Influenza A and B virus nucleic acid detection primer and kit

Abstract

The invention relates to influenza A and B virus nucleic acid detection primers and a kit, and belongs to the technical field of biological detection. The method aims at solving the problems that an existing virus detection method is long in consumed time, low in sensitivity, prone to pollution and incapable of adapting to automation. The primer, the fluorescent probe, the internal standard primer and the probe are designed for specific conserved regions of the influenza A virus and the influenza B virus, and the enzyme dosage, the primer and probe concentration and the like are optimized. The method is adaptive to automatic detection, operation transfer and aerosol pollution are reduced, and operation is simple, convenient and time-saving; the sensitivity of influenza A throat swab sample detection is 100%, the specificity is 99.65%, the sensitivity of influenza B detection is 100%, the specificity is 99.66%, cross reaction is avoided, and rapid and accurate detection is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology detection, and relates to an influenza A and B virus nucleic acid detection primer and kit. BACKGROUND

[0002] Influenza virus belongs to Orthomyxoviridae, is a single-stranded, negative-stranded, segmented RNA virus, and is an envelope virus with a diameter of 80-120 nm. According to the differences in antigenicity of nucleoprotein (NP) and matrix protein (MP), influenza virus can be divided into four virus strains, i.e., type A, type B, type C and type D. Both type A and type B influenza viruses have eight different RNA segments, encoding at least 10-11 proteins, while type C influenza virus lacks the sixth segment. According to the differences in hemagglutinin (HA) and neuraminidase (NA) on the surface of the viral envelope, type A influenza virus can be divided into various subtypes, and 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) have been found. According to the differences in antigenicity and genetic characteristics (mainly based on HA), type B influenza virus is divided into two lineages: B / Victoria / 2 / 87-like lineage (i.e., B / Victoria) and B / Yamagata / 16 / 88-like lineage (i.e., B / Yamagata), and the two lineages are currently co-transmitted globally.

[0003] Influenza virus infection is distributed globally, and appears in the forms of pandemic, small epidemic, outbreak and isolated sporadic cases, and the highly contagious respiratory diseases caused by the virus occur repeatedly every year, among which both type A and type B influenza viruses can infect humans and animals, while type C influenza virus can only infect humans, and is mainly responsible for seasonal epidemics caused by type A and type B influenza viruses, causing serious public health problems. Influenza virus is mainly transmitted through droplets, contact and aerosols, and is more easily transmitted in rooms with high population density and poor ventilation. The main sources of infection include infected patients and asymptomatic carriers, and the main clinical symptoms include acute fever, cough, headache, muscle pain, etc., which are mostly self-limiting, and some patients can develop into severe / critical cases due to complications such as pneumonia or exacerbation of underlying diseases. Human population is generally susceptible to influenza virus, and the elderly, patients with chronic lung disease, heart disease, diabetes, and immunocompromised individuals are at high risk of developing more severe diseases, hospitalization, and even death.

[0004] Influenza viruses have high variability, and can undergo antigenic drift and antigenic shift. Influenza A viruses are prone to replication mismatch during transmission, causing genetic mutations, and are prone to genome "reassortment" during infection of the same host by different subtypes, resulting in high-frequency variation of the virus. This makes traditional virus culture and immunological methods have certain limitations in detecting influenza A viruses, resulting in many patients being unable to determine the infecting pathogen in diagnosis. Therefore, rapid and accurate identification of influenza viruses can reduce the use of unnecessary antiviral drugs, and plays a crucial role in preventing virus epidemics, shortening the course of the disease, reducing the pressure on the medical system, and saving lives.

[0005] The detection methods of viruses mainly include virus isolation culture, immunological diagnosis and molecular biology diagnosis. Among them, isolation culture and immunological diagnosis are the conventional methods for laboratory diagnosis of influenza, and many rapid and specific methods have appeared in molecular biology diagnosis.

[0006] Due to the time-consuming and laborious isolation culture and immunological diagnosis method, in order to improve the detection speed, the nucleic acid of the virus can be directly detected from the clinical specimen (throat swab, nasal swab, nasopharyngeal or tracheal aspirate). Virus nucleic acid detection has the advantages of rapidity, accuracy and high sensitivity, and can make early diagnosis of influenza virus infection, and provide strong technical support for rapid analysis and control of the epidemic. Among them, reverse transcription PCR / real-time quantitative PCR has high sensitivity and specificity, and is the preferred method for rapid diagnosis of respiratory virus infection such as influenza virus and avian influenza virus. Although molecular detection methods (such as PCR) perform well in diagnosis, false negative or false positive results may occur in some cases. In particular, in the early stage of the disease or in the case of low viral load, the sensitivity may be limited. SUMMARY

[0007] Therefore, one of the purposes of the present application is to provide an influenza A and B virus nucleic acid detection primer and kit, and the second purpose of the present application is to provide an influenza A and B virus nucleic acid detection kit.

[0008] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0009] The present application provides an influenza A and B virus nucleic acid detection primer, which comprises an influenza A virus nucleic acid detection primer and a fluorescent probe, and is composed of:

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

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

[0012] The sequence of the fluorescent probe FluA-P is shown in SEQ ID NO: 3; the fluorescent excitation group FAM is inserted at the 5' end of the fluorescent probe FluA-P, and the fluorescent quenching group BHQ1 is inserted at the 3' end;

[0013] The influenza B virus nucleic acid detection primers and the fluorescent probe also comprise the following components:

[0014] The sequence of FluB-F is shown in SEQ ID NO: 4;

[0015] The sequence of FluB-R is shown in SEQ ID NO: 5;

[0016] The sequence of the fluorescent probe FluB-P is shown in SEQ ID NO: 6; the fluorescent excitation group ROX is inserted at the 5' end of the fluorescent probe FluB-P, and the fluorescent quenching group BHQ2 is inserted at the 3' end;

[0017] Further, the present application also provides an influenza A and B virus nucleic acid detection kit, which further comprises: an enzyme mixture containing reverse transcriptase, UNG enzyme, and DNA polymerase; positive control containing influenza A / B virus gene fragment pseudovirus and internal standard gene fragment pseudovirus; negative control containing internal standard gene fragment pseudovirus; main reaction solution containing influenza A and B virus nucleic acid detection primers, containing internal standard primers and probes, and further containing Tris-HCl, MgCl2, and dNTP;

[0018] The internal standard primers IC-F, IC-R, and the fluorescent probe IC-P comprise the following components:

[0019] The nucleic acid sequence of IC-F is shown in SEQ ID NO: 7;

[0020] The nucleic acid sequence of IC-R is shown in SEQ ID NO: 8;

[0021] The nucleic acid sequence of IC-P is shown in SEQ ID NO: 9; the fluorescent excitation group CY5 is inserted at the 5' end of the fluorescent probe IC-P, and the fluorescent quenching group BHQ2 is inserted at the 3' end;

[0022] Preferably, the concentrations of FluA-F, FluA-R, FluB-F, and FluB-R are 400 nM; the concentrations of FluA-P and FluB-P are 200 nM; the concentrations of IC-F and IC-R are 100 nM, and the concentration of IC-P is 50 nM;

[0023] Preferably, the kit fluorescence PCR detection procedure is: 50 DEG C for 5 min, 95 DEG C for 3 min; then according to 95 DEG C for 5 sec, 60 DEG C for 15 sec, cycle 45 times, collect fluorescence at 60 DEG C, and the detection channels are FAM, ROX, CY5 respectively;

[0024] Further, the application of the influenza A and B virus nucleic acid detection primer in the preparation of an influenza A and B virus nucleic acid detection kit.

[0025] Further, the application of the influenza A and B virus nucleic acid detection kit in the detection of influenza A and B virus.

[0026] The application has the following beneficial effects:

[0027] The influenza A and B virus nucleic acid detection primer and kit provided by the application are based on real-time fluorescence PCR technology, and the qualitative analysis of a starting template is realized through the real-time detection of fluorescence information of a product of each cycle in a PCR amplification reaction. The kit optimizes the enzyme dosage, the influenza A and B virus primer probe concentration, and the internal standard primer probe concentration, can be adapted to automatic detection, reduces the transfer operation and aerosol pollution risk in the experimental process, is simple and time-saving to operate, has good sensitivity and specificity, has a sensitivity of 100% and a specificity of 99.65% for an influenza A virus throat swab sample, has a sensitivity of 100% and a specificity of 99.66% for an influenza B virus throat swab sample, and realizes rapid and accurate detection of influenza A and B virus RNA. In the specificity verification, there is no cross reaction with other related pathogens, including meningococcus, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, rubella virus, mumps virus, respiratory adenovirus (type 3), respiratory adenovirus (type 7), respiratory syncytial virus B, parainfluenza virus 2, and high-concentration human genomic DNA. The application can realize more rapid and high-sensitivity detection of influenza A and B virus RNA, can be adapted to automatic detection, and meets the application requirements of detection.

[0028] Other advantages, objects, and features of the application will be apparent from the following specification and appended claims, and from the foregoing description. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to make the objectives, technical solutions, and advantages of the application clearer, the following will describe the preferred embodiments of the application in detail with reference to the drawings, in which:

[0030] Figure 1 The specificity test results of the kit of the application;

[0031] Figure 2 The minimum detection limit of the kit of the present application (H1N1 (2009)) is shown in the following table:

[0032] Figure 3 The minimum detection limit of the kit of the present application (Victoria) is shown in the following table. DETAILED DESCRIPTION

[0033] Other advantages and benefits of the present application will become apparent to those skilled in the art upon consideration of the disclosure, or can be learned by practice of the application. The present application can be realized and achieved by means of the structures and combinations described in this specification and claims. Various modifications and changes in the specification and claims can be made by those skilled in the art without departing from the spirit and scope of the application. It should be noted that the drawings provided in the following examples are only used to illustrate the basic concept of the present application, and the features in the following examples and embodiments can be combined with each other without conflict.

[0034] The drawings are only used for illustrative purposes, and the representation is only a schematic diagram, not a physical diagram, and should not be understood as a limitation of the present application. In order to better illustrate the embodiments of the present application, some components in the drawings may be omitted, enlarged or reduced, and do not represent the actual size of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions in the drawings may be omitted.

[0035] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components; in the description of the present application, it should be understood that the orientation or position relationship indicated by the terms "upper", "lower", "left", "right", "front", "back" and the like is based on the orientation or position relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore the terms describing the position relationship in the drawings are only used for illustrative purposes, and cannot be understood as a limitation of the present application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0036] Design and synthesis of primers and probes in Example 1

[0037] The specific primers FluA-F, FluA-R, FluB-F, FluB-R and the specific fluorescent probes FluA-P, FluB-P are designed in the specific conserved sequence region of the influenza A / B virus, the fluorescent excitation group FAM is inserted into the 5' end of the fluorescent probe FluA-P, the fluorescent quenching group BHQ1 is inserted into the 3' end of the fluorescent probe FluA-P, the fluorescent excitation group ROX is inserted into the 5' end of the fluorescent probe FluB-P, the fluorescent quenching group BHQ2 is inserted into the 3' end of the fluorescent probe FluB-P, the internal standard primers IC-F, IC-R and the probe IC-P, the fluorescent excitation group CY5 is inserted into the 5' end of the fluorescent probe IC-P, and the fluorescent quenching group BHQ2 is inserted into the 3' end of the fluorescent probe IC-P. The specific sequences are shown in Table 1:

[0038] Table 1 primer and probe sequence list

[0039]

[0040]

[0041] Example 2 influenza A / B virus nucleic acid fluorescent probe PCR detection kit

[0042] The kit includes:

[0043] (1) PCR master reaction solution;

[0044] (2) enzyme mixture;

[0045] (3) positive control;

[0046] (4) negative control.

[0047] The PCR master reaction solution contains specific primers and probes, internal standard primers and probes, Tris-HCl, MgCl2 and dNTP.

[0048] The enzyme addition amount in the reaction solution is 5 μL / T, the PCR master reaction solution contains primers and fluorescently labeled probes designed in the specific conserved region of influenza A / B virus, internal standard gene primers and probes, and the sequences of the primers and probes are shown in Table 1. The concentration of FluA-F and FluA-R in the PCR master reaction solution is 400 nM, the concentration of FluB-F and FluB-R in the PCR master reaction solution is 400 nM, the concentration of FluA-P and FluB-P in the PCR master reaction solution is 200 nM, and the concentration of IC-F, IC-R and IC-P in the PCR master reaction solution is 100 nM, 100 nM and 50 nM, respectively.

[0049] Example 3 reaction system optimization

[0050] The enzyme addition amount in the enzyme mixture was optimized, and the specific amplification primers and probes of the specific conserved region of influenza A / B virus and the internal standard gene were optimized.

[0051] (1) Enzyme dosage optimization: Four different test group reaction systems (different enzyme mixture addition amounts in different test groups) were established, and related influenza A / B positive samples and negative samples were tested simultaneously using different reaction systems. The results showed that all positive samples could be normally amplified and no non-specific amplification was observed in negative samples. Under the condition of test group 3 (enzyme mixture 5 μL / T), the Ct values of each pathogen positive channel were lower than those of test group 1 and test group 2, and the PCR amplification did not improve with the increase of enzyme mixture concentration. Based on the test results and actual production cost, 5 μL / T was selected as the optimal addition amount of enzyme mixture in the reaction system.

[0052] (2) Influenza A / B primer probe concentration optimization: Five different test group reaction systems (different primer probe concentrations of target in different test groups) were established, and the same influenza A / B positive samples were tested simultaneously using different reaction systems. The test results showed that the Ct values of the influenza A test group 3 (FluA-F 400 nM, FluA-R 400 nM, FluA-P 200 nM) did not significantly improve, and the Ct average difference of influenza B test group 1 to test group 5 was less than 0.5, with no significant difference. Based on the test results, performance and cost, the optimal primer probe concentration combination of influenza A / B was determined as FluA-F / FluB-F 400 nM, FluA-R / FluB-R 400 nM, FluA-P / FluB-P 200 nM.

[0053] (3) Internal standard primer probe concentration optimization: The internal standard primer probe concentration was optimized in the same way. Three different test group reaction systems (different primer probe concentrations of internal standard in different test groups) were established for influenza A / B positive samples and negative samples, and the corresponding pathogen channel Ct values and internal standard channel Ct values were tested and compared. The group test results showed that the endogenous gene Ct values could be normally detected after adjusting the concentration of internal standard primer probe, and different concentrations of internal standard primer probe had no effect on the detection of pathogen channel. Therefore, according to the principle of minimizing non-pathogen primer probe, the concentration combination of test group 1 was selected as the optimal concentration of internal standard primer, i.e. IC-F 100 nM, IC-R 100 nM, IC-P 50 nM.

[0054] (4) The detection system of the kit is:

[0055] PCR master reaction solution 10 μL

[0056] Enzyme mixture 5 μL

[0057] Sample RNA to be detected (or positive quality control, or negative quality control) 10 μL

[0058] (5) The preferred PCR amplification conditions of the kit are as follows: the reaction tube is placed in the fluorescent PCR instrument in a certain order, 50°C for 5 min, 95°C for 3 min; then 95°C for 5 sec, 60°C for 15 sec, for 45 cycles. The fluorescence is collected at 60°C, and the detection channels are FAM, ROX, and CY5, respectively.

[0059] Example 4 Specificity evaluation of the influenza A / B virus nucleic acid detection kit

[0060] The specificity of the reaction system was verified using the negative reference of the second generation of national reference of influenza A and B, which contains meningococcal, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, rubella virus, mumps virus, respiratory adenovirus (type 3), respiratory adenovirus (type 7), respiratory syncytial virus type B, and parainfluenza virus type 2, numbered NC01-NC10. No positive signal was found, indicating that the reaction system has no non-specific amplification to common respiratory pathogens, and has good specificity.

[0061] Example 5 Sensitivity evaluation of the influenza A / B virus nucleic acid detection kit

[0062] The detection limit reference of the second generation of national reference of influenza A and B was used to verify the reaction system, which contains 5 subtypes of influenza B Victoria, influenza B Yamagata, influenza A H1N1, influenza A H3N2, and influenza A H1N1 (2009). The national detection limit reference was diluted to 200 copies / mL concentration according to the requirements of the instruction manual for detection, and the results are shown in Table 2.

[0063] Table 2 Sensitivity detection results

[0064]

[0065]

[0066] The results show that the reaction system can detect the national minimum detection limit reference L1-L5 with a concentration of 200 copies / mL, and the performance meets the testing requirements.

[0067] Example 6 Detection of clinical samples by the kit

[0068] The present embodiment includes 392 cases of influenza A / B virus throat swab samples. The kit detection results show that the positive coincidence rate of the kit for detecting influenza A throat swab samples is 100%, the negative coincidence rate is 99.65%, and the total coincidence rate is 99.74%; the positive coincidence rate of detecting influenza B throat swab samples is 100%, the negative coincidence rate is 99.66%, and the total coincidence rate is 99.74%, which shows that the kit has high detection accuracy. The results are shown in Tables 3 and 4 as follows:

[0069] Table 3 Influenza A clinical test results

[0070]

[0071] Table 4 Influenza B clinical test results

[0072]

[0073] Example 7 Exploration and optimization of kit use steps and analysis method

[0074] The kit use steps and analysis method are as follows:

[0075] (1) Sample preparation

[0076] Extract the RNA of the sample to be detected. The extracted RNA sample can be immediately used for detection, or can be stored at -70°C or below for standby use if not detected immediately after extraction.

[0077] (2) Preparation of reaction system

[0078] Place the PCR master reaction solution and enzyme mixture on ice or at 2-8°C, melt and mix well, and centrifuge at 2000 rpm for 10 sec. Calculate the number of reaction reagents required [n = sample number + 2 (number of control samples)].

[0079] System dispensing:

[0080] According to the addition amount of 10 μL of PCR master reaction solution and 5 μL of enzyme mixture, dispense into PCR tubes suitable for fluorescence PCR instrument.

[0081] Sample detection:

[0082] Add 10 μL of the sample RNA extracted in step (1), negative quality control, and positive quality control to the PCR tube containing the system, respectively, with a final volume of 25 μL / tube. Tighten the tube cap, mix well, and centrifuge momentarily at low speed, and then transfer to the PCR amplification area.

[0083] (3) Machine detection

[0084] Put the reaction tube obtained in step (2) into a fluorescence PCR detector, and detect on machine according to the following program, and the program parameters are set as shown in the following table:

[0085]

[0086] Note: collect fluorescence at 60 DEG C, and the detection channels are FAM, ROX and CY5.

[0087] (4) result analysis

[0088] After the reaction is completed, the instrument automatically saves the results, the Ct value of the negative control FAM and ROX channel is all no or Ct>40, the Ct value of the CY5 channel is less than or equal to 35, and there is a typical amplification curve, at the same time, the Ct value of the positive control FAM, ROX and CY5 channels is less than or equal to 35, and there is a typical amplification curve, the experiment is established, otherwise the result is invalid;

[0089] 1) the amplification curve of the sample to be detected is a typical S-shaped curve, and has the following conditions, and is judged as positive.

[0090] passage Ct value Result interpretation FAM Ct < 40 Influenza A virus positive ROX Ct < 40 Influenza B virus positive

[0091] 2) the sample to be detected has the following conditions, and is judged as negative.

[0092]

[0093] 3) the sample to be detected has the following conditions, and is judged as invalid, and needs to be re-sampled for detection.

[0094]

[0095]

[0096] In summary, the application discloses an influenza A / B virus nucleic acid fluorescence probe PCR detection kit, the kit optimizes the enzyme dosage, the influenza A / B primer probe concentration and the internal standard primer probe concentration, can be adapted to automatic detection, reduces the transfer operation and aerosol pollution risk in the experimental process, is simple and time-saving to operate, realizes rapid and accurate detection of influenza A / B virus RNA, and has good sensitivity and specificity, the sensitivity of detecting influenza A pharyngeal swab samples is 100%, and the specificity is 99.65%, the sensitivity of detecting influenza B pharyngeal swab samples is 100%, and the specificity is 99.66%.

[0097] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions, and all should be covered in the scope of the claims of the present application.

Claims

1. An influenza A and B virus nucleic acid detection primer characterized by, The primer comprises an influenza A virus nucleic acid detection primer and a fluorescent probe, and is composed of the following: The sequence of FluA-F is shown in SEQ ID NO: 1; The sequence of FluA-R is shown in SEQ ID NO: 2; The sequence of the fluorescent probe FluA-P is shown in SEQ ID NO: 3; the 5' end of the fluorescent probe FluA-P is inserted with a fluorescence excitation group FAM, and the 3' end is inserted with a fluorescence quenching group BHQ1; The primer comprises an influenza A virus nucleic acid detection primer and a fluorescent probe, and is composed of the following: The sequence of FluB-F is shown in SEQ ID NO: 4; The sequence of FluB-R is shown in SEQ ID NO: 5; The sequence of the fluorescent probe FluB-P is shown in SEQ ID NO: 6; the 5' end of the fluorescent probe FluB-P is inserted with a fluorescence excitation group ROX, and the 3' end is inserted with a fluorescence quenching group BHQ2.

2. An influenza A and B virus nucleic acid detection kit, characterized by, The kit comprises: an enzyme mixture; a positive control; a negative control; a main reaction solution: containing the primer of claim 1, further containing internal standard primer IC-F and IC-R and probe IC-P, further containing Tris-HCl, MgCl2 and dNTP.

3. The kit of claim 2, wherein: The concentration of FluA-F, FluA-R, FluB-F and FluB-R is 400nM; the concentration of FluA-P and FluB-P is 200nM; the concentration of IC-F and IC-R is 100nM, and the concentration of IC-P is 50nM.

4. The kit of claim 3, wherein: The fluorescence PCR detection procedure of the kit is: 50℃ for 5min, 95℃ for 3min; then according to 95℃ for 5sec, 60℃ for 15sec, cycle 45 times, collect fluorescence at 60℃, and the detection channels are FAM, ROX and CY5 respectively.

5. The application of the influenza A and B virus nucleic acid detection primer in claim 1 in the preparation of an influenza A and B virus nucleic acid detection kit.

6. The application of the influenza A and B virus nucleic acid detection kit in any of claims 2-4 in the detection of influenza A and B virus.

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