CN117106976A - A quantitative detection method for human immunodeficiency virus type 2 (HIV-2) nucleic acid - Google Patents

Abstract

The invention belongs to the technical field of nucleic acid detection, and discloses a quantitative detection method of human immunodeficiency virus type 2 (HIV-2) nucleic acid, in particular to a reagent, wherein the reagent comprises a primer group and a probe group, the primer group comprises a primer 1 or a primer 2, and the probe group comprises a probe 1 or a probe 2. The primer probe provided by the invention has high specificity, can ensure that the quantitative nucleic acid detection of human immunodeficiency virus type 2 (HIV-2) can obtain a reliable and stable detection result, and can solve the problem of poor specificity in the prior detection technology. The sensitivity of the invention can reach 20copies/mL, and the method can quantify HIV-2 nucleic acid, and the linear quantification range can reach 20-1.0X10 ⁷ Copies/mL, supplementing the currentThe quantitative technology in the field of HIV-2 detection is empty.

Description

A quantitative detection method for human immunodeficiency virus type 2 (HIV-2) nucleic acid

Technical field

The invention belongs to the technical field of nucleic acid detection, and specifically relates to a quantitative detection method for human immunodeficiency virus type 2 (HIV-2) nucleic acid.

Background technique

There are two main types of human immunodeficiency virus (HIV): HIV-1 and HIV-2. The gene homology at the amino acid level is about 40% to 60%. HIV-1 is the most widespread and infected strain in the world. According to the United Nations Program on HIV/AIDS, more than 79.3 million people have been infected since the first case of HIV-1 infection was discovered. In 2020, 36.3 million people worldwide died from HIV-1-related diseases, and more than 37.7 million people were infected. HIV-2 was first isolated from sex workers and AIDS patients in West Africa in 1985 and then spread to other countries around the world. Currently, HIV-2 infection has spread to other countries in Europe, Asia, and North America. There are approximately 2 million people infected with HIV-2 around the world, most of whom are in West Africa, such as Guinea-Bissau, Gambia, Senegal and other countries. Co-infection with HIV-1 and HIV-2 is common in West Africa, accounting for 0.3% to 1% of HIV infections. Early research suggests that compared with HIV-1, HIV-2 is less pathogenic, spreads less efficiently, and progresses more slowly after infection, with only about 20% to 30% of HIV-2 infections eventually progressing to acquisition. Immunodeficiency Syndrome (AIDS). However, a recent study published in The Lancet HIV suggests that HIV-2 is more pathogenic than previously demonstrated. New research shows that early treatment should be available to all people with HIV, not just those with HIV-1. The study provides the first reliable estimate of the time to death associated with HIV-1 and HIV-2 infection, following 4,900 individuals in a cohort study conducted in Guinea-Bissau from 1990 to 2013. Research shows that people with HIV-2 develop HIV-related infections and AIDS in much the same way as people with HIV-1, although the process slows over time. If left untreated, HIV-2 infection can Resulting in continued risk of HIV transmission, increased mortality, and higher medical management costs. People infected with HIV-1 can take highly active antiretroviral therapy, which can effectively suppress viral replication. Four or five categories of antiviral drugs have been developed, but these drugs are all developed for HIV-1, and not all of them are effective against HIV-2, such as first-line treatment drugs non-nucleoside reverse transcriptase inhibitors and fusion drugs The inhibitor enfuvirtide is very effective against HIV-1 but is naturally resistant to HIV-2. HIV-2 is mainly prevalent in West Africa, where large-scale clinical randomized drug trials are difficult to conduct, and there are few studies on evaluating its therapeutic effects.

At present, more than 10 subtypes of HIV-1 strains have been discovered in my country. The few HIV-2 infections reported in the past were imported cases, all of which had returned from West Africa and had a history of local prostitution. Qiu Maofeng and others from the Chinese Center for Disease Control and Prevention analyzed 16 suspected HIV-2 samples and tested them with Western blot reagents, linear immunoreagents and nucleic acid tests. All 16 cases were HIV-1 infections, not HIV-2 infections. This shows that in domestic HIV testing work, most of the samples with HIV-2 or HIV-1/HIV-2 mixed infection are false positive results.

In 2017, two non-imported HIV-2 infection cases were confirmed in Hunan Province, which were the first local HIV-2 cases reported in my country. Relevant studies show that the HIV-2 infection epidemic has lasted for a long time in Hunan Province, and there are already cases of local transmission. The infected people are concentrated in a certain city, and there is a possibility of cluster infection. Although HIV-2 is less virulent than HIV-1, some infected people can still develop AIDS and even die. Some drugs that can effectively inhibit HIV-1, such as non-nucleoside reverse transcriptase inhibitors, are ineffective in treating HIV-2. There are very few studies and published literature on HIV-2 in the world, and its diagnosis and treatment mostly rely on experience rather than standard diagnosis and treatment plans. However, in my country, the research on the etiology, molecular biology and epidemiology of HIV-2 has always been a blank field, and there are no standardized requirements for detection technology, methods and strategies. Although the "National AIDS Testing Technical Specifications (2020 Revised Edition)" requires the use of antibody confirmation reagents that can distinguish HIV-1 and HIV-2 to diagnose HIV-2, the specifications focus on HIV-1 testing technology. 2 Diagnosis has less space, relatively simple content, and is difficult to operate in specific implementation.

Most of the AIDS serological in vitro diagnostic reagents approved for sale in my country are mixed detection reagents for HIV-1 and HIV-2. In fact, the HIV-2 component involved is only an antigen component synthesized through genetic engineering and cannot be used to diagnose HIV- 2 infection. The nucleic acid supplement test only has qualitative or quantitative reagents for HIV-1, and there are no commercialized nucleic acid quantitative reagents for HIV-2 in China. Moreover, these actual conditions bring great difficulties to the quantitative diagnosis of HIV-2. Therefore, there is an urgent need for an HIV-2 quantitative detection method with high sensitivity, strong specificity, and accurate quantification.

Contents of the invention

The object of the first aspect of the present invention is to provide a reagent.

The second object of the present invention is to provide a kit.

The object of the third aspect of the present invention is to provide the application of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention.

The purpose of the fourth aspect of the present invention is to provide a quantitative detection method for HIV-2 nucleic acid.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

A first aspect of the present invention is to provide a reagent, characterized in that the reagent includes a primer set and a probe set, the primer set includes primer 1 or primer 2, and the probe set includes probe 1 or probe set. Needle 2;

Wherein, the sequence of primer 1 is:

F: 5’-TGGCAGCTTTATTAAGAGGTCT-3’, or the complementary sequence of this sequence;

R: 5’-CAAGCACTGGTGAGAGTCTAGC-3’, or the complementary sequence of this sequence;

The sequence of primer 2 is:

F: 5’-GAGGTTTCTCCAGCACTAGC-3’, or the complementary sequence of this sequence;

R: 5’-GCGGCGACTAGGAGAGAT-3’, or the complementary sequence of this sequence;

The sequence of probe 1 is:

5’-AGAGGCTGGCAGATYGAG-3’, or the complement of this sequence;

The sequence of probe 2 is:

5’-CAGACGGCTCCACGCTT-3’, or the complement of this sequence.

Preferably, both ends of the probe sequence are labeled with fluorescent groups and quenching groups respectively.

Preferably, the fluorescent group is at least one of FAM, HEX, VIC, TET, ROX and CY5.

Preferably, the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

Further preferably, the fluorescent group is connected to the 5' end of the probe.

Further preferably, the quenching group is connected to the 3’ end of the probe.

A second aspect of the present invention is to provide a kit containing the reagent of the first aspect of the present invention.

Preferably, the kit further includes PCR reaction solution, enzyme, quantitative reference material, negative reference material and positive reference material.

Preferably, the enzymes include thermostable DNA polymerase, reverse transcriptase and ribonuclease inhibitors.

Preferably, the PCR reaction solution includes reaction buffer, magnesium ions and deoxyribonucleoside triphosphate.

Preferably, the quantitative reference product includes pseudoviral particles containing the target amplified fragment of HIV-2, and the pseudoviral particles are synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Preferably, the negative reference material and the positive reference material are physiological saline and the above-mentioned artificially synthesized pseudoviral particles containing the HIV-2 target amplification fragment respectively.

The third aspect of the present invention is to provide the application of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention in any one of (1) to (6):

(1) Quantitative detection of HIV-2;

(2) Prepare products for quantitative detection of HIV-2;

(3) Detect whether the sample to be tested is HIV-2;

(4) Prepare whether the sample to be tested is an HIV-2 product;

(5) Detect whether the sample to be tested is infected with HIV-2;

(6) Prepare products for testing whether the sample to be tested is infected with HIV-2;

The above applications are used for non-disease diagnosis and treatment.

The fourth aspect of the present invention is to provide a quantitative detection method of HIV-2 nucleic acid, which is carried out using the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention; the above detection method is used for the diagnosis and treatment of non-diseases.

Preferably, the detection method includes the following steps:

1) Extract nucleic acid from the sample to be tested;

2) Using the nucleic acid and quantitative reference material of step 1) as templates, use the described reagent or the described kit to perform RT-PCR amplification to obtain the Ct value;

3) HIV-2 can be quantitatively detected based on the standard curve fitted to the Ct value and copy number of the quantitative reference product.

Preferably, the RT-PCR amplification reaction system is: PCR reaction solution (including reaction buffer, 3-6nM magnesium ions and 1-4nM deoxyribonucleoside triphosphate (dNTP)), 3-6U enzyme system, 30 ~35nM up/downstream primers, 1~5nM probe and nucleic acid of the sample to be tested/quantitative reference 1-4/negative control/positive control sample nucleic acid.

Preferably, the RT-PCR amplification reaction program in step 2) is: 42~45°C, 15~45min; 94~96°C, 2~10min; 90~95°C 15~30s, 45~65°C 10~ 60s, 32~36 cycles; 70~72℃ 0.5~5min.

Preferably, the qualitative judgment method for the results of the detection method is: on the basis of the establishment of the experiment, when the Ct value of the test sample is less than or equal to 32.0, and the curve has an obvious exponential growth period, the test sample is judged to be positive; when the Ct value of the test sample is not detected The sample Ct value or Ct value is greater than or equal to 32.0, then it is judged as negative.

The beneficial effects of the present invention are:

The primer probe provided by the invention has high specificity, can obtain reliable and stable detection results in quantitative nucleic acid detection of human immunodeficiency virus type 2 (HIV-2), and can solve the problem of poor specificity in existing detection technology.

The kit provided by the invention can effectively detect HIV-2 qualitatively or quantitatively, has high sensitivity, strong specificity, good repeatability, low cost, can detect a large number of samples at the same time, and has no cross-reaction with other pathogens and is sensitive to potential endogenous substances. It has a certain degree of anti-interference and is very suitable for the detection of a large number of clinical samples and epidemic monitoring. At the same time, the reagents in the kit provided by the invention have low infectivity, no potential biological safety hazard components, and are highly safe to use.

The detection method provided by the invention is a detection method for quantitative detection of HIV-2. The sensitivity can reach 20 copies/mL, and the linear quantitative range can reach 20~1.0×10 ⁷ copies/mL, which solves the problem that other detection methods in the same field cannot perform It can solve the problems of quantitative detection and analysis, and at the same time, it can supplement the current vacancies in quantitative technology in the field of HIV-2 detection.

The detection method provided by the invention has successfully quantitatively detected most of the HIV-2 infected plasma samples found in China, has good quantitative performance in various concentration areas, and can output stable amplification curves and accurate quantitative data results. It provides a powerful tool for auxiliary diagnosis, medication selection and efficacy evaluation for HIV-2 infected patients.

Description of drawings

Figure 1 is a specific experimental analysis diagram of the human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method.

Figure 2 is a standard curve diagram of the linear analysis standard in the human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method.

Figure 3 is a standard amplification curve chart of linear analysis in the human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method.

Figure 4 is a graph showing the amplification curve of positive samples detected in the human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method.

Detailed ways

The present invention will be described in detail with reference to specific embodiments, but the scope of the present invention will not be limited.

The materials and reagents used in this example are materials and reagents obtained from commercial sources unless otherwise specified.

Example 1

The present invention designs primer probes for the conserved sequences of the HIV-2 genome LTR region and gag region (reference sequence NCBI registration number: AF082339.1). The technical team designed a total of 12 sets of primer-probe combinations, and finally selected 2 sets of primer-probe sets designed for the LTR region based on the performance experimental results. The specific sequences are shown in Table 1. The probe may be the complementary sequence of the nucleotide sequence of the probe in Table 1. Any group selected can be used to detect human immunodeficiency virus type 2 (HIV-2) nucleic acid quantification and meet the specific requirements of RT-PCR detection. The above nucleotide sequences were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Table 1 RT-PCR amplification primer and probe sequences

The fluorescent group labeled at the 5' end of the probe is FAM, and the quenching group labeled at the 3' end of the probe is BHQ1, which is used to monitor the extraction and amplification process of the target sequence to avoid false negatives.

Example 2

A human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method, including the following steps:

(1) Nucleic acid extraction of samples to be tested: Collect samples from HIV-2 infected persons, and the sample type is serum/plasma sample. The serum sample collection process is as follows: use a collection tube without anticoagulant to draw venous blood, let it sit naturally at room temperature for 1 to 2 hours, wait until the blood clots, centrifuge at 1500 to 3000 rpm for 15 minutes, collect the upper serum, and transfer it to 1.5 mL of frozen Store for later use; the process for collecting plasma samples is as follows: use a collection tube containing EDTA-Na ₂ (disodium ethylenediaminetetraacetate) or sodium citrate anticoagulant to draw venous blood, and immediately invert the collection tube gently for 5 to Mix the anticoagulant and venous blood thoroughly 10 times, centrifuge at 1500-3000rpm for 15 minutes, collect the upper plasma, and transfer it to a 1.5mL cryopreservation tube for later use. Use the nucleic acid extraction or purification reagent of Guangzhou Hailit Biotechnology Co., Ltd. (Cat. No. SUPI-1010-1), process the above serum/plasma sample (300 μL) according to the instructions, and obtain nucleic acid for later use; process the quantitative reference material in the same way 1-4 (pseudoviral particles containing HIV-2 target amplified fragments artificially synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.), negative control (physiological saline) and positive control (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. artificially synthesized pseudoviral particles containing HIV-2 target amplified fragments) samples.

(2) Use the ABI 7500 fluorescence quantitative PCR instrument to perform real-time fluorescence reverse transcription PCR (Real time RT-PCR) to detect the nucleic acid in (1). Specifically, PCR amplification reaction system (100 μL): 44 μL PCR reaction solution (including PCR reaction buffer, magnesium ions (10nM) and deoxyribonucleoside triphosphate (dNTP) (5nM)). The PCR reaction solution was obtained from Guangzhou Hainan University. Lite Biotechnology Co., Ltd. provides 3μL enzyme system (150U), 2.5μL up/downstream primers (1300nM), 0.5μL probe (250nM), 50μL nucleic acid of the sample to be tested/quantitative reference 1-4/negative control /Positive control sample nucleic acid. The PCR reaction solution was dissolved at room temperature, vortexed and mixed evenly before use, and centrifuged momentarily before use. The enzyme system was shaken and mixed before use, centrifuged momentarily, and set aside. Add the above prepared reaction solution into a 0.2 mL PCR tube or PCR reaction plate, cover the tube, mix and centrifuge immediately to eliminate air bubbles at the bottom of the tube, and place it in an ABI 7500 fluorescence quantitative PCR instrument for detection. The PCR amplification reaction procedure is as follows: 1) Synthesis of cDNA catalyzed by reverse transcriptase, the process temperature is generally 42°C, and the time is 30 minutes; 2) cDNA pre-denaturation, the time and length depend on the length and base composition of the target nucleotide, The pre-denaturation temperature is 95°C and the time is 6 minutes. The purpose of pre-denaturation is to completely separate the double-stranded nucleotide sequence into single strands; 3) Denaturation, the temperature is 95°C and the time is 25s; 4) Annealing to make each primer Anneal to HIV-2 target sequence. The annealing temperature is usually 55°C, and the annealing time is 35 seconds; 5) extension, the primer is combined with the template, and new double-stranded DNA is synthesized. The extension temperature is 72°C, the extension time is 2 minutes, and the cycle is 35 times, and finally the fluorescence is collected.

(3) Result analysis

After the reaction is completed, the results are automatically saved and the target gene amplification curve is analyzed. Users can adjust the Threshold Value according to the actual situation, adjust the amplification curve of the negative quality control product to be flat or lower than the threshold line, click Analyze to analyze, and export the sample test results.

(4) Positive judgment value:

According to the test results of clinical samples, the FAM channel reference value Ct value of this kit is determined to be 32, the detection limit: 20 copies/mL; the linear range: 20 ~ 1.0×10 ⁷ copies/mL.

VIC channel result judgment: If the VIC channel Ct is <20.0, the test tube result can be judged to be positive. If the VIC channel has no amplification curve or Ct≥20.0, the test tube result can be judged to be negative.

(5)Interpretation of test results:

1. For samples with test results between 20 and 1.0×10 ⁷ copies/mL, report the corresponding test results;

2. For samples with test results >1.0×10 ⁷ copies/mL, the report shall indicate >1.0×10 ⁷ copies/mL. If accurate test results are required, the sample to be tested can be diluted to within the linear range before testing;

3. For samples with test results <20copies/mL, the report shall indicate <20copies/mL;

4. When the test result is "NoCt" or "undet.", the test result of the sample is invalid, and the sample will be retested.

Example 3

A kit for quantitative detection of human immunodeficiency virus type 2 (HIV-2) nucleic acid, including the following components: the primer probe in Example 1, PCR reaction solution, enzyme, quantitative reference materials 1-4 (made by Pseudoviral particles containing HIV-2 target amplified fragments artificially synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.), positive control substances (artificially synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. containing HIV-2 target Pseudoviral particles for amplified fragments), negative control substance (physiological saline); among them, the PCR reaction solution includes reaction buffer, magnesium ions and deoxyribonucleoside triphosphates (dNTPs), and the enzyme includes thermostable DNA polymerase, reverse transcription enzymes and ribonuclease inhibitors.

Example 4 Specific Experimental Analysis

This example is used to evaluate the specificity of the human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method described in Example 2. The first set of primers and probes described in Example 1 are used in this embodiment. The same effect can also be produced by using the second set of primers.

Plasma samples from 2 HIV-1 infected patients (clinically determined to be positive) collected from the Hunan Provincial Center for Disease Control and Prevention were used, RNA was extracted according to the operating procedures in Example 2, and PCR amplification was performed. The results As shown in Figure 1, the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method of Example 2 did not detect HIV-1 and had no amplification curve, indicating that the human immunodeficiency virus type 2 (HIV-2) of Example 2 ( HIV-2) quantitative nucleic acid detection method can effectively distinguish HIV-1 and HIV-2. At the same time, during the specificity test, it was found that when the plasma sample contained hemoglobin (<2g/dL), total bilirubin (<28mg/dL), triglyceride (<3000mg/dL), total IgG (<40g/L) ) and the final concentration of EDTA is (<300 mg/dL), the sensitivity of the detection method is not interfered with, indicating that the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method provided in Example 2 is sensitive to potential endogenous Substances have certain resistance to interference.

The detection method of Example 2 was further used to detect human cytomegalovirus, E-B virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, and influenza A virus samples collected from the Hunan Provincial Center for Disease Control and Prevention. The results show that there is no amplification curve for human cytomegalovirus, Epstein-Barr virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, and influenza A virus, indicating that the human immunodeficiency virus type 2 (HIV-2 ) The quantitative nucleic acid detection method has no cross-reaction with the above-mentioned viruses.

In summary, the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method provided in Example 2 has excellent specificity.

Example 5 Sensitivity and Repeatability Experimental Analysis

This example is used to evaluate the sensitivity and repeatability of the human immunodeficiency virus type 2 (HIV-2) nucleic acid quantitative detection method provided in Example 2. The primer probe in this embodiment uses the second set described in Example 1, and the same effect can be produced by using the first set.

HIV-2 standard substance (synthesized and provided by Sangon Bioengineering (Shanghai) Co., Ltd.) was diluted into different concentrations (concentrations are: 1.0×10 ⁶ , 1.0×10 ⁵ , 1.0×10 ⁴ , 1.0×10 ³ , 1.0×10 ² , 5.0×10 ¹ , 2.0×10 ¹ copies/mL), the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method of Example 2 was used for detection, and each concentration was repeated three times. The obtained data were subjected to linear analysis. The relationship between the above standard concentration, repeatability, and Ct value (ie cycle threshold, which refers to the cycle value experienced when the fluorescence signal in the PCR reaction tube reaches the set threshold) is shown in Table 2 below. The linear regression line established by 7 different concentrations of standards has a slope of -3.23270, an intercept of 28.53213, a correlation coefficient of -0.99631, and an amplification efficiency of 1.03863, showing a good linear correlation (results in Figures 2 and 3). It can be seen that the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method has good repeatability, and the linear range is 20-1.0×10 ⁷ copies/mL. The correlation coefficient of establishing the HIV-2 RNA standard curve is r≥0.980. .

Table 2 RT-PCR detection results

Standard concentration Log value of standard concentration Repeated measurement results 1 Repeated measurement results 2 Repeated measurement results 3 Average Ct value 1.0×10 ⁶ 6 8.08 8.23 8.04 8.117 1.0×10 ⁵ 5 11.3 11.47 11.27 11.35 1.0×10 ⁴ 4 14.31 14.51 14.36 14.39 1.0×10 ³ 3 17.65 17.72 17.63 17.67 1.0×10 ² 2 20.77 20.78 21.62 21.06 5.0×10 ¹ 1.699 21.71 21.18 22.35 21.75 2.0×10 ¹ 1.301 22.15 23.56 24.28 23.33

Furthermore, the plasma samples (recorded as sample 1 and sample 2) of 2 HIV-2 infected persons (clinically determined to be positive) were tested through the quantitative nucleic acid detection method of human immunodeficiency virus type 2 (HIV-2) in Example 2. Perform quantitative testing. Plasma samples from two HIV-2 infected patients were collected, RNA was extracted according to the operation process of Example 2, and PCR amplification was performed. The results are shown in Table 3 and Figure 4. In Figure 4, Group 3 and Group 6 They are the amplification curves of sample 1 and sample 2 respectively. Group 1, group 2, group 4, and group 5 are respectively the amplification curves of quantitative reference products 1-4 (pseudoviral particles containing HIV-2 target fragments), with a concentration of 1.0 ×10 ⁶ , 1.0 × 10 ⁵ , 1.0 × 10 ⁴ and 1.0 × 10 ³ copies/mL (amplification efficiency is 108%), it can be seen that the quantitative nucleic acid of human immunodeficiency virus type 2 (HIV-2) in Example 2 The detection method has good detection repeatability.

Table 3 RT-PCR detection results

The above results show that the sensitivity of the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method in Example 2 can reach 20 copies/mL, and the linear quantitative range can reach 20-1.0×10 ⁷ copies/mL. This method can It solves the problem that other detection methods in the field of HIV-2 detection cannot perform quantitative detection and analysis, and can supplement the current quantitative technology gap in the field of HIV-2 detection.

Example 6 Detection of clinical samples

For 10 samples of HIV-2 infected patients collected from the Hunan Provincial Center for Disease Control and Prevention, the second set of primer probe combinations were selected and carried out according to the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method in Example 2. detection. The test results of the 10 positive samples are shown in Table 4. The results show that the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method of Example 2 is excellent in the detection interval of the HIV-2 positive samples. From Quantitative values can be measured in each range from low to high. The current distribution of HIV-2 infected people in my country is currently unclear, and there are very few samples for quantitative testing. However, the load levels of positive samples collected through the Hunan Provincial Center for Disease Control and Prevention range from low to high, and all can be quantitatively detected by repeating the human immunodeficiency virus type 2 (HIV-2) quantitative nucleic acid detection method twice. Give specific values. It has good quantitative performance in all concentration regions.

Table 4 RT-PCR

The above results show that compared with other detection methods in the same field, the detection method provided by the present invention has the advantage of being able to successfully quantitatively detect the samples of most current HIV-2 patients in China, and being able to output stable amplification curves and quantitative data results. , can solve the practical problem that HIV-2 infected people in my country cannot receive effective treatment due to the lack of effective quantitative detection methods, leading to the progression and deterioration of the disease. On the basis of having quantitative detection data, clinicians can better evaluate the treatment of HIV-2-infected patients and guide HIV-2 drug treatment selection, helping HIV-2-infected patients obtain better diagnosis and disease management.

The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various modifications can be made without departing from the purpose of the present invention. kind of change. In addition, the embodiments of the present invention and the features in the embodiments may be combined with each other without conflict.

Claims (10)

1. A reagent, characterized in that the reagent includes a primer set and a probe set, the primer set includes primer 1 or primer 2, and the probe set includes probe 1 or probe 2; Wherein, the sequence of primer 1 is: F: 5’-TGGCAGCTTTATTAAGAGGTCT-3’, or the complementary sequence of this sequence; R: 5’-CAAGCACTGGTGAGAGTCTAGC-3’, or the complementary sequence of this sequence; The sequence of primer 2 is: F: 5’-GAGGTTTCTCCAGCACTAGC-3’, or the complementary sequence of this sequence; R: 5’-GCGGCGACTAGGAGAGAT-3’, or the complementary sequence of this sequence; The sequence of probe 1 is: 5’-AGAGGCTGGCAGATYGAG-3’, or the complement of this sequence; The sequence of probe 2 is: 5’-CAGACGGCTCCACGCTT-3’, or the complement of this sequence. 2. The reagent according to claim 1, wherein both ends of the probe sequence are labeled with fluorescent groups and quenching groups respectively. 3. The reagent according to claim 2, characterized in that the fluorescent group is at least one of FAM, HEX, VIC, TET, ROX and CY5; and/or the quenching group is TAMRA, At least one of MGB, BHQ1, BHQ2 and BHQ3. 4. A kit, characterized in that the kit contains the reagent according to any one of claims 1 to 3. 5. The kit according to claim 4, wherein the kit further includes PCR reaction solution, enzyme, quantitative reference, negative reference and positive reference; preferably, the quantitative reference includes Pseudoviral particles of HIV-2 target amplified fragments. 6. The kit according to claim 5, wherein the enzyme includes thermostable DNA polymerase, reverse transcriptase and ribonuclease inhibitor; and/or, the PCR reaction solution includes reaction buffer, Magnesium ions and deoxyribonucleoside triphosphates. 7. Application of the reagent according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 6 in any one of (1) to (6); (1) Quantitative detection of HIV-2; (2) Prepare products for quantitative detection of HIV-2; (3) Detect whether the sample to be tested is HIV-2; (4) Prepare whether the sample to be tested is an HIV-2 product; (5) Detect whether the sample to be tested is infected with HIV-2; (6) Prepare products for testing whether the sample to be tested is infected with HIV-2; The above applications are used for non-disease diagnosis and treatment. 8. An HIV-2 nucleic acid quantitative detection method, using the reagent according to any one of claims 1 to 3 or the kit according to claims 4 to 6; the above detection method is used for the diagnosis and treatment of non-diseases . 9. The detection method according to claim 8, characterized in that it includes the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Using the nucleic acid and quantitative reference material of step 1) as templates, use the described reagent or the described kit to perform RT-PCR amplification to obtain the Ct value; 3) HIV-2 can be quantitatively detected based on the standard curve fitted to the Ct value and copy number of the quantitative reference product. 10. The detection method according to claim 9, characterized in that the RT-PCR amplification reaction program in step 2) is: 42~45°C, 15~45min; 94~96°C, 2~10min; 90 ~95℃ 15~30s, 45~65℃ 10~60s, 32~36 cycles; 70~72℃ 0.5~5min.