Analysis method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR (quantitative polymerase chain reaction) method

Abstract

The invention relates to an analysis method for detecting human immunodeficiency virus type 1 by a reverse transcription qPCR (quantitative polymerase chain reaction) method, which is characterized in that single-stranded RNA (ribonucleic acid) containing a specific RNA (ribonucleic acid) sequence (env gene) of H IV (human immunodeficiency virus) 1 is used as a standard substance, the sequence refers to an NCBI database JN024499.1, and gene drugs designed aiming at the env gene and modified by the env gene are used for detecting the human immunodeficiency virus type 1 by the reverse transcription qPCR method. The method can effectively distinguish whether the component contained in the medicine is the residual HIV carrier or the harmful wild HIV virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunodeficiency virus analysis methods, and particularly relates to an analysis method for detecting human immunodeficiency virus type 1 using a reverse transcription qPCR method. Background Art

[0002] AIDS is the final stage of HIV infection. HIV infection can lead to various opportunistic infections and tumors. These complications can affect a person's health and well-being, and can even be life-threatening. Therefore, HIV can cause panic.

[0003] The human immunodeficiency virus type 1 (HIV 1) genome contains three structural genes: gag, pol, and env. Most existing methods detect gag or pol, while residual HIV 1 vectors in gene drugs produced using HIV 1 transgenes often carry gag and pol. The modified HIV 1 vectors are inactivated and harmless, while wild HIV 1 viruses are harmful to the human body. During release testing, current methods often cannot distinguish whether the drug contains residual HIV 1 vectors or wild HIV 1 viruses. Summary of the Invention

[0004] The purpose of the present invention is to provide a simple and rationally designed reverse transcription qPCR method for detecting human immunodeficiency virus type 1 in order to solve the above problems.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0006] The reverse transcription qPCR method for detecting human immunodeficiency virus type 1 comprises the following steps:

[0007] S1, preparation of experimental consumables and experimental equipment;

[0008] S2, preparation of primer and probe stock solutions;

[0009] S3, synthetic HIV 1 RNA standard stock solution;

[0010] S4, evaluation and validation of HIV 1 synthetic RNA working stocks;

[0011] S5, prepare samples and quality controls;

[0012] S6, preparation of interference control group (IC);

[0013] S7, sample and quality control nucleic acid extraction;

[0014] S8, reagent preparation;

[0015] S9, sample lysis;

[0016] S10, binding and washing RNA / DNA;

[0017] S11, elution of RNA / DNA;

[0018] S12, detection sample loading;

[0019] S13, setting up a reaction plate;

[0020] S14, instrumental data analysis;

[0021] S15, data calculation.

[0022] Furthermore, in step S1, the experimental consumables include enzyme-free water, anhydrous ethanol, carrier RNA, DPBS and RNaseAway enzyme-free water, and the experimental equipment includes qPCR instrument, biological safety cabinet, vortexer, mini centrifuge, micro desktop centrifuge, desktop centrifuge, dry box, -80℃ refrigerator, 24-well PCR low-temperature indicator ice box, PCR-Cooler 0.2mL, electric pipette, single-channel pipette, 1000μL tip, 200μL tip, 20μL tip, 10μL tip, 1.5mL sterile centrifuge tube, 10mL pipette, 15mL centrifuge tube, MicroAmp TM 96-well reaction plates and MicroAmp TM Optical adhesive sealing film.

[0023] Furthermore, in step S2, the primers are in the form of DNA dry powder, and enzyme-free water is used to redissolve the primers to a storage stock solution with a concentration of 100 μM and store them for a long term.

[0024] Furthermore, in step S3, the standard stock solution needs to be introduced into a vortex centrifuge tube to ensure sufficient resolubilization to obtain the RNA initial stock solution, and the standard is quantified using digital PCR to obtain the concentration of the RNA initial stock solution, and the standard copy number needs to be doubled when converted into the number of virus particles.

[0025] Furthermore, in step S5, sample preparation must be separated from the QuantStudio™ 5 Real-Time PCR System and reagent preparation to avoid cross-contamination.

[0026] Furthermore, in step S9, the sample was lysed using a 1.5 mL centrifuge tube, vortexed for 15 seconds, and incubated at room temperature for 10 minutes.

[0027] Furthermore, in step S13, there are three markers on the reaction plate, "U" represents an unknown sample, "S" represents a standard, and "N" represents a negative control.

[0028] Furthermore, in step S15, the intermediate value is calculated to 4 significant digits, the calculation result is calculated to 3 significant digits, non-zero numbers are always valid, the relative standard deviation and recovery rate <100% are retained to 3 significant digits, the relative standard deviation and recovery rate ≥100% are retained to 4 significant digits, if the number in the smallest position is greater than or equal to 5, then the number is added +1, and any number after the number becomes zero, the determination coefficient (R2) is retained to 3 significant digits, and the fourth significant digit is discarded.

[0029] The beneficial effects of the present invention are as follows: in this method, single-stranded RNA containing HIV-1-specific RNA sequence (env gene) is used as a standard, the sequence is referenced to NCBI database JN024499.1, and is designed for the env gene. For gene drugs modified with the env gene, it can effectively distinguish whether the components contained in the drug are residual HIV-1 vectors or harmful wild HIV-1 viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0031] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0032] Example

[0033] like Figure 1 As shown, the reverse transcription qPCR method for detecting human immunodeficiency virus type 1 includes the following steps:

[0034] S1. Preparation of experimental consumables and experimental equipment; experimental consumables include enzyme-free water, anhydrous ethanol, carrier RNA, DPBS and RNase Away enzyme-free water. The suppliers and product numbers of experimental consumables are shown in Table 1 below:

[0035] Table 1

[0036]

[0037] The experimental equipment includes qPCR instrument, biological safety cabinet, vortex instrument, mini centrifuge, micro desktop centrifuge, desktop centrifuge, dry block thermostat, -80℃ refrigerator, 24-well PCR low temperature indicator ice box, PCR-Cooler 0.2mL, electric pipette, single-channel pipette, 1000μL pipette tip, 200μL pipette tip, 20μL pipette tip, 10μL pipette tip, 1.5mL sterile centrifuge tube, 10mL pipette, 15mL centrifuge tube, MicroAmpTM 96-well reaction plates and MicroAmp TM The brands and models of optical adhesive sealing films and experimental equipment are shown in Table 2 below:

[0038] Table 2

[0039]

[0040] The low temperature indicator ice box must be pre-cooled at -20°C for at least 2 hours to ensure that the sample placed in it remains at room temperature for 1 hour;

[0041] S2, preparation of primer and probe storage solutions; information of standards, primers, and probes is shown in Table 3 below:

[0042] Table 3

[0043]

[0044] Primer and probe powders should be stored at -10°C to -25°C or -60°C to -80°C before reconstitution. 100 μM fluorescently labeled TaqMan probes should be stored at -60°C to -80°C in the dark. Synthetic HIV-1 ssRNA powder should be stored at -60°C to -80°C before reconstitution. The reconstituted RNA is used as the standard for this method.

[0045] The primer and probe sequences are as follows:

[0046] EBPTPF0077:5'-AGAGTCCTGGCTGTAGAAAGA-3'

[0047] EBPTPR0078:5'-AGATGAGTTTTCCAGAGCAAC-3'

[0048] EBPTPB0079:5'-FAM-CCTAAAGGATCAACAGCTCCTG-3'-TAMRA

[0049] Primers are received as dry DNA powder. Redissolve the primers in enzyme-free water to a 100 μM stock solution for long-term storage.

[0050] Confirm the molecular weight of the primer in nmoles by adding 10 times the nmoles value in μL of enzyme-free water. For example, for 100 nmol of primer, add 1,000 μL of enzyme-free water to the primer powder to obtain a 100 μM stock solution.

[0051] Do not use the primer storage solution to directly dilute the primer working solution for qPCR detection; the primer storage solution should be stored at -60℃ to -80℃;

[0052] Then, dilute the 100 μM primer stock solution to a 10 μM primer working solution (50×) by adding enzyme-free water at a ratio of 1:10. The working solution can be directly used for RT-qPCR detection. The primer working solution is stored at -60°C to -80°C, and the probe primer is reconstituted to a 100 μM stock solution for long-term storage.

[0053] S3, synthetic HIV 1 RNA standard stock solution; designed and synthesized HIV 1 RNA EBPTRS0026 sequence:

[0054]

[0055] The tube containing HIV-1 synthetic RNA was then centrifuged briefly to allow all lyophilized RNA to settle to the bottom of the tube. An appropriate volume of DB1 (160 ng / μL carrier RNA working solution) was added to the original tube (preparing a 1 μg / μL carrier RNA stock solution and a 160 ng / μL carrier RNA working solution). The solution was incubated at room temperature for approximately 15 minutes to ensure full dissolution of the lyophilized RNA powder. The tube was then vortexed to ensure complete reconstitution, resulting in the RNA initial stock solution. The standard was quantified using digital PCR to determine the concentration of the RNA initial stock solution. Because HIV-1 is a virus with two single-stranded RNA genomes, the standard copy number required a 2-fold correction to convert the number of viral particles. The RNA initial stock solution was diluted with DB1 to 2 x 108 genome copies / μL as the HIV-1 RNA standard RNA working stock solution. The tube was vortexed to ensure thorough mixing. The RNA working stock solution was aliquoted into multiple tubes (approximately 50 μL / tube), with one tube used for each experiment. Store at -60°C to -80°C.

[0056] S4. Evaluate and validate the HIV 1 synthetic RNA working stock solution; the evaluation method is as follows:

[0057] Thaw an aliquot of HIV 1 synthetic RNA standard RNA working solution (2x108 genome copies / μL) for confirmation experiments.

[0058] The quality of the HIV 1 synthetic RNA standard should be confirmed by testing the standard curve and no-template control (three replicates for each sample);

[0059] S5, prepare samples and quality control; to prevent cross contamination, sample preparation should be separate from the QuantStudio™ 5 Real-Time PCR System and reagent preparation. The quality control steps are as follows:

[0060] Add an appropriate amount of DB1 to a 1.5 mL centrifuge tube as diluent. Label the tube with the appropriate sample name.

[0061] For example, if you want to dilute 50 μL of sample 10 times, you can add 450 μL of DB1.

[0062] Prepare a 1.5 mL centrifuge tube, mark it with "NEC", and add 200 μL of DB1 to the tube for later use.

[0063] Prepare one NEC sample per 96-well plate assay;

[0064] Prepare 1.5 mL centrifuge tubes labeled "PEC" for positive quality control and add 180 μL of DB1 to each tube for later use.

[0065] If the concentration of the standard used as a positive control is greater than 2x103 gc / μL, prepare a series of 1.5 mL centrifuge tubes with an appropriate volume of DB1 for "standard dilution";

[0066] To the sample centrifuge tube in the above step, add an appropriate amount of sample to complete the sample dilution; for example, to dilute 50 μL of sample (the sample addition process must be performed in the designated PCR positive area) 10 times, add 450 μL of DB1 and vortex each sample for 20 seconds to mix;

[0067] If the concentration of the virus stock solution or standard used as a positive quality control is greater than 2x103gc / μL, add the virus stock solution to the "Standard Dilution" centrifuge tube and dilute it to obtain a virus dilution solution with a final concentration of 2x103gc / μL. Vortex mix thoroughly at each dilution step;

[0068] Then add 20 μL of HIV1 synthetic RNA standard with a concentration of 2x103 gc / μL to the centrifuge tube marked "PEC" and finally mix by vortexing the centrifuge tube marked "PEC";

[0069] S6, preparation of interference control group (IC); the interference control group should be prepared in the designated PCR positive area, and the preparation process is as follows:

[0070] Prepare a 1.5 mL centrifuge tube, mark it with "IC", and add 180.0 μL of sample or diluted sample to the tube for later use;

[0071] Add 20.0 μL of the 2 x 105 gc / μL Std 3 standard dilution prepared in step 9.4.3.4 to the centrifuge tube labeled "IC"; vortex to mix thoroughly to obtain a 2 x 104 gc / μL interference control group.

[0072] S7, sample and quality control nucleic acid extraction; RNA extraction procedures include all samples as well as NEC, PEC, and IC;

[0073] S8, reagent preparation; the reagent preparation comprises the following steps: adding Buffer AVE to the tube containing the lyophilized carrier RNA to obtain a 1 μg / μL solution (i.e., adding 310 μg of lyophilized carrier RNA to 310 μL of Buffer AVE or adding 1550 μg of lyophilized carrier RNA to 1550 μL of Buffer AVE; check the contents on the tube label);

[0074] Vortex for 15 seconds to completely dissolve the carrier RNA, aliquot into conveniently sized aliquots, and store at -30°C to -15°C. The carrier RNA aliquots should be frozen and thawed no more than three times. Check Buffer AVL for precipitates. If necessary, incubate at 80°C until the precipitate dissolves. Calculate the volume of Buffer AVL carrier RNA mixture required for each batch of samples. For larger numbers of samples, use the following sample calculation:

[0075] nx 0.56mL=y mL

[0076] y mL x 10 μL / mL = z μL

[0077] in:

[0078] n = number of samples processed simultaneously

[0079] y = calculated volume of Buffer AVL

[0080] z = Volume of carrier RNA-Buffer AVE added to Buffer AVL

[0081] Buffer AW1 is supplied as a concentrate. Before first use, add 25 mL of anhydrous ethanol as indicated on the bottle. Buffer AW1 is stable for one year when stored tightly closed at room temperature, but only until the expiration date of the kit. Buffer AW2 is supplied as a concentrate. Before first use, add 30 mL of anhydrous ethanol to the Buffer AW2 concentrate as indicated on the bottle. Buffer AW2 is also stable for one year when stored tightly closed at room temperature, but only until the expiration date of the kit.

[0082] S9, sample lysis; for sample lysis, first pipette 560 μL of the pre-prepared Buffer AVL carrier RNA mixture into a 1.5 mL centrifuge tube, then add 140 μL of sample to the Buffer AVL carrier RNA mixture centrifuge tube, vortex mix for 15 seconds, and finally incubate at room temperature for 10 minutes;

[0083] S10, binding and washing RNA / DNA; first, centrifuge to remove droplets inside the lid, then add 560 μL of anhydrous ethanol to the sample, vortex mix for 15 seconds, and then centrifuge briefly to remove droplets inside the lid;

[0084] Carefully apply 630 μL of the solution from the previous step to the QIAamp Mini column (in a 2 mL collection tube), avoiding wetting the rim. Cap the tube and centrifuge at 6000 x g for 1 minute. Place the QIAamp Mini column in a clean 2 mL collection tube. Discard the collection tube containing the filtrate and carefully open the QIAamp Mini column. Repeat the above steps until all the lysed sample has been applied to the column. Then, open the QIAamp Mini column and add 500 μL of Buffer AW1. Cap the tube and centrifuge at 6000 x g for 1 minute. Place the QIAamp Mini column in a clean 2 mL collection tube (provided with the kit) and discard the tube containing the filtrate. Next, open the QIAamp Mini column, add 500 μL of Buffer AW2, cap the tube, and centrifuge at 20,000 x g for 3 minutes. Finally, place the QIAamp Mini column in a new 1.5 mL centrifuge tube, discard the old collection tube with the filtrate, and centrifuge at 20,000 x g for 1 minute.

[0085] S11, elute RNA / DNA; place the QIAamp Mini column in a clean 1.5 mL centrifuge tube; discard the old collection tube containing the filtrate; carefully open the QIAamp Mini column and add 60 μL of Buffer AVE equilibrated at room temperature; cap the tube, incubate at room temperature for 1 minute, and then centrifuge at 6000 x g for 1 minute; then open the QIAamp Mini column and add 80 μL of Buffer AVE equilibrated at room temperature; cap the tube, incubate at room temperature for 1 minute, and then centrifuge at 6000 x g for 1 minute;

[0086] After elution is complete, prepare the RT-qPCR assay mix. The NTC and carrier RNA should be prepared in the designated PCR-negative area. Determine the total number of reactions for preparing the assay mix and multiply this number by 1.1.

[0087] example:

[0088] Number of reactions (N) 24 × 1.1 = 26.4 (Y)

[0089] Prepare the RT-qPCR test premix system as shown in Table 4 below:

[0090] Table 4

[0091]

[0092] Then prepare carrier RNA (1 μg / μL) as follows:

[0093] 1. Add 1350 μL of enzyme-free water to the carrier RNA lyophilized powder tube to prepare a 1 μg / μL carrier RNA stock solution and vortex to mix thoroughly.

[0094] 2. Incubate at room temperature for about 15 minutes to ensure that the carrier RNA is fully dissolved. Aliquot into 5 tubes of 270 μL / tube.

[0095] 3. 1 μg / μL carrier RNA stock solution can be stored at -60℃ to -80℃ for up to 3 years from the time of reconstitution;

[0096] 4. Dilute the 1 μg / μL carrier RNA stock solution with enzyme-free water to a final concentration of 160 ng / μL carrier RNA working solution (DB1) for use as a dilution for RNA standards.

[0097] Then, prepare a standard curve in the PCR-positive area. The preparation steps are as follows: First, take a tube of HIV 1 RNA working solution (2x108 genomic copies / μL) from the refrigerator and place it at room temperature for reconstitution. Then, prepare eight 1.5mL centrifuge tubes and label them Std 0 to Std 7. Then, add 180μL of DB1 to Std 0 to Std 6. Add 160μL of DB1 to Std 7, and add 20μL of HIV 1 RNA working solution (2x108 genomic copies / μL) to Std 0. Next, vortex the RNA solution in Std 0 and extract 20μL of the liquid in Std 0 to Std 1. Finally, vortex and mix thoroughly, and then continue to prepare Std 2 to Std 7 with a 10-fold serial dilution. The specific preparation is shown in Table 5 below:

[0098] Table 5

[0099]

[0100] S12, sample loading; in the titer detection experiment, sample loading is performed according to Table 6 below:

[0101] Table 6

[0102]

[0103] Among them, Std is HIV 1 synthetic RNA standard; PEC is positive quality control, HIV 1 synthetic RNA standard is added to DB1; NEC is negative quality control; NTC is no template control, IC is sample plus HIV synthetic RNA standard as interference control. The operation steps are as follows:

[0104] First, add 15 μL of RT-qPCR detection mixture to each PCR plate reaction well;

[0105] Then add 10 μL of the corresponding standards, samples, and quality controls to the corresponding PCR plate reaction wells, and add 10 μL of DB1 to the PCR plate reaction wells corresponding to the NTC.

[0106] Next, seal all standards, samples, internal quality control, positive quality control, negative quality control, and no-template control wells with lids to ensure airtightness. The PCR plate must be completely sealed with a sealing film.

[0107] Finally, centrifuge the PCR plate at 1000 x g for 1 min at 4°C to ensure that all the mixed solution is at the bottom of the wells;

[0108] And set the qPCR instrument parameters. When setting, open QuantStudio TM Design & Analysis software, PCR system operation according to BU-EQ-CB-022;

[0109] Then click "Create New Experiment" to enter the main interface of the experiment setting;

[0110] Next, set the properties and set the instrument type to "QuantStudio TM 5System”, the module type is set to “96-Well 0.2-mL Block”, the experiment type is set to “Standard Curve”, and the reagent type is set to “ In the Reagents section, set the speed mode to "Standard"; enter 25 (μL) in the Volume field and set the PCR reaction steps as shown in Table 7 below:

[0111] Table 7

[0112]

[0113] S13, set up the reaction plate; first, in the Target window, enter the HIV 1 name, select the reporter gene FAM and the quencher gene None;

[0114] Then in Sample Name, enter the names of different standards, test samples, and quality controls;

[0115] Next, select the well on the reaction plate on the right, and choose between "Target" and "Sample" in the middle. Check "√" to select the target gene and sample number for the well.

[0116] There are three markers on the reaction plate: "U" represents unknown samples, "S" represents standard samples, and "N" represents negative quality control samples. Select the correct marker after adding the targeted gene and sample.

[0117] After completing the setup, place the loaded 96-well plate on the module of the PCR instrument, ensuring that well A1 is in the upper left corner. After placing it, click "RUN" to start the program; save first, then start the run. After the run, the analysis button will be clickable and a message box will indicate whether the run was successful. When the run is complete, click "OK" and remove the PCR plate from the PCR instrument and discard it.

[0118] S14, instrument data analysis, first set the threshold line, (the threshold line needs to be adjusted to the appropriate value according to different software), confirm that all baselines are set to automatic mode. Analyze and save the data, and the data file is named automatically by the software. After the analysis is completed, close the main software program first, then close QuantStudio TM 5. Turn off the instrument and close the computer. Finally, print, sign and date all the analysis data. The printed data will be attached to the tester's record.

[0119] S15, data calculation; the average value calculation formula is: Where n is the number of test samples, x1, x2, x3, ..., x n Detection value for each sample;

[0120] The deviation calculation formula is:

[0121] Where n is the number of samples tested, x i Detect the value for a sample. is the sample average value AVG; the relative standard deviation is calculated as

[0122] The recovery rate calculation formula is:

[0123] The formula for calculating the amplification efficiency is: Eff = 10 -1 / slope-1, where slope is the slope of the standard curve;

[0124] The formula for calculating the human immunodeficiency virus type 1 (HIV 1) genome copy number is: Concentration (gc / mL) = Q × D, where Q is the sample detection value and D is the sample dilution factor;

[0125] When performing numerical calculations, the calculation rules are as follows: the intermediate values ​​are calculated to 4 significant figures, the results are calculated to 3 significant figures, non-zero figures are always valid, the relative standard deviation and recovery rate <100% are retained to 3 significant figures, the relative standard deviation and recovery rate ≥100% are retained to 4 significant figures, if the digit in the smallest position is greater than or equal to 5, then the digit is added +1, and any digits after the digit become zero, the determination coefficient (R2) is retained to 3 significant figures, and the fourth significant figure is discarded;

[0126] The suitability criteria for the PCR detection system were as follows: the standard curve determination coefficient (R2) must be ≥0.990. If it does not meet the criteria, retest;

[0127] The amplification efficiency of the standard curve must be no greater than 110.0% and no less than 90.0%. If it does not meet the judgment criteria, retest;

[0128] The relative standard deviation between the back-calculated concentrations of the three replicate wells of the standard curve must be ≤30.0%. If it does not meet the judgment criteria, retest;

[0129] The average Ct value of NTC must be greater than the point of 2gc / μL in the standard curve. If it does not meet the judgment criteria, retest;

[0130] The relative standard deviation of the concentration between the three replicate wells of the sample to be tested must be ≤20.0%. If it does not meet the judgment standard, retest;

[0131] The spike recovery of PEC must be no greater than 150.0% and no less than 50.0%;

[0132] HAV RT-qPCR result report of sample

[0133] Interference control group acceptance criteria

[0134] The relative standard deviation (%RSDIC) of the concentrations between the three replicate wells of the interference control group must be ≤20.0%. If it does not meet the judgment criteria, retesting is required;

[0135] When the unspiked test sample result is greater than or equal to the LLOQ 20gc / μL, the recovery concentration of the interference control group is calculated according to the following formula:

[0136] IC recovery concentration (gc / μL) = IC spike average concentration – sample average concentration × 0.9;

[0137] The spike recovery (PrIC) of the interference control group must be between 50.0% and 150.0%, inclusive. If the PrIC does not meet the criteria, the sample must be diluted and retested.

[0138] IC spike recovery (PrIC) = IC recovery concentration (gc / μL) ÷ 2 × 104 gc / μL × 100%;

[0139] The relative standard deviation of the concentration between the three replicate wells of the sample to be tested must be ≤20.0%. If it does not meet the judgment standard, retesting is required;

[0140] The relative standard deviation is not calculated when the average Ct value (Cts) of the sample to be tested is greater than the Ct value of the standard curve Std 8, 2gc / μL (CtStd8), or when there is no amplification (UND) in the sample to be tested, the relative standard deviation is reported as: <LOD(2gc / μL)”;

[0141] If the sample to be tested is reported as a diluted sample, the sample result must be multiplied by the corresponding dilution factor, and the LOD must also be multiplied by the dilution factor accordingly. For example, the LOD of a sample diluted 100 times should be 200gc / μL, and the measurement should be reported accordingly as " <LOD(200gc / μL)”。

[0142] This absolute quantitative TaqMan probe-based qPCR method offers excellent specificity and a sensitivity of up to 2 gc / μL. An interference control group is included within the method to simultaneously eliminate interference from undesired samples, making it suitable for a wide range of samples. Both the test sample and the control are amplified using TaqMan probes. Because HIV-1 is a double-stranded RNA virus, the first step in quantifying HIV-1 genome titers using qPCR is the complete reverse transcription of the RNA molecule into cDNA. This step uses reverse transcriptase to synthesize the genomic RNA into cDNA. This synthesized cDNA serves as the template for absolute quantitative qPCR. Through the continuous formation of double-stranded DNA, it is further amplified in the subsequent qPCR reaction. Specific oligonucleotide primers are designed to bind to both strands of the HIV-1 template. During PCR, the primers are repeatedly extended to form complementary DNA strands containing the specific sequence recognized by the oligonucleotide probe. The probe consists of an oligonucleotide and a 5'-reporter dye, typically FAM (6-carboxyfluorescein), covalently linked to the 5' end of the oligonucleotide. Each reporter gene is quenched at the 3' end of the probe by a second dye, typically TAMRA (6-carboxytetramethylrhodamine). A dilution of a standard control RNA carrying a specific HIV-1 RNA sequence is measured in parallel with the test material. Single-stranded RNA containing an HIV-1-specific RNA sequence (env gene) is used as a standard. During PCR, if the target sequence is present, the probe binds specifically between the forward and reverse primer sites, triggering cleavage of the reporter gene and emitting a fluorescent signal. The fluorescence intensity generated by amplification in the test sample is calculated using linear regression of the standard curve to determine the corresponding concentration. Single-stranded RNA containing an HIV-1-specific RNA sequence (env gene) is used as a standard. The sequence is referenced to NCBI database JN024499.1 and is designed specifically for the env gene. For genetically modified env gene drugs, this can effectively distinguish whether the drug contains residual HIV-1 vector or harmful wild-type HIV-1 virus.

[0143] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR, characterized in that: The following steps are involved: S1, preparation of experimental consumables and experimental equipment; S2, preparation of primer and probe stock solutions; S3, synthetic HIV 1RNA standard stock solution; S4, evaluation and validation of HIV 1 synthetic RNA working stocks; S5, prepare samples and quality controls; S6, preparation of interference control group (IC); S7, sample and quality control nucleic acid extraction; S8, reagent preparation; S9, sample lysis; S10, binding and washing RNA / DNA; S11, elution of RNA / DNA; S12, detection sample loading; S13, setting up the reaction plate; S14, instrumental data analysis; S15, data calculation.

2. The method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR according to claim 1, characterized in that: In step S1, the experimental consumables include enzyme-free water, anhydrous ethanol, carrier RNA, DPBS and RNase Away enzyme-free water. The experimental equipment includes qPCR instrument, biological safety cabinet, vortexer, mini centrifuge, micro desktop centrifuge, desktop centrifuge, dry box, -80℃ refrigerator, 24-well PCR low-temperature indicator ice box, PCR-Cooler 0.2mL, electric pipette, single-channel pipette, 1000μL pipette tip, 200μL pipette tip, 20μL pipette tip, 10μL pipette tip, 1.5mL sterile centrifuge tube, 10mL pipette, 15mL centrifuge tube, MicroAmp TM 96-well reaction plates and MicroAmp TM Optical adhesive sealing film.

3. The reverse transcription qPCR analysis method for detecting human immunodeficiency virus type 1 according to claim 2, characterized in that: In step S2, the primers are in the form of DNA dry powder. Enzyme-free water is used to redissolve the primers to a storage solution with a concentration of 100 μM and store them long-term.

4. The method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR according to claim 3, characterized in that: In step S3, the standard stock solution needs to be introduced into a vortex centrifuge tube to ensure sufficient resolubilization to obtain the RNA initial stock solution, and the standard is quantified using digital PCR to obtain the concentration of the RNA initial stock solution. The copy number of the standard needs to be doubled when converted into the number of viral particles.

5. The method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR according to claim 1, characterized in that: In step S5, sample preparation must be separate from the QuantStudio™ 5 Real-Time PCR System and reagent preparation to avoid cross-contamination.

6. The method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR according to claim 1, characterized in that: In step S9, the sample was lysed in a 1.5 mL centrifuge tube, vortexed for 15 seconds, and incubated at room temperature for 10 minutes.

7. The method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR according to claim 1, characterized in that: In step S13, there are three markers on the reaction plate: "U" represents unknown samples, "S" represents standard samples, and "N" represents negative quality control samples.

8. The method for detecting human immunodeficiency virus type 1 by reverse transcription qPCR according to claim 1, characterized in that: In step S15, the intermediate value is calculated to 4 significant digits, the calculation result is calculated to 3 significant digits, non-zero digits are always valid, the relative standard deviation and recovery rate <100% are retained to 3 significant digits, the relative standard deviation and recovery rate ≥100% are retained to 4 significant digits, if the digit in the smallest position is greater than or equal to 5, the digit is added +1, and any digit after the digit becomes zero, the determination coefficient (R2) is retained to 3 significant digits, and the fourth significant digit is discarded.

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