HIV quantitative composition, kit, method and application

Abstract

The invention belongs to the field of molecular biological detection, particularly relates to a quantitative composition, a kit, a method and application, and more particularly relates to an HIV quantitative composition, a kit, a method and application. The present invention provides a quantitative composition comprising at least three homologous vectors and quantitative vectors of at least one part of at least one of at least one of a segment LTR region, a GAG region, a POL region, an ENV region, a VIF region, a VPR region, a VPU region, a TAT region, a REV region, or an NEF region of HIV, as well as primers and probes for amplifying the two vectors. The homologous vector is creatively introduced, and the concentration of the sample to be detected is artificially increased, so that the stability and accuracy of PCR amplification reaction are improved. More particularly, a plurality of homologous vectors are creatively introduced, so that the detection of the highly mutated HIV virus is more accurate, and the detection omission of the HIV virus is prevented.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to a quantitative composition, a kit, a method and a use thereof, and more specifically, relates to an HIV quantitative composition, a kit, a method and a use thereof. Background Art

[0002] Human Immunodeficiency Virus (HIV), also known as AIDS virus, is a retrovirus that causes human immune system defects. This virus attacks and gradually destroys the human immune system.

[0003] Quantitative RNA testing also plays a very important role in diagnosing HIV and evaluating the effectiveness of HIV treatment. It can be used to understand the number of viruses in the body, replication level, infectivity, drug treatment effects, and formulation of treatment strategies, and can serve as an evaluation indicator. However, the high variability of RNA itself makes HIV detection more difficult and there is a risk of missed detection.

[0004] Therefore, there is a need in the art for accurate quantitative detection of HIV, especially low viral load HIV, and in particular, for preventing missed detection. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a HIV quantitative composition, comprising:

[0006] A first reagent, comprising at least 3 sets of primers and probes, wherein the at least 3 sets of primers and probes are respectively used to amplify and detect at least one portion of at least one of the segments LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region of HIV;

[0007] A second reagent, comprising at least three homologous vectors, wherein the homologous vectors respectively comprise at least one part of at least one of the HIV segments LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region, and the HIV segments in the second reagent respectively correspond one-to-one to the HIV segments amplified in the first reagent;

[0008] A third reagent, comprising M quantitative carriers; and

[0009] The fourth reagent includes M groups of primers and probes, wherein the M groups of primers and probes are used to amplify and detect segments of the quantitative carrier, respectively.

[0010] Furthermore, M is a positive integer ≥1.

[0011] In some specific embodiments, the third reagent may include M quantitative carriers. Preferably, the M quantitative carriers are mixed in a ratio of 1:1:1, that is, mixed in equal amounts.

[0012] In some specific embodiments, the third reagent may include one quantitative vector, that is, M segments of the quantitative vector are integrated into one vector, preferably, integrated into one segment at an equal copy ratio, for example, 1:1:..:1 (a total of M 1s). Any segments may also be integrated into any vector, as long as each segment has an equal copy.

[0013] The present invention creatively introduces homologous vectors to artificially increase the concentration of HIV in the sample, thereby improving the stability and accuracy of the PCR amplification reaction. In particular, HIV can be better detected in samples with low viral loads. More particularly, by creatively introducing multiple homologous vectors, the detection of highly mutated viruses such as HIV is more accurate, preventing the missed detection of HIV virus.

[0014] Furthermore, the quantitative vector can be any vector used for quantitative analysis in the prior art, as long as it does not interfere with the amplification of the corresponding pathogen in the PCR system. For example, it can be a vector containing GAPDH, RNASE gene fragments, etc.

[0015] Furthermore, the quantitative vector comprises a nucleotide sequence, which is a sequence of the HIV segment LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region in the homologous vector, which is transformed into the LTR' region, GAG' region, POL' region, ENV' region, VIF' region, VPR' region, VPU' region, TAT' region, REV' region, or NEF' region, and the transformation comprises replacing the adenine deoxyribonucleotide (A) in the original sequence with a thymine deoxyribonucleotide (T), and replacing the thymine deoxyribonucleotide (T) in the original sequence with an adenine deoxyribonucleotide (A).

[0016] This design aims to ensure that the amplification efficiency of the target segment of the quantitative vector is similar to that of the target segment of the homologous vector, thereby effectively simplifying the process of calculating the concentration of the target segment of the homologous vector based on the quantitative vector constant.

[0017] Furthermore, the nucleotide sequence of the HIV segment LTR region is shown as SEQ ID NO.4.

[0018] Furthermore, the nucleotide sequence of the POL region of the HIV is shown as SEQ ID NO.12.

[0019] Furthermore, the nucleotide sequence of the GAG ​​region of HIV is shown in SEQ ID NO.20.

[0020] In some specific embodiments, the forward primer for amplifying the HIV segment LTR region is shown as SEQ ID NO.1, the reverse primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO.3.

[0021] In some specific embodiments, the forward primer for amplifying the POL region of HIV is shown as SEQ ID NO.9, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO.11.

[0022] In some specific embodiments, the forward primer for amplifying the HIV GAG region is shown as SEQ ID NO.17, the reverse primer is shown as SEQ ID NO.18, and the probe is shown as SEQ ID NO.19.

[0023] In the present invention, the term "vector" refers to all substances in the art that can carry nucleic acids and can be used in the process of nucleic acid amplification. For example, common vectors include plasmids, recombinant viruses containing plasmids (lentiviruses, adenoviruses, pseudoviruses, etc.), liposomes containing plasmids, artificial chromosomes, etc.

[0024] In the present invention, the term "group" refers to at least one upstream primer, at least one downstream primer, and at least one probe that match each other for detecting a target. Specifically, for example, it can be one upstream primer, one downstream primer, and one probe that match each other for detecting a target; it can also be two upstream primers, one downstream primer, and one probe that match each other for detecting a target; it can also be one upstream primer, two downstream primers, and one probe that match each other for detecting a target; it can also be one upstream primer, one downstream primer, and two probes that match each other for detecting a target, etc.

[0025] In the present invention, the term "LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, NEF region" refers to at least one nucleotide sequence in the HIV long terminal repeat region, GAG gene, POL gene, ENV gene, VIF gene, VPR gene, VPU gene, TAT gene, REV gene, or NEF gene. In some specific embodiments, the vector can be a plasmid.

[0026] Furthermore, the backbone plasmid source of the homologous plasmid and the quantitative plasmid can be any plasmid in the prior art, for example, pUC57 plasmid, pBluescript II SK+ plasmid, pUC-SP plasmid, etc., which are used to carry target segments, such as LTR region, POL region and / or GAG region, etc.

[0027] In some specific embodiments, the backbone plasmid of the homologous plasmid and the quantitative plasmid is a pUC57 plasmid, that is, the sequence of the LTR region is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the sequence after A in the LTR region is replaced by T and T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0028] In some specific embodiments, the vector may be a recombinant virus comprising the above-mentioned plasmid.

[0029] In some specific embodiments, the vector can be a liposome comprising the above-mentioned plasmid.

[0030] In some specific embodiments, the vector can be an artificial chromosome carrying the target segment.

[0031] Furthermore, the quantitative plasmid includes an LTR' region, and the nucleotide sequence of the LTR' region is as shown in SEQ ID NO.5.

[0032] Furthermore, the quantitative plasmid includes a POL' region, and the nucleotide sequence of the POL' region is as shown in SEQ ID NO.13.

[0033] Furthermore, the quantitative plasmid includes a GAG' region, and the nucleotide sequence of the GAG' region is as shown in SEQ ID NO.21.

[0034] In some specific embodiments, the forward primer for amplifying the LTR' region is shown as SEQ ID NO.6, the reverse primer is shown as SEQ ID NO.7, and the probe is shown as SEQ ID NO.8.

[0035] In some specific embodiments, the forward primer for amplifying the POL' region is shown as SEQ ID NO.14, the reverse primer is shown as SEQ ID NO.15, and the probe is shown as SEQ ID NO.16.

[0036] In some specific embodiments, the forward primer for amplifying the GAG' region is shown as SEQ ID NO.22, the reverse primer is shown as SEQ ID NO.23, and the probe is shown as SEQ ID NO.24.

[0037] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the lowest quantitative concentration, preferably, 30% to 60%.

[0038] Using the above homologous vector concentrations allows for more accurate quantification of low concentrations of HIV.

[0039] The term "lowest quantitative concentration" refers to the limit of quantitation (LoQ), which refers to the ability to stably and accurately test samples of known specific concentration values, that is, the lowest concentration of the analyte that can be measured when the preset accuracy requirements are met.

[0040] Furthermore, the composition further comprises a fifth reagent, the fifth reagent comprising a primer and a probe, wherein the primer and the probe in the fifth reagent are used to amplify and detect a segment of a pathogen. Furthermore, the segment of the pathogen amplified by the fifth reagent is different from the segment of the pathogen amplified by the first reagent and the segment of HIV in the second reagent.

[0041] By introducing the fifth reagent, the positive or negative nature of the sample can be determined, that is, whether the pathogen exists in the sample can be determined.

[0042] In some specific embodiments, the composition further comprises a fifth reagent that amplifies the segment LTR of the pathogen 2 The segment is different from the nucleotide sequence shown in the LTR region, POL region, or GAG region of the segment amplified by the first reagent of the pathogen.

[0043] In some specific embodiments, the LTR 2 The nucleotide sequence of the region is shown in SEQ ID NO.25.

[0044] In some specific embodiments, amplifying the LTR 2 The forward primer of the region is shown as SEQ ID NO.26, the reverse primer is shown as SEQ ID NO.27, and the probe is shown as SEQ ID NO.28.

[0045] In some specific embodiments, the first reagent, the fourth reagent and the fifth reagent in the quantitative composition of the present invention can be modified. Further, the modification can be one or more of locked nucleic acid (LNA), MGB, or ZNA (Zip Nucleic Acids); further, the modification can be locked nucleic acid modification.

[0046] In some specific embodiments, the HIV quantitative composition of the present invention is used for fluorescent PCR.

[0047] Furthermore, the fluorescent groups of the probes mentioned in the present invention are different from each other and do not interfere with each other.

[0048] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in the probes of the first reagent, the fourth reagent and the fifth reagent are different from each other and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO 425, Quasar705, FAM, HEX, ROX, CY5 and CY5.5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0049] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0050] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, BHQ1 or BHQ2.

[0051] In a specific embodiment, each component of the HIV quantitative composition of the present invention is present in a separate package.

[0052] In a specific embodiment, the components of the HIV quantitative composition of the present invention are present in the same package.

[0053] Furthermore, the components of the HIV quantitative composition of the present invention are present in a mixed form.

[0054] In a second aspect, the present invention provides use of the above HIV quantitative composition for preparing an HIV quantitative kit.

[0055] In a third aspect, the present invention provides an HIV quantitative kit, comprising the HIV quantitative composition as described above.

[0056] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0057] Furthermore, the amplification reagent includes dNTP, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0058] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0059] In some specific embodiments, the composition further comprises at least one of a nucleic acid amplification reagent, a nucleic acid release reagent, or a nucleic acid extraction reagent.

[0060] Furthermore, the concentration of the DNA polymerase is 3U / μL to 15U / μL, for example, the DNA polymerase may be Taq enzyme.

[0061] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , Mn 2+ , dNTPs, and PCR buffer.

[0062] Common PCR buffers consist of Tris-HCl, MgCl 2 The total volume of a single PCR reaction tube is generally 20 μL to 200 μL.

[0063] In a fourth aspect, the present invention provides a method for HIV quantification, the method comprising the following steps:

[0064] 1) Extracting nucleic acid from the sample to be tested;

[0065] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1); and

[0066] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0067] Furthermore, the quantitative formula is Among them, P 1 is the probe of the first reagent, P 2 It is the probe of the fourth reagent.

[0068] Further, a method for quantifying HIV for non-diagnostic purposes is provided, the method comprising the following steps:

[0069] 1) Extracting nucleic acid from the sample to be tested;

[0070] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1); and

[0071] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0072] Furthermore, the quantitative formula is Among them, P 1 is the probe of the first reagent, P 2 It is the probe of the fourth reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figures 1-2 is an exemplary detection diagram of the composition of the present invention;

[0074] Figure 3 This is a graph showing the results of testing negative samples of the composition of the present invention;

[0075] Figure 4This is a graph showing the test results of a sample of a comparative example composition of the present invention;

[0076] Figure 5 This is a graph showing the results of testing negative samples using the composition of the present invention. DETAILED DESCRIPTION

[0077] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0078] Measurement of minimum quantitative concentration (limit of quantitation):

[0079] HIV national standard was used, and newborn calf serum was used to dilute to 40 IU / mL, 30 IU / mL, 25 IU / mL, 20 IU / mL, and 15 IU / mL. The diluted low-concentration samples were used as test samples. Each test sample was tested 20 times each time, and it was repeated 3 times in a row, for a total of 60 times. The accuracy requirement is that the logarithmic deviation of the test result is within ±0.5, and the lowest quantitative concentration (LOQ) is the lowest concentration of the test substance that can be measured when the 100% accurate measurement requirement is met. Through the above method, it was finally determined that the LOQ of HIV under this patented PCR system is 30 IU / mL.

[0080] Example 1: Sequences used in the present invention

[0081] Some exemplary sequences used in the present invention are shown in Table 1 below, where + indicates that the position is modified with a locked nucleic acid.

[0082] Table 1

[0083]

[0084]

[0085] In Table 1, “+A” means LNA-A; “+T” means LNA-T; “+C” means LNA-C; and “+G” means LNA-G.

[0086] Among them, the fluorescent group of the probe of the first reagent is FAM; the fluorescent group of the probe of the fourth reagent is HEX; and the fluorescent group of the probe of the fifth reagent is CY5.

[0087] Example 2: HIV quantification method

[0088] Exemplary methods of the present invention are as follows Figures 1-2This project uses Shengxiang Biotechnology S1001 nucleic acid extraction kit for HIV nucleic acid extraction, and Chinese national HIV standard diluted with newborn calf serum is used to draw HIV concentration curves or as a sample to be tested to test the performance of the internal standard quantitative system.

[0089] PCR reaction conditions:

[0090] Baorui MMLV RT enzyme (5U); Kande Taq DNA polymerase (5U); Shengxiang Bio S15 Buffer; primers / probes synthesized in Bio-Tech and diluted to a working concentration of 40pmol / μL by dissolving in TE; 1mol / L Mg 2+ ; HIV-1 and IC target sequence plasmids were synthesized at Bioengineering.

[0091] The amplification system for a single person of 50 μL includes: 17 μL PCR buffer (S15), 1 μL 100mmol / L dNTP (T), 0.24 μL 40pmol / μL PCR upstream and downstream primers, 0.1 μL 40pmol / μL probe, 1.5 μL 5UTaq DNA polymerase, 0.5 μL 5U MMLV RT enzyme, 0.4 μL 1mol / L Mg 2+ , 25.54 μL purified water.

[0092] PCR amplification program: 50°C for 30 min, 1 cycle; 95°C for 8 min, 1 cycle; 95°C for 15 s, 57°C for 30 s, 45 cycles, reading the fluorescence signal once after each cycle; 25°C for 10 s, 1 cycle, and the reaction system was set to 50 μL.

[0093] Sample processing:

[0094] This project uses Shengxiang Bio's S1001 nucleic acid extraction kit to extract HIV-1 nucleic acid, and the Chinese national HIV-1 standard diluted with newborn calf serum is used to draw HIV-1 concentration curves or as test samples to test the performance of the internal standard quantitative system.

[0095] 10 μL of 1.00E-7 μg / mL internal standard plasmid or HIV-1 homologous plasmid is added to 800 μL of newborn calf serum, and the HIV-1 concentration curve is drawn by diluting the Chinese national HIV-1 standard with newborn calf serum for determination, and the average of the determined concentrations measured by three independent repeated tests is set as the HIV-1 determined concentration of the plasmid. In this scheme, illustratively, 10 μL of 1.00E-7 μg / mL quantitative plasmid is determined in 800 μL of the sample system to be tested, and the determined concentration is diluted to a working concentration of 600 IU / mL by TE, and 10 μL of 1.00E-7 μg / mL HIV homologous plasmid solution (mixed by the third reagent with each plasmid in a ratio of 1:1:1) is determined in 800 μL of the sample system to be tested, and the determined concentration is diluted to a working concentration of 1000 IU / mL by TE.

[0096] After completing the plasmid determination, add 10 μL of internal standard plasmids with a final concentration of 600 IU / mL (three internal standard plasmids containing sequences SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.21, respectively) and 10 μL of HIV-1 homologous plasmids with corresponding fixed concentrations (three homologous plasmids containing sequences SEQ ID NO.4, SEQ ID NO.12, and SEQ ID NO.20, respectively) to 800 μL of the sample to be tested, mix evenly, add a mixed solution containing 500 μL of lysate and 20 μL of proteinase K to the centrifuge tube, shake and mix, centrifuge instantly, and stand at room temperature for 30 minutes. After instantaneous centrifugation, place it on a magnetic separator for 3 minutes. When the magnetic beads are aggregated on the wall of the centrifuge tube, discard the liquid in the tube, add 500 μL of nucleic acid washing solution 1 and 200 μL of nucleic acid washing solution 2 to the centrifuge tube at the same time, shake and mix, centrifuge instantly, and place the centrifuge tube on the magnetic separator again. After about 3 minutes, discard the liquid in the tube again. Pipette 50μL PCR reaction solution to elute the magnetic beads and place them on a magnetic separator. Transfer the separated clear liquid to the PCR reaction tube, shake and mix, and centrifuge instantly. Place them in the PCR instrument in order, set the running program and related parameters, save the file, and run the reaction program.

[0097] For example, the fixed concentration of the quantitative plasmid is 600 IU / mL, so according to the formula Calculate the HIV load in the sample to be tested.

[0098] Example 3: Test results of the test samples of the composition of the present invention

[0099] The comparative example and the example scheme (sequence shown in Table 1) were used to detect the Chinese national HIV standard diluted with newborn calf serum. The comparative example only added 10 μL of a quantitative plasmid with a total concentration of 600 IU / mL; on this basis, the example additionally added a homologous plasmid with a final concentration of 10 IU / mL.

[0100] The results of the two systems for the Chinese National HIV Standard diluted with 25 IU / mL newborn calf serum are as follows Figures 3-4 As shown, compared with the comparative example, the amplification curve of the HIV target sequence of the HIV dual-channel dual-plasmid internal standard quantitative technology (implementation) is more concentrated, and the Ct value variance is significantly lower than that of the comparative example, that is, the detection result is more stable. As shown in Table 2, the quantitative accuracy of the comparative example is 78.57% (11 / 14), and the logarithmic deviation of the concentration of 11 detected samples is within ±0.5; the quantitative accuracy of the embodiment is 100% (14 / 14), and the logarithmic deviation of the concentration of 14 detected samples is within ±0.4. The HIV dual-channel dual-plasmid internal standard quantitative technology shows a lower quantitative lower limit and better detection accuracy for low HIV load samples. It can also be seen from the result diagram that the system of the present invention ( Figure 3 ) is more concentrated and has a smaller deviation. Figure 3 and Figure 4 It can be seen that there is an amplification curve of the fifth reagent (red, CY5 channel), indicating that HIV virus exists in the sample. When the combination is used to detect negative samples, Figure 5 As shown, no amplification curve was observed for the fifth reagent.

[0101] Table 2

[0102]

[0103] *The quantitative analysis is inaccurate, and the deviation of the logarithmic value of the sample to be tested exceeds ±0.5 compared with the actual sample concentration.

[0104] Example 4: Test results of the test sample of the composition 1 of the present invention

[0105] HIV dual-channel dual-plasmid internal standard quantitative technology (Example) was used to detect the Chinese national HIV standard diluted with 25IU / mL newborn calf serum, and μL of quantitative plasmid with a fixed value of 600IU / mL was added to the sample to be tested; on this basis, homologous plasmids with a final concentration of 7 / 8 / 12 / 18 / 20 / 22IU / mL HIV fixed values ​​were added to each group. Under this test system, the lowest quantitative concentration (LOQ) of the HIV single plasmid internal standard system is about 30IU / mL. As shown in Table 3, the optimal addition amount of HIV homologous plasmid is approximately between 7.5IU / mL and 20IU / mL, that is, 25% to 66.7% of the lowest quantitative concentration (LOQ) of the HIV single plasmid internal standard system.

[0106] Table 3

[0107]

[0108] *The quantitative analysis is inaccurate, and the deviation of the logarithmic value of the sample to be tested exceeds ±0.5 compared with the actual sample concentration.

Claims

1. A HIV quantitative composition, comprising: A first reagent, comprising at least 3 sets of primers and probes, wherein the at least 3 sets of primers and probes are respectively used to amplify and detect at least one portion of at least one of the segments LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region of HIV; A second reagent, comprising at least three homologous vectors, wherein the homologous vectors respectively comprise at least one part of at least one of the HIV segments LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region, and the HIV segments in the second reagent respectively correspond one-to-one to the HIV segments amplified in the first reagent; A third reagent, comprising M quantitative carriers; and The fourth reagent includes M groups of primers and probes, wherein the M groups of primers and probes are used to amplify and detect segments of the quantitative carrier, respectively.

2. The HIV quantitative composition according to claim 1, wherein The quantitative vector includes a nucleotide sequence, which is a sequence modified for the HIV segment LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region in the homologous vector, and the modification includes replacing the adenine deoxyribonucleotide in the homologous vector sequence with thymine deoxyribonucleotide, and replacing the thymine deoxyribonucleotide in the homologous vector sequence with adenine deoxyribonucleotide. The quantitative vector in the fourth reagent includes the LTR' region, GAG' region, POL' region, ENV' region, VIF' region, VPR' region, VPU' region, TAT' region, REV' region, or NEF' region of HIV, respectively, and the segment of HIV in the fourth reagent corresponds one to one to the segment of HIV amplified in the second reagent.

3. The HIV quantitative composition according to claim 1, characterized in that Any primer and / or probe in the first reagent and the fourth reagent is modified with one or more of locked nucleic acid, MGB, or ZNA.

4. The HIV quantitative composition according to claim 1, characterized in that The nucleotide sequence of the HIV LTR region is shown as SEQ ID NO.4; the nucleotide sequence of the HIV POL region is shown as SEQ ID NO.12; or the nucleotide sequence of the HIV GAG region is shown as SEQ ID NO.

20.

5. The HIV quantitative composition according to claim 4, characterized in that The forward primer for amplifying the LTR region of the HIV segment is shown as SEQ ID NO.1, the reverse primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO.3; the forward primer for amplifying the POL region of the HIV segment is shown as SEQ ID NO.9, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO.11; or the forward primer for amplifying the GAG ​​region of the HIV segment is shown as SEQ ID NO.17, the reverse primer is shown as SEQ ID NO.18, and the probe is shown as SEQ ID NO.

19.

6. The HIV quantitative composition according to claim 2, characterized in that The nucleotide sequence of the LTR' region is shown as SEQ ID NO.5; the nucleotide sequence of the POL' region is shown as SEQ ID NO.13; or the nucleotide sequence of the GAG' region is shown as SEQ ID NO.

21.

7. The HIV quantitative composition according to claim 6, characterized in that The forward primer for amplifying the LTR' region is shown as SEQ ID NO.6, the reverse primer is shown as SEQ ID NO.7, and the probe is shown as SEQ ID NO.8; the forward primer for amplifying the POL' region is shown as SEQ ID NO.14, the reverse primer is shown as SEQ ID NO.15, and the probe is shown as SEQ ID NO.16; or the forward primer for amplifying the GAG' region is shown as SEQ ID NO.22, the reverse primer is shown as SEQ ID NO.23, and the probe is shown as SEQ ID NO.

24.

8. The HIV quantitative composition according to claim 7, characterized in that The composition also includes a fifth reagent, which includes primers and probes, wherein the primers and probes in the fifth reagent are used to amplify and detect HIV segments, wherein the HIV segments amplified by the fifth reagent are different from the HIV segments amplified by the first reagent and the HIV segments in the second reagent.

9. The HIV quantitative composition according to claim 8, characterized in that The concentration of the homologous vector is 25% to 66.7% of the lowest quantitative concentration.

10. An HIV quantitative kit comprising the HIV quantitative composition according to any one of claims 1 to 9.

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