CN112063764A - Multiplex real-time fluorescent RT-PCR primer probe composition and kit for novel coronavirus nucleic acid detection - Google Patents
Multiplex real-time fluorescent RT-PCR primer probe composition and kit for novel coronavirus nucleic acid detection Download PDFInfo
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Abstract
The invention discloses a multiple real-time fluorescent RT-PCR primer probe composition and a kit for detecting novel coronavirus nucleic acid. The kit comprises multiplex real-time fluorescence RT-PCR primers and probes for detecting ORF1ab gene and N gene of SARS-CoV-2; wherein the upstream primer for amplifying ORF1ab gene is shown as SEQ ID NO: 1, and the downstream primer is shown as SEQ ID NO: 2, the probe sequence for detecting ORF1ab gene is shown as SEQ ID NO: 3 is shown in the specification; the upstream primer for amplifying the N gene is shown as SEQ ID NO: 4, and the downstream primer is shown as SEQ ID NO: 5, the probe sequence for detecting the N gene is shown as SEQ ID NO: and 6. Through clinical verification of China and America, the detection of clinical samples at home and abroad can be met.
Description
Technical Field
The patent belongs to the field of medical in-vitro diagnosis, and relates to a multiple real-time fluorescent RT-PCR primer probe composition and a kit for detecting novel coronavirus nucleic acid.
Background
The new coronavirus pneumonia diagnosis and treatment scheme released by Weijian Commission of China (updated to the eighth trial), which definitely lists the real-time fluorescence RT-PCR detection of the new coronavirus SARS-CoV-2 nucleic acid positive as one of the diagnosis-confirming standards, and accurately identifies SARS-CoV-2 in time, thereby being the prerequisite basis for reasonable clinical treatment and scientific prevention and control of epidemic situation of the new coronavirus pneumonia (COVID-19).
However, at the beginning of epidemic outbreak, the national drug administration starts the "medical instrument emergency approval procedure" to meet the needs of a large number of tests, so that reagent manufacturers complete the research and development and approval of nucleic acid test kits in the shortest time, and the detection principle and performance parameters of the kits are inevitably flawed. The continuous report of the cases of continuous negative of nucleic acid detection with clinical manifestation and reduction of clinical symptoms but 'recovering positive' of nucleic acid detection reflects certain problems of the nucleic acid detection kit. The design of PCR primers and probes for detecting virus nucleic acid and the setting of system parameters of multi-target synchronous amplification are key factors influencing the specificity and sensitivity of the detection method. Currently, in the design of PCR primers and probes of commercial kits which are put into clinical use, only Wuhan-Hu-1 virus strain (NC-045512) which is originally reported in China is mostly used as a design template, and SARS-CoV-2 is easy to mutate as an RNA virus, so that certain degree of omission is caused. At present, domestic epidemic situation is basically controlled, but global epidemic situation is still spreading, epidemic-resistant emphasis is shifted to control and input, and under the new situation, whether the kit can meet the detection of virus epidemic strains outside the country is very important.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a multiplex real-time fluorescence RT-PCR primer probe composition and a kit for SARS-CoV-2 nucleic acid detection, so as to realize the detection of reported SARS-CoV-2 at home and abroad and solve the problem of missed detection of the existing kit.
The technical scheme of the invention is as follows:
a multiplex real-time fluorescent RT-PCR primer probe composition for detecting novel coronavirus comprises multiplex real-time fluorescent RT-PCR primers and probes for detecting ORF1ab gene and N gene of SARS-CoV-2; wherein the upstream primer for amplifying ORF1ab gene is shown as SEQ ID NO: 1, and the downstream primer is shown as SEQ ID NO: 2, the probe sequence for detecting ORF1ab gene is shown as SEQ ID NO: 3 is shown in the specification; the upstream primer for amplifying the N gene is shown as SEQ ID NO: 4, and the downstream primer is shown as SEQ ID NO: 5, the probe sequence for detecting the N gene is shown as SEQ ID NO: and 6.
As a preferable preference of the invention, the multiplex real-time fluorescent RT-PCR primer probe composition also comprises a multiplex real-time fluorescent RT-PCR primer probe composition for detecting human beta-Actin genes.
As a further optimization of the invention, the upstream primer for amplifying the human beta-Actin gene is shown as SEQ ID NO: 7, the downstream primer is shown as SEQ ID NO: 8, and the probe sequence for detecting the human beta-Actin gene is shown as SEQ ID NO: shown at 9.
Two ends of the probe are respectively marked with a reporter fluorophore and a quenching fluorophore. The 5' modifications of the probes of the invention are selected from the reporter fluorophores commonly used in the art, such as FAM, Texas Red, JOE; the 3' modification of the probe of the invention is selected from quenching fluorescent groups commonly used in the field, such as BHQ1, BHQ2, ECLIPSE. And the reporter fluorophore and the quencher fluorophore of each gene are different.
As a preferable aspect of the present invention, the probe for detecting ORF1ab gene is 5 '-modified FAM, 3' -modified BHQ 1; the probe for detecting the N gene is 5 'modified Texas Red and 3' modified BHQ 2; the probe for detecting the human beta-Actin gene is 5 'modified JOE and 3' modified ECLIPSE.
The invention relates to application of a multiplex real-time fluorescent RT-PCR primer probe composition in preparation of a novel coronavirus detection reagent.
A novel coronavirus nucleic acid detection kit comprises the multiple real-time fluorescent RT-PCR primer probe composition.
The PCR primer pair and the probe aiming at the ORF1ab and the N gene are designed to have a template sequence which is not one of the Wuhan-Hu-1 virus strains (NC-045512), but comprehensively refer to all virus strain sequences which are publicly reported 6, 25 and earlier in 2020. Specifically, all sequences with Complete sequence and High quality evaluation in the national bioinformatics center-2019 novel coronavirus information base (https:// bigd.big.ac.cn/ncov/release _ genome) were downloaded and saved as local files, and after removing sequences except ORF1ab and N gene, the files were submitted to European molecular biology laboratories-European bioinformatics institute (EMBL-EBI), and a Clustal Omega (https:// www.ebi.ac.uk/Tools/msa/clustalo /) multiple sequence alignment tool was used to obtain the consistent sequences and the consistent rates of the bases in the sequences, and the consistent sequences were used as design templates of primers and probes. Thus, the detection of all virus strains in the database is realized, and the omission caused by virus nucleic acid variation is avoided to the maximum extent.
The primers and probes of ORF1ab and N gene have multiple design results, 3 design results are optimized for each pair of primers and each probe according to the general principle of PCR primer design, and a plurality of real-time fluorescent RT-PCR primer probe compositions are synthesized by a pair of primers and a probe of ORF1ab gene, a pair of primers and a probe of N gene, and a pair of primers and a probe of human beta-Actin gene, and a probe set, so as to form 9 sets of primer probe compositions (see Table 1 for an example of combination). And analyzing the formation condition of the dimer among 9 primer probes in each set of composition, and selecting the optimal 2 sets of compositions for experimental performance comparison.
TABLE 1 PCR primer Probe combination example
As a further preferred embodiment of the present invention, the sequences of the primer and probe composition for ORF1ab and N gene are shown in Table 2, and the composition is highly specific to SARS-CoV-2 detection.
TABLE 2 PCR primer and Probe sequences for SARS-CoV-2 ORF1ab and the N Gene
Preferably, the ratio of the final concentration of each primer to the final concentration of the probe involved in the reaction is 2: 1.
Preferably, the kit further comprises an RT-PCR reaction solution, a positive quality control product and a negative quality control product.
In a further preferred embodiment of the present invention, the RT-PCR reaction solution comprises RT-PCR buffer, reverse transcriptase and hot start Taq enzyme.
As a further preferred aspect of the present invention, the positive quality control material is an in vitro transcription RNA fragment containing a PCR amplification target sequence (the sequence is shown in Table 3). Therefore, the consistency of the chemical properties of the quality control product and the tested object of the sample is ensured, the clinical sample is simulated to the maximum extent to monitor the RT-PCR whole-process reaction, and meanwhile, the infection risk caused by using inactivated virus or false virus is avoided.
TABLE 3 in vitro transcribed RNA sequences in Positive quality controls
In a further preferred embodiment of the present invention, the negative quality control material is a solvent matrix of the positive quality control material, and does not contain a PCR amplifiable target substance.
The kit comprises a component A and a component B, wherein the component A is the primer probe composition, the component B comprises concentrated RT-PCR buffer solution, reverse transcriptase and hot start Taq enzyme, the preparation of reaction solution is completed only by mixing A, B components in clinical application, and after an RNA sample is added, one-step multiplex fluorescence RT-PCR is carried out, so that ORF1ab and N gene of SARS-CoV-2 virus and human source sampling quality control gene beta-Actin can be detected simultaneously, and the operation is simple, convenient and quick.
The invention is suitable for real-time fluorescent quantitative PCR instruments, such as ABI 7500 series, Roche 480 series, Bio-Rad CFX96, and Hongshite SLAN series.
Due to the implementation of the technical scheme, the invention has the beneficial effects that: the invention discloses a multiple real-time fluorescent RT-PCR primer probe composition and a kit for detecting novel coronavirus nucleic acid, wherein the primer probe in the kit is designed based on a virus information base, so that the omission caused by virus nucleic acid variation is avoided to the greatest extent; the kit of the optimized primer probe composition has high specificity on SARS-CoV-2 detection, the lowest detection limit is 400 copies/mL, and the Ct value variation coefficient<5 percent; the tested object of the positive quality control product is RNA in nature and participates in the RT-PCR whole-process reaction, so that the accuracy and effectiveness of the detection result are ensured. Through clinical verification of China and America, the kit, the Huada kit and the application thereof
6800 the consistency between the results of the automatic detection equipment is uniform>95 percent, can meet the detection of clinical samples at home and abroad.
Drawings
FIG. 1 is a graph showing PCR amplification curves for 2 primer probe concentrations in an embodiment of the present invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly shown below in conjunction with the detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
In the embodiment of the present invention, Lasergene primer select software (DNASTAR, inc. ver.7.1.0) is used to analyze the formation of primer-to-probe dimer on 9 sets of candidate primer probe compositions, and preferably 2 sets of primer probe compositions are selected, and the worst Δ G value (free energy) formed by pairing of more than 3 consecutive bases in the dimer is compared as shown in table 4.
TABLE 4 comparison of dimer formation between primer probes
The 2 sets of primer probe compositions are respectively assembled into corresponding kit 1 and kit 5, and then the detection performances are compared, so as to further preferably select the primer probe composition of the invention. Specifically, the novel coronavirus ribonucleic acid genome standard substance (China institute of metrology science; accession number: GBW (E) 091099; lot number: 2020-02; ORF1 ab: 6.89X 10)2Copy/. mu.L; n: 1.36X 103Copy/. mu.L) was diluted in pharyngeal swab virus stock (Haishi Gene technology, cat #: XB06013, batch number: 200518001), obtaining simulated virus samples with ORF1ab of 10000 copies/mL, 1000 copies/mL and 100 copies/mL and N of 10000 copies/mL, 1000 copies/mL and 100 copies/mL respectively. And (3) according to the requirements of the clinical sample RNA extraction kit specification, simultaneously carrying out RNA extraction on the simulated virus sample, the virus preservation solution, the positive quality control substance and the negative quality control substance.
The RT-PCR reaction system is prepared by respectively adopting the kit 1 and the kit 5, the final concentration of each primer and the final concentration of each probe in the 2 reaction systems are respectively 200nmol/L and 100nmol/L, and the volumes of the used reagents are shown in a table 5:
preparation of RT-PCR reaction system of Table 52 kits
The reaction system is loaded into PCR reaction tubes according to 20 mul per tube, 5 mul of the RNA extracted sample is added into the corresponding reaction tubes, the tube covers are covered, the reaction tubes are instantly put on a machine by instant centrifugation, the RT-PCR reaction is carried out according to the set parameters in the table 6, and the detection channels are FAM, JOE and Texas Red.
TABLE 6 RT-PCR reaction parameter settings
The data analysis was performed according to the instructions of the instrument used and the results are shown in Table 7:
TABLE 72 comparison of detection Performance of primer Probe compositions
Therefore, on the premise that the quality control product result is correct, the kit 1 is superior to the kit 5 in both positive detection rate and Ct value. The primer probe composition 1 contained in the kit 1 is the primer probe composition of the present invention.
Further, the detection performance of 2 concentrations of the primer probes in the primer probe composition 1 was compared to further optimize the concentration of the primer probes of the present invention.
Specifically, preparation of a virus-simulated sample and extraction of sample RNA were carried out as described above, and RT-PCR reaction systems were prepared using 2 primer probe concentrations, respectively, using the reagent volumes shown in Table 8:
preparation of RT-PCR reaction system with concentration of 82 primer probes in table
The reaction system was divided into PCR reaction tubes of 20. mu.L/tube, 5. mu.L of the RNA-extracted sample was added to each reaction tube, and RT-PCR was performed as described above, and the results are shown in Table 9:
comparison of detection Performance for the concentrations of the primers probes in Table 92
Therefore, on the premise of controlling the quality control result, the concentration 2 of the primer probe is superior to the concentration 1 of the primer probe in both positive detection rate and Ct value, and the PCR amplification reaction curve is shown in figure 1. The concentration of the primer probe 2 is the concentration of the primer probe composition in the kit of the invention.
Further, the minimum detection limit of the kit of the present invention was evaluated.
Specifically, mock virus samples were prepared as described above such that the concentrations of ORFs 1ab and N were 800 copies/mL, 400 copies/mL, 200 copies/mL, and 100 copies/mL, respectively. As described above, RNA in the sample was extracted and the RT-PCR reaction was performed on the sample, and the results are shown in Table 10:
TABLE 10 lowest detection limit of the kit
Note: and the Ct <40 of any gene of ORF1ab or N is positive.
Therefore, the minimum detection limit of the kit is 400 copies/mL, the Ct value variation coefficient of positive results is less than 5%, and the kit meets the requirements of new versions of reagents (kits) for nucleic acid amplification detection (YY/T1182-2020) of the PRC industry standard to be implemented.
Further, the sequence BLAST was used to analyze common pathogens of the respiratory tract to evaluate the detection specificity of the kit of the present invention. Specifically, the primer probe sequence of the present invention and the clinically common pathogen are subjected to BLAST alignment analysis, and the results are shown in table 11:
TABLE 11 BLAST alignment analysis of primer probes of the invention with common respiratory pathogens
As can be seen, BLAST analysis showed that none of the above common respiratory pathogens could be amplified by the primer probes of the present invention.
Further, the nucleic acid positive samples of the common clinical microorganisms (from the microbiology laboratory of the national institute of medical examination of Jiangsu province) were tested by using the kit of the present invention to evaluate their cross-reactivity, and the test results are shown in Table 12:
TABLE 12 detection results of nucleic acid positive samples of clinically common microorganisms
Therefore, the bioinformatics BLAST analysis and the detection of nucleic acid positive samples of common microorganisms both show that the kit has high specificity and no cross reaction to common clinical microorganisms.
In example 1 of the present invention, a total of 90 clinical specimens (from the first hospital affiliated to Guangdong medical university) were subjected to parallel assay comparison (the assay was performed in the first hospital affiliated to Guangdong medical university) using the kit of the present invention and a novel coronavirus 2019-nCoV nucleic acid detection kit (national institutes Care 20203400060, lot No.: 6020200217, minimum detection limit: 100 copies/mL) produced by Huada Biotechnology (Wuhan) Co., Ltd., and the results are shown in Table 13:
TABLE 13 parallel detection comparison of the kit of the present invention and Huada kit
Note: positive detection is achieved by Ct <40 of any gene of ORF1ab or N; # and Ct less than or equal to 38 are positive.
It can be seen that the clinical sample detection results of the kit of the present invention and the huada kit are 100% identical (95% confidence interval: 96.7% -100%), the Ct value of ORF1ab gene is not statistically different from that of the huada kit (paired t test, bilateral α ═ 0.05, N ═ 14, t ═ 1.676, and P ═ 0.118), and the Ct value of N gene is significantly smaller than that of the huada kit (paired t test, unilateral α ═ 0.05, N ═ 15, t ═ 4.997, and P < 0.001).
Inventive example 2, use
6800 automatic nucleic acid analyzer and detection reagent therefor
SARS-CoV-2Test, a total of 90 clinical specimens (from Wisconsin Medical school of Milwaukee, Wisconsin, USA) were tested in parallel (testing was done at Wisconsin Medical school), and the results are shown in Table 14:
TABLE 14 kits and methods of the invention
6800 comparison of full-automatic detection system
Thus, the kit of the present invention and
6800 the consistency between the results of the full-automatic detection is 96.7% (95% confidence interval: 90.6% -99.3%).
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
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Claims (10)
1. A multiple real-time fluorescence PCR primer probe composition for detecting novel coronavirus is characterized by comprising multiple real-time fluorescence PCR primers and probes for detecting ORF1ab gene and N gene of SARS-CoV-2; wherein the upstream primer for amplifying ORF1ab gene is shown as SEQ ID NO: 1, and the downstream primer is shown as SEQ ID NO: 2, the probe sequence for detecting ORF1ab gene is shown as SEQ ID NO: 3 is shown in the specification; the upstream primer for amplifying the N gene is shown as SEQ ID NO: 4, and the downstream primer is shown as SEQ ID NO: 5, the probe sequence for detecting the N gene is shown as SEQ ID NO: and 6.
2. A multiple real-time fluorescent PCR primer probe composition for detecting novel coronavirus is characterized by also comprising a multiple real-time fluorescent PCR primer probe composition of human beta-Actin gene.
3. The multiplex real-time fluorescent PCR primer probe composition according to claim 2, characterized in that the upstream primer for amplifying human β -Actin gene is as shown in SEQ ID NO: 7, the downstream primer is shown as SEQ ID NO: 8, and the probe sequence for detecting the human beta-Actin gene is shown as SEQ ID NO: shown at 9.
4. Use of the multiplex real-time fluorescent PCR primer probe composition according to any one of claims 1 to 3 for the preparation of novel coronavirus detection reagents.
5. A novel coronavirus detection kit comprising the multiplex real-time fluorescent PCR primer probe composition of any one of claims 1-3.
6. The novel coronavirus detection kit according to claim 5, wherein the final concentration ratio of each primer to the probe participating in the reaction is 2: 1.
7. The novel coronavirus detection kit according to claim 5 or 6, wherein the kit further comprises an RT-PCR reaction solution, a positive quality control substance and a negative quality control substance.
8. The novel coronavirus detection kit according to claim 7, wherein the RT-PCR reaction solution comprises RT-PCR buffer solution, reverse transcriptase and hot start Taq enzyme.
9. The novel coronavirus detection kit of claim 7, wherein the positive quality control substance is an in vitro transcription RNA fragment containing a PCR amplification target sequence, and the RNA fragment of ORF1ab gene is shown as SEQ ID NO: 10, and the RNA segment of the N gene is shown as SEQ ID NO: shown at 11.
10. The novel coronavirus detection kit according to claim 7, wherein the negative quality control substance is a solvent matrix of the positive quality control substance and does not contain a PCR amplification target sequence.
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| CN113817868A (en) * | 2021-07-08 | 2021-12-21 | 成都峰际生物技术有限公司 | Primer, probe composition and kit for detecting novel coronavirus and variant thereof |
| WO2022089550A1 (en) * | 2020-10-28 | 2022-05-05 | Jiangsu Code Biomedical Technology Co., Ltd. | Novel compositions and methods for coronavirus detection |
| EP4043588A1 (en) * | 2021-02-10 | 2022-08-17 | Procomcure Biotech GmbH | Assays for the detection of sars-cov-2 mutants |
| WO2022171584A1 (en) * | 2021-02-10 | 2022-08-18 | Procomcure Biotech Gmbh | Assays for the detection of sars-cov-2 mutants |
| WO2023135350A1 (en) | 2022-01-13 | 2023-07-20 | Fundación Para La Investigación E Innovación Biosanitaria En El Principado De Asturias | SYSTEM FOR DETECTING SARS-COV-2 VARIANTS USING RT-gPCR |
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