A specific extraction reagent, extraction and detection method and application of viral nucleic acid RNA

Abstract

The present invention relates to a specific extraction reagent, extraction detection method and application of viral nucleic acid RNA, wherein the specific extraction reagent of viral nucleic acid RNA comprises a magnetic microsphere containing γFe2O3 magnetic particles or Fe3O4 magnetic particles, and a capture probe targeting influenza A virus nucleic acid RNA; the surface of the magnetic microsphere is modified with a connecting group connected to the end of the capture probe, and the capture probe comprises a specific oligonucleotide chain targeting influenza A virus nucleic acid RNA, and the specific oligonucleotide chain can produce specific binding with the target influenza A virus nucleic acid RNA through base complementary pairing. Compared with the prior art, the present invention improves the specificity of nucleic acid extraction, is suitable for low-abundance samples, and can be used for extracting viral nucleic acids in large-volume swabs, plasma, serum, urine and other samples.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a specific extraction reagent, extraction and detection method and application of viral nucleic acid RNA. Background Art

[0002] Influenza A virus is a common influenza virus, while avian influenza (abbreviated as avian influenza) is an acute respiratory infectious disease caused by some strains of certain subtypes of avian influenza A virus. After the virus gene mutates, it can infect humans. The symptoms after infection are mainly high fever, cough, runny nose, myalgia, etc. Influenza A virus is most likely to mutate, that is, the surface antigen will often undergo small mutations, thereby evading the recognition of the human immune system. The influenza pandemic is caused by the emergence of new subtypes of influenza A virus or the reappearance of old subtypes. The strains that cause influenza may be different every year, and people need to be re-vaccinated with influenza vaccines every year for prevention. Influenza viruses usually rely on the combination of a part of the viral protein with a specific protein in the human body to invade the human body, because through such a combination, the influenza virus can suppress the human body's own natural defense system against viral infection and help the virus to replicate effectively in the human body.

[0003] Influenza A virus is divided into many subtypes according to the difference of H and N antigens. H can be divided into 18 subtypes (H1~H18), and N has 11 subtypes (N1~N11). Among them, only H1N1, H2N2, and H3N2 mainly infect humans, and the natural hosts of many other subtypes are various poultry and animals. After 1997, there have been several incidents of avian influenza virus infecting humans in the world. Once the highly pathogenic avian influenza viruses such as H5N1, H7N7, H7N9, and H9N2 mutate and have the ability to spread from person to person, they will cause the spread of avian influenza in humans, indicating that the avian influenza virus has a great potential threat to humans. Humans are the main source of infection for this virus, and asymptomatic infected people are also contagious. It is mainly transmitted through droplets through the respiratory tract, and can also be transmitted through direct or indirect contact with mucous membranes in the mouth, nose, eyes, etc., and can also be indirectly transmitted through virus-contaminated tea sets, tableware, towels, etc. The speed and breadth of transmission are related to population density. People are generally susceptible to this virus. Pregnant women, obese people, children under 5 years old, people over 65 years old, people with chronic underlying diseases and people with low immune function are more likely to become high-risk groups for severe cases. After infection, such people are more likely to develop complications such as viral pneumonia, secondary bacterial pneumonia, and acute respiratory distress syndrome. In severe cases, multiple organ failure such as heart and kidney failure leads to death. If such people have flu-like symptoms, they should be taken seriously and undergo influenza A (H1N1) virus nucleic acid testing and other necessary examinations as soon as possible.

[0004] The existing commercial nucleic acid extraction methods mainly include organic solvent extraction method (TRIzol), silica membrane adsorption column method (Qiagen), silica-based magnetic bead nucleic acid extraction method and immunomagnetic bead specific capture method. The phenol / chloroform extraction method is inexpensive, but the organic reagent is highly toxic, requires manual operation and has poor repeatability, and is not suitable for trace nucleic acid extraction. The silica membrane adsorption column method has high extraction efficiency, but it has requirements for equipment. It needs to be equipped with a centrifuge or even a vacuum pump. There is a risk of clogging by sample impurities, and the automation cost is high. It is suitable for processing a small amount of samples. The silica-based magnetic bead nucleic acid extraction method adsorbs total nucleic acid in a certain ionic environment. The magnetic beads can be quickly separated by a magnetic field, and no centrifugation equipment is required, which is easy to automate; however, the target sequence is scarce in the total nucleic acid of a large number of samples or low-abundance samples, and other nucleic acids will isolate or hinder the target sequence from contacting the amplification system, reducing the amplification efficiency and reagent sensitivity; and the loss of various types of DNA or RNA or the extraction efficiency in each step is uneven.

[0005] Patents CN101985665A, CN103131794A and CN103255231B use affinity principle to extract influenza A virus nucleic acid. The capture probe sequences used are different from those of the present invention. In addition, these three patents add a pre-amplification step before or after extraction to reduce the detection limit. At the same time, pre-amplification uses asymmetric primers to introduce biotin labels to the ends of nucleic acids, and then combines streptavidin-phycoerythrin to produce detectable fluorescent signals. The detection process is relatively complicated.

[0006] The purpose of the present invention is to develop a simple, rapid, highly sensitive and highly specific method for detecting viral nucleic acids. Summary of the invention

[0007] The purpose of the present invention is to provide a specific extraction reagent, extraction detection method and application of viral nucleic acid RNA in order to improve the efficiency of extracting viral RNA.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide a specific extraction reagent for viral nucleic acid RNA, including magnetic microspheres containing γFe2O3 magnetic particles or Fe3O4 magnetic particles, and a capture probe targeting influenza A virus nucleic acid RNA;

[0010] The surface of the magnetic microsphere is modified with a linking group connected to the end of the capture probe, and the linking group is selected from any one of streptavidin, -NH2, -SH, -N3, -COOH, -C=C, alkynyl, p-toluenesulfonyl, epoxy, biotin and derivative groups thereof;

[0011] The capture probe comprises a specific oligonucleotide chain targeting influenza A virus nucleic acid RNA, and the specific oligonucleotide chain can specifically bind to the target influenza A virus nucleic acid RNA through complementary base pairing.

[0012] In some specific embodiments, the specific oligonucleotide chain is selected from any one or more of CP1-1, CP1-2, CP1-3, CP2-1, CP2-2, CP2-3, CP3-1, CP3-2, and CP3-3 whose base sequences are shown in SEQ ID NO.1-9.

[0013] The base sequence of the above-mentioned specific oligonucleotide chain is as follows:

[0014]

[0015] In some specific embodiments, the particle size of the magnetic microspheres is 0.2-10 μm, and the binding capacity of the magnetic microspheres is at least 100 pmol / mg.

[0016] The second technical solution of the present invention is to provide a method for extracting and detecting viral nucleic acid RNA, using the specific extraction reagent as described in one of the above technical solutions, comprising the following steps:

[0017] S1. Add lysis solution to the sample to be tested to react and obtain a lysis mixture for lysing viral nucleic acid in the sample;

[0018] S2, mixing the capture probe with the magnetic microspheres to obtain magnetic microspheres coated with the capture probe;

[0019] S3, mixing the lysis mixture in step S1 with the magnetic microspheres coated with capture probes in step S2 to allow the capture probes to hybridize with the viral nucleic acid in the lysis mixture;

[0020] S4. Magnetic separation of the magnetic microspheres and the lysis mixture in step S3, discarding the supernatant; adding an eluent to elute the magnetic microspheres, taking the supernatant for fluorescent quantitative PCR to detect the viral nucleic acid RNA content, or directly performing fluorescent quantitative PCR on the magnetic microspheres to detect the viral nucleic acid RNA content.

[0021] In some embodiments, in step S1, the ratio of the sample to be tested to the lysate is 1:0.5-2.5.

[0022] In some specific embodiments, in step S1, the reaction temperature is 50°C-95°C, the reaction time is 5-30 min, and then naturally cooled to room temperature.

[0023] In some specific embodiments, in step S3, the ratio between the amount of magnetic microspheres coated with capture probes added and the sample to be tested is (1-50) μg:100 μL.

[0024] In some specific embodiments, in step S3, the reaction temperature is room temperature and the reaction time is 10-30 min.

[0025] In some specific embodiments, in step S4, the volume ratio of the added amount of the eluent to the sample to be tested is (1-10): (20-100), and the elution condition is: heating at 70° C.-95° C. for 5-30 min.

[0026] The third technical solution of the present invention is to provide an application of a specific extraction reagent for viral nucleic acid RNA as described in one of the above technical solutions, wherein the specific extraction reagent for viral nucleic acid RNA is used to prepare a kit for extracting viral nucleic acid RNA, and the kit includes a specific extraction reagent for viral nucleic acid RNA, a lysis solution, a cleaning solution and an elution solution.

[0027] In some specific embodiments, the lysate is composed of an RNase inhibitor, a surfactant, a reducing agent, a metal ion chelator, a proteinase K and a buffer.

[0028] The RNase inhibitor is selected from any one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, vanadyl ribonucleoside complex or 8-hydroxyquinoline;

[0029] The surfactant is selected from any one or more of PEG200, Triton X-100, Triton X-114, Tween-20, SDS, LDS, SLS or NP-40;

[0030] The reducing agent is selected from any one or more of dithiothreitol, cysteine, glutathione, ammonium thioglycolate, 2-mercaptoethanol or tris(2-formylethyl)phosphine hydrochloride;

[0031] The buffer is selected from any one or more of Tris-HCl, citrate, borate, or phosphate;

[0032] The metal ion chelating agent is selected from any one or more of ethylenediaminetetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid and their salts.

[0033] In some specific embodiments, the final concentration of the RNase inhibitor is 0.01-10.00 M, the mass percentage concentration of the surfactant is 0.05%-40%, the final concentration of the reducing agent is 0.1-1000 mM, the final concentration of the buffer is 1-1000 mM, the final concentration of the metal ion chelator is 1-100 mM, and the final concentration of the proteinase K is 50-100 mg / L.

[0034] In some specific embodiments, the final concentration of the RNase inhibitor is preferably 1.00-5.00 M, the mass percentage concentration of the surfactant is preferably 0.5%-20%, the final concentration of the reducing agent is preferably 1-100 mM, and the final concentration of the buffer is preferably 5-300 mM.

[0035] In some specific embodiments, the cleaning solution is composed of the following raw materials with final concentrations: 1-50 mM Tris-HCl buffer, 0.05-0.5 M NaCl, and 0.5-10 mM EDTA.

[0036] In some embodiments, the eluent is composed of the following raw materials at final concentrations: 1-20 mM Tris-HCl buffer, 1 mM EDTA, and enzyme-free water.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention improves the specificity of nucleic acid extraction, is suitable for low-abundance samples, and can be used for extracting viral nucleic acids from large-volume swabs, plasma, serum, urine and other samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The concentration of 10 was extracted from Example 2. 4 / 10 3 / 10 2 Amplification curve of influenza A virus with 10 copies / mL.

[0040] Figure 2 The concentration of 10 is extracted from Example 6 4 / 10 3 / 10 2 Amplification curve of influenza A virus at 10 copies / mL. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0042] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0043] The capture probe targeting influenza A virus nucleic acid RNA was synthesized by a biological company.

[0044] Influenza A virus: provided by Shanghai Daolong Biotechnology Co., Ltd.

[0045] Complementary sequences of nucleic acids in the conserved regions of influenza A viruses of different lengths and positions were selected as capture probes, and their sequences are shown in Table 1:

[0046] Table 1 Biotin-labeled specific capture probe sequence list

[0047]

[0048] According to the product Dynabeads TM The instructions provided by M-270 Streptavidin (Cat. No. 65305) are to coat the biotin-labeled specific capture probe on the magnetic microspheres with streptavidin bound to the surface.

[0049] Embodiment 1:

[0050] 1. Add 200 μL of 3×10 4 100 μg of magnetic beads coated with specific capture probes were added to the lysed plasma sample and incubated at room temperature for 15 minutes to allow the influenza A virus target nucleic acid in the sputum sample to fully bind to the magnetic beads.

[0051] 2. Place the volume tube on the magnetic rack, discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, and centrifuge briefly; place it on the magnetic rack, discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge briefly, and remove the supernatant by magnetic separation.

[0052] 3. Add 60 μL of elution buffer and heat at 70°C for 10 min to elute the influenza A virus nucleic acid bound to the magnetic beads.

[0053] 4. Take 10 μL of supernatant for qPCR amplification and detect the viral nucleic acid RNA content, i.e., the viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCR Probe Kit.

[0054] Lysis buffer: 2 M guanidine hydrochloride, 4 M guanidine isothiocyanate, 5% Triton X-100, 200 mM dithiothreitol, 50 mM Tris-HCl, 50 mM EDTA;

[0055] Washing solution: 200 mM Tris-HCl buffer, 10 mM NaCl, 20 mM EDTA;

[0056] Elution buffer: 100 mM Tris-HCl buffer, 0.5 mM EDTA, enzyme-free water.

[0057] The results are shown in Table 2:

[0058] Table 2 Virus content detected by fluorescence qPCR

[0059]

[0060] From the results in Table 2, it can be seen that capture probes of different lengths and sequences, whether used alone or in combination, can achieve similar influenza A virus extraction effects.

[0061] Embodiment 2:

[0062] Compared with Example 1, most of the above are the same, except that in step (1), the samples used are nasal swab samples, and the influenza A virus concentrations of the nasal swab samples are 10 4 / 10 3 / 10 2 copies / mL, the specific capture probe used was a combination probe (CP2-1 / CP2-2 / CP2-3), and it was heated at 75°C for 30 min; in step (3), 100 μL of eluent was added and heated at 80°C for 20 min.

[0063] Lysis buffer: 6 M guanidine hydrochloride, 35% Tween-20, 2 mM TCEP, 200 mM Tris-HCl, 2 mM EDTA;

[0064] Washing solution: 20 mM Tris-HCl buffer, 300 mM NaCl, 10 mM EDTA;

[0065] Elution buffer: 5 mM Tris-HCl buffer, 5 mM EDTA, enzyme-free water.

[0066] The results of amplification of influenza A virus nucleic acid RNA are as follows Figure 1 As shown, the extraction results of samples of the same volume but with different virus concentrations are roughly linear.

[0067] Embodiment 3:

[0068] Compared with Example 1, most of the above are the same, except that in step 1, the amount of magnetic beads coated with CP2-3 specific capture probes is changed to 10-50 μg, and the influenza A virus concentration of the sample is 10 6 copies / mL. The results are shown in Table 3:

[0069] Table 3 Virus content detected by fluorescence qPCR

[0070] Magnetic beads dosage 10 μg 20 μg 30 μg 40 μg 50μg Ct value 24.789 23.800 23.702 23.953 23.661

[0071] As shown in Table 3, the addition amount of magnetic beads coated with specific capture probes has little effect on the extraction efficiency when it is within the range of 20-50 μg.

[0072] Embodiment 4:

[0073] 1. Add 0.2 mL, 0.4 mL, 1 mL, 2 mL and 4 mL of influenza A virus with a concentration of 1×10 3 The mixed saliva sample with 100 copies / mL and the lysis buffer with 2 times the volume of the sample were heated at 65℃ for 10 minutes after mixing, and then cooled naturally to room temperature. 100μg, 100μg, 200μg, 400μg and 800μg of magnetic beads coated with specific capture probes (CP1-1 / CP1-2 / CP1-3 combination probes) were added to the lysed blood samples, and then incubated at room temperature for 30 minutes to allow the target nucleic acid in the sample to fully bind to the magnetic beads.

[0074] 2. Place the volume tube on the magnetic rack and discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, and centrifuge instantly; place it on the magnetic rack and discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge instantly, and remove the supernatant by magnetic separation.

[0075] 3. Add 60 μL of elution buffer and heat at 70°C for 30 min to elute the influenza A virus nucleic acid bound to the magnetic beads.

[0076] 4. Take 10 μL of supernatant for qPCR amplification and detect viral nucleic acid RNA, i.e., viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCR Probe Kit.

[0077] Lysis buffer: 2M guanidine thiocyanate, 20% Triton X-114, 500mM ammonium thioglycolate, 10mMTris-HCl, 100mM EDTA;

[0078] Washing solution: 450 mM Tris-HCl buffer, 500 mM NaCl, 2 mM EDTA;

[0079] Elution buffer: 50 mM Tris-HCl buffer, 1 mM EDTA, enzyme-free water.

[0080] The results are shown in Table 4. The present invention is applicable to a wide range of sample volumes. In the range of 0.2-4 mL, the specific magnetic beads coated with the CP1-1 / CP1-2 / CP1-3 combination probes can extract influenza A samples of the same concentration but different sample volumes, and the extraction results have a good linear relationship.

[0081] Table 4 Virus content detected by fluorescence qPCR

[0082] Sample volume mL 0.2 0.4 1 2 4 Ct value 35.91 34.89 33.02 31.69 30.76

[0083] Embodiment 5:

[0084] 1. Add 200 μL (containing 10 5 / 10 4 / 10 3 / 10 2 The throat swab samples containing 100 copies / mL influenza A virus were then added with 200 μL of lysis solution, mixed and heated at 95°C for 5 minutes, and then naturally cooled to room temperature. 50 μg of magnetic beads coated with specific capture probes (CP2-1) were added to the lysed blood samples, and then incubated at room temperature for 15 minutes to allow the influenza A virus target nucleic acid in the sample to fully bind to the magnetic beads.

[0085] 2. Place the volume tube on the magnetic rack, discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, centrifuge instantly; place it on the magnetic rack, discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge instantly, and magnetically separate and remove the supernatant;

[0086] 3. Add all the magnetic beads cleaned in step 2 to the amplification tube for PCR amplification, and detect the viral nucleic acid RNA content, i.e., the viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCRProbe Kit.

[0087] Lysis buffer: 5M guanidine hydrochloride, 1M guanidine isothiocyanate, 2% SDS, 20mM cysteine, 50mM Tris-HCl, 25mM EDTA;

[0088] Washing solution: 200mM Tris-HCl buffer, 100mM NaCl, 15mM EDTA.

[0089] Comparative Example 1:

[0090] Compared with Example 5, most of the contents are the same, except that RNA is extracted by centrifugal adsorption column, Qiagen-52904 kit QIAamp Viral RNA Mini Kit (Cat. No.: 52904) is used to extract influenza A virus RNA in throat swab samples, RNA is used as an amplification template, and the PCR amplification method exactly the same as that in Example 5 is used for detection.

[0091] Comparative Example 2:

[0092] Compared with Example 5, most of them are the same, the difference is that the magnetic bead virus RNA extraction kit is used to extract RNA, the Shengxiang nucleic acid extraction or purification kit S10015 (pre-packaged 32-T) is selected, and the nucleic acid extractor Natch-32A is used to extract influenza A virus RNA in the throat swab sample, and RNA is used as the amplification template, and the PCR amplification method exactly the same as in Example 5 is used for detection.

[0093] Comparative Example 3:

[0094] Compared with Example 5, most of them are the same, the difference is that the RNA is extracted by using a magnetic bead method virus RNA extraction kit, and the Daan Gene nucleic acid extraction or purification reagent (magnetic bead method) DA0623 pre-packaged 32T is selected, and it is matched with the Tianlong fully automatic nucleic acid extractor NP068-C to extract influenza A virus RNA from the throat swab sample, and RNA is used as an amplification template, and the PCR amplification method exactly the same as in Example 5 is used for detection.

[0095] The results of fluorescent qPCR detection of virus content in Example 5 and Comparative Examples 1 to 3 are shown in Table 5:

[0096] Table 5 Virus content detected by fluorescence qPCR

[0097]

[0098] As shown in Table 5, after all the magnetic beads bound to influenza A virus were loaded for PCR, the Ct value of the present invention was further reduced than that of the partial supernatant PCR after thermal elution, and was significantly better than other commercial extraction kits, with a lower detection limit.

[0099] Embodiment 6:

[0100] 1. Add 1 mL of 10 4 / 10 3 / 10 2 / 10 copies / mL nasal swab samples of influenza A virus were added with 1.5 mL of lysis solution, mixed and heated at 65 ° C for 10 minutes, and then naturally cooled to room temperature. 300 μg of magnetic beads coated with specific capture probes (CP2-1, CP2-2, CP2-3 combination probes) were added to the lysed nasal swab samples, and then incubated at room temperature for 15 minutes to allow the influenza A virus target nucleic acid in the sample to fully bind to the magnetic beads.

[0101] 2. Place the volume tube on the magnetic rack, discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, centrifuge instantly; place it on the magnetic rack, discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge instantly, and magnetically separate and remove the supernatant;

[0102] 3. Add all the magnetic beads cleaned in step 2 to the amplification tube for PCR amplification, and detect the viral nucleic acid RNA content, i.e., the viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCRProbe Kit.

[0103] Lysis buffer: 2 M guanidine hydrochloride, 4 M guanidine isothiocyanate, 15% Triton X-114, 50 mM dithiothreitol, 150 mM Tris-HCl, 50 mM EDTA;

[0104] Washing solution: 30mM Tris-HCl buffer, 50mM NaCl, 25mM EDTA.

[0105] The results of amplification of influenza A virus nucleic acid RNA are as follows Figure 2 As shown, the method of this embodiment is used to load all viral nucleic acid-bound magnetic beads for qPCR, and a viral nucleic acid concentration effect similar to that of Example 5 is achieved;

[0106] Compared with Example 2, the sample volume was increased from 200 μL to 1 mL. This example can detect influenza A virus nucleic acid at a lower concentration (10 copies / mL).

[0107] Embodiment 7:

[0108] 1. Add 200 μL of 3×10 4 300 μL of lysis buffer was added to the clinical throat swab samples containing 100 copies / mL influenza A virus, mixed and heated at 65°C for 10 minutes, and then naturally cooled to room temperature. 100 μg of magnetic beads coated with specific capture probes (CP2-3) were added to the lysed throat swab samples, and then incubated at room temperature for 15 minutes to allow the influenza A virus target nucleic acid in the sample to fully bind to the magnetic beads.

[0109] 2. Place the volume tube on the magnetic rack, discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, centrifuge instantly; place it on the magnetic rack, discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge instantly, and magnetically separate and remove the supernatant;

[0110] 3. Add all the magnetic beads cleaned in step 2 to the amplification tube for PCR amplification, and detect the viral nucleic acid RNA content, i.e., the viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCRProbe Kit.

[0111] Lysis buffer: 6 M guanidine hydrochloride, 35% Tween-20, 2 mM TCEP, 200 mM Tris-HCl, 2 mM EDTA;

[0112] Washing solution: 20mM Tris-HCl buffer, 300mM NaCl, 10mM EDTA.

[0113] Comparative Example 4:

[0114] Compared with Example 7, most of them are the same, except that RNA is extracted by centrifugal adsorption column, the kit QIAamp Viral RNA Mini Kit (Cat. No.: 52904) is used to extract influenza A virus RNA in clinical throat swab samples, and the PCR amplification method exactly the same as Example 7 is used for amplification detection.

[0115] The results of the fluorescent qPCR detection of virus content in Example 7 and Comparative Example 4 are shown in Table 6:

[0116] Table 6 Virus content detected by fluorescence qPCR

[0117] Sample No. Example 7 Comparative Example 4 Sample No. Example 7 Comparative Example 4 1 32.75 35.94 11 29.11 31.75 2 21.14 23.75 12 26.15 28.63 3 32.16 34.57 13 35.02 / 4 26.65 28.52 14 22.49 25.22 5 19.62 22.03 15 27.22 29.21 6 33.59 36.45 16 28.47 31.90 7 31.33 34.98 17 24.41 27.54 8 20.35 22.78 18 34.78 / 9 32.58 35.82 19 23.61 26.58 10 25.41 27.84 20 25.08 27.56

[0118] From the results in Table 6, it can be seen that the method of Example 7 has a better extraction effect than the Qiagen separation column in clinical throat swab samples, two false negative samples were detected, and the method of the present invention is more suitable for subsequent automated extraction than the column method.

[0119] Embodiment 8:

[0120] 1. Add 200 μL of 3×10 4300 μL of lysis buffer was added to the pharyngeal swab samples of influenza A virus subtypes H1N1, H3N2, H5N1, and H7N9, mixed and heated at 65°C for 10 minutes, and then naturally cooled to room temperature. 10 μg of magnetic beads coated with specific capture probes (CP1-1, CP2-2, CP3-3) were added to the lysed blood samples, and then incubated at room temperature for 15 minutes to allow the influenza A virus subtype target nucleic acid in the sample to fully bind to the magnetic beads.

[0121] 2. Place the volume tube on the magnetic rack, discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, centrifuge instantly; place it on the magnetic rack, discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge instantly, and magnetically separate and remove the supernatant;

[0122] 3. Add all the magnetic beads cleaned in step 2 to the amplification tube for PCR amplification, and detect the viral nucleic acid RNA content, i.e., the viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCRProbe Kit.

[0123] Lysis buffer: 2M guanidine thiocyanate, 20% Triton X-114, 500mM ammonium thioglycolate, 10mM Tris-HCl, 100mM EDTA;

[0124] Washing solution: 450mM Tris-HCl buffer, 500mM NaCl, 2mM EDTA.

[0125] The results of the fluorescent qPCR detection of the virus content of each subtype of influenza A are shown in Table 7:

[0126] Table 7 Virus content detected by fluorescence qPCR

[0127]

[0128] As can be seen from Table 7, the capture probe used in the present invention can specifically bind to influenza A virus nucleic acid and is suitable for the extraction and diagnosis of various common subtypes.

[0129] Comparative Example 5:

[0130] 1. Add 200 μL of 1×10 5100 μg of magnetic beads coated with specific capture probes (CP1-1, CP1-2, CP1-3, CP2-1, CP2-2, CP2-3, CP3-1, CP3-2, CP3-3) were added to the lysed blood samples, and then incubated at room temperature for 15 minutes to allow the target nucleic acid of the new coronavirus in the sample to fully bind to the magnetic beads.

[0131] 2. Place the volume tube on the magnetic rack, discard the supernatant after magnetic separation; add 300 μL of cleaning solution, shake and mix, centrifuge instantly; place it on the magnetic rack, discard the supernatant after magnetic separation; then add 150 μL of cleaning solution, shake and mix, centrifuge instantly, and magnetically separate and remove the supernatant;

[0132] 3. Add all the magnetic beads cleaned in step 2 to the amplification tube for PCR amplification, and detect the viral nucleic acid RNA content, i.e., the viral content, by fluorescent qPCR. The reaction kit is V C58P2 Multiplex One Step RT-qPCRProbe Kit.

[0133] No qPCR signal was detected in each group, indicating that the capture probe of the present invention has good specificity and the detection result of influenza A virus is not interfered by other nucleic acids.

[0134] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A specific extraction reagent for viral nucleic acid RNA, characterized in that: It includes magnetic microspheres containing γFe2O3 magnetic particles or Fe3O4 magnetic particles, and a capture probe targeting influenza A virus nucleic acid RNA; The surface of the magnetic microsphere is modified with a linking group connected to the end of the capture probe, and the linking group is selected from any one of streptavidin, -NH2, -SH, -N3, -COOH, -C=C, alkynyl, p-toluenesulfonyl, epoxy, biotin and derivative groups thereof; The capture probe comprises a specific oligonucleotide chain targeting influenza A virus nucleic acid RNA, and the specific oligonucleotide chain can specifically bind to the target influenza A virus nucleic acid RNA through complementary base pairing.

2. A specific extraction reagent for viral nucleic acid RNA according to claim 1, characterized in that: The specific oligonucleotide chain is selected from any one or more of CP1-1, CP1-2, CP1-3, CP2-1, CP2-2, CP2-3, CP3-1, CP3-2, and CP3-3 whose base sequences are shown in SEQ ID NO.1-9.

3. A specific extraction reagent for viral nucleic acid RNA according to claim 1, characterized in that: The particle size of the magnetic microspheres is 0.2-10 μm, and the binding capacity of the magnetic microspheres is at least 100 pmol / mg.

4. A method for extracting and detecting viral nucleic acid RNA, characterized in that: Using the specific extraction reagent for viral nucleic acid RNA according to any one of claims 1 to 3, comprising the following steps: S1. Add lysis solution to the sample to be tested to react and obtain a lysis mixture for lysing viral nucleic acid in the sample; S2, mixing the capture probe with the magnetic microspheres to obtain magnetic microspheres coated with the capture probe; S3, mixing the lysis mixture in step S1 with the magnetic microspheres coated with capture probes in step S2 to allow the capture probes to hybridize with the viral nucleic acid in the lysis mixture; S4, magnetically separate the magnetic microspheres and the lysis mixture in step S3, and discard the supernatant; add an eluent to elute the magnetic microspheres, and then use the eluent to perform fluorescence quantitative PCR to detect the viral nucleic acid RNA content, or directly perform fluorescence quantitative PCR on the magnetic microspheres to detect the viral nucleic acid RNA content.

5. The extraction and detection method according to claim 4, characterized in that: In step S1, the volume ratio of the sample to be tested to the lysis solution is 1:0.5-2.

5.

6. The extraction and detection method according to claim 4, characterized in that: In step S1, the reaction temperature is 50°C-95°C, the reaction time is 5-30 minutes, and then naturally cooled to room temperature.

7. The extraction and detection method according to claim 4, characterized in that: In step S3, the ratio between the amount of magnetic microspheres coated with capture probes and the sample to be tested is (1-50) μg: 100 μL.

8. The extraction and detection method according to claim 4, characterized in that: In step S3, the reaction temperature is room temperature and the reaction time is 10-30 min.

9. The extraction and detection method according to claim 4, characterized in that: In step S4, the volume ratio of the added amount of the eluent to the sample to be tested is (1-10): (20-100), and the elution conditions are: heating at 70° C.-95° C. and elution time of 5-30 min.

10. A use of a specific extraction reagent for viral nucleic acid RNA as claimed in any one of claims 1 to 3, characterized in that: The specific extraction reagent of viral nucleic acid RNA is used to prepare a kit for extracting viral nucleic acid RNA. The kit comprises the specific extraction reagent of viral nucleic acid RNA, a lysing solution, a cleaning solution and an eluting solution.

Images