Freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection

Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection. Positive control; performing negative control; the reaction freeze-dried powder comprises an optimized buffer system, Taq DNA polymerase, reverse transcriptase, dNTP / dUTP Mix, a freeze-drying protective agent, a specific primer and a probe; the specific primer and the probe comprise an oligonucleotide upstream primer for specifically amplifying an M gene conserved region on an influenza A virus genome; by preparing reaction freeze-dried powder, an oligonucleotide primer, a probe, Taq DNA polymerase, reverse transcriptase, dNTP, an optimized buffer system, a freeze-drying protective additive and the like are prepared into components which stably coexist at normal temperature, the detection performance is consistent with that of a liquid reagent, normal-temperature transportation and storage can be realized, the transportation and storage cost of the fluorescent PCR reagent is greatly reduced, the shelf life is prolonged, and the fluorescent PCR reagent is suitable for industrial production. And operation steps are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, in particular to a freeze-dried PCR reagent and a kit for respiratory virus nucleic acid detection. Background Art

[0002] Respiratory viral infections are one of the most common clinical diseases, which seriously affect human health and quality of life, especially infants and the elderly with low immunity. It has been proven that 95% of upper respiratory tract infections and most lower respiratory tract infections are caused by pathogens other than bacteria, among which respiratory viruses are the most common, including influenza A virus, influenza B virus and respiratory syncytial virus.

[0003] The current respiratory virus diagnosis methods mainly include virus isolation and culture, serological diagnosis and molecular diagnosis. Virus isolation and culture is the "gold standard" for respiratory virus detection, but this method has high requirements for specimen collection, transportation and storage conditions, and is complex and time-consuming to operate, and is not suitable for early diagnosis; although serological diagnosis is simple to operate, it has low sensitivity, is prone to false positives and false negatives, and is often not used for early diagnosis of the disease.

[0004] The Chinese Influenza Diagnosis and Treatment Guidelines recommend the use of nucleic acid detection methodology for the diagnosis of respiratory viruses. Respiratory virus antigen and nucleic acid detection can be used for early diagnosis, and viral antibody detection can be used for retrospective studies, but is not suitable for early diagnosis of the disease. The multiplex fluorescent RT-PCR method uses specific conserved sequences of respiratory viruses as detection targets and conducts in vitro qualitative detection of respiratory virus nucleic acids from human samples. It can accurately, sensitively and quickly detect viral infections. This method is simple to operate and can obtain results in just one step. It is simple, fast and accurate.

[0005] In the prior art, the reagent kits for detecting respiratory virus nucleic acid by multiplex fluorescent RT-PCR are all liquid reagents, containing temperature-sensitive Taq enzyme, primers, probes and other components, which need to be stored below -20°C to maintain detection activity. The increase in temperature and repeated freezing and thawing during storage, transportation and use will reduce the detection performance of the reagents. The configuration is cumbersome during use, and the cost of product storage and transportation is high. In addition, the detection time is long, which is not conducive to quickly obtaining test results and delays the time for diagnosis and treatment.

[0006] To this end, the present invention provides a freeze-dried PCR reagent and a kit for respiratory virus nucleic acid detection. Summary of the invention

[0007] In order to make up for the deficiencies of the prior art, at least one technical problem raised in the background technology is solved.

[0008] The technical solution adopted by the present invention to solve the technical problem is: a freeze-dried PCR reagent for respiratory virus nucleic acid detection described in the present invention comprises:

[0009] Reaction lyophilized powder;

[0010] Positive control;

[0011] Negative control;

[0012] The reaction lyophilized powder includes an optimized buffer system, Taq DNA polymerase, reverse transcriptase, dNTP / dUTPMix, a lyophilization protectant, and specific primers and probes;

[0013] The specific primers and probes include oligonucleotide upstream primers for specifically amplifying the conserved region of the M gene on the influenza A virus genome, the sequences of which are SEQ ID NOs: 1-2; oligonucleotide downstream primers, the sequences of which are SEQ ID NOs: 3-4; and oligonucleotide fluorescent probes, the sequences of which are SEQ ID NOs: 5;

[0014] An oligonucleotide upstream primer for specifically amplifying the conserved region of the NS gene on the influenza B virus genome, the sequence of which is SEQ ID NO: 6; an oligonucleotide downstream primer, the sequence of which is SEQ ID NO: 7; and an oligonucleotide fluorescent probe, the sequence of which is SEQ ID NO: 8;

[0015] An oligonucleotide upstream primer for specifically amplifying the conserved region of the M gene on the respiratory syncytial virus genome, the sequence of which is SEQ ID NO: 9; an oligonucleotide downstream primer, the sequence of which is SEQ ID NO: 10-11; an oligonucleotide fluorescent probe, the sequence of which is SEQ ID NO: 12;

[0016] The oligonucleotide upstream primer for specifically amplifying the human ribonuclease gene has a sequence of SEQ ID NO: 13; the oligonucleotide downstream primer has a sequence of SEQ ID NO: 14; and the oligonucleotide fluorescent probe has a sequence of SEQ ID NO: 15.

[0017] Preferably, the two ends of the oligonucleotide fluorescent probe are respectively labeled with a fluorescent group and a quenching group, the fluorescent group is one of FAM, VIC, CY5, ROX or other fluorescent groups with luminescent properties, and the quenching group is one of BHQ1, BHQ2, BHQ3, TAMRA, MGB or other chemical groups with fluorescence quenching function.

[0018] Preferably, each tube of the reaction lyophilized powder comprises: 0.1 μM to 1 μM oligonucleotide upstream primer; 0.1 μM to 1 μM oligonucleotide downstream primer; 0.1 μM to 1 μM oligonucleotide fluorescent probe; 0.1 to 1 unit Taq DNA polymerase; m / v 1% to 10% lyophilization protectant; 0.1 to 1 mM dNTP and optimized 1 to 10× buffer system.

[0019] Preferably, the Taq DNA polymerase is a mutant Taq DNA polymerase, and the Taq DNA polymerase is one of chemically modified, antibody modified, and ligand modified.

[0020] Preferably, the reverse transcriptase is a mutant reverse transcriptase.

[0021] Preferably, the lyoprotectant is a mixture of one or more of pullulan, glycine, HPMC-E5, trehalose, PEG-20000, sucrose, maltodextrin, gelatin, CMC-Na, PVP-10000, etc.

[0022] Preferably, the reaction lyophilized powder is prepared by the following lyophilization procedure: 4°C, 20 min; -45°C, 5 h; -40°C, 14 bar, 5 h; -20°C, 14 bar, 4 h; 0°C, 14 bar, 2 h; 10°C, 14 bar, 2 h; 30°C, 14 bar, 2 h.

[0023] Preferably, the positive control is a pseudovirus containing a specific fragment of influenza A virus M gene, a pseudovirus containing a specific fragment of influenza B virus NS gene, a pseudovirus containing a specific fragment of respiratory syncytial virus M gene, and a pseudovirus containing a specific fragment of human RNaseP gene, Tris buffer, EDTA, Triton X-100, and gelatin; the negative control is a pseudovirus containing a specific fragment of human RNaseP gene, Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudovirus is 10 5 copies / mL.

[0024] Preferably, the fluorescent quantitative PCR program is set as follows: reverse transcription at 55°C for 30s, pre-denaturation at 95°C for 10s, pre-amplification for 12 cycles, amplification for 30 cycles, FluA detection in the FAM channel, FluB detection in the ROX channel, RSV detection in the CY5 channel, and human RNaseP detection in the VIC channel.

[0025] Preferably, a kit for respiratory virus nucleic acid detection is provided, wherein the kit is used for reacting lyophilized powder, positive control and negative control, and the kit is provided with multiple components for the entire detection process of sample collection, nucleic acid extraction and amplification detection.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection described in the present invention prepares reaction freeze-dried powder, and makes oligonucleotide primers and probes, Taq DNA polymerase, reverse transcriptase, dNTP, optimized buffer system, freeze-dried protective agent, etc. into components that can coexist stably at room temperature. The detection performance is consistent with that of liquid reagents, and can be transported and stored at room temperature. The transportation and storage costs of fluorescent PCR reagents are greatly reduced, the shelf life is extended, the use operation steps are reduced, the clinical detection process is simplified, and it is more convenient for practical use and promotion.

[0028] 2. The freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection described in the present invention use a mutant reverse transcriptase, which has a high enzyme activity at 50°C to 60°C and can complete reverse transcription in a very short time. At the same time, the mutant antibody-modified Taq DNA polymerase can quickly release enzyme activity at 95°C, and with a shorter amplicon, the detection time can be greatly shortened, and the detection can be completed in about 30 minutes.

[0029] 3. A freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection according to the present invention can be used for in vitro qualitative detection of influenza A virus, influenza B virus and respiratory syncytial virus nucleic acid in human throat swab samples through the prepared kit, and can distinguish respiratory pathogens from pathogens, and can guide clinical medication and treatment, with a detection sensitivity of 3 copies / reaction and strong specificity. At the same time, the conservative region sequence of the RNaseP gene on the human genome is used as an internal standard, and the entire detection process including sampling, nucleic acid extraction and amplification detection can be monitored throughout the whole process to avoid false negative results due to sampling, extraction or amplification failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 is a schematic diagram of a reaction freeze-dried powder in one embodiment of the present invention;

[0032] Figure 2 is a comparison chart of the detection performance of the reaction lyophilized powder and the liquid control in one embodiment of the present invention;

[0033] Figure 3 is a schematic diagram of a clinical sample test result in one embodiment of the present invention;

[0034] Figure 4 is a schematic diagram of the stability test results of a kit in one embodiment of the present invention; DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0036] like Figures 1 to 4 As shown, a freeze-dried PCR reagent for respiratory virus nucleic acid detection described in an embodiment of the present invention includes: a reaction freeze-dried powder; a positive control; a negative control; the reaction freeze-dried powder includes an optimized buffer system, TaqDNA polymerase, reverse transcriptase, dNTP / dUTP Mix, a freeze-dried protective agent, and specific primers and probes; the specific primers and probes include oligonucleotide upstream primers for specifically amplifying the conserved region of the M gene on the influenza A virus genome, the sequence of which is SEQ ID NO: 1-2; oligonucleotide downstream primers, the sequence of which is SEQ ID NO: 3-4; oligonucleotide fluorescent probes, the sequence of which is SEQ ID NO: 5; oligonucleotide upstream primers for specifically amplifying the conserved region of the NS gene on the influenza B virus genome, the sequence of which is SEQ ID NO: 6; oligonucleotide downstream primers, the sequence of which is SEQ ID NO: 7; oligonucleotide fluorescent probes, the sequence of which is SEQ ID NO: 8; oligonucleotide upstream primers for specifically amplifying the conserved region of the M gene on the respiratory syncytial virus genome, the sequence of which is SEQ ID NO: 9; oligonucleotide downstream primers, the sequence of which is SEQ ID NO: 10-11; oligonucleotide fluorescent probe, the sequence is SEQ ID NO: 12; oligonucleotide upstream primer for specific amplification of human ribonuclease gene, the sequence is SEQ ID NO: 13; oligonucleotide downstream primer, the sequence is SEQ ID NO: 14; oligonucleotide fluorescent probe, the sequence is SEQ ID NO: 15.

[0037] The reaction freeze-dried powder provided by the present invention is a component that can stably coexist at room temperature by making oligonucleotide primers and probes, Taq DNA polymerase, reverse transcriptase, dNTP, optimized buffer system, freeze-drying protective agent, etc., and the detection performance is consistent with that of liquid reagents, and can be transported at room temperature. It greatly reduces the transportation and storage costs of fluorescent PCR reagents, extends the shelf life, reduces the use of operating steps, simplifies the clinical detection process, and is more convenient for practical use and promotion.

[0038] Furthermore, the two ends of the oligonucleotide fluorescent probe are respectively labeled with a fluorescent group and a quenching group, the fluorescent group is one of FAM, VIC, CY5, ROX or other fluorescent groups with luminescent properties, and the quenching group is one of BHQ1, BHQ2, BHQ3, TAMRA, MGB or other chemical groups with fluorescence quenching function.

[0039] Furthermore, each tube of the reaction lyophilized powder includes: 0.4 μM oligonucleotide upstream primer, 0.4 μM oligonucleotide downstream primer, 0.2 μM oligonucleotide fluorescent probe, 0.5 unit Taq DNA polymerase, 0.5 unit reverse transcriptase, m / v 2% lyophilization protectant, 0.8 mM dNTP and optimized 5× buffer system.

[0040] Furthermore, the Taq DNA polymerase is a mutant antibody-modified Taq DNA polymerase.

[0041] Furthermore, the reverse transcriptase is a mutant reverse transcriptase.

[0042] The present invention uses a mutant reverse transcriptase, which has a high enzyme activity at 50°C to 60°C and can complete reverse transcription in a very short time. At the same time, the mutant antibody-modified Taq DNA polymerase can quickly release enzyme activity at 95°C, and with a shorter amplicon, the detection time can be greatly shortened, and the detection can be completed in about 30 minutes.

[0043] Furthermore, the freeze-drying protective agent is 2% trehalose, 2% gelatin, and 2% maltodextrin.

[0044] Furthermore, the reaction freeze-dried powder is prepared by the following freeze-drying procedure: 4°C, 20 min; -45°C, 5 h; -40°C, 14 bar, 5 h; -20°C, 14 bar, 4 h; 0°C, 14 bar, 2 h; 10°C, 14 bar, 2 h; 30°C, 14 bar, 2 h.

[0045] Furthermore, the positive control is a pseudovirus containing a specific fragment of influenza A virus M gene, a pseudovirus containing a specific fragment of influenza B virus NS gene, a pseudovirus containing a specific fragment of respiratory syncytial virus M gene, and a pseudovirus containing a specific fragment of human RNaseP gene, Tris buffer, EDTA, Triton X-100, and gelatin; the negative control is a pseudovirus containing a specific fragment of human RNaseP gene, Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudovirus is 10 5 copies / mL.

[0046] Furthermore, the fluorescence quantitative PCR program was set as follows: reverse transcription at 55°C for 30s, pre-denaturation at 95°C for 10s, pre-amplification for 12 cycles, amplification for 30 cycles, FluA was detected in the FAM channel, FluB was detected in the ROX channel, RSV was detected in the CY5 channel, and human RNaseP was detected in the VIC channel.

[0047] Furthermore, a kit for respiratory virus nucleic acid detection is provided, wherein the kit is used for reacting lyophilized powder, positive control and negative control, and the kit is provided with multiple components for the entire detection process of sample collection, nucleic acid extraction and amplification detection.

[0048] The kit provided by the present invention is used for in vitro qualitative detection of influenza A virus, influenza B virus and respiratory syncytial virus nucleic acid in human throat swab samples, can distinguish respiratory pathogens, can guide clinical medication and treatment, and has a detection sensitivity of 3 copies / reaction and strong specificity. At the same time, the conservative region sequence of the RNaseP gene on the human genome is used as an internal standard, and the entire detection process including sampling, nucleic acid extraction and amplification detection can be fully monitored to avoid false negative results caused by sampling, extraction or amplification failure.

[0049] The embodiments of the present invention will be described in detail below with reference to specific examples;

[0050] Example 1: Design of primers and probes for target and internal standard;

[0051] Search the influenza A virus M gene sequence, influenza B virus NS gene sequence, respiratory syncytial virus M gene sequence, and human RNaseP gene sequence in the past 10 years on NCBI, and design specific primers and probe sequences according to the primer and probe design principles. After sequence synthesis, quality inspection and quantification are performed, and then diluted to the required concentration as needed. The primer and probe sequences of the target and internal standard used in the present invention are shown in the table below.

[0052]

[0053] Example 2: Preparation of freeze-dried PCR reagents and kits for respiratory virus nucleic acid detection; (1) Reaction freeze-dried powder

[0054] The reaction lyophilized powder was prepared as follows:

[0055] Components Amount per person (μL) Final concentration FluA-F1 (100 μM) 0.1 0.4μM FluA-F2 (100 μM) 0.1 0.4μM FluA-R1 (100 μM) 0.1 0.4μM FluA-R1 (100 μM) 0.1 0.4μM FluA-P (100 μM) 0.05 0.2μM FluB-F (100 μM) 0.1 0.4μM FluB-R (100 μM) 0.1 0.4μM FluB-P (100 μM) 0.05 0.2μM RSV-F (100 μM) 0.1 0.4μM RSV-R1 (100 μM) 0.1 0.4μM RSV-R2 (100 μM) 0.1 0.4μM RSV-P (100 μM) 0.05 0.2μM RP-F (100 μM) 0.1 0.4μM RP-R (100 μM) 0.1 0.4μM RP-P (100 μM) 0.05 0.2μM Lyophilized Protectant Stock Solution (20%) 2.5 2％ TaqDNA polymerase (5 units) 2.5 0.5 units Reverse transcriptase (200 units) 0.0625 0.5 units dNTP(10mM) 2 0.8mM Optimized 5× buffer system 5 1× ddH2O 11.6375 / Total 25

[0056] After mixing the above components evenly, a reaction lyophilized powder solution with a final reaction volume of 25 μL is prepared, and the required volume of the mixed solution 25 μL×N is prepared according to the needs. After mixing evenly, it is divided into 8 strips of PCR tubes and placed in a freeze dryer without covering the tube caps to prepare MP reaction lyophilized powder. The following procedure is followed: 4°C, 20 min; -45°C, 5 h; -40°C, 14 bar, 5 h; -20°C, 14 bar, 4 h; 0°C, 14 bar, 2 h; 10°C, 14 bar, 2 h; 30°C, 14 bar, 2 h; after the freeze-drying is completed, the PCR tube cap is covered, and the reaction lyophilized powder is obtained by vacuum packaging with an aluminum foil bag with a desiccant inside.

[0057] (2) Positive control: pseudovirus containing influenza A virus M gene-specific fragment, pseudovirus containing influenza B virus NS gene-specific fragment, pseudovirus containing respiratory syncytial virus M gene-specific fragment, and pseudovirus containing human RNaseP gene-specific fragment, Tris buffer, EDTA, TritonX-100, gelatin, pseudovirus concentration of 10 5 copies / mL.

[0058] (3) Negative control: pseudovirus containing human RNaseP gene-specific fragment, Tris buffer, EDTA, TritonX-100, gelatin, pseudovirus concentration of 10 5 copies / mL.

[0059] The components of the kit (48 servings / box) are as follows:

[0060]

[0061] Example 3: Detection process of freeze-dried PCR reagents and kits for respiratory virus nucleic acid detection;

[0062] (1) Sample processing: Take 200 μL of the sample to be tested, positive control, and negative control, and perform nucleic acid extraction according to the instructions;

[0063] (2) Sample addition:

[0064] ① Preparation of reaction solution: Calculate the required number of reactions according to the number of samples to be tested. If the number of samples is n, the total number of reactions is N = n + 2. Take N tubes of reaction lyophilized powder and centrifuge them for 30 seconds to ensure that the reaction lyophilized powder is at the bottom of the tube and set aside.

[0065] ② Sample addition: Gently open the cap of the eight-tube containing the lyophilized powder, transfer 25 μL of the extracted sample, positive control and negative control nucleic acid into the eight-tube, and close the cap tightly;

[0066] ③ Vortex for 30 seconds until completely dissolved and the solution is clear with no white lyophilized residue remaining, remove bubbles and centrifuge for 30 seconds for later use.

[0067] (3) PCR amplification: The present invention uses Applied Biosystems QuantStudioTM 5 real-time fluorescence quantitative PCR instrument for detection. In Fast mode, reverse transcription is performed at 55°C for 30 s, pre-denaturation is performed at 95°C for 10 s, pre-amplification is performed for 12 cycles (denaturation at 95°C for 1 s, annealing and extension at 58°C for 3 s), and amplification is performed for 30 cycles (denaturation at 95°C for 1 s, annealing and extension at 58°C for 13 s to collect fluorescence); FluA is detected in the FAM channel, FluB is detected in the ROX channel, RSV is detected in the CY5 channel, and human RNaseP is detected in the VIC channel.

[0068] (4) Result analysis:

[0069] ①Quality control standards:

[0070] Positive control: The Ct value of the corresponding target of FAM, ROX, VIC and CY5 channels should be ≤25;

[0071] Negative control: 5.2FAM, ROX and CY5 channels have no amplification curve or Ct value>29, and the Ct value of the corresponding target in the VIC channel should be ≤25;

[0072] The above requirements must be met simultaneously in the same experiment, otherwise the experiment will be invalid and must be retested.

[0073] ②Result determination

[0074]

[0075] Note: For samples with positive test results for FluA, FluB, and RSV, there is no requirement for the internal standard test results. For negative samples, the internal standard test should be positive. If the internal standard test is negative, the test result of the sample is invalid. The cause should be found and eliminated, and the sample should be retested and the experiment repeated.

[0076] Example 4: Comparison of the detection performance of freeze-dried PCR reagents and liquid PCR reagents;

[0077] The freeze-dried PCR reagent used in this example is the freeze-dried PCR reagent for respiratory virus nucleic acid detection prepared in Example 2, and the control reagent is a liquid PCR reagent (consistent with the freeze-dried PCR reagent formula).

[0078] Lyophilized PCR reagent: lyophilized reaction powder + 10μL sample to be tested + 15μL ddH2O

[0079] Liquid amplification system: Prepare the liquid amplification system according to the table below, vortex to mix, and dispense 15 μL / well.

[0080] Components Amount per person (μL) Final concentration FluA-F1 (100 μM) 0.1 0.4μM FluA-F2 (100 μM) 0.1 0.4μM FluA-R1 (100 μM) 0.1 0.4μM FluA-R1 (100 μM) 0.1 0.4μM FluA-P (100 μM) 0.05 0.2μM FluB-F (100 μM) 0.1 0.4μM FluB-R (100 μM) 0.1 0.4μM FluB-P (100 μM) 0.05 0.2μM RSV-F (100 μM) 0.1 0.4μM RSV-R1 (100 μM) 0.1 0.4μM RSV-R2 (100 μM) 0.1 0.4μM RSV-P (100 μM) 0.05 0.2μM RP-F (100 μM) 0.1 0.4μM RP-R (100 μM) 0.1 0.4μM RP-P (100 μM) 0.05 0.2μM Lyophilized Protectant Stock Solution (20%) 2.5 2％ TaqDNA polymerase (5 units) 2.5 0.5 units Reverse transcriptase (200 units) 0.0625 0.5 units dNTP(10mM) 2 0.8mM Optimized 5× buffer system 5 1× ddH2O 1.6375 / template 10 / Total 25

[0081] The templates are the positive control and negative control prepared in Example 2 and the throat swab samples of normal persons.

[0082] The detection method is the same as in Example 3. The positive control and negative control meet the quality control standards, and this detection is valid.

[0083] Reaction freeze-dried powder Figure 1 As shown, the test results are Figure 2 As shown: the prepared freeze-dried PCR reagent for respiratory virus nucleic acid detection has basically the same detection performance as the liquid PCR reagent (consistent with the freeze-dried PCR reagent formula).

[0084] Example 5: Application of freeze-dried PCR reagents and kits for respiratory virus nucleic acid detection in clinical samples;

[0085] The reagents used were the freeze-dried PCR reagents and kits for respiratory virus nucleic acid detection prepared in Example 2, and the samples to be tested were 2 influenza A virus-positive throat swab samples, 2 influenza B virus-positive throat swab samples, 2 respiratory syncytial virus-positive throat swab samples, and 5 negative throat swab samples remaining from the hospital laboratory.

[0086] The detection method was the same as in Example 3. The positive control and negative control met the quality control standard, and the detection was effective. The detection results are shown in the following table: 2 samples were FluA positive (such as Figure 3 ); 2 samples were FluB positive (as shown); Figure 3 2 samples were RSV positive (as shown); Figure 3 5 samples were negative (as shown); Figure 3 The diagnosis was consistent with that of the hospital.

[0087]

[0088] Note: Undet means Undetermined.

[0089] Example 6: Detection limit test of freeze-dried PCR reagents and kits for respiratory virus nucleic acid detection;

[0090] The templates were influenza A virus / influenza B virus / respiratory syncytial virus cultures of 5 different concentrations, and 25 μL RNase-Free Water was used as a no-template control. All templates were replicated in 20 wells, and the detection method was the same as in Example 3;

[0091] The results are shown in the following table: The detection sensitivity of influenza A virus / influenza B virus / respiratory syncytial virus is 3 copies / reaction.

[0092] Respiratory Virus Detection Limit Test Results:

[0093]

[0094] Example 7: Stability test of freeze-dried PCR reagents and kits for respiratory virus nucleic acid detection;

[0095] The kit used in this example is the freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection prepared in Example 2. The kit was stored at room temperature for 12 months and compared with the newly prepared freeze-dried PCR kit (preparation method is the same as in Example 2). The samples to be tested are 1 influenza A virus positive throat swab sample, 1 influenza B virus positive throat swab sample, 1 respiratory syncytial virus positive throat swab sample and 1 negative throat swab sample remaining from the hospital laboratory. The detection method is the same as in Example 3.

[0096] The test results are as follows Figure 4 As shown: the amplification performance of the freeze-dried PCR kit stored at room temperature for 12 months is consistent with the amplification performance of the newly prepared freeze-dried PCR kit in the control group. Therefore, the freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection prepared in Example 2 can be stored at room temperature for 12 months.

[0097] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A freeze-dried PCR reagent for respiratory virus nucleic acid detection, comprising: Reaction lyophilized powder; Positive control; Negative control; The invention is characterized in that: the reaction lyophilized powder comprises an optimized buffer system, Taq DNA polymerase, reverse transcriptase, dNTP / dUTP Mix, a lyophilization protective agent, and specific primers and probes; The specific primers and probes include oligonucleotide upstream primers for specifically amplifying the conserved region of the M gene on the influenza A virus genome, the sequences of which are SEQ ID NOs: 1-2; oligonucleotide downstream primers, the sequences of which are SEQ ID NOs: 3-4; and oligonucleotide fluorescent probes, the sequences of which are SEQ ID NOs: 5; An oligonucleotide upstream primer for specifically amplifying the conserved region of the NS gene on the influenza B virus genome, the sequence of which is SEQ ID NO: 6; an oligonucleotide downstream primer, the sequence of which is SEQ ID NO: 7; and an oligonucleotide fluorescent probe, the sequence of which is SEQ ID NO: 8; An oligonucleotide upstream primer for specifically amplifying the conserved region of the M gene on the respiratory syncytial virus genome, the sequence of which is SEQ ID NO: 9; an oligonucleotide downstream primer, the sequence of which is SEQ ID NO: 10-11; an oligonucleotide fluorescent probe, the sequence of which is SEQ ID NO: 12; The oligonucleotide upstream primer for specifically amplifying the human ribonuclease gene has a sequence of SEQ ID NO: 13; the oligonucleotide downstream primer has a sequence of SEQ ID NO: 14; and the oligonucleotide fluorescent probe has a sequence of SEQ ID NO:

15.

2. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 1, characterized in that: The two ends of the oligonucleotide fluorescent probe are respectively labeled with a fluorescent group and a quenching group, wherein the fluorescent group is one of FAM, VIC, CY5, ROX or other fluorescent groups with luminescent properties, and the quenching group is one of BHQ1, BHQ2, BHQ3, TAMRA, MGB or other chemical groups with fluorescence quenching function.

3. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 2, characterized in that: Each tube of the reaction freeze-dried powder includes: 0.1μM-1μM oligonucleotide upstream primer; 0.1μM-1μM oligonucleotide downstream primer; 0.1μM-1μM oligonucleotide fluorescent probe; 0.1-1 unit Taq DNA polymerase; m / v 1%-10% freeze-dried protective agent; 0.1-1mM dNTP and optimized 1-10× buffer system.

4. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 3, characterized in that: The Taq DNA polymerase is a mutant Taq DNA polymerase, and the Taq DNA polymerase is one of chemically modified, antibody modified, and ligand modified.

5. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 4, characterized in that: The reverse transcriptase is a mutant reverse transcriptase.

6. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 5, characterized in that: The lyophilization protective agent is a mixture of one or more of pullulan, glycine, HPMC-E5, trehalose, PEG-20000, sucrose, maltodextrin, gelatin, CMC-Na, PVP-10000, etc.

7. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 6, characterized in that: The reaction lyophilized powder was prepared by the following lyophilization procedure: 4°C, 20 min; -45°C, 5 h; -40°C, 14 bar, 5 h; -20°C, 14 bar, 4 h; 0°C, 14 bar, 2 h; 10°C, 14 bar, 2 h; 30°C, 14 bar, 2 h.

8. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 7, characterized in that: The positive control is a pseudovirus containing a specific fragment of influenza A virus M gene, a pseudovirus containing a specific fragment of influenza B virus NS gene, a pseudovirus containing a specific fragment of respiratory syncytial virus M gene, and a pseudovirus containing a specific fragment of human RNaseP gene, Tris buffer, EDTA, Triton X-100, and gelatin; the negative control is a pseudovirus containing a specific fragment of human RNaseP gene, Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudovirus is 10 5 copies / mL.

9. A freeze-dried PCR reagent for respiratory virus nucleic acid detection according to claim 8, characterized in that: The fluorescence quantitative PCR program was set as follows: reverse transcription at 55°C for 30 s, pre-denaturation at 95°C for 10 s, pre-amplification for 12 cycles, amplification for 30 cycles, FluA was detected in the FAM channel, FluB was detected in the ROX channel, RSV was detected in the CY5 channel, and human RNaseP was detected in the VIC channel.

10. A kit for detecting nucleic acid of respiratory viruses, the kit being applicable to the freeze-dried PCR reagent for detecting nucleic acid of respiratory viruses according to any one of claims 1 to 9, characterized in that: The kit is used for reacting freeze-dried powder, positive control and negative control, and the kit is provided with multiple components for the entire detection process of sample collection, nucleic acid extraction and amplification detection.

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