A primer set, kit, and application for detecting respiratory syncytial virus based on isothermal amplification technology.

Abstract

This invention discloses a primer set, kit, and application for detecting respiratory syncytial virus (RSV) based on isothermal amplification (ASA). The primer set of this invention, combined with ASA technology, is used to detect RSV type A and RSV type B. The primers provided by this invention exhibit good specificity, enabling accurate detection of RSV type A (RSV-A) and RSV type B (RSV-B). The detection sensitivity for the target viruses reaches 10. 2 It has copies / uL, high specificity, no cross-reactivity, and low inter-batch coefficient of variation.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a primer set and kit for detecting respiratory syncytial virus based on isothermal amplification technology and its application. Background Technology

[0002] Respiratory syncytial virus (RSV) is a common and highly contagious pathogen that primarily infects the human respiratory system. It is one of the leading causes of acute lower respiratory tract infections (such as bronchiolitis and pneumonia) in infants, the elderly, and immunocompromised individuals. Globally, RSV infection results in a large number of outpatient visits, hospitalizations, and even deaths each year, placing a heavy burden on public health systems. Therefore, establishing a rapid, accurate, easy-to-use, and suitable RSV testing method for primary healthcare settings is of great significance for early diagnosis, epidemic monitoring, and clinical intervention.

[0003] Currently, laboratory testing methods for RSV mainly include virus isolation and culture, antigen detection, serological detection, and nucleic acid detection. Traditional virus isolation and culture methods are time-consuming, complex, and have limited sensitivity; antigen detection (such as immunochromatographic test strips), while rapid and convenient, has relatively low sensitivity, especially in early-stage infection or low-viral-load samples, where false negatives are common. Nucleic acid detection technology, represented by polymerase chain reaction (PCR), has become the gold standard for clinical diagnosis due to its high sensitivity and specificity; however, it relies on sophisticated temperature cycling equipment, professional operators, and long testing times, making it difficult to promote and apply in resource-constrained primary healthcare institutions or point-of-care testing (POCT) scenarios.

[0004] Isothermal amplification technology, as an emerging nucleic acid amplification method, can rapidly and efficiently amplify target nucleic acid sequences at a constant temperature without the need for complex thermal cycling equipment, significantly reducing the detection threshold and operational complexity. This technology has demonstrated enormous application potential in the field of rapid on-site pathogen detection. However, the core key to isothermal amplification detection methods for RSV lies in designing primer sets with high specificity and high amplification efficiency to ensure accuracy, sensitivity, and interference resistance. Currently, there is a lack of fully optimized, stable, and reliable primer sets and matching kits suitable for various isothermal amplification platforms (such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA)). Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the purpose of this invention is to provide a primer set, kit and application for detecting respiratory syncytial virus based on isothermal amplification technology.

[0006] This invention provides a carefully designed specific primer set targeting conserved regions of the respiratory syncytial virus (RSV) genome. This primer set has undergone rigorous bioinformatics screening and experimental validation, enabling efficient and specific amplification of RSV nucleic acid under isothermal conditions. Furthermore, this invention also provides a kit containing this primer set and its application in the preparation of RSV detection products.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0008] A primer set for detecting respiratory syncytial virus based on isothermal amplification technology, comprising:

[0009] Primer set for detecting respiratory syncytial virus type A;

[0010] And, a primer set for detecting respiratory syncytial virus type B;

[0011] The primer set used for detecting respiratory syncytial virus type A is shown in seq no. 1-6 below:

[0012]

[0013] The primer set used for detecting respiratory syncytial virus type B is shown in seq no. 7-11 below:

[0014] .

[0015] A kit for detecting respiratory syncytial virus based on isothermal amplification technology, the kit comprising:

[0016] A microfluidic chip comprising at least one of the primer sets described above;

[0017] And the amplification system reactants used to amplify the primer set.

[0018] In a preferred embodiment of the present invention, the reactants of the amplification system are:

[0019] 10mM dNTP;

[0020] 5% BSA;

[0021] 5M MgSO4;

[0022] 100μM fluorescent dye;

[0023] 5M betaine;

[0024] The reverse transcriptase used in the amplification system is a nucleic acid polymerase.

[0025] The preferred nucleic acid polymerase is Bst DNA polymerase with a concentration of 8 U / μL, and its amplification concentration is 1.5-3 U / μL.

[0026] An application of a kit for detecting respiratory syncytial virus (RSV) based on isothermal amplification technology, wherein the application involves using a microfluidic chip containing at least one of the primer sets described above in conjunction with isothermal amplification technology to amplify RSV type A viral nucleic acid and / or RSV type B viral nucleic acid.

[0027] The beneficial effects of this invention are as follows:

[0028] The technical solution of this invention is expected to overcome the shortcomings of the prior art and provide a more convenient, sensitive and specific detection tool for the rapid diagnosis of RSV infection. It is especially suitable for promotion and use in primary healthcare institutions, port quarantine and on-site emergency testing, and has important clinical value and socio-economic benefits. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the detection in Example 1. Figure 1 .

[0030] Figure 2 This is a schematic diagram of the detection in Example 1. Figure 2 .

[0031] Figure 3 This is a schematic diagram of the detection in Example 2. Figure 1 .

[0032] Figure 4 This is a schematic diagram of the detection in Example 2. Figure 2 .

[0033] Figure 5 This is a schematic diagram of the verification results for Example 3.

[0034] Figure 6 This is a schematic diagram of the verification results for Example 4. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, in the following descriptions, well-known structures and technologies are omitted to avoid unnecessarily obscuring the concept of the invention.

[0036] The purpose of this invention is to provide a primer set and kit for detecting respiratory syncytial virus (RSV) types A and B using LAMP technology. The primers provided by this invention have good specificity and can accurately detect RSV-A and RSV-B. The technical solution of this invention is achieved through the following steps:

[0037] 3.1 Primer set

[0038] 3.1.1 By querying the gene sequences of the above pathogens in GeneBank, the gene sequences from various sources were compared and analyzed using MEGA software. Appropriate target fragments were selected for the next stage of primer design based on the comparison.

[0039] (1) The target genes of each pathogen were introduced into the primer design software. The DNA sequences with similar Tm values ​​of F1c and B1c, similar Tm values ​​of F3 / B3 / F2 / B2, and Tm values ​​of F1c and B1c greater than Tm values ​​of F3 / B3 / F2 / B2, and absolute values ​​of 5'dG and 3'dG less than 4 were selected as candidate primers.

[0040] (2) Primer synthesis: The designed primer sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd. for later use.

[0041] (3) Primer confirmation: After dissolving the synthesized primers, primer screening was performed to finally obtain the high-specificity and sensitive primer set required for preparing the microfluidic chip of the present invention. The screened primers were dried and placed in the reaction cell of the microfluidic chip.

[0042] The isothermal amplification detection primer set provided by this invention includes outer primers F3 and B3, inner primers FIP and BIP, and a loop primer LB, the nucleotide sequences of which are shown below:

[0043]

[0044] 3.2 Sample Types and Processing

[0045] Synthesized plasmids were used for sensitivity verification; products obtained from nucleic acid extraction of respiratory specimens such as nasopharyngeal swabs, bronchoalveolar lavage fluid, and sputum were used to verify the primers and kits against real samples; viral RNA extraction kits were used to extract RNA, following the ShuoShi nucleic acid extraction method (nucleic acid extraction or purification reagents, viral nucleic acid extraction kit, magnetic bead method, catalog number SDK60104, ShuoShi, China).

[0046] 3.3 Components of the reagent kit

[0047] 3.3.1 Microfluidic Chips

[0048] By integrating multiple steps such as sample amplification and detection through an integrated microfluidic detection chip, and by combining the equipment's zoned heating system, microchannel system, and active centrifugation force, sample detection is automated, achieving advantages such as high detection throughput, convenient operation, short detection time, and low contamination.

[0049] 3.3.2 Spotting process: Microfluidic chip spotting is used. Small air bubbles are removed from the prepared primer set, and then the sample is spotted using a needle chip spotting instrument: each needle is pre-sprayed to balance the pressure difference inside and outside the spotting needle; then the prepared primer set is spotted into the chip reaction well in multiple drops at a dose of 2µL per person.

[0050] 3.3.3 Drying process: Spread the microfluidic chip with the above-mentioned constant temperature amplification reaction solution evenly and place it in a vacuum drying oven for 10-30 minutes at room temperature and vacuum ≤100Pa, with an ambient humidity of 40-60%. Finally, attach a backing film to the back of the chip and seal it in a sealing bag.

[0051] 3.3.4 Isothermal amplification premix: The amplification system contains 10mM dNTP, 5% BSA, 5M MgSO4, 100μM fluorescent dye, and 5M betaine. The reverse transcriptase is Bst DNA polymerase with a concentration of 8U / μL and an amplification concentration of 1.5-3U / μL.

[0052] 3.4 Testing Procedures

[0053] Transfer 32 μL and 8 μL of the isothermal amplification premix to the sample wells of the chip, seal tightly with the sealing film, place in the instrument, and react at 60-65 °C for 30 min.

[0054]

[0055] 3.5 Result Judgment and Analysis

[0056] The Dt value is calculated, and the results are judged and analyzed, as shown in the example.

[0057] This invention enables rapid and efficient amplification of RSV-A and RSV-B viral nucleic acids under isothermal conditions. It also provides a microfluidic chip integrating this primer combination. The chip employs a multilayer structure design, integrating multiple independent reaction units, each pre-loaded with a LAMP primer set, supporting multiplex detection. This microfluidic chip features high throughput, low reagent consumption, and rapid detection, completing the process from nucleic acid input to result output within 30 minutes; the detection sensitivity for the target virus reaches 10-10. 2 It has copies / uL, high specificity, no cross-reactivity, and low inter-batch coefficient of variation.

[0058] 4. Example 1: Display of RSV-A primer set validation results

[0059] (1) Specificity of the RSV-A primer set: The RSV-A primer set can achieve specific amplification of RSV-A, excluding amplification of RSV-B, influenza A / H1N1, A / H3N2, B / Victoria, and COVID-19, as detailed below. Figure 1 As shown.

[0060] (2) Sensitivity of RSV-A primer set: The RSV-A primer set has a detection sensitivity of 10 for the sample. 2 copies / ml, combined Figure 2 It can be seen that it is in 10 2 A concentration of copies / ml or higher indicates sensitivity.

[0061] (3) The intra-day and inter-day precision of the RSV-A primer set is shown in Table 1 below:

[0062] Table 1:

[0063]

[0064] The intraday CV value of 1.686% indicates that the LAMP primer combination has excellent reproducibility, and the stability of the isothermal equipment and the sample loading method were not problematic within a single day.

[0065] The daytime CV value remains excellent and does not affect qualitative testing (such as clinical judgment of positive / negative), and is also generally acceptable for semi-quantitative tests.

[0066] (4) The intraday precision of the RSV-A primer set at different sample concentrations is shown in Table 2 below:

[0067] Table 2

[0068]

[0069] 5. Example 2: Display of RSV-B primer set validation results

[0070] (1) The specificity curve of the RSV-B primer set is as follows Figure 3 As shown, the RSV-B primer set can achieve specific amplification of RSV-A, excluding amplification of RSV-A, influenza A / H1N1, A / H3N2, B / Victoria, and COVID-19, as detailed below. Figure 3 As shown.

[0071] (2) The sensitivity curve of the RSV-B primer set is as follows Figure 4 As shown, by Figure 4 It can be seen that the RSV-B primer set has a detection sensitivity of 10 for the sample. 2 copies / ml, which is in 10 2 A concentration of copies / ml or higher indicates sensitivity.

[0072] (3) The intra-day and inter-day precision of the RSV-B primer set is shown in Table 3:

[0073] Table 3

[0074]

[0075] In Table 3, the intraday CV of the RSV-B LAMP primer set is 2.098%, and the interday CV is 3.166%, both of which are at an excellent level (usually <5%). The interday CV is slightly higher than the intraday CV, indicating that the primer has excellent reproducibility in the same experiment.

[0076] Compared with the RSV-A primer set (1.686% intraday and 3.091% interday), the two primer sets showed highly consistent performance, indicating that both primer sets are robust in design and stable in performance.

[0077] In practical applications, simply maintaining consistent daytime experimental conditions (such as using the same batch of reagents and avoiding repeated freeze-thaw cycles) can keep daytime variations at extremely low levels and not affect the reliable detection of clinical samples.

[0078] (4) The intraday precision of the RSV-B primer set at different sample concentrations is shown in Table 4:

[0079] Table 4

[0080]

[0081] Table 4 shows that the RSV-B primer set exhibits good stability at low concentrations (CV less than 2.1%).

[0082] 6. Example 3: Validation of suspected RSV-A clinical specimens

[0083] Sixty-nine nasopharyngeal swab specimens suspected of RSV-A infection were validated and compared with Real-time PCR. The results showed that for samples with Cq values ​​less than 30, the concordance rate between the two methods was 97.7%, and the detection time of the microfluidic chip method was significantly shorter than that of the Real-time PCR method. For samples with Cq values ​​greater than or equal to 30, the concordance rate was 92.0%. (Details are as follows...) Figure 5 As shown.

[0084] 7. Example 4: Validation of suspected RSV-B clinical specimens

[0085] Sixty-nine nasopharyngeal swab specimens suspected of RSV-B infection were validated and compared with Real-time PCR. The results showed that for samples with Cq values ​​less than 30, the concordance rate between the two methods was 100%, and the detection time using the microfluidic chip method was significantly shorter than that of the Real-time PCR method. For samples with Cq values ​​greater than or equal to 30, the concordance rate was 88.9%. (Details are as follows...) Figure 6 As shown.

[0086] The foregoing has shown and described the basic principles and main features of the invention and the advantages of the invention.

[0087] Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope. All such changes and modifications fall within the scope of the present invention as claimed, which is defined by the appended claims and their equivalents.

Claims

1. A primer set for detecting respiratory syncytial virus based on isothermal amplification technology, characterized in that, include: Primer set for detecting respiratory syncytial virus type A; And, a primer set for detecting respiratory syncytial virus type B; The primer set used for detecting respiratory syncytial virus type A is shown in seq no. 1-6 below: The primer set used for detecting respiratory syncytial virus type B is shown in seq no. 7-11 below: 。 2. A kit for detecting respiratory syncytial virus based on isothermal amplification technology, characterized in that, The kit includes: A microfluidic chip comprising at least one primer set as described in claim 1; And the amplification system reactants used to amplify the primer set as described in claim 1.

3. The kit for detecting respiratory syncytial virus based on isothermal amplification technology as described in claim 2, characterized in that, The reactants in the amplification system are: 10mM dNTP; 5% BSA; 5M MgSO4; 100μM fluorescent dye; 5M betaine; The reverse transcriptase used in the amplification system is a nucleic acid polymerase.

4. The kit for detecting respiratory syncytial virus based on isothermal amplification technology as described in claim 2, wherein the nucleic acid polymerase is Bst DNA polymerase with a concentration of 8 U / μL and an amplification concentration of 1.5-3 U / μL.

5. An application of a kit for detecting respiratory syncytial virus (RSV) based on isothermal amplification technology, wherein the application involves using a microfluidic chip containing at least one primer set as described in claim 1 in conjunction with isothermal amplification technology to amplify RSV type A viral nucleic acid and / or RSV type B viral nucleic acid.

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