LAMP (loop-mediated isothermal amplification) primer composition, HRV (human rhinovirus) detection system, kit for rapidly detecting human rhinovirus and application

Abstract

The invention relates to the technical field of biological detection, in particular to an LAMP primer composition, an HRV detection system, a kit for rapidly detecting human rhinoviruses and application, the LAMP primer composition comprises an outer primer pair, an inner primer pair and a loop primer pair, the outer primer pair comprises the human rhinoviruses F3 and the human rhinoviruses B3, the inner primer pair comprises the human rhinoviruses FIP and the human rhinoviruses BIP, and the loop primer pair comprises the human rhinoviruses F3 and the human rhinoviruses B3. The loop primer pair comprises a human rhinovirus LF and a human rhinovirus LB, the HRV detection system comprises the LAMP primer composition, and the kit comprises the HRV detection system. The LAMP primer composition with the optimal performance, the HRV detection system and the kit for rapidly detecting the human rhinovirus are provided for the first time, when the LAMP primer composition is used for detecting the HRV, the reaction time is only 15 minutes, the detection sensitivity is 200 copies / mL, and the LAMP primer composition has the advantages of being easy and convenient to operate, low in cost and the like and is suitable for rapid on-site detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically a LAMP primer composition, an HRV detection system, a rapid detection kit for human rhinovirus, and their applications. Background Technology

[0002] Human rhinovirus (HRV) is one of the main pathogens causing acute upper respiratory tract infections, especially in children, the elderly, and immunocompromised individuals, where it can easily lead to serious complications such as bronchitis and pneumonia. Rapid and accurate early diagnosis is crucial for controlling the spread of infection and guiding clinical treatment.

[0003] Currently, HRV detection methods mainly include virus isolation and culture, antigen detection, and reverse transcription polymerase chain reaction (RT-PCR). Virus isolation and culture is cumbersome and time-consuming, taking several days, making it difficult to meet the needs of rapid diagnosis; antigen detection has low sensitivity and is prone to false negative results; although RT-PCR has high sensitivity and specificity, it relies on sophisticated instruments, is complex to operate, and has a long detection cycle (usually 1 to 2 hours), making it unsuitable for primary healthcare institutions or on-site rapid testing scenarios.

[0004] Loop-mediated isothermal amplification (LAMP), as a novel nucleic acid amplification method, has been widely used in pathogen detection due to its advantages such as simple operation, rapid reaction (typically 30 to 60 minutes), and lack of complex instruments. In recent years, HRV detection methods based on RT-LAMP technology have gradually gained attention. For example, the HRV-C type RT-LAMP detection method developed by Wong YP et al. (Int Microbiol. 2024 Dec 2; doi: 10.1007 / s10123-024-00621-8) has better sensitivity and efficiency than traditional RT-PCR, but the reaction time still requires more than 30 minutes, and the detection sensitivity in some studies only reaches 1000 copies / mL, which is insufficient to meet the rapid detection requirements of low-load samples. Furthermore, existing LAMP detection systems suffer from insufficient primer specificity and inadequate optimization of reaction conditions, which may lead to non-specific amplification or unstable detection performance, further limiting its application in rapid on-site detection.

[0005] Patent document CN111363848A discloses an internal reference gene for PCR detection of respiratory RNA viruses and its detection product, wherein the internal reference gene is the DDX5 gene. This invention also designs primers and probes targeting the internal reference gene. Compared with the internal reference gene RNase P (RPP30) and corresponding primers and probes used by the US CDC, the primers and probes designed and screened for the internal reference gene of this invention are highly specific, amplifying only mRNA and not genomic DNA; and the internal reference gene of this invention can be stably detected in the human population. However, this technology does not provide specific primer design for LAMP (such as loop primers, combinations of inner and outer primers), nor has it verified its amplification efficiency under isothermal conditions, nor has it independently verified its cross-reactivity with other respiratory viruses, thus limiting its application scenarios.

[0006] Patent document CN109996888A discloses systems, methods, and devices for sensing and / or identifying pathogens, genomic materials, proteins, and / or other small molecules or biomarkers. In some embodiments, miniaturized, low-cost devices provide rapid and robust sensing and identification. Such devices can utilize microfluidics, biochemistry, and electronics to detect one or more targets simultaneously in the field and at or near the bedside. However, this approach cannot achieve isothermal (no PCR instrument required) and rapid (typically <1 hour) field detection.

[0007] To address the aforementioned issues, there is an urgent need in this field to develop an HRV detection system with shorter reaction time, higher sensitivity, stronger specificity, and ease of operation, for rapid and accurate detection of human rhinovirus, especially suitable for on-site detection in primary healthcare units or during public health emergencies. Summary of the Invention

[0008] This invention provides a LAMP primer composition, an HRV detection system, a rapid detection kit for human rhinovirus, and its application, overcoming the shortcomings of the prior art. It can effectively solve the problems of complex operation, long detection time, low detection sensitivity, and high detection cost in existing human rhinovirus detection methods.

[0009] One of the technical solutions of the present invention is achieved through the following measures: a LAMP primer composition comprising an outer primer pair, an inner primer pair and a circular primer pair, wherein the outer primer pair comprises human rhinovirus F3 and human rhinovirus B3, the inner primer pair comprises human rhinovirus FIP and human rhinovirus BIP, and the circular primer pair comprises human rhinovirus LF and human rhinovirus LB.

[0010] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: The base sequence of human rhinovirus F3 is shown in SEQ ID NO: 1, and the base sequence of human rhinovirus B3 is shown in SEQ ID NO: 2.

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

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

[0013] The second technical solution of the present invention is achieved through the following measures: an HRV detection system, comprising the aforementioned LAMP primer composition, wherein the detection method is SYBR Green fluorescence detection and electrophoretic analysis, and the test conditions include: The inner primer pairs are 0.4 μM, 0.8 μM, 1.2 μM, 1.6 μM, and 2.0 μM; The outer primer pairs were 0.2 μM, 0.2 μM, 0.2 μM, 0.2 μM, and 0.2 μM. The loop primer pairs were 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, and 1.0 μM. The enzyme dosages are 4U, 6U, 8U, 10U, and 12U; Magnesium ion concentrations were 4 mM, 6 mM, 8 mM, 10 mM, and 12 mM. The dNTP concentrations were 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, and 1.8 mM. The reaction temperature ranged from 60℃ to 68℃, with a gradient of 1℃. The PCR instrument temperature zones were: 60.0℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃, 65.4℃, 66.4℃, 67.2℃, and 68.0℃. The reaction times were 15 min, 20 min, 25 min, and 30 min.

[0014] The following are further optimizations and / or improvements to the second technical solution of the above invention: The above test conditions include: The inner primer pair is 1.2 μM; The outer primer pair is 0.2 μM; The circular primer pair is 0.6 μM; The enzyme dosage is 8U; The magnesium ion concentration is 8 mM; The dNTP concentration was 1.4 mM; The reaction temperature was 64.4℃; The reaction time is 30 minutes.

[0015] The third technical solution of the present invention is achieved through the following measures: a rapid detection kit for human rhinovirus, comprising the aforementioned HRV detection system, wherein the HRV detection system comprises the aforementioned LAMP primer composition, and the LAMP primer composition comprises an outer primer pair, an inner primer pair, and a loop primer pair.

[0016] The following are further optimizations and / or improvements to the third technical solution of the above invention: The aforementioned outer primer pairs include human rhinovirus F3 and human rhinovirus B3, the inner primer pairs include human rhinovirus FIP and human rhinovirus BIP, and the circular primer pairs include human rhinovirus LF and human rhinovirus LB.

[0017] The fourth technical solution of the present invention is achieved through the following measures: the application of a LAMP primer composition in an HRV detection system.

[0018] The fifth technical solution of the present invention is achieved through the following measures: the application of an HRV detection system in a kit for rapid detection of human rhinovirus.

[0019] This invention presents for the first time a high-performance LAMP primer composition, HRV detection system, and kit. When used to detect HRV, the reaction time is only 15 minutes and the detection sensitivity is 200 copies / mL. It has the advantages of simple operation and low cost, and is suitable for rapid on-site detection. Attached Figure Description

[0020] Appendix Figure 1 The images show the amplification curves and turbidity photographs of the four LAMP primer compositions in Example 10 of this invention.

[0021] Appendix Figure 2 This is an amplification curve showing the optimized ratio of inner primer pairs to outer primer pairs in the HRV detection system of Example 10 of the present invention.

[0022] Appendix Figure 3 This is an optimized amplification curve of different loop primer pairs in the HRV detection system of Example 10 of the present invention.

[0023] Appendix Figure 4 This is an optimized amplification curve of different enzyme dosages in the HRV detection system of Example 10 of the present invention.

[0024] Appendix Figure 5 This is an optimized amplification curve of different magnesium ion concentrations in the HRV detection system in Example 10 of the present invention.

[0025] Appendix Figure 6 This is an optimized amplification curve of different dNTP concentrations in the HRV detection system in Example 10 of the present invention.

[0026] Appendix Figure 7 This is an optimized amplification curve of the HRV detection system in Example 10 of the present invention at different reaction temperatures (60℃, 60.8℃, 61.6℃, and 62.6℃).

[0027] Appendix Figure 8 This is an optimized amplification curve of the HRV detection system in Example 10 of the present invention at different reaction temperatures (reaction temperatures of 64.4℃, 65.4℃, 66.4℃, 67.2℃, and 68℃).

[0028] Appendix Figure 9 This is an optimized amplification curve of different reaction times in the HRV detection system in Example 10 of the present invention.

[0029] Appendix Figure 10 This refers to the specific amplification Ct value of the HRV detection system in Example 10 of this invention.

[0030] Appendix Figure 11 This is a specific amplification curve of the HRV detection system in Example 10 of the present invention.

[0031] Appendix Figure 12 This is a diagram showing the sensitivity test items of the HRV detection system in Embodiment 10 of the present invention.

[0032] Appendix Figure 13 The graph shows the sensitivity test results of the HRV detection system in Embodiment 10 of the present invention. Detailed Implementation

[0033] This invention is not limited to the following embodiments; specific implementation methods can be determined based on the technical solution of this invention and actual circumstances. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art.

[0034] The present invention will be further described below with reference to embodiments: Example 1: The LAMP primer composition includes an outer primer pair, an inner primer pair, and a circular primer pair, wherein the outer primer pair includes human rhinovirus F3 and human rhinovirus B3, the inner primer pair includes human rhinovirus FIP and human rhinovirus BIP, and the circular primer pair includes human rhinovirus LF and human rhinovirus LB.

[0035] Example 2: As an optimization of the above example, the base sequence of human rhinovirus F3 is shown in SEQ ID NO: 1, and the base sequence of human rhinovirus B3 is shown in SEQ ID NO: 2.

[0036] Example 3: As an optimization of the above examples, the base sequence of human rhinovirus FIP is shown in SEQ ID NO: 3, and the base sequence of human rhinovirus BIP is shown in SEQ ID NO: 4.

[0037] Example 4: As an optimization of the above examples, the base sequence of human rhinovirus LF is shown in SEQ ID NO: 5, and the base sequence of human rhinovirus LB is shown in SEQ ID NO: 6.

[0038] Example 5: This HRV detection system includes the aforementioned LAMP primer composition. The detection method is SYBR Green fluorescence detection and electrophoresis analysis. The test conditions include: The inner primer pairs are 0.4 μM, 0.8 μM, 1.2 μM, 1.6 μM, and 2.0 μM; The outer primer pairs were 0.2 μM, 0.2 μM, 0.2 μM, 0.2 μM, and 0.2 μM. The loop primer pairs were 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, and 1.0 μM. The enzyme dosages are 4U, 6U, 8U, 10U, and 12U; Magnesium ion concentrations were 4 mM, 6 mM, 8 mM, 10 mM, and 12 mM. The dNTP concentrations were 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, and 1.8 mM. The reaction temperature ranged from 60℃ to 68℃, with a gradient of 1℃. The PCR instrument temperature zones were: 60.0℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃, 65.4℃, 66.4℃, 67.2℃, and 68.0℃. The reaction times were 15 min, 20 min, 25 min, and 30 min.

[0039] Example 6: As an optimization of the above examples, the test conditions include: The inner primer pair is 1.2 μM; The outer primer pair is 0.2 μM; The circular primer pair is 0.6 μM; The enzyme dosage is 8U; The magnesium ion concentration is 8 mM; The dNTP concentration was 1.4 mM; The reaction temperature was 64.4℃; The reaction time is 30 minutes.

[0040] Example 7: The rapid detection kit for human rhinovirus (hereinafter referred to as HRV) includes the HRV detection system, which includes the LAMP primer composition. The LAMP primer composition includes an outer primer pair, an inner primer pair, and a circular primer pair. The outer primer pair includes human rhinovirus F3 and human rhinovirus B3, the inner primer pair includes human rhinovirus FIP and human rhinovirus BIP, and the circular primer pair includes human rhinovirus LF and human rhinovirus LB.

[0041] Example 8: Application of this LAMP primer composition in HRV detection system.

[0042] Example 9: Application of this HRV detection system in a rapid detection kit for human rhinovirus.

[0043] Example 10: Design of the HRV detection system.

[0044] I. Determination of HRV Conservative Sequence (a) Downloading gene sequences The HRV gene sequence was downloaded from the GenBank nucleic acid sequence database established by NCBI (GenBank:MK858737.1).

[0045] The selected target sequence is shown in SEQ ID NO: 7: The human rhinovirus 5'UTR gene was found to be a relatively conserved sequence and could be used to design LAMP primer compositions.

[0046] (II) Determination of Conservative Sequences The 5' UTR gene sequence of human rhinovirus (total 390 bp) was compared with the NCBI database, and typical alignment results (180 bp to 360 bp) were extracted. The results showed that the sequence is conserved, with a sequence similarity greater than 70%, and exists only in human rhinovirus. Except for the conserved sequence from 1 bp to 360 bp, the downstream sequence is not conserved. Therefore, sequences from 1 bp to 360 bp were selected for designing LAMP primer compositions.

[0047] The plasmid sequence of the HRV 5'UTR gene is shown in SEQ ID NO: 8, and the T7 promoter sequence is shown in SEQ ID NO: 9.

[0048] II. Design and Screening of LAMP Primer Compositions (a) Design of LAMP primer compositions Based on the conserved sequence of the HRV 5'UTR gene identified above, LAMP primers were designed using Primer Explorer V5 (http: / / primerexplorer.jp / e / ). Four sets of LAMP primers with good function were selected according to factors such as primer range, Tm value, and GC value. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0049] The information of the four sets of LAMP primer compositions is shown in Tables 1 to 4. Among them, in the first set of LAMP primer compositions, the base sequence of human rhinovirus F3 is shown in SEQ ID NO: 10, the base sequence of human rhinovirus B3 is shown in SEQ ID NO: 11, the base sequence of human rhinovirus FIP is shown in SEQ ID NO: 12, the base sequence of human rhinovirus BIP is shown in SEQ ID NO: 13, the base sequence of human rhinovirus LF is shown in SEQ ID NO: 14, and the base sequence of human rhinovirus LB is shown in SEQ ID NO: 15. In the second group of LAMP primer compositions, the base sequence of human rhinovirus F3 is shown in SEQ ID NO: 16, the base sequence of human rhinovirus B3 is shown in SEQ ID NO: 17, the base sequence of human rhinovirus FIP is shown in SEQ ID NO: 18, the base sequence of human rhinovirus BIP is shown in SEQ ID NO: 19, the base sequence of human rhinovirus LF is shown in SEQ ID NO: 20, and the base sequence of human rhinovirus LB is shown in SEQ ID NO: 21. In the third group of LAMP primer compositions, the base sequence of human rhinovirus F3 is shown in SEQ ID NO: 22, the base sequence of human rhinovirus B3 is shown in SEQ ID NO: 23, the base sequence of human rhinovirus FIP is shown in SEQ ID NO: 24, the base sequence of human rhinovirus BIP is shown in SEQ ID NO: 25, the base sequence of human rhinovirus LF is shown in SEQ ID NO: 26, and the base sequence of human rhinovirus LB is shown in SEQ ID NO: 27. In the fourth group of LAMP primer compositions, the base sequence of human rhinovirus F3 is shown in SEQ ID NO: 1, the base sequence of human rhinovirus B3 is shown in SEQ ID NO: 2, the base sequence of human rhinovirus FIP is shown in SEQ ID NO: 3, the base sequence of human rhinovirus BIP is shown in SEQ ID NO: 4, the base sequence of human rhinovirus LF is shown in SEQ ID NO: 5, and the base sequence of human rhinovirus LB is shown in SEQ ID NO: 6.

[0050] (II) Screening of LAMP primer compositions Using Bst2.0 isothermal amplification reagent, plasmids of the HRV 5'UTR gene were selected as samples to test the performance of four LAMP primer combinations. Finally, the optimal LAMP primer combination (the fourth LAMP primer combination) was selected. Figure 1 (As shown).

[0051] Figure 1 In the diagram, the red curve represents a 10 fg / μL plasmid sample; the pink curve represents a 1 fg / μL plasmid sample; the yellow curve represents a 100 ag / μL plasmid sample; and the green curve represents an NTC sample.

[0052] Turbidity photos: From left to right, the samples are 10 fg / μL, 10 fg / μL, 1 fg / μL, 1 fg / μL, 100 ag / μL, 100 ag / μL, NTC, NTC.

[0053] Figure 1 In the image, A represents the amplification curve and turbidity image of the first LAMP primer combination; B shows the amplification curve and turbidity image of the second group of LAMP primers; C shows the amplification curve and turbidity image of the third group of LAMP primers; D shows the amplification curve and turbidity image of the fourth group of LAMP primers.

[0054] Test conclusion: The LAMP primer composition in group 4 is the optimal LAMP primer composition.

[0055] III. Optimization of the HRV Detection System (I) Optimization of the ratio of inner primer pairs to outer primer pairs in the HRV detection system Using the fourth group of LAMP primer compositions, the optimal ratio of inner and outer primer pairs was tested, and the detection methods were SYBR Green fluorescence detection and electrophoresis analysis (e.g., Figure 2 (As shown).

[0056] The measurement ratios are as follows: Ratio 1 (2:1): Inner primer pair 0.4 μM, outer primer pair 0.2 μM; Ratio 2 (4:1): Inner primer pair 0.8 μM, outer primer pair 0.2 μM; Ratio 3 (6:1): Inner primer pair 1.2 μM, outer primer pair 0.2 μM; Ratio 4 (8:1): Inner primer pair 1.6 μM, outer primer pair 0.2 μM; Ratio 5 (10:1): Inner primer pair 2.0 uM, outer primer pair 0.2 uM.

[0057] Figure 2In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0058] Test conclusion: The optimal selection ratio of inner primer pairs and outer primer pairs in the HRV detection system is: 1.2 μM for inner primer pairs and 0.2 μM for outer primer pairs.

[0059] (II) Optimization of the ratio of loop primer pairs in the HRV detection system The ratio of loop primer pairs in the HRV detection system was tested using SYBR Green fluorescence detection and electrophoresis analysis. The inner primer pair was 1.2 μM, and the outer primer pair was 0.2 μM (e.g., ...). Figure 3 (As shown).

[0060] The measurement ratios are 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; Figure 3 In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0061] Test conclusion: The optimal choice of loop primer pair in the HRV detection system is: 0.6 μM loop primer pair.

[0062] (III) Optimization of enzyme dosage in HRV detection system The optimal enzyme dosage was determined using SYBR Green fluorescence detection and electrophoresis analysis, with the inner primer pair at 1.2 μM, the outer primer pair at 0.2 μM, and the loop primer pair at 0.6 μM (e.g., ...). Figure 4 (As shown).

[0063] 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.

[0064] Figure 4 In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0065] Test conclusion: The optimal enzyme dosage in the HRV detection system is 8U.

[0066] (iv) Optimization of magnesium ion concentration in the HRV detection system: The optimal magnesium ion concentration was screened, and the detection method was SYBR Green fluorescence detection and electrophoresis analysis. The inner primer pair was 1.2 uM, the outer primer pair was 0.2 uM, the loop primer pair was 0.6 uM, and the enzyme dosage was 8 U (e.g., ...). Figure 5 (As shown).

[0067] The measurement ratios are as follows: System 1: Magnesium ion concentration 4 mM; System 2: Magnesium ion concentration 6 mM; System 3: Magnesium ion concentration 8 mM; System 4: Magnesium ion concentration 10 mM; System 5: Magnesium ion concentration 12mM.

[0068] Figure 5 In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0069] Test conclusion: The optimal magnesium ion concentration in the HRV detection system is 8 mM.

[0070] (v) Optimization of dNTP concentration in HRV detection system The optimal dNTP concentration was screened, and the detection method was SYBR Green fluorescence detection and electrophoresis analysis. The inner primer pair was 1.2 uM, the outer primer pair was 0.2 uM, the loop primer pair was 0.6 uM, the enzyme dosage was 8 U, and the magnesium ion concentration was 8 mM (e.g., ...). Figure 6 (As shown).

[0071] 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; Figure 6 In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0072] Test conclusion: The optimal concentration of dNTP in the HRV detection system is 1.4 mM.

[0073] (vi) Optimization of reaction temperature in HRV detection system The optimal reaction temperature was screened, and the detection method was SYBR Green fluorescence detection and electrophoresis analysis. The inner primer pair was 1.2 μM, the outer primer pair was 0.2 μM, the loop primer pair was 0.6 μM, the enzyme dosage was 8 U, the magnesium ion concentration was 8 mM, and the dNTP concentration was 1.4 mM (e.g., ...). Figure 7 and Figure 8 (As shown).

[0074] The measurement ratios are as follows: Test reaction temperature: 60℃ to 68℃, gradient 1℃.

[0075] The PCR instrument temperature zones are: 60℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃, 65.4℃, 66.4℃, 67.2℃, and 68℃.

[0076] Figure 7 and Figure 8 In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0077] Test conclusion: The optimal reaction temperature in the HRV detection system is 64.4℃.

[0078] (vii) Optimization of reaction time The optimal reaction time was screened, and the detection methods were SYBR Green fluorescence detection and electrophoresis analysis. The inner primer pair was 1.2 μM, the outer primer pair was 0.2 μM, the loop primer pair was 0.6 μM, the enzyme dosage was 8 U, the magnesium ion concentration was 8 mM, the dNTP concentration was 1.4 mM, and the reaction temperature was 64.4℃ (e.g., ...). Figure 9 (As shown).

[0079] The measurement ratios are as follows: Test reaction times: 15 min, 20 min, 25 min, 30 min.

[0080] Figure 9 In the diagram, the red curve represents the amplification curve of a 1fg / ul sample; the blue curve represents the amplification curve of an NTC sample.

[0081] Test conclusion: The optimal reaction time in the HRV detection system is 30 min.

[0082] IV. Specificity Detection of HRV Detection System The test items are: using 11 viral standards, extracted and used as specific samples, to test the specificity of the HRV detection system (e.g., Figure 10 and Figure 11 (As shown).

[0083] Reaction system: inner primer pair 1.2 uM, outer primer pair 0.2 uM, loop primer pair 0.6 uM, enzyme dosage 8 U, magnesium ion concentration 8 mM, dNTP concentration 1.4 mM, reaction temperature 64.4℃, 30 min.

[0084] Figure 10 Among them, 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: HRV standard.

[0085] PC: Adenovirus uses 1fg / uL plasmid, human rhinovirus, parainfluenza virus, respiratory syncytial virus, influenza A virus, SARS-CoV-2, and influenza B virus use 1fg / uL in vitro transcribed RNA, and human genomic DNA is used as the internal control.

[0086] Results show that the human rhinovirus system was able to detect human rhinovirus sample No. 11 (HRV standard) and PC, with a detection rate of 3 / 3, which meets the requirements.

[0087] V. Sensitivity Testing of the HRV Detection System The test item is: to test the sensitivity of the HRV detection system.

[0088] Reaction system: inner primer pair 1.2 uM, outer primer pair 0.2 uM, loop primer pair 0.6 uM, enzyme dosage 8 U, magnesium ion concentration 8 mM, dNTP concentration 1.4 mM, reaction temperature 64.4℃, 30 min.

[0089] Test items such as Figure 12 As shown, the test results are as follows: Figure 13 As shown.

[0090] Results Explanation: Red: 10 6 copies / mL; Orange: 10 5 Copy / mL; Yellow: 10 4 Copy / mL; Green: 10 3 Copy / mL; Blue: 10 2 Copy / mL; Blue: 10 1 Copy / mL; Blue: 10 0 Copy / mL; Blue: 0 copies / mL.

[0091] Experimental results: Red 1000 copies / mL, detection rate 3 / 3; Orange 500 copies / mL, detection rate 3 / 3; Yellow 200 copies / mL, detection rate 2 / 3; Blue 100 copies / mL and 0 copies / mL, detection rate 0 / 3.

[0092] Therefore, when the LAMP primer composition of the present invention is used for rapid detection of human rhinovirus, the reaction time for HRV detection is only 15 minutes, which is much shorter than the currently reported LAMP methodologies. Moreover, the detection sensitivity is 200 copies / mL, which is superior to the detection sensitivity of some LAMP studies. It can be widely used in HRV detection systems and kits for rapid detection of human rhinovirus.

[0093] In summary, this invention presents for the first time a high-performance LAMP primer composition, HRV detection system, and kit. When used to detect HRV, it not only has a short reaction time but also high detection sensitivity. It also has the advantages of simple operation and low cost, making it suitable for rapid on-site detection.

[0094] 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 LAMP primer composition, characterized in that... It includes outer primer pairs, inner primer pairs, and loop primer pairs. The outer primer pairs include human rhinovirus F3 and human rhinovirus B3, the inner primer pairs include human rhinovirus FIP and human rhinovirus BIP, and the loop primer pairs include human rhinovirus LF and human rhinovirus LB.

2. The LAMP primer composition according to claim 1, characterized in that... The base sequence of human rhinovirus F3 is shown in SEQ ID NO: 1, and the base sequence of human rhinovirus B3 is shown in SEQ ID NO:

2.

3. The LAMP primer composition according to claim 1 or 2, characterized in that... The base sequence of human rhinovirus FIP is shown in SEQ ID NO: 3, and the base sequence of human rhinovirus BIP is shown in SEQ ID NO:

4.

4. The LAMP primer composition according to claim 1, characterized in that... The base sequence of human rhinovirus LF is shown in SEQ ID NO: 5, and the base sequence of human rhinovirus LB is shown in SEQ ID NO:

6.

5. An HRV detection system, characterized in that... The LAMP primer composition according to any one of claims 1 to 4 is included, and the detection method is SYBR Green fluorescence detection and electrophoresis analysis, wherein the test conditions include: The inner primer pairs are 0.4 μM, 0.8 μM, 1.2 μM, 1.6 μM, and 2.0 μM; The outer primer pairs were 0.2 μM, 0.2 μM, 0.2 μM, 0.2 μM, and 0.2 μM. The loop primer pairs were 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, and 1.0 μM. The enzyme dosages are 4U, 6U, 8U, 10U, and 12U; Magnesium ion concentrations were 4 mM, 6 mM, 8 mM, 10 mM, and 12 mM. The dNTP concentrations were 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, and 1.8 mM. The reaction temperature ranged from 60℃ to 68℃, with a gradient of 1℃. The PCR instrument temperature zones were: 60.0℃, 60.8℃, 61.6℃, 62.6℃, 64.4℃, 65.4℃, 66.4℃, 67.2℃, and 68.0℃. The reaction times were 15 min, 20 min, 25 min, and 30 min.

6. The HRV detection system according to claim 5, characterized in that... Test conditions include: The inner primer pair is 1.2 μM; The outer primer pair is 0.2 μM; The circular primer pair is 0.6 μM; The enzyme dosage is 8U; The magnesium ion concentration is 8 mM; The dNTP concentration was 1.4 mM; The reaction temperature was 64.4℃; The reaction time is 30 minutes.

7. A rapid detection kit for human rhinovirus, characterized in that... The system includes the HRV detection system according to claim 5 or 6, wherein the HRV detection system includes the LAMP primer composition according to any one of claims 1 to 4, and the LAMP primer composition includes an outer primer pair, an inner primer pair, and a loop primer pair.

8. The rapid detection kit for human rhinovirus according to claim 7, characterized in that... The outer primer pairs include human rhinovirus F3 and human rhinovirus B3, the inner primer pairs include human rhinovirus FIP and human rhinovirus BIP, and the loop primer pairs include human rhinovirus LF and human rhinovirus LB.

9. The application of a LAMP primer composition according to claim 1, 2 or 3 in an HRV detection system.

10. The application of the HRV detection system according to claim 5 or 6 in a kit for rapid detection of human rhinovirus.

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