Primer probe combination, kit and application for vaccine strain identification

Abstract

The present invention discloses a primer-probe combination, a kit and an application for vaccine strain identification, and belongs to the field of biological detection technology. The primer-probe combination is a specific primer-probe combination 1 and / or a specific primer-probe combination 2; the specific primer-probe combination 1 includes a forward primer with a sequence as shown in SEQ ID NO.1, a reverse primer with a sequence as shown in SEQ ID NO.2, and a probe with a sequence as shown in SEQ ID NO.5; the specific primer-probe combination 2 includes a forward primer with a sequence as shown in SEQ ID NO.6, a reverse primer with a sequence as shown in SEQ ID NO.7, and a probe with a sequence as shown in SEQ ID NO.11. The present invention innovatively combines specific primers and probes for identifying human rabies vaccine strain PV, and utilizes one-step droplet digital PCR technology to solve the complexity of reverse transcription operation in the traditional method and the problem of easy contamination, showing specificity and sensitivity, improving detection efficiency, and ensuring the effectiveness and safety of human rabies vaccine in clinical medication.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a primer-probe combination, a kit and an application for vaccine strain identification. Background Art

[0002] Rabies is a serious zoonosis caused by the rabies virus, which severely attacks the central nervous system and causes progressively worsening, even fatal, inflammation of the brain and spinal cord. The rabies virus is an RNA virus that first replicates in the bitten muscle tissue after entering the human body, then invades the nervous system and enters the brain, further damaging the respiratory and circulatory systems. Rabies has a long incubation period, and once it occurs, the mortality rate is close to 100%. Therefore, ensuring the effectiveness and safety of human rabies vaccines is of great significance in reducing the mortality rate of rabies.

[0003] The rabies virus strain used in a large number of human rabies vaccines produced in my country is PV. The variation of rabies virus strains will affect the process stability and product consistency of vaccine production, and may also lead to differences in biological characteristics, pathogenicity, etc., which in turn affect the protective effect of the vaccine. Therefore, accurate and efficient detection of vaccine strains of different generations is crucial to improve the quality control level of vaccines, ensure the safety of vaccines and the best protective effect. PV RNA encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA polymerase (L). Among them, G protein is the most important antigen of rabies virus, which can effectively stimulate the proliferation of specific helper T cells and cytotoxic T cells, and induce the body to produce specific antibodies. The N gene is relatively constant in the PV genome, with fewer point mutations, so it provides better quantitative resolution and is the main basis for genotyping.

[0004] One-step digital PCR can directly count the number of RNA molecules without separate reverse transcription amplification, achieving absolute quantification of the starting sample. Compared with qPCR, digital PCR does not rely on the cycle threshold (Ct) of the amplification curve or the standard curve, and the results are more accurate and reliable, with higher sensitivity, and has more prominent advantages in detecting rare mutations in complex backgrounds and analyzing small differences in expression.

[0005] The method for identifying strains of rabies vaccines for human use included in the third volume of the "Chinese Pharmacopoeia" uses a neutralization test in the mouse brain. This method requires the sacrifice of animals and is not conducive to animal protection. Traditional ordinary RT-PCR testing has the risk of cross-contamination and is complicated to operate. Real-time fluorescence quantitative RT-PCR needs to rely on standard samples when quantifying the virus and is easily affected by the standard samples. Summary of the invention

[0006] In response to the problems existing in the prior art, the present invention provides a primer-probe combination, a kit and an application for vaccine strain identification, which improves the speed, specificity, accuracy and sensitivity of detection and can play an important role in strain identification.

[0007] The present invention is achieved through the following technical solutions:

[0008] A primer-probe combination for vaccine strain identification, wherein the primer-probe combination is a specific primer-probe combination 1 and a specific primer-probe combination 2;

[0009] The specific primer-probe combination 1 comprises a forward primer with a sequence as shown in SEQ ID NO.1, a reverse primer with a sequence as shown in SEQ ID NO.2, and a probe with a sequence as shown in SEQ ID NO.5;

[0010] The specific primer-probe combination 2 comprises a forward primer having a sequence as shown in SEQ ID NO.6, a reverse primer having a sequence as shown in SEQ ID NO.7, and a probe having a sequence as shown in SEQ ID NO.11.

[0011] Furthermore, the probe with a sequence as shown in SEQ ID NO.5 and the probe with a sequence as shown in SEQ ID NO.11 are labeled with a fluorescent group FAM at the 5' end and a quenching group at the 3' end.

[0012] Furthermore, the fluorescent group is FAM; the quenching group is VIC.

[0013] The present invention discloses a kit for identifying vaccine strains, comprising a specific primer-probe combination 1 and a specific primer-probe combination 2.

[0014] In the present invention, the primer-probe combination for vaccine strain identification is used in the preparation of a rabies vaccine strain detection reagent for human use.

[0015] Furthermore, the method for using the human rabies vaccine strain detection reagent comprises the following steps:

[0016] (1) Obtaining RNA template of the sample to be tested;

[0017] (2) Prepare digital PCR reaction system;

[0018] (3) Perform digital PCR amplification on the digital PCR reaction system prepared in step (2), read the droplet signal, and determine whether the sample to be tested is a human rabies vaccine strain.

[0019] Furthermore, according to the droplet signal in step (3), if the fluorescence of the droplet signal is detected by both sets of specific primer-probe combinations, it is proved that the sample to be tested is a human rabies vaccine strain; the copy number of rabies virus DNA in the droplet PCR reaction system is calculated according to the Poisson distribution principle to obtain the quantitative result of the corresponding virus.

[0020] Furthermore, the composition of the PCR reaction system described in step (2) is: Absolute Q™ 1-Step RT-dPCR Master Mix (4X) 10.0 μL, 10 μmol / L forward primer 0.5 μL, 10 μmol / L reverse primer 0.5 μL, 10 μmol / L probe 1 μL, RNA template 2.0 μL, and RNA-free water to 40 μL.

[0021] Furthermore, the digital PCR amplification program described in step (3) is: 55°C for 10 min; 96°C for 10 min; 96°C for 5 s, 60°C for 10 s, 40 cycles; stored at 4°C, with a heating and cooling rate of 2°C / s.

[0022] Beneficial Effects

[0023] The present invention innovatively combines specific primers and probes for identifying human rabies vaccine strain PV, and utilizes one-step droplet digital PCR technology to solve the complexity and easy contamination problems of the traditional method of first performing reverse transcription, thereby achieving accurate detection of human rabies vaccine strains. This method not only exhibits excellent specificity, but also has extremely high sensitivity, can achieve absolute quantification of viral load, eliminates the cumbersome standard curve construction steps in the traditional method, greatly simplifies the operation process, improves detection efficiency, and helps to significantly improve the quality control level of human rabies vaccines, ensuring the effectiveness and safety of human rabies vaccines in clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a graph showing the amplification results of digital PCR detection using different primer-probe combinations;

[0025] Figure 2 This is the sensitivity detection standard curve diagram of Example 2;

[0026] Figure 3 This is a digital PCR test result diagram of specific primer-probe combination 1 in passaged PV strains;

[0027] Figure 4 This is a graph showing the results of digital PCR detection of specific primer-probe combination 2 in passaged PV strains. DETAILED DESCRIPTION

[0028] The following description further describes the specific details of the present invention for a full understanding of the present invention. The terms used in the present invention are only used to illustrate the advantages and features of the present invention and are not intended to limit the present invention.

[0029] Unless otherwise defined, all professional and scientific terms used in the present invention have the same meanings as those understood by technicians in the technical field of the present invention. Unless otherwise specified, the drugs or reagents used in the present invention are used in accordance with the product instructions or the conventional methods of use in the field. The technical solution of the present invention is further described according to the drawings and specific embodiments of the specification.

[0030] Example 1

[0031] Design of primer-probe combination:

[0032] 1. Primers and probe sequences were designed based on the N gene and G gene sequences of the human rabies vaccine strain PV. The designed primers and probe sequences are shown in Tables 1 and 2 respectively:

[0033] Table 1 Primer and probe sequences designed for the N gene of human rabies vaccine strain PV

[0034]

[0035] Table 2 Primer and probe sequences designed for the G gene of human rabies vaccine strain PV

[0036]

[0037] 2. Primer screening

[0038] (1) Reagents and consumables

[0039] Viral RNA extraction kit (Tiangen, batch number: DP315); TaqMan™ Fast Virus 1-step MasterMix (Thermo, batch number: 4444434); Fluorescence quantitative PCR instrument (Thermo; Quantstudio 5);

[0040] (2) Extract sample RNA according to the instructions of the RNA extraction kit;

[0041] (3) Primer probe combination targeting the N gene of human rabies vaccine strain PV: F1R1P1, F1R2 P1, F1R3P1;

[0042] Primer probe combination targeting the G gene of human rabies vaccine strain PV: F2R4P2, F3R5P2, F2R6P2;

[0043] The 5'-end fluorescent reporter of the probe is FAM, and the 3'-end quencher is VIC;

[0044] (4) Prepare the digital PCR (RT-qPCR) reaction system: RT-qPCR system formula: 4×TaqMan® FastVirus 1-Step Master Mix 5μL, forward primer 500nmol, reverse primer 500nmol, probe 200nmol, template 5μL, RT-PCR Grade Water to 20μL;

[0045] (5) Perform digital PCR amplification. The PCR amplification program is: 50°C, 5 min; 95°C, 20 sec; 95°C, 15 sec; 60°C, 60 sec to collect fluorescence, a total of 40 cycles; end the reaction; then read the droplet signal. The digital PCR detection amplification results of different primer probe combinations are shown in the figure. Figure 1 As shown by Figure 1 It can be seen that F1R1P1 and F2R4P2 are the best primer probe combinations for detecting human rabies vaccine strain PV, which are recorded as specific primer probe combination 1 and specific primer probe combination 2 respectively. The use of these two sets of primers in combination makes the identification results more accurate and avoids false positives caused by amplification of a single primer combination.

[0046] Example 2

[0047] Establishment of a method for identifying rabies vaccine strains for human use

[0048] (1) Reagents and consumables: Viral RNA extraction kit (Tiangen, batch number: DP315), Absolute Q™ 1-StepRT-dPCR Master Mix (4X) (Thermo, batch number: A55146), digital PCR instrument Applied Biosystems™ QuantStudio™ Absolute Q™ (Thermo), droplet generation card slot and gel pad and other related reagents and materials were purchased from Thermo Fisher Scientific, USA;

[0049] (2) Extracting RNA from the sample to be tested using a commercially available kit;

[0050] (3) Prepare a one-step digital PCR (RT-qPCR) reaction system as shown in Table 3 below;

[0051] Table 3 Digital PCR (RT-qPCR) reaction system

[0052] Reagents Addition amount Absolute Q™ 1-Step RT-dPCR Master Mix (4X) 10μL Forward Primer 0.5μL Reverse primer 0.5μL Probe 1μL RNA template 5μL RNAse-free water Make up to 40 μL

[0053] (4) Add the digital PCR reaction system of step (3) to the QS Absolute Q MAP16 chip plate, add 15 μL of separation blocking buffer, and cover the MAP chip plate with 5 chip pads; select the loaded MAP chip column and perform PCR amplification reaction. The conditions of the PCR amplification reaction are: 55°C for 10 min; 96°C for 10 min; 96°C for 5 s, 60°C for 10 s, 40 cycles; store at 4°C, and the heating and cooling rate is 2°C / s;

[0054] (5) Reading fluorescence signals: Use software to directly read and analyze results; automatically calculate the copy number of human rabies vaccine strains in the microdroplet PCR reaction system according to the Poisson distribution principle;

[0055] (6) Result judgment: If both sets of primers amplify droplets with fluorescent signals, the amplified product is positive, otherwise it is negative. The software automatically calculates the number of virus copies in the droplet PCR reaction system according to the Poisson distribution principle to quantify the virus.

[0056] (7) Sensitivity test

[0057] The concentration is 3.01×10 5 copies / μL of PV viral nucleic acid, at a 10-fold ratio (10 0 ~10 5 ) was diluted as a template, and 3 replicates were set for each concentration. The detection was performed according to the established one-step ddPCR method. The logarithm of the sample dilution was used as the horizontal axis (x) and the logarithm of the copy number amplified by the F1R1P1 primer was used as the vertical axis (y) to draw a standard curve. The detection limit was determined and the sensitivity of the detection was evaluated. The logarithm of the sample dilution was used as the horizontal axis (x) and the logarithm of the copy number amplified by the F1R1P1 primer was used as the vertical axis (y) to draw a standard curve. The results are shown in Figure 2. Figure 2 shown by Figure 2 It can be seen that the linear regression equation is y=0.9958x-0.0062, R 2 The value is 0.9998, showing a good linear relationship. 5 The corresponding copy number after 10-fold dilution was 3.14 copies / μL, and the detection limit was 3.14 copies / μL.

[0058] (8) Specificity detection

[0059] The optimized one-step digital PCR method was used to detect nucleic acids extracted from positive samples of PV vaccine strains, CTN-1 vaccine strains, and αG vaccine strains, and added to the optimized ddPCR reaction system for detection to verify the specificity of the established method. The results showed that only the PV vaccine strain viral nucleic acid was positive, and the rest were negative, indicating that the method has good specificity.

[0060] (9) Repeatability test

[0061] The same PV-positive sample was repeatedly tested 10 times using the one-step digital PCR method. The repeatability test results of the F1R1P1 primer amplification are shown in Table 4 below. The coefficient of variation was 2.4%, which was less than 5%. This indicates that this method has good repeatability and the test results are stable and reliable.

[0062] Table 4

[0063]

[0064] (10) Testing of actual samples

[0065] The method of this embodiment was used to test 3 strain samples of each passage for 4 generations. The results showed that the digital PCR test results of the specific primer probe combination 1 in the passaged PV strains were as shown in the figure Figure 3 As shown, the digital PCR detection results of the specific primer probe combination 2 are shown in FIG. Figure 4 shown by Figure 3 and Figure 4 It can be seen that the primer-probe combination and method of the present invention can detect PV vaccine strains of all generations.

[0066] (11) Conclusion

[0067] The design of primer probes directly affects the efficiency and specificity of PCR amplification. Good primer probe design can ensure the efficient conduct of PCR reaction, reduce nonspecific amplification and the formation of primer dimers, and the specificity of the probe can significantly improve the sensitivity of digital PCR, thereby improving the accuracy and reliability of the experiment. The present invention determined a group of specific primer probe groups for identifying human rabies vaccine strains through analytical experiments, and based on this primer probe group, established a one-step digital PCR method for qualitative and quantitative analysis of human rabies vaccine strains. This method has good specificity and high sensitivity, and can directly obtain the number of viral nucleic acid copies from the sample without the need for a separate reverse transcription operation, and does not rely on standard curve quantification, which overcomes the detection defects of traditional methods and provides a new idea for the identification of vaccine strains.

[0068] The above description only illustrates several embodiments of the present invention, and it cannot be understood as limiting the scope of the present invention. It should be pointed out that other persons in the art may make modifications, substitutions, improvements, etc. without departing from the concept and scope of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of the present invention shall be based on the described claims.

Claims

1. Application of a primer-probe combination for vaccine strain identification in the preparation of a human rabies vaccine strain PV detection reagent; The primer-probe combination is a specific primer-probe combination 1 and a specific primer-probe combination 2; The specific primer-probe combination 1 comprises a forward primer with a sequence as shown in SEQ ID NO.1, a reverse primer with a sequence as shown in SEQ ID NO.2, and a probe with a sequence as shown in SEQ ID NO.5; The specific primer-probe combination 2 comprises a forward primer with a sequence as shown in SEQ ID NO.6, a reverse primer with a sequence as shown in SEQ ID NO.7, and a probe with a sequence as shown in SEQ ID NO.11; The probe with the sequence shown in SEQ ID NO.5 and the probe with the sequence shown in SEQ ID NO.11 are labeled with a fluorescent group FAM at the 5' end and a quenching group VIC at the 3' end.

2. The use according to claim 1, characterized in that: The method for using the human rabies vaccine strain PV detection reagent includes the following steps: (1) Obtaining RNA template of the sample to be tested; (2) Prepare digital PCR reaction system; (3) Performing digital PCR amplification on the digital PCR reaction system prepared in step (2), reading the droplet signal, and determining whether the sample to be tested is the human rabies vaccine strain PV.

3. The use according to claim 2, characterized in that: According to the droplet signal in step (3), if the two sets of specific primer probe combinations both detect the fluorescence of the droplet signal, it proves that the sample to be tested is the human rabies vaccine strain PV; the copy number of rabies virus DNA in the droplet PCR reaction system is calculated according to the Poisson distribution principle to obtain the quantitative result of the corresponding virus.

4. The use according to claim 2, characterized in that: The composition of the PCR reaction system described in step (2) is: AbsoluteQ™ 1-Step RT-dPCR Master Mix (4X) 10.0 μL, 10 μmol / L forward primer 0.5 μL, 10 μmol / L reverse primer 0.5 μL, 10 μmol / L probe 1 μL, RNA template 2.0 μL, and RNA-free water to 40 μL.

5. The use according to claim 2, characterized in that: The digital PCR amplification program described in step (3) is: 55°C for 10 min; 96°C for 10 min; 96°C for 5 s, 60°C for 10 s, 40 cycles; stored at 4°C, with a heating and cooling rate of 2°C / s.

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