Respiratory syncytial virus nucleic acid detection primer and kit

Abstract

The invention relates to a respiratory syncytial virus nucleic acid detection primer and a kit, and belongs to the technical field of biological detection. The invention aims to solve the technical problems of tedious operation, easy pollution, low sensitivity, easy influence by variants, high false negative risk and the like in the existing RSV detection method. The invention provides an RSV-containing PCR (Polymerase Chain Reaction) detection primer group and a kit, the RSV-containing PCR detection primer group comprises a specific primer and a probe designed aiming at an RSV matrix protein M gene conserved region, and an internal standard primer probe, and the concentration of each component and a reaction system are optimized. The kit has high specificity and sensitivity, RSV RNA as low as 200 copies / mL can be detected, automatic detection is adapted, aerosol pollution and false negative results are effectively avoided, the detection sensitivity to clinical samples reaches 97.12%, the specificity reaches 99.53%, and the detection accuracy and efficiency are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to primers and kits for detecting respiratory syncytial virus nucleic acid. Background Technology

[0002] Respiratory syncytial virus (RSV) is a medium-sized (120-300 nm in diameter) polymorphic enveloped virus belonging to the genus *Orthopneumovirus* of the family Pneumoviridae. It possesses a non-segmented, negatively directed, single-stranded RNA genome (15-16 kb) encoding 11 proteins: two non-structural proteins and nine structural proteins. The two non-structural proteins, NS1 and NS2, play crucial roles in RSV replication and pathogenesis, inhibiting type I interferon activity. Fusion proteins (F), adsorption glycoproteins (G), and small hydrophobic proteins (SH) are hydrophobic transmembrane surface glycoproteins important for infectivity; the most efficient fusion requires the participation of all three surface glycoproteins. G protein mediates the binding of host cells and viral particles, while F protein mediates the fusion of virus and host cells, facilitating viral entry into host cells. Both G and F proteins can stimulate the production of specific antibodies, serving as important viral antigen proteins; SH protein can induce inflammatory responses and inhibit apoptosis. Matrix proteins (M) accumulate on the inner surface of the viral envelope and play an important role in viral morphogenesis. Nucleocapsid proteins (N), phosphoproteins (P), polymeric proteins (L), and matrix proteins (M1-M2) are viral transcription factors.

[0003] RSV can infect people of all ages, but infants, the elderly, and immunocompromised individuals are at high risk. Symptoms mainly include local respiratory symptoms such as cough, nasal congestion, runny nose, and sore throat, as well as systemic symptoms such as fever, fatigue, headache, and muscle aches. The immune response acquired after RSV infection is short-lived, and reinfection is possible, resulting in a severe disease burden and placing a significant economic burden on healthcare systems. Therefore, early diagnosis and identification of RSV infection are crucial for its prevention and control.

[0004] The clinical manifestations of RSV infection are difficult to distinguish from acute respiratory infections caused by other respiratory viruses, requiring differentiation based on etiological testing results. Currently, commonly used RSV detection methods in clinical practice, basic research, and public health laboratories mainly include: virus isolation and culture, antigen detection, antibody detection, and nucleic acid detection.

[0005] Virus isolation and culture is the gold standard for detecting RSV infection, with high specificity. However, virus isolation usually requires the collection of fresh specimens, bedside inoculation, and blind passage for several generations. The operation is cumbersome and time-consuming, and has high requirements for personnel and laboratories. The positive rate is affected by a variety of factors, and it cannot meet the needs of processing a large number of samples at the same time during a virus epidemic. Its clinical application is limited, and it is currently mostly used for experimental research.

[0006] Nucleic acid testing is applicable throughout the entire infection process, from early infection to viral clearance. Currently, the most widely used methods are real-time fluorescent RT-PCR, multiplex PCR, and gene amplification sequencing. Real-time fluorescent RT-PCR is a rapid, specific, and sensitive method that uses fluorescently labeled probes. It can be used for RSV positivity identification and differentiation between A and B subtypes, and is superior to virus isolation methods and serological testing. This type of method is currently the most widely used, but RSV nucleic acid detection is highly sensitive, prone to contamination, and requires high standards for environmental conditions and operator skills. Furthermore, this method is susceptible to the influence of variant strains; if primers or probes are incompatible, false negative results may occur. Summary of the Invention

[0007] In view of this, one objective of the present invention is to provide a primer for detecting respiratory syncytial virus (RSV) nucleic acid, and another objective is to provide a reagent kit for detecting RSV nucleic acid.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a primer for detecting respiratory syncytial virus (RSV) nucleic acid. The primer comprises a primer designed specifically for the conserved region of the RSV matrix protein M gene and a fluorescent probe, with the following composition:

[0010] The sequence of RSV-F is shown in SEQ ID NO: 1;

[0011] The sequence of RSV-R1 is shown in SEQ ID NO: 2;

[0012] The sequence of RSV-R2 is shown in SEQ ID NO: 3;

[0013] The sequence of the fluorescent probe RSV-P is shown in SEQ ID NO: 4; the fluorescent probe RSV-P has a fluorescent excitation group FAM inserted at its 5' end and a fluorescent quencher group BHQ1 inserted at its 3' end;

[0014] Furthermore, a respiratory syncytial virus (RSV) nucleic acid detection kit is provided, comprising: an RSV enzyme mixture containing reverse transcriptase, UNG enzyme, and DNA polymerase; an RSV positive control containing RSV gene fragment pseudovirus and internal standard gene fragment pseudovirus; an RSV negative control containing internal standard gene fragment pseudovirus; and a main reaction solution containing RSV nucleic acid detection primers, internal standard primers IC-F and IC-R, and probe IC-P, as well as Tris-HCl, MgCl2, and dNTPs.

[0015] The internal standard primers IC-F and IC-R and the fluorescent probe IC-P are composed of the following:

[0016] The nucleic acid sequence of IC-F is shown in SEQ ID NO: 5;

[0017] The nucleic acid sequence of IC-R is shown in SEQ ID NO: 6;

[0018] The nucleic acid sequence of IC-P is shown in SEQ ID NO: 7; the fluorescent probe IC-P has a fluorescent excitation group CY5 inserted at the 5' end and a fluorescent quencher group BHQ2 inserted at the 3' end;

[0019] Preferably, the concentrations of RSV-F, RSV-R1, and RSV-R2 are 400 nM; the concentration of RSV-P is 200 nM; the concentrations of IC-F and IC-R are 100 nM; and the concentration of IC-P is 50 nM.

[0020] Preferably, the fluorescence PCR detection program of the kit is: 50℃ for 5 min, 95℃ for 3 min; then 95℃ for 5 sec, 60℃ for 15 sec, for 45 cycles, with fluorescence collected every 60℃, and the detection channels are FAM and CY5 respectively;

[0021] Further: The application of the respiratory syncytial virus (RSV) nucleic acid detection primers in the preparation of RSV nucleic acid detection kits;

[0022] Furthermore, the respiratory syncytial virus nucleic acid detection kit is used in the detection of respiratory syncytial virus.

[0023] The beneficial effects of this invention are as follows:

[0024] The respiratory syncytial virus (RSV) nucleic acid detection primers and kit of this invention are based on real-time fluorescence PCR technology. By real-time detection of the fluorescence information of each cycle product in the PCR amplification reaction, the RSV nucleic acid in the sample can be detected. This kit optimizes the enzyme dosage and primer / probe concentration, enabling specific detection of RSV nucleic acid. It is also adaptable to automated detection, offering simple and rapid operation, effectively avoiding the risks of multi-step transfer operations and aerosol contamination for laboratory personnel, and achieving rapid detection of RSV RNA as low as 180 copies / mL. In specificity verification, no cross-reactivity was observed with other respiratory pathogens, including adenovirus type 7, influenza A (H1N1 (2009)), influenza B, coronavirus 229E, rhinovirus, and Streptococcus pneumoniae, as well as high concentrations of human genomic DNA. This kit achieves faster and more accurate detection, is adaptable to automated detection, and has a sensitivity of 97.12% and a specificity of 99.53% for clinical pharyngeal swab samples, demonstrating good specificity and meeting the application requirements for detection.

[0025] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0027] Figure 1 This is the specific detection result of the kit of the present invention;

[0028] Figure 2 The results are the detection limits of the reagent kit of this invention. Detailed Implementation

[0029] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0030] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0031] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0032] Example 1: Design and Synthesis of Primers and Probes

[0033] Specific primers RSV-F, RSV-R1, and RSV-R2, and a specific fluorescent probe RSV-P were designed for the conserved region of the RSV viral matrix protein M gene. The fluorescent probe RSV-P has a fluorescent excitation group FAM inserted at its 5' end and a fluorescent quencher group BHQ1 inserted at its 3' end. Internal standard primers IC-F and IC-R, and probe IC-P were also designed. The fluorescent probe IC-P has a fluorescent excitation group CY5 inserted at its 5' end and a fluorescent quencher group BHQ2 inserted at its 3' end. The specific sequences are shown in Table 1.

[0034] Table 1 Primer and probe sequence list

[0035]

[0036]

[0037] Example 2: A Respiratory Syncytial Virus Nucleic Acid Fluorescent Probe PCR Detection Kit

[0038] The kit includes:

[0039] (1) RSV PCR master reaction solution;

[0040] (2) RSV enzyme mixture;

[0041] (3) RSV positive control sample;

[0042] (4) RSV negative control sample;

[0043] The RSV PCR master reaction solution contains specific primers and probes, internal standard primers and probes, Tris-HCl, MgCl2, and dNTPs.

[0044] The RSV PCR master reaction solution contains specific primers and fluorescent probes designed for the conserved region of the respiratory syncytial virus matrix protein M gene, as well as internal control gene primers and probes. The sequences of each primer and probe are shown in Table 1. The concentrations of RSV-F, RSV-R1, and RSV-R2 in the RSV PCR master reaction solution are all 400 nM, the concentration of RSV-P in the RSV PCR master reaction solution is 200 nM, the concentrations of IC-F and IC-R in the RSV PCR master reaction solution are all 100 nM, and the concentration of IC-P in the RSV PCR master reaction solution is 50 nM.

[0045] Example 3: Optimization of the Reaction System

[0046] This embodiment progressively optimizes the amount of enzyme added to the enzyme mixture, the primers and probes for specific amplification of the respiratory syncytial virus matrix protein M gene, and the concentrations of primers and probes for specific amplification of the internal control gene.

[0047] (1) Optimization of enzyme dosage: The enzyme mixture mainly contains reverse transcriptase and Taq DNA polymerase, both of which play an important role in the sensitivity and specificity of the PCR reaction. Reverse transcriptase reverse transcribes RNA in the sample into cDNA as the initial template for PCR, and then Taq DNA polymerase is started at high temperature to begin PCR amplification. To confirm the optimal dosage of the enzyme mixture used in this kit, four different test group reaction systems were established (enzyme mixture addition volumes of 1 μL, 2.5 μL, 5 μL, and 7.5 μL, respectively), and positive and negative samples were tested simultaneously using different reaction systems. The test results showed that all positive samples could be amplified normally, and the RSV channel of negative samples showed no non-specific amplification. Under the condition of 5 μL / T enzyme mixture (test group 3), the Ct value of the RSV positive channel was lower than that of the other three test group reaction systems. As the enzyme mixture concentration increased, PCR amplification was inhibited. Therefore, based on the test results and considering the actual production cost, 5 μL / T was chosen as the final dosage of enzyme mixture in the reaction system.

[0048] (2) Optimization of target primer and probe concentration: The concentration of primers and probes is closely related to the detection sensitivity and specificity in real-time fluorescence PCR. Five different test group reaction systems were established (the target primer and probe concentrations were different among the different test groups). The same positive and negative samples were tested simultaneously using different reaction systems to screen out the optimal target primer and probe concentration. Based on the test results and the overall detection effect and cost, the optimal combination of target primer and probe concentrations was RSV-F 400nM, RSV-R1 400nM, RSV-R2 400nM, and RSV-P 200nM.

[0049] (3) Optimization of internal standard primer and probe concentration: Similarly, the concentration of internal standard primers and probes was optimized, and three test group reaction systems with different primer and probe concentrations were established. There was no significant difference in the mean Ct values ​​of the internal standard channel and the syncytial virus channel in the three test groups. Considering that the interference of the internal standard channel on the positive target should be minimized, the amount of internal standard product should be reduced without affecting the detection of the target channel. Therefore, based on the test results and the overall detection effect and cost, the internal standard primer and probe concentration combination was set as IC-F 100nM, IC-R 100nM, and IC-P 50nM.

[0050] (4) The detection system of this kit is as follows:

[0051] 10 μL RSV PCR master reaction solution

[0052] 5 μL RSV enzyme mixture

[0053] 10 μL of RNA sample to be tested (or RSV positive control, or RSV negative control)

[0054] (5) The preferred PCR amplification conditions for this kit are as follows: Place the reaction tubes in a fluorescence PCR instrument in a certain order, 50℃ for 5 min, 95℃ for 3 min; then cycle 45 times at 95℃ for 5 sec and 60℃ for 15 sec. Collect fluorescence every 60℃, and detect the FAM and CY5 channels respectively.

[0055] Example 4: Specificity evaluation of the respiratory syncytial virus nucleic acid detection kit

[0056] The specificity of the reaction system was validated using negative reference materials from the National Reference Catalog for Respiratory Syncytial Virus (RSV) Nucleic Acid Detection Reagent. These reference materials included adenovirus type 7, influenza A (H1N1, 2009), influenza B, coronavirus 229E, rhinovirus, parainfluenza virus (mixed types 2 and 3), human metapneumovirus, Staphylococcus aureus, Neisseria meningitidis, and Streptococcus pneumoniae, numbered N1 to N10 respectively. No positive signals were observed in any of the results, indicating that the reaction system did not provide non-specific amplification for other common respiratory pathogens and exhibited good specificity.

[0057] Example 5: Sensitivity evaluation of the respiratory syncytial virus nucleic acid detection kit

[0058] The sensitivity of the reaction system was validated using the detection limit reference standard L from the National Reference Catalogue for Respiratory Syncytial Virus Nucleic Acid Detection Reagent. The National Detection Limit Reference Standard was diluted to a concentration of 200 copies / mL according to the instructions for use. The results are shown in Table 2 below:

[0059] Table 2 Sensitivity Detection Results

[0060]

[0061]

[0062] The results showed that, after 20 repeated tests, the reaction system was able to detect the national minimum detection limit reference standard L at a concentration of 200 copies / mL, and the performance met the testing requirements.

[0063] Example 6: Detection of clinical samples using the kit.

[0064] A comparative study design was used, employing a method that compared the kit with similar products already on the market. Clinical trials were conducted at three clinical facilities, testing a total of 533 oropharyngeal swab samples. The positive concordance rate with the control kit was 97.12%, the negative concordance rate was 99.53%, and the overall concordance rate was 99.06%, indicating that the kit of this invention has high detection accuracy. The results are shown in Table 3 below:

[0065] Table 3. Results of clinical trials for respiratory syncytial virus (RSV)

[0066]

[0067] Example 7: Optimization of reagent kit usage procedures and analytical methods

[0068] This example demonstrates the usage steps and analysis methods of the optimized kit. Details are as follows:

[0069] (1) Sample preparation

[0070] Extract RNA from the sample to be tested. The extracted RNA sample can be used for testing immediately. If it is not to be tested immediately after extraction, it can be stored at -70℃ or below for later use.

[0071] (2) Preparation of reaction system

[0072] Place the RSV PCR master reaction solution and RSV enzyme mixture on ice or at 2℃-8℃ until thawed, then vortex to mix and centrifuge at 2000 rpm for 10 seconds. Calculate the number of reaction reagents required [n = number of samples + 2 (number of controls)].

[0073] System repackaging:

[0074] Dispense 10 μL of RSV PCR master reaction solution and 5 μL of RSV enzyme mixture into PCR tubes suitable for the fluorescence PCR instrument.

[0075] Sample testing:

[0076] Add 10 μL each of the RNA sample to be tested, negative control, and positive control extracted in step (1) to the PCR tube containing the system, with a final volume of 25 μL / tube. Tighten the cap, vortex to mix, and then centrifuge briefly at low speed to transfer to the PCR amplification area.

[0077] (3) On-machine testing

[0078] Place the reaction tube obtained in step (2) into a fluorescent PCR detector and perform the detection according to the following procedure. The program parameters are set as shown in the table below:

[0079]

[0080] Note: Fluorescence is collected at 60℃, and the detection channels are FAM and CY5.

[0081] (4) Results Analysis

[0082] After the reaction is completed, the instrument automatically saves the results. If the negative control FAM channel has no Ct value or Ct > 40, and the CY5 channel Ct value is ≤ 35 and has a typical amplification curve, and the positive control FAM and CY5 channels have Ct values ​​≤ 35 and have a typical amplification curve, the experiment is considered valid; otherwise, the results are considered invalid.

[0083] If the Ct value of the FAM channel is ≤40 and there is a typical amplification curve, then the respiratory syncytial virus is considered positive.

[0084] If FAM > 40 or there is no Ct value, and the Ct value of the CY5 channel is ≤ 40 and there is a typical amplification curve, then the respiratory syncytial virus is considered negative.

[0085] If FAM > 40 or there is no Ct value, or if the Ct value of the CY5 channel is > 40 or there is no Ct value, then the test result of this sample is invalid and resampling is required for testing.

[0086] In summary, this invention discloses a fluorescent probe PCR detection primer and kit for respiratory syncytial virus (RSV) nucleic acid. The kit optimizes enzyme dosage, target primer / probe concentration, and internal standard primer / probe concentration, enabling specific detection of RSV nucleic acid, including RSV types A and B. It is also adaptable to automated detection, offering simple and time-saving operation, effectively avoiding contamination from transfer procedures and PCR products, preventing false negatives, ensuring accurate results, and achieving rapid detection of RSV RNA down to 200 copies / mL. The kit demonstrates a sensitivity of 97.12% and a specificity of 99.53% for detecting pharyngeal swab samples.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Primers for respiratory syncytial virus nucleic acid detection, characterized in that, The primers comprise primers and fluorescent probes designed specifically for the conserved region of the respiratory syncytial virus matrix protein M gene, and are composed as follows: The sequence of RSV-F is shown in SEQ ID NO: 1; The sequence of RSV-R1 is shown in SEQ ID NO: 2; The sequence of RSV-R2 is shown in SEQ ID NO: 3; The sequence of the fluorescent probe RSV-P is shown in SEQ ID NO: 4; the fluorescent probe RSV-P has a fluorescent excitation group FAM inserted at its 5' end and a fluorescent quencher group BHQ1 inserted at its 3' end.

2. A respiratory syncytial virus nucleic acid detection kit, characterized in that, The kit includes: RSV enzyme mixture; RSV positive control; RSV negative control; master reaction solution containing the respiratory syncytial virus nucleic acid detection primers as described in claim 1, internal standard primers IC-F and IC-R and probe IC-P, and also contains Tris-HCl, MgCl2 and dNTP.

3. The reagent kit according to claim 2, characterized in that: The concentrations of RSV-F, RSV-R1, and RSV-R2 are 400 nM; the concentration of RSV-P is 200 nM; the concentrations of IC-F and IC-R are 100 nM; and the concentration of IC-P is 50 nM.

4. The reagent kit according to claim 3, characterized in that: The fluorescence PCR detection program of the kit is as follows: 50℃ for 5 min, 95℃ for 3 min; then 95℃ for 5 sec, 60℃ for 15 sec, for 45 cycles, collecting fluorescence every 60℃, with the detection channels being FAM and CY5.

5. The application of the respiratory syncytial virus nucleic acid detection primers according to claim 1 in the preparation of respiratory syncytial virus nucleic acid detection kits.

6. The application of the respiratory syncytial virus nucleic acid detection kit according to any one of claims 2-4 in the detection of respiratory syncytial virus.

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