Primer composition for detecting respiratory syncytial virus and application

Abstract

The invention relates to the technical field of respiratory syncytial virus detection, in particular to a primer composition for respiratory syncytial virus detection and application, the former comprises an outer primer pair, an inner primer pair and a loop primer pair, the outer primer pair comprises a respiratory syncytial virus F3 and a respiratory syncytial virus B3, and the inner primer pair comprises a loop primer pair. The inner primer pair comprises a respiratory syncytial virus FIP and a respiratory syncytial virus BIP, and the loop primer pair comprises a respiratory syncytial virus LF and a respiratory syncytial virus LB. According to the primer composition for detecting the respiratory syncytial virus and the application, the respiratory syncytial virus (RSV) can be rapidly, sensitively and specifically detected, the detection time is short, and the primer composition has the advantages of being rapid, efficient and easy and convenient to operate and is suitable for on-site rapid detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of respiratory syncytial virus detection, and is a primer composition for respiratory syncytial virus detection and application thereof. BACKGROUND

[0002] Respiratory infection is a common disease, mainly caused by viruses, bacteria, mycoplasma and chlamydia, among which respiratory syncytial virus (RSV) is a common virus in clinical practice. The symptoms of viral infection are similar to those of influenza, and it is difficult to determine the cause solely based on symptoms. Since accurate identification of pathogens is crucial for developing the correct treatment plan and controlling the epidemic, rapid and effective identification of respiratory pathogen types is of great significance for treatment and public health management.

[0003] Currently, there are various methods for detecting respiratory syncytial virus, and existing detection methods include: 1. Antigen detection, which utilizes the principle of specific binding between antigen and antibody to detect RSV antigen in the sample, but this method has relatively low sensitivity and may not be able to detect low viral load samples; 2. Nucleic acid detection, which detects RSV nucleic acid in the sample through molecular biology techniques (such as PCR, real-time fluorescent quantitative PCR), but this method requires professional equipment and operation techniques, and the detection cost is high; 3. Virus isolation culture, which inoculates the sample into a cell culture system and observes the growth of the virus, but this method is complex and time-consuming, and is not suitable for large-scale screening; 4. Immunofluorescence method, which uses fluorescently labeled antibodies to detect viral antigens in the sample, but this method requires high technical requirements for operators, and the sample needs to be fresh and contain sufficient cells.

[0004] Currently, RT-LAMP technology (reverse transcription loop-mediated isothermal amplification technology) is a highly efficient, rapid and specific nucleic acid detection method, especially suitable for the detection of respiratory viruses and other RNA pathogens. This method mainly combines reverse transcription and loop-mediated isothermal amplification technology to rapidly amplify RSV nucleic acid under constant temperature conditions. This method is simple and efficient, with a detection time of usually 30 minutes to 1 hour, and has high requirements for primer design.

[0005] The Chinese patent document with the publication number CN116103438A discloses a LAMP detection respiratory virus kit and primer composition. The LAMP detection respiratory virus kit includes a first detection system for detecting respiratory syncytial virus, the first detection system includes a first primer set for detecting respiratory syncytial virus type A and a second primer set for detecting respiratory syncytial virus type B; the first primer set includes the nucleotide sequences of the outer primer pair F3 and B3, the inner primer pair FIP and BIP, and the loop primer pair LF and LB, which are shown in SEQ ID NO. 1 to SEQ ID NO. 6 in order; the second primer set includes the nucleotide sequences of the outer primer pair F3 and B3, the inner primer pair FIP and BIP, and the loop primer LB, which are shown in SEQ ID NO. 7 to SEQ ID NO. 11 in order. The application can accurately detect two subtypes of respiratory syncytial virus A and B.

[0006] At present, there are still many research results on finding RSV detection based on RT-LAMP technology, which are concentrated in two stages. The first stage is concentrated around 2013, mainly to establish RT-LAMP technology to detect RSV; the second stage is mainly concentrated around 2019-2020, which is mainly based on LAMP to detect RSV alone, based on LAMP to detect RSV and other viruses, and the specificity of the detection primer is poor, and the time is long.

[0007] Therefore, the existing LAMP technology for detecting respiratory syncytial virus (RSV) has the problems of poor primer specificity and long detection time. SUMMARY

[0008] The present application provides a primer composition for respiratory syncytial virus detection and application, which overcomes the shortcomings of the prior art and effectively solves the problems of poor primer specificity and long detection time of the existing LAMP technology for detecting respiratory syncytial virus (RSV).

[0009] One of the technical solutions of the present application is achieved by the following measures: a primer composition for respiratory syncytial virus detection, including an outer primer pair, an inner primer pair and a loop primer pair, the outer primer pair includes respiratory syncytial virus F3 and respiratory syncytial virus B3, the inner primer pair includes respiratory syncytial virus FIP and respiratory syncytial virus BIP, and the loop primer pair includes respiratory syncytial virus LF and respiratory syncytial virus LB.

[0010] The following is a further optimization or / and improvement of the above-mentioned one of the technical solutions: The base sequence of the above-mentioned respiratory syncytial virus F3 is shown in SEQ ID NO: 1, and the base sequence of the respiratory syncytial virus B3 is shown in SEQ ID NO: 2.

[0011] The base sequence of the respiratory syncytial virus FIP is shown in SEQ ID NO: 3, and the base sequence of the respiratory syncytial virus BIP is shown in SEQ ID NO: 4.

[0012] The base sequence of the respiratory syncytial virus LF is shown in SEQ ID NO: 5, and the base sequence of the respiratory syncytial virus LB is shown in SEQ ID NO: 6.

[0013] The second technical solution of the present application is realized by the following measures: a primer composition for respiratory syncytial virus detection based on the application of the RT-LAMP method in detecting respiratory syncytial virus.

[0014] The following is a further optimization or / and improvement of the above-mentioned first technical solution of the application: In the above-mentioned RT-LAMP method, the molar concentration ratio of the inner primer pair, the outer primer pair and the loop primer pair in the primer composition is (0.4 to 2.0):0.2:(0.2 to 1.0), the enzyme dosage is 4U to 12U, the magnesium ion concentration is 4mM to 12mM, the dNTP concentration is 1.0mM to 1.8mM, the reaction temperature is 60℃ to 68℃, and the reaction time is 15min to 30min.

[0015] In the above-mentioned RT-LAMP method, the molar concentration ratio of the inner primer pair, the outer primer pair and the loop primer pair in the primer composition is 0.8:0.2:0.4, the enzyme dosage is 8U, the magnesium ion concentration is 8mM, the dNTP concentration is 1.4mM, the reaction temperature is 64.4℃, and the reaction time is 30min.

[0016] The above-mentioned respiratory syncytial virus is respiratory syncytial virus type A.

[0017] The present application provides a primer composition for respiratory syncytial virus detection and application, which can quickly, sensitively and specifically detect respiratory syncytial virus (RSV), has the advantages of short detection time, fast and efficient operation, and is suitable for on-site rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the amplification curve and turbidity chart of the primer composition 1 of the present application.

[0019] Figure 2 It is the amplification curve and turbidity chart of the primer composition 2 of the present application.

[0020] Figure 3 It is the amplification curve chart of the primer composition for respiratory syncytial virus detection of the present application under each system (different proportions of inner primer pair and outer primer).

[0021] Figure 4 The amplification curve diagram of each system (different proportions of loop primer pairs) in the primer composition for respiratory syncytial virus detection of the present application.

[0022] Figure 5 The amplification curve diagram of each system (different enzyme dosages) in the primer composition for respiratory syncytial virus detection of the present application.

[0023] Figure 6 The amplification curve diagram of each system (different magnesium ion concentrations) in the primer composition for respiratory syncytial virus detection of the present application.

[0024] Figure 7 The amplification curve diagram of each system (different dNTP concentrations) in the primer composition for respiratory syncytial virus detection of the present application.

[0025] Figure 8 The amplification curve diagram of each system (different reaction temperatures 60℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃) in the primer composition for respiratory syncytial virus detection of the present application.

[0026] Figure 9 The amplification curve diagram of each system (different reaction temperatures 65.4℃, 66.4℃, 67.2℃, 68℃) in the primer composition for respiratory syncytial virus detection of the present application.

[0027] Figure 10 The amplification curve diagram of each system (different reaction times) in the primer composition for respiratory syncytial virus detection of the present application.

[0028] Figure 11 The respiratory syncytial virus (RSV) specific detection diagram.

[0029] Figure 12 The respiratory syncytial virus (RSV) sensitivity detection item diagram.

[0030] Figure 13 The respiratory syncytial virus (RSV) sensitivity detection result diagram. DETAILED DESCRIPTION

[0031] The present application is not limited by the following examples, and the specific implementation can be determined according to the technical solution of the present application and the actual situation. The various chemical reagents and chemical supplies mentioned in the present application are well-known and commonly used chemical reagents and chemical supplies in the prior art unless otherwise specified; the percentages in the present application are mass percentages unless otherwise specified; the solutions in the present application are aqueous solutions with water as the solvent unless otherwise specified, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution; the normal temperature and room temperature in the present application generally refer to a temperature of 15℃ to 25℃, and is generally defined as 25℃.

[0032] The present invention will be further described below with reference to embodiments: Example 1: The primer composition for respiratory syncytial virus (RSV) detection includes an outer primer pair, an inner primer pair, and a circular primer pair. The outer primer pair includes RSV F3 and RSV B3, the inner primer pair includes RSV FIP and RSV BIP, and the circular primer pair includes RSV LF and RSV LB.

[0033] Example 2: As an optimization of the above example, the base sequence of respiratory syncytial virus F3 is shown in SEQ ID NO: 1, and the base sequence of respiratory syncytial virus B3 is shown in SEQ ID NO: 2.

[0034] Example 3: As an optimization of the above examples, the base sequence of respiratory syncytial virus FIP is shown in SEQ ID NO: 3, and the base sequence of respiratory syncytial virus BIP is shown in SEQ ID NO: 4.

[0035] Example 4: As an optimization of the above examples, the base sequence of respiratory syncytial virus LF is shown in SEQ ID NO: 5, and the base sequence of respiratory syncytial virus LB is shown in SEQ ID NO: 6.

[0036] Example 5: As an optimization of the above examples, the primer composition for respiratory syncytial virus detection is applied to the detection of respiratory syncytial virus based on the RT-LAMP method.

[0037] Example 6: As an optimization of the above examples, in the RT-LAMP method, the molar concentration ratio of the inner primer pair, outer primer pair and loop primer pair in the primer composition is (0.4 to 2.0):0.2:(0.2 to 1.0), the enzyme amount is 4 U to 12 U, the magnesium ion concentration is 4 mM to 12 mM, the dNTP concentration is 1.0 mM to 1.8 mM, the reaction temperature is 60°C to 68°C, and the reaction time is 15 min to 30 min.

[0038] Example 7: As an optimization of the above example, in the RT-LAMP method, the molar ratio of the inner primer pair, outer primer pair and loop primer pair in the primer composition is 0.8:0.2:0.4, the enzyme amount is 8U, the magnesium ion concentration is 8mM, the dNTP concentration is 1.4mM, the reaction temperature is 64.4℃, and the reaction time is 30min.

[0039] Example 8: As an optimization of the above examples, the respiratory syncytial virus is respiratory syncytial virus type A.

[0040] I. Determination of conserved sequences of respiratory syncytial virus (RSV) (1) Downloading gene sequences The RSV gene sequence was downloaded from the GenBank nucleic acid sequence database established by NCBI (GenBank: OQ595372.1). The selected target sequence is shown in SEQ ID NO: 7.

[0041] (2) Determination of conserved sequences The respiratory syncytial virus (RSV) matrix protein gene sequence (771 bp in total) was compared with the NCBI database. Typical alignment results (0 to 190 bp) were extracted, and the alignment results for the remaining regions were consistent. The results showed that the RSV matrix gene plasmid sequence is highly conserved, with a sequence similarity exceeding 95%, and is present only in RSV. Therefore, the RSV matrix gene plasmid sequence was selected for primer design. The RSV matrix gene plasmid sequence is shown in SEQ ID NO: 8.

[0042] II. Design and screening of primer compositions for respiratory syncytial virus detection (1) Design of primer compositions for respiratory syncytial virus detection Based on the conserved RSV sequence identified above, primer compositions for respiratory syncytial virus detection based on the RT-LAMP method were designed using Primer Explorer V5 (http: / / primerexplorer.jp / e / ). Two sets of primer compositions with good function were selected according to factors such as the primer composition's range, Tm value, and GC value (parameters are shown in Table 1), and synthesized by Shanghai Sangon Biotech Co., Ltd.

[0043] Table 1 .

[0044] (2) Screening of primer compositions for respiratory syncytial virus detection Using Bst2.0 isothermal amplification reagent, plasmids containing the RSV matrix gene were selected as samples to test the performance of the two RSV primer combinations listed in Table 1. The amplification curves and turbidity photographs of these two RSV primer combinations are shown below. Figure 1 and 2 As shown, Figure 1 The amplification curve and turbidity graph of primer composition 1 are shown. Figure 2Figure A shows the amplification curve and turbidity graph of primer combination 2. Figure A is the amplification curve, and Figure B is a turbidity comparison image. Red curve: 10 fg / μL plasmid sample; pink curve: 1 fg / μL plasmid sample; yellow curve: 100 ag / μL plasmid sample; green curve: NTC sample. Turbidity images: from left to right, samples are 10 fg / μL, 10 fg / μL, 1 fg / μL, 1 fg / μL, 100 ag / μL, 100 ag / μL, NTC, NTC. Finally, from... Figure 1 and 2 As can be seen from the table, the optimal primer composition is primer composition 1 in Table 1 (i.e., the primer composition used for respiratory syncytial virus detection).

[0045] III. Optimization of respiratory syncytial virus (RSV) detection methods.

[0046] The primer composition of this invention for respiratory syncytial virus (RSV) detection is used based on the RT-LAMP method. The detection conditions for the RT-LAMP method include the molar ratio of the inner primer pair, outer primer pair, and loop primer pair, enzyme dosage, magnesium ion concentration, dNTP concentration, and reaction temperature. Detection is performed using SYBR Green fluorescence detection and electrophoresis analysis.

[0047] Experiment 1: Optimization of the ratio of inner primer pairs to outer primer pairs in primer compositions for respiratory syncytial virus detection.

[0048] The ratio of inner primer pairs to outer primer pairs was determined as follows: System 1 (2:1): 0.4 μM inner primer pair, 0.2 μM outer primer pair System 2 (4:1): 0.8 μM inner primer pair, 0.2 μM outer primer pair System 3 (6:1): 1.2 μM inner primer pair, 0.2 μM outer primer pair System 4 (8:1): 1.6 μM inner primer pair, 0.2 μM outer primer pair System 5 (10:1): 2.0 μM inner primer pair, 0.2 μM outer primer pair The amplification curves of samples in various systems (with different ratios of inner primer pairs and outer primers) are as follows: Figure 3 As shown, note: red curve: amplification curve of 1fg / ul sample; blue curve: amplification curve of NTC sample. Figure 3 It is known that in the RT-LAMP method, the optimal ratio of inner primer pair to outer primer pair in the primer composition for respiratory syncytial virus detection is 4:1, that is, 0.8 μM inner primer pair and 0.2 μM outer primer pair.

[0049] Experiment 2: Optimization of the ratio of loop primer pairs in primer compositions for respiratory syncytial virus detection.

[0050] Using the optimal ratio of inner primer pairs to outer primer pairs (0.8 μM / 0.2 μM) from Experiment 1, we investigated the optimal ratio of loop primer pairs.

[0051] The determination ratio of the loop primer pairs is as follows: System 1: 0.2 μM circular primer pair System 2: 0.4 μM circular primer pair System 3: 0.6 μM circular primer pair System 4: 0.8 μM circular primer pair System 5: 1.0 μM circular primer pair The amplification curves of samples in different systems (with different ratios of loop primer pairs) are as follows: Figure 4 As shown, note: red curve: amplification curve of 1fg / ul sample; blue curve: amplification curve of NTC sample. Figure 4 It is known that the optimal ratio of circular primer pairs in the primer composition for respiratory syncytial virus detection in the RT-LAMP method is 0.4 μM.

[0052] Experiment 3: Optimization of enzyme dosage.

[0053] The optimal ratio of inner primer pairs, outer primer pairs, and loop primer pairs used in Experiments 1 and 2 was 0.8 uM / 0.2 uM / 0.4 uM, and the enzyme dosage was investigated.

[0054] The measurement ratios are as follows: System 1: Enzyme dosage 4U System 2: Enzyme dosage 6U System 3: Enzyme dosage 8U System 4: Enzyme dosage 10U System 5: Enzyme dosage 12U The amplification curves of samples in different systems (with different enzyme dosages) are as follows: Figure 5 As shown, note: red curve: amplification curve of 1fg / ul sample; blue curve: amplification curve of NTC sample. Figure 5 It is known that the optimal enzyme dosage in the RT-LAMP method is 8 U.

[0055] Experiment 4: Optimization of magnesium ion concentration.

[0056] Using the detection conditions in Experiments 1 to 3, the optimal ratio of inner primer pair, outer primer pair, and loop primer pair was 0.8 uM / 0.2 uM / 0.4 uM, and the enzyme dosage was 8 U. The magnesium ion concentration was investigated.

[0057] The measurement ratios are as follows: System 1: Magnesium ion concentration 4mM System 2: Magnesium ion concentration 6mM System 3: Magnesium ion concentration 8mM System 4: Magnesium ion concentration 10 mM System 5: Magnesium ion concentration 12mM The amplification curves of samples in different systems (with different magnesium ion concentrations) are as follows: Figure 6 As shown, note: red curve: amplification curve of 1fg / ul sample; blue curve: amplification curve of NTC sample. Figure 6 It is known that the magnesium ion concentration is preferably 8 mM in the RT-LAMP method.

[0058] Experiment 5: Optimization of dNTP concentration.

[0059] Using the detection conditions in Experiments 1 to 4, the optimal ratio of inner primer pair, outer primer pair, and loop primer pair was 0.8 uM / 0.2 uM / 0.4 uM, the enzyme dosage was 8 U, and the magnesium ion concentration was 8 mM. The dNTP concentration was investigated.

[0060] The measurement ratios are as follows: System 1: dNTP concentration 1.0 mM System 2: dNTP concentration 1.2 mM System 3: dNTP concentration 1.4 mM System 4: dNTP concentration 1.6 mM System 5: dNTP concentration 1.8 mM The amplification curves of samples in different systems (with different dNTP concentrations) are as follows: Figure 7 As shown, note: red curve: amplification curve of 1fg / ul sample; blue curve: amplification curve of NTC sample. Figure 7 It is known that the optimal dNTP concentration in the RT-LAMP method is 1.4 mM.

[0061] Experiment 6: Optimization of reaction temperature.

[0062] Using the detection conditions in Experiments 1 to 5, the optimal ratio of inner primer pair, outer primer pair, and loop primer pair was 0.8 uM / 0.2 uM / 0.4 uM, the enzyme dosage was 8 U, the magnesium ion concentration was 8 mM, the dNTP concentration was 1.4 mM, and the optimal reaction temperature was screened.

[0063] The reaction temperatures were measured as follows: The test reaction temperature ranged from 60℃ to 68℃, with a gradient of 1℃. The PCR instrument temperature zones were: 60℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃, 65.4℃, 66.4℃, 67.2℃, and 68℃.

[0064] The amplification curves of samples at different reaction temperatures (60℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃) are shown below. Figure 8 As shown, the amplification curves of samples at different reaction temperatures (65.4℃, 66.4℃, 67.2℃, 68℃) are as follows. Figure 9 As shown, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample. Figure 8 and Figure 9 It can be seen that the reaction temperature in the RT-LAMP method is 64.4℃.

[0065] Experiment 7: Optimization of reaction time.

[0066] Using the detection conditions in Experiments 1 to 6, the optimal ratio of inner primer pair, outer primer pair, and loop primer pair was 0.8 uM / 0.2 uM / 0.4 uM, the enzyme dosage was 8 U, the magnesium ion concentration was 8 mM, the dNTP concentration was 1.4 mM, the reaction temperature was 64.4 ℃, and the reaction time was investigated.

[0067] The reaction times were measured as follows: Test reaction times: 15 min, 20 min, 25 min, 30 min.

[0068] The amplification curves of samples at various reaction times (15 min, 20 min, 25 min, 30 min) are shown below. Figure 10 As shown, note: red curve: amplification curve of 1fg / ul sample; blue curve: amplification curve of NTC sample. Figure 10 It is known that the optimal reaction time in the RT-LAMP method is 30 min, but in practice, it can be detected in 15 min.

[0069] Based on experiments 1 to 7, the optimal detection conditions for the primer composition of this invention for detecting respiratory syncytial virus (RSV) using the RT-LAMP method are as follows: the molar ratio of the inner primer pair, outer primer pair, and loop primer pair in the primer composition is 0.8:0.2:0.4; the enzyme dosage is 8 U; the magnesium ion concentration is 8 mM; the dNTP concentration is 1.4 mM; the reaction temperature is 64.4 °C; and the reaction time is 30 min.

[0070] IV. Specific detection of respiratory syncytial virus (RSV).

[0071] The test item is: using 11 viral standards, extracted and used as specific samples, to test the specificity of the RSV detection method.

[0072] Test conditions: The concentrations of the inner primer pair, outer primer pair, and loop primer pair were 0.8 uM, 0.2 uM, and 0.4 uM, respectively; the enzyme dosage was 8 U; the magnesium ion concentration was 8 mM; the dNTP concentration was 1.4 mM; the reaction temperature was 64.4 ℃; and the reaction time was 30 min.

[0073] The 11 virus standards are: 1. Influenza A H1N1 standard; 2. Influenza A H3N2 standard; 3. Influenza A H7N9 standard; 4. Influenza B Victoria standard; 5. Influenza B Yamagata standard; 6. COVID-19 standard; 7. Parainfluenza type 1 standard; 8. Syncytial virus A standard; 9. Syncytial virus B standard; 10. Adenovirus type 7 standard; 11. Rhinovirus standard.

[0074] PC: Adenovirus uses 1 fg / uL plasmid, rhinovirus, parainfluenza virus, respiratory syncytial virus, influenza A virus, SARS-CoV-2, and influenza B virus use 1 fg / uL in vitro transcribed RNA, and the internal control uses human genomic DNA.

[0075] Respiratory syncytial virus (RSV) specific test results as follows Figure 11 As shown, by Figure 11 It can be seen that the primer composition for respiratory syncytial virus detection selected by the present invention, combined with the optimal detection conditions of the RT-LAMP method, can detect sample No. 8 (syncytial A standard) and PC, with a detection rate of 3 / 3, and the results meet the requirements.

[0076] V. Respiratory syncytial virus (RSV) sensitivity test.

[0077] The test item is: testing the sensitivity of RSV.

[0078] Test conditions: The concentrations of the inner primer pair, outer primer pair, and loop primer pair were 0.8 uM, 0.2 uM, and 0.4 uM, respectively; the enzyme dosage was 8 U; the magnesium ion concentration was 8 mM; the dNTP concentration was 1.4 mM; the reaction temperature was 64.4 ℃; and the reaction time was 30 min.

[0079] Respiratory syncytial virus (RSV) sensitivity testing items such as Figure 12 As shown in the figure, the sensitivity test results for respiratory syncytial virus (RSV) are as follows. Figure 13 As shown, Figure 12 and 13 In the middle, red: 10 6 copies / mL; Orange: 10 5 Copy / mL; Yellow: 10 4 Copy / mL; Green: 103 Copy / mL; Blue: 10 2 Copy / mL; Blue: 10 1 Copy / mL; Blue: 10 0 copies / mL; blue: 0 copies / mL, from Figure 8 It can be seen that the detection rates are 3 / 3 for 1000 copies / mL, 3 / 3 for 500 copies / mL, 0 / 3 for 200 copies / mL, 0 / 3 for 100 copies / mL, and 0 / 3 for 0 copies / mL. Therefore, the primer composition of this invention for respiratory syncytial virus (RSV) detection based on the RT-LAMP method exhibits high sensitivity, capable of detecting concentrations above 500 copies / mL.

[0080] Meanwhile, the amplification curve shows that the detection results can be obtained in 15 minutes. Compared with the prior art, the primer composition of the present invention for respiratory syncytial virus detection is based on the RT-LAMP method for respiratory syncytial virus detection, and the detection time is shorter.

[0081] In summary, this invention provides a primer composition and its application for respiratory syncytial virus (RSV) detection, which can rapidly, sensitively, and specifically detect RSV with short detection time, and has the advantages of being fast, efficient, and easy to operate, making it suitable for rapid on-site detection.

[0082] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A primer composition for detecting respiratory syncytial virus, characterized in that... It includes outer primer pairs, inner primer pairs, and loop primer pairs. The outer primer pairs include respiratory syncytial virus F3 and respiratory syncytial virus B3, the inner primer pairs include respiratory syncytial virus FIP and respiratory syncytial virus BIP, and the loop primer pairs include respiratory syncytial virus LF and respiratory syncytial virus LB.

2. The primer composition for respiratory syncytial virus detection according to claim 1, characterized in that... The base sequence of respiratory syncytial virus F3 is shown in SEQ ID NO: 1, and the base sequence of respiratory syncytial virus B3 is shown in SEQ ID NO:

2.

3. The primer composition for respiratory syncytial virus detection according to claim 1 or 2, characterized in that... The base sequence of respiratory syncytial virus (RSV) FIP is shown in SEQ ID NO: 3, and the base sequence of respiratory syncytial virus (RSV) BIP is shown in SEQ ID NO:

4.

4. The primer composition for respiratory syncytial virus detection according to claim 1, 2, or 3, characterized in that... The base sequence of respiratory syncytial virus LF is shown in SEQ ID NO: 5, and the base sequence of respiratory syncytial virus LB is shown in SEQ ID NO:

6.

5. The application of a primer composition for respiratory syncytial virus (RSV) detection according to any one of claims 1 to 4 based on the RT-LAMP method in the detection of RSV, characterized in that... In the RT-LAMP method, the molar ratio of the inner primer pair, outer primer pair, and loop primer pair in the primer composition is 0.4 to 2.0:0.2:0.2 to 1.0, the enzyme amount is 4 U to 12 U, the magnesium ion concentration is 4 mM to 12 mM, the dNTP concentration is 1.0 mM to 1.8 mM, the reaction temperature is 60 °C to 68 °C, and the reaction time is 15 min to 30 min.

6. The application of the primer composition for respiratory syncytial virus detection according to claim 5 in the detection of respiratory syncytial virus based on the RT-LAMP method, characterized in that... In the RT-LAMP method, the molar ratio of the inner primer pair, outer primer pair, and loop primer pair in the primer composition is 0.8:0.2:0.4, the enzyme dosage is 8U, the magnesium ion concentration is 8mM, the dNTP concentration is 1.4mM, the reaction temperature is 64.4℃, and the reaction time is 30min.

7. The application of the primer composition for respiratory syncytial virus detection according to claim 5 or 6 in the detection of respiratory syncytial virus based on the RT-LAMP method, characterized in that... Respiratory syncytial virus (RSV) is a type A respiratory syncytial virus.

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