A nucleic acid detection reagent for coronavirus pathogens, its detection method and application

Abstract

The present invention discloses a nucleic acid detection reagent for coronavirus pathogens, its detection method and application, which relates to the technical field of biological detection. The present invention designs specific primer-probes for 229E and NL63 in the genus Alphacoronavirus. The sequence of the target region of 229E is shown as SEQ ID No: 1, and the sequence of the target region of NL63 is shown as SEQ ID No: 2. The primer-probe combinations designed for these target regions are all compatible with real-time fluorescence quantitative PCR analyzers, and can achieve rapid detection of results on the basis of ensuring the specificity and sensitivity of detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular, to a nucleic acid detection reagent for coronavirus pathogens, a detection method thereof, and an application thereof. Background Art

[0002] Coronaviruses are spherical and polymorphic, with a diameter of about 80-160 nm, an envelope, and contain a non-segmented, linear single-stranded positive-sense RNA genome, which is the longest among known RNA viruses, about 27-32 kb. This RNA contains a 5' cap structure and a 3' polyadenylate, and also contains multiple open reading frames. Currently, coronaviruses are divided into four major categories: α, β, γ, and δ. Human coronaviruses are mainly distributed in the α and β categories, and there are a total of seven coronaviruses that infect humans. The less pathogenic ones are HCoV-229E and HCoV-NL63 in the genus α coronavirus, and HCoV-OC43 and HCoV-HKU1 in the genus β coronavirus; the more pathogenic ones are SARS-CoV, MERS-CoV, and SARS-CoV-2 in the genus β coronavirus.

[0003] Coronaviruses are highly contagious viruses that can be transmitted through direct, indirect contact, or aerosols. Their pathogenic process generally consists of three stages, namely, the virus invasion and replication stage, the excessive immune response stage, and the acute lung injury stage. They first reach the body through routes such as droplet transmission, aerosol transmission, and contact transmission, and adhere to the outer surface of cells by electrostatic action. Subsequently, the spike protein (S) or hemagglutinin-esterase protein (HE) on the virus surface recognizes and binds to specific cell receptors on the surface of sensitive cell membranes, and uses various methods to successfully escape the host's natural immune response and initiate the virus life cycle. At the same time, in this game between the body and the virus, a large number of pro-inflammatory cytokines and chemokines are induced, triggering the "cytokine storm" effect, causing damage or death of target cells, and then causing local tissue and organ lesions or necrosis. Finally, the released progeny viruses continuously infect other uninfected cells, and as the virus replicates continuously, the condition of the body deteriorates and the disease course intensifies. The main pathogenic characteristics of coronavirus infection are respiratory tract infection and an overactive host immune system.

[0004] It takes about two weeks for the onset of the disease after being infected with coronaviruses. The early symptoms are similar to those of a cold. There is also research showing that patients infected with SARS-CoV-2 or asymptomatic infected individuals are infectious during the incubation period. Therefore, timely and accurate diagnosis of suspected cases, research and development of effective therapeutic drugs and antibodies, and development of safe and effective vaccines are important means to prevent the further spread of the epidemic.

[0005] At present, there are methods for detecting coronaviruses such as serological testing, nucleic acid testing, electron microscopy technology, and virus isolation and culture. Among them, serological testing and nucleic acid testing are the main methods for detecting coronaviruses. Electron microscopy technology can be used for the morphological identification and observation of coronaviruses, but it depends on high-tech electron microscopy equipment and is suitable for morphological research in the laboratory. Virus isolation and culture is the gold standard for virus detection, but the isolation of viruses is highly dependent on technology and is related to sample quality, virus cell tropism, and isolation system. Serological testing includes IgM antibody detection method, IgG antibody detection method, and IgM / IgG double antibody detection method, which have the advantages of strong operability and wide application range; however, the test samples are easily contaminated or hemolyzed, and false positive or false negative test results may occur.

[0006] At present, nucleic acid testing is mostly used for the detection of coronaviruses. Nucleic acid testing includes methods such as whole-genome high-throughput sequencing, real-time fluorescence quantitative PCR, multiplex PCR technology, and reverse transcription loop-mediated isothermal amplification. Among them, whole-genome high-throughput sequencing can detect the entire genome of coronaviruses, but it is costly and highly dependent on equipment; real-time fluorescence quantitative PCR is the most commonly used nucleic acid testing method at present, but it also requires expensive equipment for assistance; the detection efficiency of multiplex PCR technology is relatively high, and it can detect multiple viruses simultaneously; reverse transcription loop-mediated isothermal amplification has the advantages of convenience, time-saving, and independence from equipment, and is suitable for on-site detection, but the detection accuracy and repeatability need to be improved, and there may be a risk of false negatives.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The purpose of the present invention is to provide a nucleic acid detection reagent for coronavirus pathogens, its detection method, and application.

[0009] The present invention is implemented as follows:

[0010] In the first aspect, an embodiment of the present invention provides a nucleic acid detection reagent for coronavirus pathogens, which includes a nucleic acid composition for detecting the target regions of alpha coronavirus 229E and NL63; wherein, the sequence of the 229E target region is as shown in SEQ ID No: 1, and the sequence of the NL63 target region is as shown in SEQ ID No: 2.

[0011] In the second aspect, an embodiment of the present invention provides the application of the nucleic acid detection reagent for coronavirus pathogens as described in the foregoing embodiment in the preparation of a kit for detecting nucleic acids of coronavirus pathogens.

[0012] In the third aspect, an embodiment of the present invention provides a kit for detecting nucleic acids of coronavirus pathogens, which includes the nucleic acid detection reagent for coronavirus pathogens as described in the foregoing embodiment.

[0013] Fourthly, an embodiment of the present invention provides a method for detecting nucleic acid of a coronavirus pathogen, which includes performing PCR detection on a sample to be tested by using the coronavirus pathogen nucleic acid detection reagent as described in the foregoing embodiments or the kit for detecting coronavirus pathogen nucleic acid as described in the foregoing embodiments; the detection method is not directly aimed at the diagnosis or treatment of diseases.

[0014] The present invention has the following beneficial effects:

[0015] The present invention designs specific primer-probe sets for 229E and NL63 in the genus Alphacoronavirus, and both of these two primer-probe sets are adapted to a real-time fluorescence quantitative PCR analyzer, which can quickly detect results on the basis of ensuring the specificity and sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 It is the amplification curve graph data for the optimization of the primer-probe dosage and system;

[0018] Figure 2 It is the amplification curve data for the detection limit detection experiment;

[0019] Figure 3 It is the amplification curve data for the specificity detection experiment;

[0020] Figure 4 It is the amplification curve data adapted to a real-time fluorescence quantitative PCR analyzer (model: P810);

[0021] Figure 5 It is the amplification curve data for the detection limit detection experiment of P810. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0023] The technical solutions provided by the present invention are specifically as follows.

[0024] An embodiment of the present invention provides a nucleic acid detection reagent for coronavirus pathogens, which includes a nucleic acid composition for detecting the target regions of alpha coronavirus 229E (Human coronavirus 229E, MN306046.1) and NL63 (Human coronavirus NL63, MK334047.1); wherein, the sequence of the 229E target region is as shown in SEQ ID No: 1, and the sequence of the NL63 target region is as shown in SEQ ID No: 2.

[0025] In some embodiments, the nucleic acid detection reagent for coronavirus pathogens detects subtypes HCoV-229E and / or HCoV-NL63 in the genus alpha coronavirus.

[0026] In some embodiments, the nucleic acid composition includes a primer pair. Among them, the nucleotide sequence of the upstream primer of the primer pair is as shown in SEQ ID No: 3-4, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No: 5. Compared with other primer pairs, the primer pair has better detection effects.

[0027] In some embodiments, in the primer pair, the molar ratio of the upstream primer to the downstream primer is (0.1-0.5):(0.1-0.5); this molar ratio can specifically be any one or the range between any two of 0.1:0.2, 0.1:0.3, 0.1:0.4, 0.1:0.5, 0.2:0.1, 0.2:0.3, 0.2:0.5, 0.3:0.1, 0.3:0.2, 0.3:0.4, 0.3:0.5, 0.4:0.1, 0.4:0.3, 0.4:0.5, 0.5:0.1, 0.5:0.2, 0.5:0.3, 0.5:0.4. Preferably, the molar ratio of the upstream primer to the downstream primer is (0.2-0.4):(0.4-0.5).

[0028] It should be noted that the molar ratio of the upstream primer to the downstream primer should be understood as the molar ratio of each / each type of upstream primer to the downstream primer. For example, the molar ratio of the upstream primer shown in SEQ ID No: 3 or 4 to the downstream primer shown in SEQ ID No: 5.

[0029] In some embodiments, the nucleic acid composition further includes a probe.

[0030] In some embodiments, the molar ratio of the downstream primer to the probe is (0.1 - 0.5):(0.1 - 0.5); specifically, the molar ratio is (0.1 - 0.5):(0.1 - 0.5); specifically, it can be any one or the range between any two of 0.1:0.2, 0.1:0.3, 0.1:0.4, 0.1:0.5, 0.2:0.1, 0.2:0.3, 0.2:0.5, 0.3:0.1, 0.3:0.2, 0.3:0.4, 0.3:0.5, 0.4:0.1, 0.4:0.3, 0.4:0.5, 0.5:0.1, 0.5:0.2, 0.5:0.3, 0.5:0.4.

[0031] Preferably, the molar ratio of the downstream primer to the probe is (0.4 - 0.5):(0.2 - 0.3).

[0032] In some embodiments, the sequence of the probe is as shown in SEQ ID No: 6.

[0033] In some embodiments, a fluorescent reporter group is connected to the 5'-end of the probe, and a fluorescent quenching group and / or MGB are connected to the 3'-end.

[0034] In some embodiments, the fluorescent reporter group is selected from any one of FAM, VIC, HEX, and TET.

[0035] In some embodiments, the fluorescent quenching group is selected from any one of BHQ2 and TAMRA.

[0036] The embodiments of the present invention also provide the application of the nucleic acid detection reagent for coronavirus pathogen as described in any of the foregoing embodiments in the preparation of a kit for detecting the nucleic acid of coronavirus pathogen.

[0037] The embodiments of the present invention also provide a kit for detecting the nucleic acid of coronavirus pathogen, which includes the nucleic acid detection reagent for coronavirus pathogen as described in any of the foregoing embodiments.

[0038] In some embodiments, the kit further includes at least one of PCR reaction solution, positive control product, negative control product, and enzyme mixture.

[0039] In some embodiments, the positive control product includes: armor RNAs containing the specific conserved sequences of alpha coronavirus 229E and alpha coronavirus NL63 respectively; the specific conserved sequence of 229E is as shown in SEQ ID No: 1, and the specific conserved sequence of NL63 is as shown in SEQ ID No: 2.

[0040] In some embodiments, the PCR reaction solution includes at least one of dNTPs, 10×BSA, and a buffer containing magnesium ions. Among them, dNTPs specifically include five nucleotides, namely dATPs, dUTPs, dGTPs, dCTPs, and dTTPs, and the magnesium ion can specifically be MgCl2.

[0041] In some embodiments, the primers and probes in the detection reagent are included in the PCR reaction solution.

[0042] In some embodiments, the enzyme mixture includes at least one of Taq enzyme, reverse transcriptase, and RNase inhibitor.

[0043] In some embodiments, the negative control product is a sterile TE buffer, which is prepared with molecular-grade water.

[0044] The embodiment of the present invention also provides a method for detecting the nucleic acid of a coronavirus pathogen, which includes performing PCR detection on a sample to be tested using the coronavirus pathogen nucleic acid detection reagent as described in any of the foregoing embodiments or the kit for detecting the nucleic acid of a coronavirus pathogen as described in any of the foregoing embodiments.

[0045] In some embodiments, the sample to be tested can be a blood sample, a tissue sample, an environmental sample, or an artificially prepared positive sample.

[0046] The detection method is not directly aimed at the diagnosis or treatment of a disease. For example, when the sample is an environmental sample, the direct purpose of the detection is to detect whether there is a coronavirus pathogen therein, rather than the diagnosis and treatment of a disease.

[0047] In some embodiments, when performing the PCR detection, the working concentration of each primer in the nucleic acid composition is independently 0.1 - 0.5 μM, and specifically can be any one of 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM or the range between any two of them.

[0048] Preferably, in the nucleic acid composition, the working concentration of the upstream primer is 0.36 μM, and the working concentration of the downstream primer is 0.48 μM.

[0049] In some embodiments, when performing the PCR detection, the working concentration of the probe in the nucleic acid composition is 0.1 - 0.5 μM, and specifically can be any one of 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM or the range between any two of them.

[0050] Preferably, when performing the PCR detection, the working concentration of the probe in the nucleic acid composition is 0.24 μM.

[0051] In some embodiments, when performing the PCR detection, the working concentration of MgCl2 is 2 - 8 mM, specifically, it can be any one of 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM or the range between any two of them.

[0052] In some embodiments, when performing the PCR detection, the working concentration of Taq enzyme is 0.01 - 1 U / μL, specifically, it can be any one of 0.01 U / μL, 0.05 U / μL, 0.1 U / μL, 0.15 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, 0.7 U / μL, 0.8 U / μL, 0.9 U / μL, 1 U / μL or the range between any two of them.

[0053] In some embodiments, when performing the PCR detection, the working concentration of reverse transcriptase is 1 - 10 U / μL, specifically, it can be any one of 1 U / μL, 1.5 U / μL, 2 U / μL, 2.5 U / μL, 3 U / μL, 3.5 U / μL, 4 U / μL, 4.5 U / μL, 5 U / μL, 5.5 U / μL, 6 U / μL, 6.5 U / μL, 7 U / μL, 7.5 U / μL, 8 U / μL, 8.5 U / μL, 9 U / μL, 9.5 U / μL, 10 U / μL or the range between any two of them.

[0054] In some embodiments, when performing the PCR detection, the working concentration of RNase inhibitor is 0.1 - 1 U / μL, specifically, it can be any one of 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, 0.7 U / μL, 0.8 U / μL, 0.9 U / μL, 1 U / μL or the range between any two of them.

[0055] In some embodiments, when performing the PCR detection, if the PCR instrument used is a conventional qPCR instrument (such as qPCR instrument ABI7500), the reaction conditions of PCR are as follows: reverse transcription at 48 - 52 °C for 13 - 17 min; pre-denaturation at 94 - 96 °C for 1 - 5 min; denaturation at 94 - 96 °C for 10 - 20 s, annealing and extension at 49 - 51 °C for 25 - 35 s, collecting fluorescence signals, and 44 - 46 cycles of denaturation - annealing and extension.

[0056] In some embodiments, when performing PCR detection, if the real-time fluorescence quantitative PCR analyzer P810 is used, the reaction conditions for PCR are as follows: reverse transcription at 48 - 52°C for 0.5 - 2 min; 0 s at 94 - 96°C; annealing and extension at 58 - 62°C for 5 - 15 s, collecting fluorescence signals, and denaturation - annealing and extension for 38 - 42 cycles.

[0057] Specifically, the detection method includes the following operating steps:

[0058] (1) Nucleic acid extraction: Use a nucleic acid extraction product, namely the virus DNA / RNA rapid extraction reagent (magnetic bead method) (product number: RK1002), to extract the nucleic acid of the sample, and the extraction product will be used for subsequent nucleic acid detection.

[0059] (2) System preparation: Take out the PCR reaction solution and enzyme mixture reagent prepared in the previous embodiment from the -20°C refrigerator, melt it at room temperature, vortex for 10 s, and centrifuge at low speed for 10 s. Take the corresponding amounts of the PCR reaction solution and enzyme mixture according to the ratio (8 μL of PCR reaction solution per person + 2 μL of enzyme mixture per person), mix well, and centrifuge at low speed for 10 s for standby.

[0060] (3) Then aliquot according to 10 μL per reaction, then add 5 μL of the above extraction product, mix well, and perform amplification detection according to the following program.

[0061] Amplification program settings on the real-time fluorescence quantitative PCR analyzer P810:

[0062]

[0063] All amplifications can be completed within 9 minutes using this program.

[0064] Amplification program settings on the conventional qPCR instrument ABI7500:

[0065]

[0066] All amplifications can be completed within 90 minutes using this program.

[0067] (4) Result analysis:

[0068] 4.1 Save the detection data file after the experiment.

[0069] 4.2 Analysis condition settings: Adjust the Start value, End value of the baseline and the Value value of the threshold according to the analyzed image (the user can adjust according to the actual situation, the Start value can be between 3 - 15, the End value can be between 5 - 20, and adjust the amplification curve of the negative control to be flat or below the threshold line) so that the instrument gives the correct result.

[0070] (5) Quality Control

[0071] 5.1 Negative control: In the FAM channel and ROX channel, the Ct value shows 0 and there is no obvious S-shaped amplification curve; in the CY5 channel, the Ct value shows 0 and there is no obvious S-shaped amplification curve;

[0072] 5.2 Positive control: In both the FAM channel and ROX channel, there is a Ct value and Ct ≤ 35, and there is an obvious S-shaped amplification curve; in the CY5 channel, there is a Ct value and Ct ≤ 35, and there is an obvious S-shaped amplification curve;

[0073] The above two requirements need to be met simultaneously in the same experiment. Otherwise, this experiment is invalid and needs to be repeated.

[0074] (6) Result Interpretation

[0075] 6.1 When there is an amplification curve in the FAM channel or ROX channel, and the Ct values are all ≤ 35, it can be determined that the nucleic acid of the coronavirus pathogen is positive;

[0076] 6.2 When there is no amplification curve in both the FAM channel and ROX channel, and the Ct value shows 0, and there is an amplification curve in the CY5 channel and the Ct values are all ≤ 35, it can be determined that the nucleic acid of the coronavirus pathogen is negative;

[0077] 6.3 When there is an amplification curve in the FAM channel or ROX channel, 35 < Ct value ≤ 40, and there is an amplification curve in the CY5 channel, and the Ct values are all ≤ 35, it is recommended to repeat the experiment once. If the results are the same as above, it can be determined as positive for the coronavirus pathogen. If there is no amplification, it can be determined as negative for the coronavirus pathogen;

[0078] 6.4 When there is no amplification curve in the FAM channel, ROX channel, and CY5 channel, and the Ct value shows 0, the experiment is invalid, and it is recommended to replace a batch of reagents and repeat the experiment.

[0079] The definition of Ct value (cycle threshold, Ct) is: the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold. The threshold is generally set to exactly cover the fluorescence values of the negative control and blank control, so that the influence of the fluorescence value of the reaction tube, that is, the background, on the test result can be well removed.

[0080] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specified detailed conditions in the following embodiments, they are generally carried out according to the conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" by Sambrook.J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0081] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0082] Example 1 Design of primers and probes

[0083] By analyzing 229E and NL63 in the genus Alphacoronavirus, multiple sets of specific primer and probe sequences were finally determined to be designed in the conserved region of the ORF1ab gene.

[0084] Table 1 Coronavirus-specific primer and probe sequences

[0085]

[0086]

[0087] Note: F is the upstream primer, R is the downstream primer, and P is the probe; R in the sequence is a degenerate base, representing A / G.

[0088] In the process of designing the above primers and probes, primer-probe fragments with fewer cases of hairpin structures, internal primer dimers, inter-primer dimers, and mismatches were selected as much as possible. Then, the designed primer-probe sequences were compared and analyzed on the NCBI Blast online database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to avoid non-specific binding and amplification with other pathogenic bacteria or human genes.

[0089] After multiple rounds of screening and optimization on a conventional real-time fluorescence quantitative PCR instrument, and then through performance comparison and confirmation on a real-time fluorescence quantitative PCR analyzer, a set of primer and probe combinations with the best sensitivity and specificity was finally determined.

[0090] In order to screen the target primer-probe combination with the best amplification efficiency, the present application synthesized the following target fragments of the coronavirus target gene, as shown in the following table, and inserted these sequences into a cloning vector. Purified plasmids containing positive fragments were obtained through monoclonal screening. Then, armor RNA preparation technology was used to produce the corresponding armor RNA for the project, making the sample closer to the state of a real sample.

[0091]

[0092] The following amplification system was used to prepare the primer-probe combination, and the amplification efficiencies of the primer-probe combinations of coronavirus 229E and NL63 were compared. Among them, the 5×PCR buffer (product number: MD013), reverse transcriptase (product number: MD301), and Taq enzyme (product number: MD099) in the preparation table are raw materials developed by PhyNexus Biotech Co., Ltd., and all the primer-probes in the preparation table were synthesized by Sangon Biotech Co., Ltd.

[0093] Table 2 Singleplex Amplification System Table of Different Combinations of Primer-Probe

[0094]

[0095]

[0096] The template used was the extraction product of the armor RNA of the coronavirus constructed above. The concentration of the product after extraction of the positive control was approximately 1.0×10 6 copies / mL. The nucleic acid product after extraction was diluted to 1 / 10 8 times of the original solution with molecular grade purified water.

[0097] The amplification program was as shown in Table 3 below.

[0098] Table 3 Amplification Program of Conventional Fluorescent Quantitative PCR Instrument - ABI7500

[0099]

[0100] The amplification results are as follows in the table:

[0101] Table 4 Amplification Data of the First Group of Primer-Probe Combinations of Coronavirus 229E / NL63 on ABI7500

[0102]

[0103] Table 5 Amplification Data of the Second Group of Primer-Probe Combinations of Coronavirus 229E / NL63 on ABI7500

[0104]

[0105] According to the above data, the amplification effect of the first group of target primer-probes of coronavirus 229E and NL63 is the best.

[0106] Example 2 Inspection Method and Optimization of Detection System

[0107] To determine the detection system of primer-probe, the effects of primer-probes at different concentrations on fluorescence PCR reaction were attempted respectively. The synthesized dry powder of primer-probe was diluted into a working solution with a concentration of 10 μM according to the instructions. In a 15-μL system, the gradient addition amounts of primers and probes are shown in Table 6. An orthogonal experiment was conducted with the gradient amounts of primers and the gradient amounts of probes, as shown in Tables 7 and 8. Using the primer-probe combination of coronavirus 229E / NL63 target and the armor RNA extract of coronavirus 229E as the template, then the above systems were all supplemented with purified water to 15 μL for amplification. The amplification system was prepared as follows:

[0108] Table 6 Dosage Debugging General Amplification System Table

[0109] Component 15 μL System / μL 5×PCR buffer 3 dNPTs (including dUTP) (10 mM) 0.3 <![CDATA[MgCl2(25mM)]]> 1.8 10×BSA 1.5 Taq enzyme (5 U / μL) 0.2 Reverse transcriptase (200 U / μL) 0.2 RNAsin (40 U / μL) 0.1 ACTB-F1 (10 μM) 0.4 ACTB-P1 (10 μM) 0.2 ACTB-R1 (10 μM) 0.4 Target table primer-probe combination Dosage number in Table 8, Nos. 10 - 18 Purified water Make up to 10 Template 5

[0110] Table 7 Variation Range of Primer-Probe Dosages

[0111]

[0112]

[0113] Table 8 Dosage Debugging Amplification System Table and Dosage Number of Coronavirus 229E / NL63 Target Primer-Probe Combination

[0114]

[0115] The amplification procedure was as follows:

[0116] Amplification program settings on a conventional qPCR instrument ABI7500:

[0117]

[0118] The amplification results are as follows in the table:

[0119] Table 9 Amplification Data of Dosage Debugging of Coronavirus 229E / NL63 Primer-Probe

[0120]

[0121] The amplification curve is as Figure 1 shown. According to the amplification curve and the amplification data results, among the primer-probe combinations of the coronavirus 229E / NL63 target, the primer-probe combination with dosage number 18 has the best amplification effect. Therefore, when the final concentration of F3 is 360 nM, the final concentration of F4 is 360 nM, the final concentration of R4 is 480 nM, and the final concentration of P3 is 240 nM, the amplification efficiency is the best.

[0122] Example 3 Detection Limit Detection

[0123] The armor RNA for the corresponding project was prepared using the armor RNA preparation technology, and then extracted using a viral DNA / RNA rapid extraction reagent (magnetic bead method) (product number: RK1002). The extracted product was quantified using Digital PCR technology (i.e., digital PCR) to obtain the concentration of the extracted product for the corresponding project. After diluting the corresponding extracted product to an appropriate concentration according to the known concentration, it was then serially diluted 2-fold, with concentrations of 400 copies / mL, 200 copies / mL, and 100 copies / mL respectively. The above-mentioned extracted product was detected using the detection system (amplification system as shown in Table 10 below), product form, and cycle parameters determined above. The amplification results are shown in Table 11. Confirmation was carried out for 4 armor RNA extraction products with a concentration of 200 copies / mL respectively. The amplification data results are shown in Table 12, and the amplification curve diagram is as Figure 2 . The results show that the detection method of the present invention has high sensitivity, and the sensitivity can reach 200 copies / mL.

[0124] Table 10 Dual amplification system for alpha coronavirus 229E / NL63

[0125] Component 15 μL System / μL 5×PCR buffer 3 dNPTs (including dUTP) (10 mM) 0.3 <![CDATA[MgCl2(25mM)]]> 1.8 10×BSA 1.5 Taq enzyme (5 U / μL) 0.2 Reverse transcriptase (200 U / μL) 0.2 RNAsin (40 U / μL) 0.1 ACTB-F1 (10 μM) 0.4 ACTB-P1 (10 μM) 0.2 ACTB-R1 (10 μM) 0.4 F3 (30 μM) 0.18 F4 (30 μM) 0.18 R4 (30 μM) 0.24 P3 (30 μM) 0.12 Purified water 1.18 Template 5

[0126] Table 11 Amplification data table for testing gradient concentration armor RNA of the detection system

[0127]

[0128]

[0129] Table 12 Amplification data table for testing the lowest detectable limit concentration armor RNA of the detection system

[0130]

[0131] Example 4 Specific detection

[0132] In this example, samples such as a mixture of respiratory syncytial virus strain (type B), Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, influenza A virus strain (type: H1N1), influenza A virus strain (type: H3N2), influenza A virus strain (type: H7N9), respiratory adenovirus (type 3), respiratory adenovirus (type 7) and saliva of healthy people were used as specific samples for detection.

[0133] The test results showed that the test results of specific quality control products for detecting respiratory syncytial virus strain (type B), Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, influenza A virus strain (type: H1N1), influenza A virus strain (type: H3N2), influenza A virus strain (type: H7N9), respiratory adenovirus (type 3), respiratory adenovirus (type 7), etc. were all negative, such as Figure 3 , and the internal standard test result was positive, indicating that the primer-probe combination (Group 1) of the present invention could be amplified with good specificity.

[0134] Example 5 Repeatability Test

[0135] In this example, armor RNA extraction products with concentrations of 1×10 5 copies / mL and 1×10 3 copies / mL were selected for testing, and the detection system and cycling parameters determined in the above example were used for repeated testing.

[0136] The test results are shown in Table 13 below. After statistical analysis, the CV values % were all less than 5%, indicating that the kit and detection method of the present invention had good repeatability.

[0137] Table 13 Amplification data table for testing the repeatability of armor RNA in the detection system

[0138]

[0139]

[0140] Example 6 Adaptation work of real-time fluorescence quantitative PCR analyzer (model: P810)

[0141] In this example, armor RNA extracts of coronavirus 229E and NL63 with concentrations of 800 copies / mL, 400 copies / mL, 200 copies / mL, and 100 copies / mL were selected for testing. The detection system determined in the above example was used, and at the same time, the detection performance of the reagent was verified using the real-time fluorescence quantitative PCR analyzer (model: P810) developed by the company.

[0142] Amplification program settings on the real-time fluorescence quantitative PCR analyzer P810:

[0143]

[0144] The amplification results are shown in Table 14, and the amplification curves are as Figure 4, a good linear relationship was obtained, and the lowest detection limit reached 200 copies / mL. Four RNA extraction products of armor with a concentration of 200 copies / mL were confirmed respectively. The amplification data results are shown in Table 15, and the amplification curve diagram is as Figure 5 , the lowest detection limit of the P810 platform is the same as that of the ABI7500.

[0145] Table 14 Amplification data table of armor RNA with gradient concentrations tested by P810 instrument

[0146]

[0147] Table 15 Amplification data table of armor RNA with the lowest detection limit concentration tested by P810

[0148]

[0149] Example 7 Detection of real samples

[0150] In this example, virus strains of coronavirus 229E and NL63 and 26 clinical samples were used as samples. All were extracted using a rapid virus DNA / RNA extraction reagent (magnetic bead method). Among them, the extraction products of the virus strains were diluted 100 times with the extraction products of the saliva of healthy people. The first group of detection reagents of the present invention were respectively tested on a real-time fluorescence quantitative PCR analyzer P810 and a conventional qPCR instrument ABI7500. The amplification effects of the reagents of the present invention on the two instruments were compared. The amplification procedures are as follows in the table, and the amplification results are shown in Table 16 below. The data show that the coincidence rate of the amplification results of the reagents of the present invention on the real-time fluorescence quantitative PCR analyzer P810 and the conventional qPCR instrument ABI7500 is 100%. Moreover, the amplification effect of the present invention on the fluorescence quantitative PCR instrument P810 is better than that on the conventional qPCR instrument ABI7500, indicating that the reagents of the present invention are more suitable for the fluorescence quantitative PCR instrument P810. The coronavirus detection kit provided by the present invention is sensitive, specific, and rapid, and can be used for the detection of clinical samples.

[0151] Amplification program settings on the real-time fluorescence quantitative PCR analyzer P810:

[0152]

[0153] Amplification program settings on the conventional qPCR instrument ABI7500:

[0154]

[0155]

[0156] Table 16 Amplification data table of real samples tested by two PCR instruments

[0157] Clinical sample number P810 ABI7500 Clinical sample number P810 ABI7500 1 28.61 28.23 16 21.38 22.52 2 36.47 37.63 17 24.29 26.60 3 20.60 20.88 18 23.02 26.03 4 0 Undetermined 19 0 Undetermined 5 0 Undetermined 20 28.21 30.76 6 28.12 30.84 21 29.60 30.36 7 29.83 29.51 22 0 Undetermined 8 37.92 39.80 23 33.90 36.81 9 0 Undetermined 24 17.85 20.33 10 36.30 37.32 25 18.18 19.69 11 34.13 36.70 26 22.61 24.73 12 37.96 39.95 Coronavirus strain 229E 21.07 23.81 13 0 Undetermined Coronavirus strain NL63 25.17 27.33

[0158] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid detection reagent for coronavirus pathogens, which comprises a nucleic acid composition for detecting the target regions of alpha coronavirus 229E and NL63; wherein, The sequence of the 229E target region is shown in SEQ ID No: 1, and the sequence of the NL63 target region is shown in SEQ ID No: 2; the nucleic acid composition includes a primer pair and a probe. The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID No: 3-4, and the nucleotide sequence of the downstream primer is shown in SEQ ID No: 5; the sequence of the probe is shown in SEQ ID No:

6.

2. The nucleic acid detection reagent for coronavirus pathogen according to claim 1, wherein, The coronavirus pathogen nucleic acid detection reagent detects HCoV-229E and / or HCoV-NL63 subtypes in the genus Alphacoronavirus.

3. The coronavirus pathogen nucleic acid detection reagent according to claim 1, wherein, The 5' end of the probe is linked with a fluorescent reporter group, and the 3' end is linked with a fluorescent quenching group.

4. The coronavirus pathogen nucleic acid detection reagent according to claim 3, wherein, The fluorescent reporter group is selected from any one of FAM, VIC, HEX and TET.

5. The coronavirus pathogen nucleic acid detection reagent according to claim 3, wherein, The fluorescent quenching group is selected from any one of BHQ2 and TAMRA.

6. The coronavirus pathogen nucleic acid detection reagent according to claim 1, wherein, The 3' end of the probe is linked with MGB.

7. The nucleic acid detection reagent for coronavirus pathogen according to any one of claims 1 to 6, wherein, The molar ratio of the upstream primer to the downstream primer is (0.2-0.4):(0.4-0.5).

8. Use of the nucleic acid detection reagent for coronavirus pathogen according to any one of claims 1 to 7 in the preparation of a kit for detecting nucleic acid of coronavirus pathogen, wherein, Detecting the nucleic acid of the coronavirus pathogen is to detect HCoV-229E and / or HCoV-NL63 subtypes in the genus Alphacoronavirus.

9. A kit for detecting nucleic acid of coronavirus pathogen, wherein, It includes the coronavirus pathogen nucleic acid detection reagent according to any one of claims 1-7.

10. The kit for detecting nucleic acid of coronavirus pathogen according to claim 9, wherein, The kit further includes at least one of PCR reaction solution, positive control product, negative control product and enzyme mixture.

11. The kit for detecting nucleic acid of coronavirus pathogen according to claim 10, wherein, The PCR reaction solution includes dNTPs, 10×BSA and a buffer containing magnesium ions.

12. The kit for detecting nucleic acid of coronavirus pathogen according to claim 10, wherein, The enzyme mixture includes Taq enzyme, reverse transcriptase and RNA enzyme inhibitor.

13. A method for detecting the nucleic acid of a coronavirus pathogen, wherein, It includes performing PCR detection on a test sample using the coronavirus pathogen nucleic acid detection reagent according to any one of claims 1-7 or the kit for detecting the nucleic acid of the coronavirus pathogen according to any one of claims 9-12; The detection method is not directly aimed at the diagnosis and treatment of diseases.

14. The detection method according to claim 13, wherein, The working concentration of the upstream primer and the downstream primer in the nucleic acid composition is 0.1-0.5 μM.

15. The detection method according to claim 13, wherein, The working concentration of the probe in the nucleic acid composition is 0.1-0.5 μM.

Images