the supernatant was harvested and the expansion process was repeated until the pseudovirus titer reached 10 8 TCID50 / mL.

[0024] Step 3: Prepare human internal standard RPP30 gene plasmid DNA. The specific steps are as follows:

[0025] (1) Gene construction: Based on the human RPP30 gene target sequence NC_006413:4-820, an RPP30 gene fragment with an MluI restriction site was designed and synthesized; the fragment was ligated with the pVSV-RNA-Amplicon or pVSV-RNA-G-Amplicon vector by in-fusion recombination to obtain a recombinant plasmid containing the RPP30 gene; the nucleotide sequence of the pVSV-RNA-Amplicon is shown in SEQ ID NO.1, and the nucleotide sequence of the pVSV-RNA-G-Amplicon is shown in SEQ ID NO.2;

[0026] (2) Transformation and screening: The recombinant plasmid of step (1) was transformed into competent Escherichia coli, positive clones were screened on a medium containing kanamycin, and positive clones were verified by colony PCR and sequencing;

[0027] (3) Plasmid extraction: The positive clones from step (2) were picked and inoculated into liquid LB medium containing kanamycin, cultured at 37°C overnight with shaking, and the recombinant plasmid was extracted and purified using a plasmid extraction kit to obtain the human internal standard RPP30 gene plasmid DNA.

[0028] Step 4. Preparation of multiple quality control products: Mix the prepared novel coronavirus ORF1ab gene pseudovirus, novel coronavirus N gene RNA molecule and human internal standard RPP30 gene plasmid DNA, and add the above-mentioned protective agent to prepare a multiple quality control product.

[0029] Furthermore, in the prepared multiple quality control products, the concentrations of the novel coronavirus ORF1ab gene pseudovirus, the novel coronavirus N gene RNA molecule, and the human internal standard RPP30 gene plasmid DNA were all 1×10 7 copies / mL.

[0030] Furthermore, the prepared multiple quality control products can be diluted to 1×10 5 ~1×10 7 copies / mL, used for various daily monitoring, precision testing and performance verification work.

[0031] Furthermore, the prepared multiple quality control products have a concentration range (Ct value) of nucleic acid detection results between 25 and 30 in daily monitoring.

[0032] The beneficial effects obtained by the present invention are:

[0033] (1) Full-process coverage monitoring Through the triple synchronous detection design of the novel coronavirus ORF1ab pseudovirus (simulated virus particles, monitoring nucleic acid extraction), the novel coronavirus N gene RNA molecule (naked RNA molecule, monitoring reverse transcription), and the human internal standard RPP30 gene plasmid DNA (monitoring nucleic acid amplification), precise layered monitoring of nucleic acid extraction (physical link), reverse transcription (template conversion), and amplification (cyclic reaction) is achieved, solving the monitoring blind spot problem of traditional single-component quality control products.

[0034] (2) No DNA residue interference. Pseudovirus preparation has no DNA residue, avoiding false positive results caused by DNA contamination. It is especially suitable for laboratory scenarios that rely on RNA extraction efficiency evaluation.

[0035] (3) Advantages of pseudovirus production and stability. The titer of VSV pseudovirus production can reach 10 8 TCID50 / mL, the single batch output can meet the needs of 10,000 tests. The gene construction, expression, purification and other steps involved all adopt conventional biotechnology means, which are easy to operate and implement, and the cost is relatively low, suitable for large-scale production and application.

[0036] (4) In terms of application, by establishing a quality control chart, it is possible to clearly judge whether each link of the nucleic acid detection is normal, providing a powerful tool for the laboratory to promptly discover problems and improve the detection process, which helps to improve the detection level of the entire new coronavirus nucleic acid detection laboratory. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A normal novel coronavirus nucleic acid detection quality control chart established for the multiple quality control products involved in the present invention.

[0038] Figure 2 This is a quality control chart showing that the nucleic acid extraction process is out of control when monitoring the multiple quality control products involved in the present invention.

[0039] Figure 3 The present invention relates to a quality control chart showing that the reverse transcription link is out of control during monitoring of the multiple quality control products.

[0040] Figure 4 The present invention relates to a quality control chart showing that the amplification link is out of control during monitoring of the multiple quality control products. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0042] Example 1: Preparation of novel coronavirus ORF1ab gene pseudovirus

[0043] Gene Construction: Based on the known novel coronavirus ORF1ab gene sequence No. NC_045512.2: 6266-9565 (SEQ ID NO. 3), an ORF1ab gene fragment with a specific restriction site (MluI) was designed and synthesized. The ORF1ab gene fragment recovered after restriction digestion was ligated with the pVSV-RNA-Amplicon (SEQ ID NO. 1) or pVSV-RNA-G-Amplicon (SEQ ID NO. 2) vector by in-fusion recombination to obtain a novel coronavirus ORF1ab gene vector plasmid.

[0044] The novel coronavirus ORF1ab gene vector plasmid was co-transfected into HEK293T cells with packaging auxiliary vectors pP, pN, pL and VSVG, respectively. 48 hours after transfection, the cell culture supernatant was collected, centrifuged at 2000 g for 10 minutes, and filtered with a 0.22 μm microfilter to obtain the novel coronavirus pseudovirus packaging rescue stock solution.

[0045] Plasmid VSVG was transfected into HEK293T cells. After 6-8 hours, the novel coronavirus amplicon rescue solution was added. After 48 hours, the cell culture supernatant was harvested and centrifuged at 2000g for 10 minutes. The remaining HEK293T cells and cell debris in the supernatant were removed by filtration with a 0.22μm microfilter. Then, the plasmid VSVG was re-transfected into new HEK293T cells and the novel coronavirus amplicon previous round of expansion solution was added. The next round of expansion was carried out until the novel coronavirus amplicon titer reached 10 8 TCID50 / mL.

[0046] Example 2: Preparation of novel coronavirus N gene RNA molecules

[0047] Preparation of novel coronavirus N gene pseudovirus: Based on the novel coronavirus N gene sequence (No. NC_045512.2: 28274-2953) (SEQ ID NO. 4), an N gene fragment with a specific restriction site (MluI) was designed and synthesized. The N gene fragment recovered after restriction digestion was ligated with the pVSV-RNA-Amplicon (SEQ ID NO. 1) or pVSV-RNA-G-Amplicon (SEQ ID NO. 2) vector by in-fusion recombination to obtain a novel coronavirus N gene vector plasmid.

[0048] The novel coronavirus N gene vector plasmid was co-transfected into HEK293T cells with packaging auxiliary vectors pP, pN, pL and VSVG, respectively. 48 hours after transfection, the cell culture supernatant was collected, centrifuged at 2000 g for 10 minutes, and filtered with a 0.22 μm microfilter to obtain the novel coronavirus N gene pseudovirus packaging rescue stock solution.

[0049] The plasmid VSVG was transfected into HEK293T cells. After 6-8 hours, a rescue stock solution of the novel coronavirus N gene pseudovirus was added. After 48 hours, the cell culture supernatant was harvested and centrifuged at 2000g for 10 minutes. Residual HEK293T cells and cell debris were removed from the supernatant by filtration using a 0.22μm microfilter. Next, the plasmid VSVG was re-transfected into new HEK293T cells, and the stock solution from the previous round of expansion of the novel coronavirus N gene pseudovirus was added. The next round of expansion was continued until the titer of the novel coronavirus N gene pseudovirus reached the required level.

[0050] Novel coronavirus N gene RNA molecule nucleic acid extraction: The obtained novel coronavirus N gene pseudovirus was subjected to nucleic acid extraction using the QIAGEN column membrane method nucleic acid extraction kit (QIAamp Viral RNA Mini Kit, QIAGEN, #52906). The product obtained after extraction is the purified molecule of the novel coronavirus N gene RNA molecule.

[0051] Example 3: Preparation of human internal standard RPP30 gene plasmid DNA

[0052] Based on the known RPP30 gene sequence No. NC_006413: 4-820 (SEQ ID NO. 5), an RPP30 gene fragment with a specific restriction enzyme cleavage site (MluI) was designed and synthesized. The pVSV-RNA-Amplicon (SEQ ID NO. 1) or pVSV-RNA-G-Amplicon (SEQ ID NO. 2) vector (CN113943832A) was selected, and the RPP30 gene fragment recovered after restriction enzyme cleavage and the vector were ligated by in-fusion recombination. The ligation product was then transformed into competent cells, and positive clones were verified by colony PCR and sequencing.

[0053] After the positive clones were identified, plasmid extraction was performed. First, the positive clones were picked and inoculated into liquid LB medium containing Kan antibiotics, and cultured at 37°C with shaking overnight to allow the bacteria to proliferate in large quantities. Then, a plasmid extraction kit was used and the instructions of the kit were followed to perform the steps of bacterial collection, lysis, neutralization, adsorption, washing and elution in sequence, and finally the purified human internal standard RPP30 gene plasmid DNA containing the RPP30 gene was obtained.

[0054] Example 4: Preparation of multiple quality control products for novel coronavirus nucleic acid detection

[0055] This novel coronavirus nucleic acid detection multiple quality control product uses the QIAGEN column membrane method nucleic acid extraction kit (QIAamp Viral RNA Mini Kit, QIAGEN, #52906) for nucleic acid extraction, and the novel coronavirus ORF1ab gene pseudovirus prepared in Examples 1-3, the novel coronavirus N gene RNA molecule, and the human internal standard RPP30 gene plasmid DNA are preliminarily determined by different digital PCR systems.

[0056] The digital PCR systems used included Stilla (fully automated microdroplet chip digital PCR system, #NaicaCrystal), BioRad (digital PCR system, #QX200), and Sniper (digital PCR all-in-one, #DQ24). Preliminary assay values ​​for the three digital PCR systems are shown in the table.

[0057] Table 1 Analysis results of Stilla digital PCR value data for multiplex quality control products for novel coronavirus nucleic acid detection

[0058]

[0059] Table 2 Analysis results of Sniper digital PCR fixed value data of multiple quality control products for novel coronavirus nucleic acid detection

[0060]

[0061] Table 3 Analysis results of BioRad digital PCR value data for multiplex quality control products for novel coronavirus nucleic acid detection

[0062]

[0063] After the initial determination, the ORF1ab gene pseudovirus, N gene RNA and internal standard RPP30 gene plasmid DNA were mixed with protective agent at a concentration of 1×10 7 copies / mL, and the concentration of N gene RNA was 1×10 7 copies / mL, and the concentration of the internal standard plasmid was 1×10 7 The samples were mixed at a ratio of 10 copies / mL.

[0064] The protective agent consists of: 10-20 mM phosphate buffer (pH 7.2), 2% to 5% (w / v) trehalose, 2% to 10% (w / v) sucrose, 1% to 5% (w / v) gelatin, 0.1% to 1% (w / v) bovine serum albumin, 5-10 mM EDTA (DNA nuclease inhibitor), and 1-5 U / mL RNasin (RNA nuclease inhibitor).

[0065] Example 5: Uniformity Test

[0066] 140 μL of each of the 30 SARS-CoV-2 nucleic acid test multiplex quality control samples was extracted and aliquoted using a Biori nucleic acid extraction system. The ORF1ab, N, and RPP30 gene targets of the extracted SARS-CoV-2 nucleic acid test multiplex quality control samples were tested. The homogeneity of the test results was assessed using analysis of variance (F test). The results of the F test for the Ct values ​​of each target gene in the SARS-CoV-2 nucleic acid test multiplex quality control samples are shown in Table 4.

[0067] Table 4 F-test statistical results of target genes of multiple quality control products for novel coronavirus nucleic acid detection

[0068]

[0069] Looking up the F distribution table, m=30, n=3, we get the critical value F0.05, (29,60)=1.66.

[0070] From the data analysis, it was found that the F value of each target gene of the multi-link quality control product for the new coronavirus nucleic acid detection was less than 1.66, indicating that there was no significant difference between the intra-group variance and inter-group variance of each target of the multi-link quality control product (P>0.05), and the uniformity was good.

[0071] Example 6: Stability Test

[0072] The experiment adopted a unified placement and regular testing method to study the stability of the new coronavirus nucleic acid detection multiple quality control products stored at -80°C. At 0 months, 1 month, 2 months, 3 months, and 6 months, three multiple quality control products were removed, and nucleic acid was extracted and packaged according to the Biori nucleic acid extraction (machine extraction). The ORF1ab, N, and RPP30 targets of the extracted multiple quality control samples were tested. Three samples were randomly selected each time, and the test was repeated 3 times. The stability was evaluated using the t-test. The test data of the long-term stability study of the new coronavirus nucleic acid detection multiple quality control products at -80°C are shown in the following table.

[0073] Table 5 Test results of the storage stability study of the novel coronavirus nucleic acid detection multiple quality control products at -80℃

[0074]

[0075]

[0076] The t-test was used to evaluate the detection data of the long-term stability study at -80°C. The results of the t-test on the Ct values of each target gene of the multiplex quality control product for novel coronavirus nucleic acid detection are shown in Table 6.

[0077] Table 6 Results of the stability assessment (t-test) of the multiplex quality control product for novel coronavirus nucleic acid detection at -80°C for 6 months

[0078] Statistics O1 N RPP30 <![CDATA[b0]]> 24.12 23.88 22.64 <![CDATA[|b1|]]> 0.03 0.05 0.02 <![CDATA[S(b1)]]> 0.02 0.02 0.01 <![CDATA[t 0.95,n-2 ×S(b1)]]> 0.04 0.07 0.04

[0079] Stability results (t-test) after storage at -80°C for 12 months: By looking up the t-distribution table, the critical value t 0.95,4 = 2.776.

[0080] As can be seen from the above table, for each target of the multiplex quality control product for novel coronavirus nucleic acid detection, |b1| < t0.95,n-2 × S(b1) is satisfied. It can be seen that after the multiplex quality control product is stored at -80°C for 6 months, each detection target is stable.

[0081] Example 6: Application of full-process monitoring of nucleic acid detection

[0082] (1) Establishment of a quality control chart for normal novel coronavirus nucleic acid detection

[0083] Within 20 working days, strictly in accordance with the laboratory standard operating procedure (SOP), the multiplex quality control product for novel coronavirus nucleic acid detection was diluted to 1×10 5 copies / mL for detection, and a total of 20 test results of the quality control product were obtained. By calculating the mean and standard deviation of all detection results, the mean was set as the target value, and the quality control limits were formulated based on the standard deviation. Specifically, with the mean as the center, control lines were drawn at the mean ± 2 times the standard deviation and the mean ± 3 times the standard deviation, thus constructing a complete quality control chart ( Figure 1 ). This quality control chart can intuitively reflect the fluctuation range of the detection results, providing a quantitative standard for subsequent judgment of whether the detection process is in a controlled state. When the detection result exceeds the set quality control limit, it can be determined that there is an abnormality in the detection process, and the cause needs to be investigated and corrected in a timely manner.

[0084] (2) Application of the multiplex quality control product for novel coronavirus nucleic acid detection in monitoring the out-of-control situation in the nucleic acid extraction link

[0085] A nucleic acid extraction kit with failed / poor effect was selected to perform extraction and detection operations on the multiplex quality control product for novel coronavirus nucleic acid detection.

[0086] From the detection results ( Figure 2), during the detection process, the Ct values ​​of the novel coronavirus N gene RNA molecule and the RPP30 internal standard did not show obvious abnormal fluctuations, indicating that these two components were not significantly affected by the failure of the test kit during the nucleic acid extraction process. However, the detection Ct value of the novel coronavirus ORF1ab pseudovirus deviated significantly from the normal range, exceeded the pre-set quality control limit, and became out of control. This result fully demonstrates that there are problems in the current nucleic acid extraction process, which is likely due to the decline in the performance of the nucleic acid extraction kit, which cannot effectively extract the nucleic acid in the sample, thereby affecting the subsequent test results. By using multiple quality control products, abnormalities in the nucleic acid extraction process can be accurately located, providing a strong basis for the laboratory to promptly replace qualified nucleic acid extraction kits and ensure the quality of detection.

[0087] (3) Application of multiplex quality control products for novel coronavirus nucleic acid detection in monitoring loss of control in the reverse transcription process

[0088] The nucleic acid reverse transcriptase was inactivated, and then the new coronavirus nucleic acid detection multiple quality control products were extracted and tested according to the normal operating procedures.

[0089] Test results ( Figure 3 ) showed that when the reverse transcriptase was inactivated, the detection Ct value of the RPP30 internal standard remained within the normal fluctuation range, indicating that the RPP30 internal standard was not affected by the inactivation of the nucleic acid reverse transcriptase. In sharp contrast, the Ct values ​​of the detection results of the new coronavirus ORF1ab pseudovirus and the new coronavirus N gene RNA molecule were out of control and significantly deviated from the normal range. This phenomenon clearly shows that the nucleic acid reverse transcription link has been out of control. Due to the inactivation of the reverse transcriptase, the RNA cannot be reverse transcribed into cDNA normally, which in turn affects the subsequent test results. Based on this, the laboratory can conduct an in-depth investigation of the nucleic acid reverse transcription link, such as checking the storage conditions of the reverse transcriptase, whether the usage method is correct, or replacing the new reverse transcriptase in time to ensure the accuracy and reliability of the reverse transcription process.

[0090] (4) Application of novel coronavirus nucleic acid detection in monitoring the loss of control in the amplification process

[0091] Select a nucleic acid detection kit that is ineffective / ineffective, and then strictly follow the normal operating procedures to extract and test the new coronavirus nucleic acid detection multiple quality control products.

[0092] Test results ( Figure 4) showed that the Ct values ​​of the three components of the novel coronavirus ORF1ab pseudovirus, the novel coronavirus N gene RNA molecule, and the RPP30 internal standard were out of control, far exceeding the pre-set quality control limits. This result indicates that the nucleic acid amplification kit has failed. Combined with the actual situation, the inactivation of the nucleic acid amplification enzyme is likely due to reasons such as the expiration of the kit and improper storage conditions. When this happens, the laboratory should immediately stop using the nucleic acid amplification kit, conduct a comprehensive inspection of other kits in stock, and contact the supplier to replace qualified products to avoid deviations in the test results due to problems in the amplification link and ensure the quality and accuracy of the novel coronavirus nucleic acid test.

[0093] By monitoring the entire testing process for the novel coronavirus nucleic acid test using these multi-link quality control products, potential risks can be systematically identified at every stage of the process, including nucleic acid extraction, reverse transcription, and amplification. Test results at each stage can be quantified using quality control charts and specific Ct value changes, providing an objective and accurate quantitative basis for laboratory quality control. This comprehensive quality monitoring system helps laboratories promptly identify and resolve issues, continuously optimize testing processes, and improve the overall quality and reliability of novel coronavirus nucleic acid testing.

[0094] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A multiple quality control product for laboratory quality control of novel coronavirus nucleic acid detection, characterized in that: The multiple quality control products include: novel coronavirus ORF1ab gene RNA pseudovirus, target gene RNA molecule, human internal standard RPP30 gene plasmid DNA and protective agent; The novel coronavirus ORF1ab gene pseudovirus is a modified vesicular stomatitis virus vector that encapsulates the novel coronavirus ORF1ab gene RNA, and its target gene sequence is NC_045512.2: 6266-9565; The target gene RNA molecule is a novel coronavirus N gene RNA molecule, and its target gene sequence is NC_045512.2: 28274-29533; The human internal standard RPP30 gene plasmid DNA, the target gene sequence is NC_006413:4-820; The protective agent comprises: phosphate buffered saline with pH 7.2, trehalose, sucrose, gelatin, bovine serum albumin, DNA nuclease inhibitor, and RNA nuclease inhibitor.

2. The multi-link quality control product according to claim 1, characterized in that: The concentrations of each component are as follows: 1×10 7 copies / mL, novel coronavirus N gene RNA molecules 1×10 7 copies / mL, human internal standard RPP30 gene plasmid DNA 1×10 7 copies / mL.

3. A method for preparing a multiple quality control product according to any one of claims 1 to 2, characterized in that: The following steps are involved: Step 1: Prepare a novel coronavirus ORF1ab gene pseudovirus; Step 2: Prepare the novel coronavirus N gene RNA molecule; Step 3: Prepare human internal standard RPP30 gene plasmid DNA; Step 4. Preparation of multiple quality control products: Mix the prepared novel coronavirus ORF1ab gene pseudovirus, novel coronavirus N gene RNA molecule and human internal standard RPP30 gene plasmid DNA, and add a protective agent to prepare a multiple quality control product; The protective agent comprises: 10-20 mM phosphate buffered saline with a pH of 7.2, 2% to 5% w / v trehalose, 2% to 10% w / v sucrose, 1% to 5% w / v gelatin, 0.1% to 1% w / v bovine serum albumin, 5-10 mM EDTA, and 1-5 U / mL RNasin.

4. The preparation method according to claim 3, characterized in that In step 1, the preparation steps of the novel coronavirus ORF1ab gene pseudovirus are as follows: (1) Construction of pseudoviral vector: Based on the ORF1ab gene target sequence NC_045512.2: 6266-9565, an ORF1ab gene fragment with an MluI restriction site was designed and synthesized; the fragment was ligated with the pVSV-RNA-Amplicon or pVSV-RNA-G-Amplicon vector by in-fusion recombination to obtain a recombinant pseudoviral vector containing the ORF1ab gene; the nucleotide sequence of the pVSV-RNA-Amplicon is shown in SEQ ID NO.1, and the nucleotide sequence of the pVSV-RNA-G-Amplicon is shown in SEQ ID NO.2; (2) Pseudovirus packaging: The recombinant plasmid of step (1) was co-transfected into HEK293T cells with the packaging auxiliary vectors pP, pN, pL, and VSVG. The cell culture supernatant was collected 48 hours after transfection, centrifuged at 2000 g for 10 minutes, and filtered through a 0.22 μm filter to obtain the pseudovirus packaging rescue stock solution; (3) Pseudovirus propagation: VSVG plasmid was transfected into HEK293T cells, and the pseudovirus stock solution from step (2) was added 6 to 8 hours later. After 48 hours, the supernatant was harvested and the propagation process was repeated until the pseudovirus titer reached 10 8 TCID50 / mL.

5. The preparation method according to claim 3, characterized in that In step 2, the purified novel coronavirus N gene RNA molecule is extracted and purified from the novel coronavirus N gene pseudovirus; The preparation steps of the novel coronavirus N gene pseudovirus are as follows: (1) Construction of pseudoviral vector: Based on the novel coronavirus N gene target sequence NC_045512.2: 28274-29533, an N gene fragment with an MluI restriction site was designed and synthesized; the fragment was ligated with the pVSV-RNA-Amplicon or pVSV-RNA-G-Amplicon vector by in-fusion recombination to obtain a recombinant pseudoviral vector containing the N gene; the nucleotide sequence of the pVSV-RNA-Amplicon is shown in SEQ ID NO.1, and the nucleotide sequence of the pVSV-RNA-G-Amplicon is shown in SEQ ID NO.2; (2) Pseudovirus packaging: The recombinant plasmid of step (1) was co-transfected into HEK293T cells with the packaging auxiliary vectors pP, pN, pL, and VSVG. The cell culture supernatant was collected 48 hours after transfection, centrifuged at 2000 g for 10 minutes, and filtered through a 0.22 μm filter to obtain the pseudovirus packaging rescue stock solution; (3) Pseudovirus expansion: VSVG plasmid was transfected into HEK293T cells. After 6 to 8 hours, the pseudovirus packaging and rescue stock solution of step (2) was added. After 48 hours, the supernatant was harvested and the expansion process was repeated until the pseudovirus titer reached 10 8 TCID50 / mL.

6. The preparation method according to claim 3, characterized in that In step 3, the preparation steps of the human internal standard RPP30 gene plasmid DNA are as follows: (1) Plasmid construction: Based on the human RPP30 gene target sequence NC_006413:4-820, an RPP30 gene fragment with an MluI restriction site was designed and synthesized; the fragment was ligated with the pVSV-RNA-Amplicon or pVSV-RNA-G-Amplicon vector by in-fusion recombination to obtain a recombinant plasmid containing the RPP30 gene; the nucleotide sequence of the pVSV-RNA-Amplicon is shown in SEQ ID NO.1, and the nucleotide sequence of the pVSV-RNA-G-Amplicon is shown in SEQ ID NO.2; (2) Transformation and screening: The recombinant plasmid of step (1) was transformed into competent Escherichia coli, positive clones were screened on a medium containing kanamycin, and positive clones were verified by colony PCR and sequencing; (3) Plasmid extraction: The positive clones from step (2) were picked and inoculated into liquid LB medium containing kanamycin, cultured at 37°C overnight with shaking, and the recombinant plasmid was extracted and purified using a plasmid extraction kit to obtain the human internal standard RPP30 gene plasmid DNA.

7. The preparation method according to claim 3, characterized in that In step 4, the concentrations of the novel coronavirus ORF1ab gene pseudovirus, novel coronavirus N gene RNA molecule, and human internal standard RPP30 gene plasmid DNA in the prepared multiple quality control products were all 1×10 7 copies / mL.

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