CN115992293A - Quality control product for RNA virus detection kit, and preparation method and application thereof - Google Patents
Quality control product for RNA virus detection kit, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a quality control product for an RNA virus detection kit, a preparation method and application thereof, and belongs to the technical field of virus detection. Including DNA fragment synthesis, PCR amplification, in vitro RNA transcription, RNA mixing, RNA freeze-drying and RNA copy number calibration. The preparation method can obtain stable quality control products of the full-length genome of the viral RNA, and can solve the problems of non-uniform standards of all kits in the market, no definite copy number quality control products, unstable transportation and the like. The quality control product is stored at normal temperature, and can be used after being dissolved again by adding water; the kit has definite copy number, can be directly diluted into quality control products with different concentrations, so that the detection limit of each kit can be well verified, and the possibility of clinical false positive is greatly reduced. The quality control product prepared by the process can be well applied to performance evaluation and clinical diagnosis of all viral nucleic acid detection kits based on PCR or NGS platforms on the market, and has good application value.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a quality control product for an RNA virus detection kit, and a preparation method and application thereof.
Background
As related pneumonia infection events frequently occur, a plurality of domestic detection companies sequentially push out related viral nucleic acid detection kits, which mainly detect viral nucleic acids based on PCR or q-PCR methods, and then subsequently have NGS method-based kits under development. Although the advent of these kits has greatly met the need in the marketplace for rapid detection of viral nucleic acids. However, the detection limit of the kit is not clear and the RNA substance with the copy number is not used as a reference, so that the detection result is difficult to confirm, and the news that the detection result is false negative is continuously reported, so that some virus carriers are secondarily transmitted, and the difficulty of epidemic prevention and control is greatly increased.
Because the virus nucleic acid fragments detected by each kit are inconsistent, it is difficult to have uniform quality control products on the market to control the quality of all the kits.
The genetic material of the related virus is RNA, so that the nucleic acid detection kit is difficult to store for a long time in vitro, and therefore, a stable RNA quality control product is difficult to monitor the performance of the nucleic acid detection kit in the transportation and storage processes, and therefore, a stable RNA quality control product is required to verify the performance of the corresponding kit and control the quality of the kit.
Disclosure of Invention
The invention aims to solve the technical problem that a nucleic acid detection kit in the prior art lacks a quality control product which is uniform, stable, reproducible and high in safety, and provides a quality control product for an RNA virus detection kit, a preparation method and application thereof.
The technical scheme adopted by the invention is as follows: a method of preparing a quality control for an RNA virus detection kit, the method comprising:
acquiring an RNA genome sequence of at least one virus to be detected, and generating a corresponding complementary sequence;
segmenting, synthesizing, amplifying and purifying transcripts of the complementary sequences;
designing a primer probe for each nucleotide fragment after treatment and determining the copy number of RNA transcripts;
mixing each RNA transcript, adding a preservation stabilizer, and freeze-drying to obtain RNA dry powder;
and adding water into the RNA dry powder for re-dissolution, calibrating the copy number, and determining parameters of the RNA quality control product to obtain the required RNA quality control product.
The above viruses to be tested are viruses whose genetic material is RNA, for example: novel coronavirus (COVID-19), HIV, hepatitis C virus, japanese encephalitis virus, all influenza viruses, ebola virus (Ebola virus), etc.
The mixing of each RNA transcript refers to mixing RNA transcript fragments after processing each nucleotide fragment with different sizes. The full-length RNA quality control product of the viral genome is prepared by mixing the above modes, and the fragments can be derived from the same virus or different viruses. If the fragments are derived from different viruses, the prepared RNA quality control product can be simultaneously applied to detection kits of various viruses.
Preferably, in the segmentation processing of the complementary sequences, the size of each fragment is not less than 3000bp;
preferably, there is an overlap region between each fragment of the complementary sequences, the overlap region being 100-500bp in size.
Specifically, the number of synthetic fragments can be designed according to the total length of the RNA genome, and the number of fragments to be synthesized is about genome total length/3000.
Preferably, the synthesizing the complementary sequence includes: RNA is synthesized in vitro by using RNA polymerase and DNA double strand as template.
Preferably, after the synthesis treatment and before the amplification treatment of the complementary sequence, the method further comprises: adopting DNase digestion reaction liquid to remove DNA template and inactivating DNase;
the amplification treatment includes: in the design of the amplification primer, the RNA transcriptase binding sequence is ligated to the forward primer.
The RNA polymerase is mainly a commercial in vitro transcriptase, such as Ambion TM T7 RNA Po lymerase，Ambion TM RNA transcriptases such as SP6 RNA Po lymerase and T3 RNA Po lymerase. When using Ambion TM In the case of T7 RNA Po lymerase, it is preferable that the time for in vitro transcription is 30 to 60 minutes.
The DNase is mainly commercial TURBO TM DNase and Ambion TM DNase I, when TURBO is used TM In DNase, preferably the DNA digestion time is 15min and the optimal temperature is 37 ℃.
The RNA transcripts may be purified by phenol chloroform extraction, column chromatography or gel electrophoresis.
Preferably, the primer probe labeled fluorescent group is FAM or 6-FAM;
the fluorescence quenching group is any one of BHQ1, TAMRA or MGB.
Preferably, the method for determining the copy number of the RNA transcript is an RT-ddPCR method.
The RT-ddPCR described above used a combination of Berle QX200 Droplet Digital PCR and One-Step RT-ddPCR Advanced Kit for Probes reagents.
Preferably, the lyophilization conditions are: treating at 2-8deg.C under 30-50Pa for 1-1.2 hr.
A quality control product of an RNA virus detection kit is obtained by the preparation method of the quality control product for the RNA virus detection kit.
An application of quality control of RNA virus detection kit in non-diagnostic purpose detection of virus detection kit.
An application of quality control product of RNA virus detection kit in preparing laboratory daily quality control product.
The quality control of the RNA virus detection kit is also suitable for application in evaluating the performance of the virus nucleic acid diagnosis kit, in particular to application in evaluating one or more of the performances of the kit, such as detection limit, specificity, sensitivity, accuracy and stability. The invention provides the RNA full-length quality control product for the first time, has definite copy number and strong stability, and can be suitable for evaluating the performance of all nucleic acid kits on the market.
The quality control of the RNA virus detection kit can also simulate a real sample clinically, and can be used for simulating the situation that the real clinical sample contains human RNA after being extracted by adding human RNA fragments into an RNA mixed solution.
The quality control product of the RNA virus detection kit contains the whole length of a virus genome, can be stored at normal temperature, has good batch-to-batch stability, is safe and renewable, has definite copy number, and can meet the requirements of most kit manufacturers and detection institutions on the market.
The beneficial effects of the invention are as follows:
the RNA virus quality control product contains the full length of the virus genome, is suitable for the performance evaluation of all virus nucleic acid kits on the market, and is suitable for both PCR platforms and NGS platforms, and the application range is wide.
And secondly, the RNA virus quality control product can be stored at normal temperature, can be used after being dissolved again by adding water, does not need to carry out RNA extraction operation, avoids the influence caused by the difference of extraction operation of different users, and is simple and convenient to use.
And thirdly, the RNA virus quality control product has definite copy number, can be directly diluted into quality control products with different concentrations, so as to well verify the detection limit of each kit, and greatly reduce the possibility of clinical false positive.
The RNA virus quality control material has simple preparation process, safety, stability and reliability, can be stored at normal temperature, and can be well applied to performance evaluation of various RNA virus nucleic acid detection kits.
And fifthly, the RNA virus quality control product can be used for mixing genome fragments of various viruses and is simultaneously applicable to various virus nucleic acid detection kits.
The RNA virus quality control product can be well applied to the aspects of research and development of RNA virus nucleic acid detection kits, performance evaluation, factory inspection, validity period monitoring, detection flow monitoring, result assurance and the like.
In conclusion, the quality control product for the RNA virus detection kit, the preparation method and the application thereof can well solve the problems of no uniform reference quality control standard, unstable transportation, unsafe use, frequent false negative detection and the like in the evaluation of different kits.
Drawings
FIG. 1 is a schematic diagram of the function of a specific case virus, a fragment synthesis profile and a schematic diagram of the length of a synthesized fragment in example 1.
Detailed Description
The invention will be further illustrated with reference to specific examples. It will be appreciated by those skilled in the art that the embodiments described below are some, but not all, of the embodiments of the present invention and are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. The reagents used were all conventional products commercially available.
Aiming at the quality control product which has no available RNA virus genome full length in the market, definite virus copy number and safe and convenient use, the invention provides the following technical scheme.
Embodiment one;
according to an exemplary embodiment of the present invention, there is provided a method for preparing a quality control for an RNA virus detection kit. The preparation method comprises the following steps: this example is specifically described in terms of the novel coronavirus (COVID-19).
A, the full-length sequence of the genome of the novel coronavirus (COVID-19) was obtained from the NCBI database (https:// www.ncbi.n lm. N ih. Gov /).
B, generating the reverse complement of the RNA virus genome from the full-length sequence of the genome of the novel coronavirus (COVID-19) using sequence analysis software (e.g., geneius, vector NTI, snapgene, etc.).
C, dividing the genome full length of the COVID-19 into 10 gene fragments according to functional region division, wherein the positions of the 10 gene fragments are shown in figure 1, the overlapping region between the fragments is 100-300bp, the sequence numbers of the fragments are SEQ01-SEQ10 respectively, and thus the applicability of the quality control product in a PCR platform and an NGS platform can be ensured. The novel coronavirus functional region of COVID-19 is shown in Table 1 below:
TABLE 1 COVID-19 novel coronavirus functional regions:
| annotating | Size (bp) | Initial | Termination position | |
| 5′- |
265 | 1 | 265 | |
| |
21290 | 266 | 21555 | |
| |
3822 | 21563 | 25384 | |
| |
828 | 25393 | 26220 | |
| |
228 | 26245 | 26472 | |
| M | 669 | 26523 | 27191 | |
| |
186 | 27202 | 27387 | |
| |
366 | 27394 | 27759 | |
| |
132 | 27756 | 27887 | |
| |
366 | 27894 | 28259 | |
| |
1260 | 28274 | 29533 | |
| ORF10 | 117 | 29558 | 29674 | |
| 3′- |
229 | 29675 | 29903 |
D, synthesizing SEQ01-SEQ10 sequences, wherein the fragment length of each sequence is shown in figure 1.
E, designing a primer, adding an RNA reverse transcriptase binding sequence to a forward primer, performing PCR amplification, and recovering and purifying the PCR primer by adopting gel cutting recovery.
The primer sequences are specifically shown in table 2, and the schematic positions of the primers are shown in fig. 1:
table 2 primer sequences:
the black bolded font in the table above represents the T7 RNA Po lymerase binding sequence.
F, use of commercial RNA polymerase Ambion TM T7 RNA Po lymerase (Cat. No. AM2082), 10 DNA double-strand in the step D was used as template to synthesize RNA in vitro. According to the requirements of the specification, a reaction system of 20u l in vitro transcription is prepared, and incubated for 60min at 37 ℃.
G, adding 2U TURBO into the reaction system TM DNase, incubated at 37℃for 15min.
H, classifying and purifying the reaction solution obtained in the step G by a phenol-chloroform method to obtain RNA solution, wherein the quality of the obtained RNA is about 2-4ug after each reaction is measured.
And I, designing and synthesizing 3-4 pairs of primers and 2-3 probes for each nucleotide fragment, verifying the primers and the probes through a Berle q-PCR platform, respectively verifying the amplification efficiency and the specificity of the primers and the specificity of the probes, and selecting and optimizing the optimal annealing temperature. The optimal annealing temperature is set by screening the optimal primer and probe combination, the raw material copy number concentration of each RNA is determined by using a QX200 Drop let Digita l PCR platform of Berle and an One-Step RT-ddPCR Advanced Kit for Probes kit, and the reaction system and reaction conditions of RT-ddPCR are operated according to the specification. The final and optimized primer and probe sequences for RT-ddPCR are shown in Table 2 below:
table 2 primer and probe sequences:
j, diluting the RNA raw materials to the same concentration, e.g., 10 8 Copies/u l. Mixing the RNA raw materials in equal volume, calculating the volume of the required added RNA raw materials according to the target copy number, and fully shaking and uniformly mixing.
K, adding a certain amount of RNA preservation stabilizer according to a proportion, calculating the split charging number of the RNA viruses according to the required copy number of each part, split charging according to the required specification, and vacuum drying for 60min at 2-8 ℃ with the vacuum strength set at 30-50Pa.
And L, after the preparation of the RNA dry powder in the step K is finished, adding a certain amount of Nuc release-free water, and then, calibrating the RNA copy number of each fragment of the novel coronavirus (COVID-19) by adopting the primer and the probe solution in the step I in a RT-ddPCR mode to determine the copy number parameter of the final RNA quality control product.
Example two
The invention provides an application of a kit for detecting RNA viruses, comprising a method for evaluating performance. The method mainly comprises the following steps:
re-dissolving the prepared RNA virus quality control material dry powder with Nuc release-free water, and diluting to desired copy number concentration, such as 1×10 7 Copies/u l; according to the judgment of the detection limit of the kit, the RNA virus quality control material solution is diluted to 10 in a gradient way 1 ,10 2 ,10 3 ,10 4 Copies/u l; detecting the RNA virus quality control substances subjected to gradient dilution by adopting a kit instruction, wherein each sample is detected for 20 times; if the concentration of the RNA virus quality control substance can be 100% detected by the kit to be evaluated in 20 assays, the detection limit of the kit can be defined as the concentration value.
Embodiment III:
the full-length quality control of the viral RNA genome in the patent is prepared into quality control products with 2 gradient (strong positive 3000copies/ml, weak positive 300 copies/ml) concentrations by selecting 6 new coronavirus detection kits which are mainstream in the market, synchronous detection is carried out by using ddwater or negative control carried by the kits, the operation and loading are carried out according to the instruction book of each kit, the detection results of the comparison kits (6 kits are respectively indicated by A, B, C, D, E, F), and the experimental results are shown in the following table:
table 3: detection result of 6 kits on full-length quality control of novel coronavirus RNA genome
The detection in the table indicates that a novel coronavirus positive signal was detected, and the undetected indicates that a novel coronavirus positive signal was not detected.
As can be seen from the above results, no positive signal was detected by the negative control, indicating that the false positive rate of the 6-format kit was 0%. When the concentration of the full-length quality control product of the viral RNA genome in the patent is higher (3000 copies/ml), the full-length quality control product can be detected by the main-stream kits on the market, which indicates that the quality control product of the viral RNA genome in the patent is applicable to most kits. However, when the concentration of the quality control product was diluted to a low concentration (300 copies/ml), 2 out of 6 kit manufacturers (manufacturers B and D) failed to detect a positive result, and the false negative rate was 33.3%. The full explanation is that the detection quality of the current commercial kits is uneven and the detection limit is inconsistent.
In summary, the invention performs steps of sectional synthesis, in vitro transcription, fragment mixing and the like on the whole length of the viral RNA genome to prepare RNA virus dry powder, and then marks the copy number of viral RNA through digital PCR, thereby providing a stable and reliable preparation process of RNA virus quality control product and application thereof in kit evaluation. Can solve the problems of non-uniform standard, lack of standard, unstable transportation, unsafe use and the like of the clinical kit.
The invention is not limited to the above-described alternative embodiments, and any person who may obtain other products in various forms under the teaching of the invention falls within the scope of protection of the invention. The above detailed description should not be construed as limiting the scope of the invention, and it should be understood by those skilled in the art that the technical solutions described in the foregoing embodiments may be modified or some or all of the technical features may be replaced equally without departing from the scope of the technical solutions of the embodiments of the invention.
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<400> 38
actatgtgag aaggcattaa 20
<210> 39
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(22)
<223> SEQ07-dd-F
<400> 39
acacttcgca tggtggacag cc 22
<210> 40
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> SEQ07-dd-R
<400> 40
acataaccat ctatttgttc gcgtggt 27
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> SEQ07-dd-P,5 '-end tag FAM,3' -end tag BHQ1
<400> 41
atgcgtcatc atctgaagca 20
<210> 42
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(23)
<223> SEQ08-dd-F
<400> 42
actgcacaga agtccctgtt gct 23
<210> 43
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(23)
<223> SEQ08-dd-R
<400> 43
agcccctatt aaacagcctg cac 23
<210> 44
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(23)
<223> SEQ08-dd-P, FAM at 5 'end, BHQ1 at 3' end
<400> 44
tcctacttgg cgtgtttatt cta 23
<210> 45
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(23)
<223> SEQ09-dd-F
<400> 45
gcttgttttg tgcttgctgc tgt 23
<210> 46
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(21)
<223> SEQ09-dd-R
<400> 46
gcgtacgcgc aaacagtctg a 21
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(21)
<223> SEQ09-dd-P, FAM at 5 'end, BHQ1 at 3' end
<400> 47
ggcttgatgt ggctcagcta c 21
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> SEQ10-dd-F
<400> 48
cgcagaaggg agcagaggcg 20
<210> 49
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> SEQ10-dd-R
<400> 49
gcatcaccgc cattgccagc 20
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(21)
<223> SEQ10-dd-P, FAM at 5 'end, BHQ1 at 3' end
<400> 50
gaaattcaac tccaggcagc a 21
Claims (10)
1. A method for preparing a quality control product for an RNA virus detection kit, the method comprising:
acquiring an RNA genome sequence of at least one virus to be detected, and generating a corresponding complementary sequence;
segmenting, synthesizing, amplifying and purifying transcripts of the complementary sequences;
designing a primer probe for each nucleotide fragment after treatment and determining the copy number of RNA transcripts;
mixing each RNA transcript, adding a preservation stabilizer, and freeze-drying to obtain RNA dry powder;
and adding water into the RNA dry powder for re-dissolution, calibrating the copy number, and determining parameters of the RNA quality control product to obtain the required RNA quality control product.
2. The method for producing a quality control product for an RNA virus detection kit according to claim 1, wherein the size of each fragment is not less than 3000bp in the segmentation process of the complementary sequences;
preferably, there is an overlap region between each fragment of the complementary sequences, the overlap region being 100-500bp in size.
3. The method for preparing a quality control product for an RNA virus detection kit according to claim 1, wherein the synthesizing the complementary sequence comprises: RNA is synthesized in vitro by using RNA polymerase and DNA double strand as template.
4. The method for preparing a quality control product for an RNA virus detection kit according to claim 1, wherein after synthesizing the complementary sequence and before amplifying the complementary sequence, further comprising: adopting DNase digestion reaction liquid to remove DNA template and inactivating DNase;
the amplification treatment includes: in the design of the amplification primer, the RNA transcriptase binding sequence is ligated to the forward primer.
5. The method for preparing a quality control product for an RNA virus detection kit according to claim 1, wherein the primer probe-labeled fluorescent group is FAM or 6-FAM;
the fluorescence quenching group is any one of BHQ1, TAMRA or MGB.
6. The method for preparing a quality control product for an RNA virus detection kit according to claim 1, wherein the method for determining the copy number of the RNA transcript is RT-ddPCR method.
7. The method for preparing a quality control product for an RNA virus detection kit according to claim 1, wherein the lyophilization conditions are: treating at 2-8deg.C under 30-50Pa for 1-1.2 hr.
8. A quality control product for an RNA virus detection kit, characterized by being obtained by the method for producing a quality control product for an RNA virus detection kit according to any one of claims 1 to 7.
9. Use of the quality control of an RNA virus detection kit of claim 8 for detection of a virus detection kit of non-diagnostic interest.
10. Use of the quality control of RNA virus detection kit of claim 8 in the preparation of laboratory daily quality control.
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| CN202210731318.1A CN115992293A (en) | 2022-06-24 | 2022-06-24 | Quality control product for RNA virus detection kit, and preparation method and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111607A (en) * | 2020-09-30 | 2020-12-22 | 菁良基因科技(深圳)有限公司 | COVID-19 virus nucleic acid quality control product and application thereof |
| WO2021180139A1 (en) * | 2020-03-10 | 2021-09-16 | 广州复能基因有限公司 | Dopable reference standard for use in detecting sample target from dna or rna organism |
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2022
- 2022-06-24 CN CN202210731318.1A patent/CN115992293A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021180139A1 (en) * | 2020-03-10 | 2021-09-16 | 广州复能基因有限公司 | Dopable reference standard for use in detecting sample target from dna or rna organism |
| CN112111607A (en) * | 2020-09-30 | 2020-12-22 | 菁良基因科技(深圳)有限公司 | COVID-19 virus nucleic acid quality control product and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 许丽;梁文;杨雪;闻艳丽;李兰英;杨镇州;李妍;邓敏;陆青;丁敏;任淑贞;孙洁林;左小磊;王丽华;曹程明;胡钧;刘刚;樊春海;: "新型冠状病毒RNA标准物质", 科学通报, no. 22, pages 2363 - 2370 * |
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