HIV quantitative composition, kit, method and use

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to a quantitative composition, a kit, a method and a use, more particularly relates to a HIV quantitative composition, a kit, a method and a use. The present application provides a quantitative composition, which comprises at least 3 homologous carriers of at least one part in 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 and a quantitative carrier, and primers and probes for amplifying the two carriers. The present application creatively introduces homologous carriers, artificially increases the concentration of the sample to be tested, and further improves the stability and accuracy of the PCR amplification reaction. More particularly, by creatively introducing multiple homologous carriers, the detection of HIV, a highly mutated virus, is more accurate, and the missed detection of HIV virus is prevented.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically, it relates to a quantitative composition, kit, method and use, and more specifically, it relates to an HIV quantitative composition, kit, method and use. Background Technology

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

[0003] RNA quantitative detection plays a vital role in diagnosing HIV and assessing the effectiveness of HIV treatment. It can reveal the amount of virus in the body, replication level, infectivity, drug treatment efficacy, and help in developing treatment strategies, serving as an evaluation indicator. However, the high variability of RNA itself further increases the difficulty of HIV detection and increases the risk of missed detection.

[0004] Therefore, there is a need in this field for accurate quantitative detection of HIV, especially low viral load HIV, and in particular, to prevent missed detection. Summary of the Invention

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

[0006] The first reagent comprises at least three sets of primers and probes, wherein the at least three sets of primers and probes are used to amplify and detect at least one portion of at least one of the following HIV regions: LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region.

[0007] The second reagent includes at least three homologous vectors, wherein each homologous vector includes at least one part of at least one of the following HIV regions: LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region. The HIV regions in the second reagent correspond one-to-one with the HIV regions amplified in the first reagent.

[0008] The third reagent includes M quantitative carriers; and

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

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

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

[0012] In some specific implementations, the third reagent may include one quantitative carrier, that is, integrating M segments of the quantitative carrier onto one carrier. Preferably, they are integrated into one segment at an equal copy ratio, for example, 1:1:..:1 (a total of M 1s). Alternatively, any number of segments can be integrated onto any number of carriers, as long as the total number of copies of each segment is equal.

[0013] This invention innovatively introduces homologous vectors to artificially increase the concentration of HIV in samples, thereby improving the stability and accuracy of PCR amplification reactions. In particular, it can detect HIV more effectively in samples with low viral loads. Even more significantly, by innovatively introducing multiple homologous vectors, the detection of HIV, a highly mutating virus, is more accurate, preventing missed detections of HIV.

[0014] Furthermore, the quantification vector can be any quantification vector used 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] Further, the quantitative vector includes a nucleotide sequence, wherein the nucleotide sequence is a sequence modified to modify 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 into an LTR' region, GAG' region, POL' region, ENV' region, VIF' region, VPR' region, VPU' region, TAT' region, REV' region, or NEF' region. The modification includes replacing adenine deoxyribonucleotide (A) in the original sequence with thymine deoxyribonucleotide (T), and replacing thymine deoxyribonucleotide (T) in the original sequence with adenine deoxyribonucleotide (A).

[0016] The purpose of this design is to ensure that the amplification efficiency of the target region of the quantitative vector is similar to that of the homologous vector, effectively simplifying the process of calculating the target region concentration of the homologous vector based on the quantitative vector.

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

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

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

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

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

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

[0023] In this invention, the term "vector" refers to any substance in the art capable of loading nucleic acids and used in the process of nucleic acid amplification. Common vectors include plasmids, recombinant viruses containing plasmids (lentiviruses, adenoviruses, pseudoviruses, etc.), liposomes containing plasmids, artificial chromosomes, etc.

[0024] In this invention, the term "set" refers to at least one upstream primer, at least one downstream primer, and at least one probe that are mutually matched to detect a target. Specifically, for example, it could be one upstream primer, one downstream primer, and one probe that are mutually matched to detect a target; it could also be two upstream primers, one downstream primer, and one probe that are mutually matched to detect a target; it could also be one upstream primer, two downstream primers, and one probe that are mutually matched to detect a target; or it could be one upstream primer, one downstream primer, and two probes that are mutually matched to detect a target, etc.

[0025] In this invention, the terms "LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, NEF region" refer to at least one nucleotide sequence from 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 may be a plasmid.

[0026] Furthermore, the backbone plasmids of the homologous plasmids and quantitative plasmids are derived from any plasmids 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 implementations, the backbone plasmid of the homologous plasmid and the quantitative plasmid is the pUC57 plasmid, that is, the LTR region sequence is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the A in the LTR region is replaced with T, and the sequence after T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0028] In some specific implementations, the vector may be a recombinant virus comprising the plasmids described above.

[0029] In some specific implementations, the vector may be a liposome comprising the plasmid described above.

[0030] In some specific implementations, the vector may be an artificial chromosome carrying a target segment.

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

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

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

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

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

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

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

[0038] Using the concentration of the aforementioned homologous vector allows for more accurate quantification of low-concentration HIV.

[0039] The term "lowest quantitation concentration" refers to the limit of quantitation (LoQ), which is the ability to stably and accurately test samples with known specific concentration values. In other words, it is the lowest concentration of the analyte that can be measured while meeting the preset accuracy requirements.

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

[0041] By introducing a fifth reagent, it is possible to determine whether a sample is positive or negative, that is, to determine whether the pathogen is present in the sample.

[0042] In some specific embodiments, the composition further includes a fifth reagent that amplifies the LTR2 region of the pathogen, the region being different from the nucleotide sequence of the LTR region, POL region, or GAG region amplified by the first reagent of the pathogen.

[0043] In some specific implementations, the nucleotide sequence of the LTR2 region is shown in SEQ ID NO.25.

[0044] In some specific implementations, the forward primer for amplifying the LTR2 region is shown in SEQ ID NO.26, the reverse primer is shown in SEQ ID NO.27, and the probe is shown in SEQ ID NO.28.

[0045] In some specific embodiments, the first, fourth, and fifth reagents in the quantitative composition of the present invention can be modified. Further, the modification can be one or more of locked nucleic acids (LNA), MGB, or ZNA (Zip Nucleic Acids); further still, 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 this invention are different from each other and do not interfere with each other.

[0048] In this paper, "dissimilar and non-interfering" means that the fluorescent groups used in the probes of the first, fourth, and fifth reagents are different and will not affect each other's detection; that is, different channels can be used for detection. For example, ATTO 425, Quasar 705, FAM, HEX, ROX, CY5, and CY5.5 can be used. These groups have different absorbance values, allowing for the selection of different channels and thus preventing mutual interference.

[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 quenching group, such as MGB, BHQ1, or BHQ2.

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

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

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

[0054] Secondly, the present invention provides the use of the above-described HIV quantitative composition for preparing an HIV quantitative kit.

[0055] Thirdly, the present invention provides an HIV quantitative kit comprising the HIV quantitative composition as described above.

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

[0057] Furthermore, the amplification reagents include dNTPs, PCR buffer, DNA polymerase, and Mg. 2+ At least one of them.

[0058] Furthermore, the kit also includes: nucleic acid release reagent and nucleic acid extraction reagent.

[0059] In some specific embodiments, the composition further includes 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 3 U / μL to 15 U / μL, for example, the DNA polymerase can be Taq polymerase.

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

[0062] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume in a single PCR reaction tube is typically 20 μL to 200 μL.

[0063] Fourthly, the present invention provides a method for quantifying HIV, the method comprising the following steps:

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

[0065] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above; 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 as follows: P1 is the probe for the first reagent, and P2 is the probe for the fourth reagent.

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

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

[0070] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above; 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 as follows: P1 is the probe for the first reagent, and P2 is the probe for the fourth reagent. Attached Figure Description

[0073] Figures 1-2 This 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 using the composition of the present invention.

[0075] Figure 4 This is a graph showing the test results of the comparative 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 Implementation

[0077] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0078] Measurement of the lowest quantitation concentration (limit of quantitation):

[0079] Using HIV national standard, newborn calf serum was diluted to 40 IU / mL, 30 IU / mL, 25 IU / mL, 20 IU / mL, and 15 IU / mL. The lowest concentration samples were used as test samples. Each test sample was subjected to 20 replicates per test, for a total of 60 replicates (3 times each). The accuracy requirement was a logarithmic deviation of within ±0.5%. The lowest quantitation level (LOQ) was the lowest measurable analyte concentration that could be measured with 100% accuracy. Using the above method, the LOQ for HIV was ultimately determined to be 30 IU / mL in this patented PCR system.

[0080] Example 1: The sequence used in this invention

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

[0082] Table 1

[0083]

[0084]

[0085] In Table 1: "+A" means LNA-A; "+T" means LNA-T; "+C" means LNA-C; "+G" means LNA-G.

[0086] The probe of the first reagent has a fluorescent group of FAM; the probe of the fourth reagent has a fluorescent group of HEX; and the probe of the fifth reagent has a fluorescent group of CY5.

[0087] Example 2: Quantitative Methods for HIV

[0088] Exemplary methods of the present invention are as follows: Figures 1-2 As shown in the figure. This project used the Sansure Biotech S1001 nucleic acid extraction kit for HIV nucleic acid extraction. Diluted newborn calf serum was used to plot HIV concentration curves or as a sample to test the performance of the internal standard quantitative system.

[0089] PCR reaction conditions:

[0090] Baorui MMLV RT enzyme (5U); Kangde Taq DNA polymerase (5U); Sansure Biotech S15 Buffer; primers / probes synthesized in Bailige and diluted to working concentration of 40 pmol / μL via TE buffer; 1 mol / L Mg 2+ HIV-1 and IC target sequence plasmids were synthesized at Sangon Biotech.

[0091] A single-sample 50 μL amplification system includes: 17 μL PCR buffer (S15), 1 μL 100 mmol / L dNTP(T), 0.24 μL 40 pmol / μL PCR forward and reverse primers, 0.1 μL 40 pmol / μL probe, 1.5 μL 5 U Taq DNA polymerase, 0.5 μL 5 U MMLV RT enzyme, and 0.4 μL 1 mol / L MgSO₄. 2+ 25.54 μL of purified water.

[0092] PCR amplification program: 50℃ for 30 min, 1 cycle; 95℃ for 8 min, 1 cycle; 95℃ for 15 s, 57℃ for 30 s, 45 cycles, with fluorescence signal read once after each cycle; 25℃ for 10 s, 1 cycle, with a reaction volume of 50 μL.

[0093] Sample processing:

[0094] This project used Sansure Biotech's S1001 nucleic acid extraction kit to extract HIV-1 nucleic acid. Diluted newborn calf serum was used to plot the HIV-1 concentration curve or as a sample to test the performance of the internal standard quantitative system.

[0095] Add 10 μL of 1.00E-7 μg / mL internal standard plasmid or HIV-1 homologous plasmid to 800 μL of newborn calf serum. Plot an HIV-1 concentration curve using diluted Chinese national HIV-1 standard from newborn calf serum for determination. The average concentration obtained from three independent replicate experiments is set as the HIV-1 determination concentration for that plasmid. In this protocol, for example, 10 μL of 1.00E-7 μg / mL quantitative plasmid is used for determination in an 800 μL sample system, and the determination concentration is diluted to a usable concentration of 600 IU / mL using TE buffer. Similarly, 10 μL of 1.00E-7 μg / mL HIV homologous plasmid solution (a 1:1:1 mixture of the three reagent plasmids) is used for determination in an 800 μL sample system, and the determination concentration is diluted to a usable concentration of 1000 IU / mL using TE buffer.

[0096] After plasmid determination, 10 μL of internal standard plasmid with a final concentration of 600 IU / mL (containing three internal standard plasmids with sequences SEQ ID NO. 5, SEQ ID NO. 13, and SEQ ID NO. 21) and 10 μL of HIV-1 homologous plasmids with corresponding concentrations (containing three homologous plasmids with sequences SEQ ID NO. 4, SEQ ID NO. 12, and SEQ ID NO. 20) were added to 800 μL of the sample to be tested. After mixing thoroughly, a mixed solution containing 500 μL of lysis buffer and 20 μL of proteinase K was added to the centrifuge tube. After vortexing and briefly centrifuging, the mixture was incubated at room temperature for 30 minutes. After brief centrifugation, the tube was placed on a magnetic separator for 3 minutes. Once the magnetic beads had aggregated on the centrifuge tube wall, the liquid in the tube was aspirated. 500 μL of nucleic acid washing buffer 1 and 200 μL of nucleic acid washing buffer 2 were added to the centrifuge tube simultaneously. After vortexing and briefly centrifuging, the centrifuge tube was placed on the magnetic separator again. After approximately 3 minutes, aspirate the liquid from the tube again. Elute the magnetic beads with 50 μL of PCR reaction solution and place it on a magnetic separator. Transfer the supernatant to the PCR reaction tube, vortex to mix, and centrifuge briefly. Place the tubes into the PCR instrument in sequence, set the program and parameters, save the file, and run the reaction program.

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

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

[0099] The comparative and example protocols (sequences shown in Table 1) were used to detect diluted Chinese national HIV standards in newborn calf serum. The comparative protocol only added 10 μL of a quantitative plasmid with a total set value of 600 IU / mL; the example protocol added an additional homologous plasmid with a final concentration of 10 IU / mL.

[0100] The results of the two systems on the Chinese national HIV standard diluted at 25 IU / mL newborn calf serum are as follows: Figures 3-4 As shown, compared to the comparative example, the HIV target sequence amplification curve of the HIV dual-channel dual-plasmid internal standard quantification technology (example) is more concentrated, and the variance of the Ct value is significantly lower than that of the comparative example, indicating more stable detection results. As shown in Table 2, the quantitative accuracy rate 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 rate of the example 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 quantification technology exhibits a lower quantitative lower limit and better detection accuracy for samples with low HIV viral load. The results also show that the system of this invention (…) Figure 3The curves of the two groups are more concentrated and have smaller deviations. On the other hand, from... Figure 3 and Figure 4 As can be seen, the amplification curves of the fifth reagent (red, CY5 channel) are present in all samples, indicating the presence of HIV virus in the samples. However, when the composition is used to test negative samples, such as... Figure 5 As shown, no amplification curve was observed for the fifth reagent.

[0101] Table 2

[0102]

[0103] *Quantitative inaccuracy; the logarithmic value of the sample being tested deviates from the actual sample concentration by more than ±0.5%.

[0104] Example 4: Detection results of test samples of composition 1 of the present invention

[0105] The HIV dual-channel dual-plasmid internal standard quantification technique (example) was used to detect the Chinese national HIV standard diluted at 25 IU / mL newborn calf serum. μL of a 600 IU / mL quantitative plasmid was added to the test sample. Based on this, homologous plasmids with final concentrations of 7 / 8 / 12 / 18 / 20 / 22 IU / mL for HIV were added to each group. In this test system, the lowest quantitation (LOQ) of the HIV single-plasmid internal standard system is approximately 30 IU / mL. As shown in Table 3, the optimal addition amount of HIV homologous plasmid is approximately between 7.5 IU / mL and 20 IU / mL, which is 25% to 66.7% of the lowest quantitation (LOQ) of the HIV single-plasmid internal standard system.

[0106] Table 3

[0107]

[0108] *Quantitative inaccuracy; the logarithmic value of the sample being tested deviates from the actual sample concentration by more than ±0.5%.

Claims

1. An HIV quantitative composition, comprising: The first reagent comprises at least three sets of primers and probes, wherein the at least three sets of primers and probes are used to amplify and detect at least one portion of at least one of the following HIV regions: LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region. The second reagent includes at least three homologous vectors, wherein each homologous vector includes at least one part of at least one of the following HIV regions: LTR region, GAG region, POL region, ENV region, VIF region, VPR region, VPU region, TAT region, REV region, or NEF region. The HIV regions in the second reagent correspond one-to-one with the HIV regions amplified in the first reagent. The third reagent includes M quantitative carriers; and The fourth reagent includes M sets of primers and probes, wherein the M sets of primers and probes are used to amplify and detect segments of the quantitative vector, respectively. Where M is a positive integer ≥ 1; The quantitative vector comprises 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. The modification includes replacing adenine deoxyribonucleotides in the homologous vector sequence with thymine deoxyribonucleotides, and replacing thymine deoxyribonucleotides in the homologous vector sequence with adenine deoxyribonucleotides. The quantitative vector in the fourth reagent comprises the HIV LTR' region, GAG' region, POL' region, ENV' region, VIF' region, VPR' region, VPU' region, TAT' region, REV' region, or NEF' region. The HIV segment in the fourth reagent corresponds one-to-one with the HIV amplification segment in the second reagent.

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

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

20.

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

19.

5. The HIV quantitative composition according to claim 1, characterized in that, The nucleotide sequence of the LTR' region is shown in SEQ ID NO. 5; the nucleotide sequence of the POL' region is shown in SEQ ID NO. 13; or the nucleotide sequence of the GAG' region is shown in SEQ ID NO.

21.

6. The HIV quantitative composition according to claim 5, characterized in that, The forward primer for amplifying the LTR' region is shown in SEQ ID NO. 6, the reverse primer is shown in SEQ ID NO. 7, and the probe is shown in SEQ ID NO. 8; the forward primer for amplifying the POL' region is shown in SEQ ID NO. 14, the reverse primer is shown in SEQ ID NO. 15, and the probe is shown in SEQ ID NO. 16; or the forward primer for amplifying the GAG' region is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the probe is shown in SEQ ID NO.

24.

7. The HIV quantitative composition according to claim 6, characterized in that, The composition further includes a fifth reagent comprising 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.

8. The HIV quantitative composition according to claim 7, characterized in that, The concentration of the homologous carrier is 25% to 66.7% of the minimum quantitative concentration.

9. An HIV quantitative kit comprising the HIV quantitative composition as described in any one of claims 1 to 8.

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