An HBV quantitative composition, kit, method and use

Abstract

This invention relates to the field of molecular biology detection, specifically to a quantitative composition, kit, method, and application; more specifically, to an HBV quantitative composition, kit, method, and application. This invention provides a quantitative composition comprising a homologous vector containing at least one portion of at least one of the C, P, X, or S regions of HBV, a quantitative vector, and primers and probes for amplifying both vectors. This invention creatively introduces a homologous vector, artificially increasing the concentration of the nucleic acid to be tested, thereby improving the stability and accuracy of the PCR amplification reaction. In particular, it can detect HBV more effectively in samples with low viral load.

Description

Technical Field

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

[0002] Hepatitis B virus (HBV) belongs to the Hepatoviridae family. HBV-infected individuals are infectious in their blood during the incubation period, acute phase, and chronic phase. The five HBV markers test cannot determine whether the virus is replicating, while HBV-DNA testing, which amplifies viral nucleic acid, is sensitive to low levels of HBV in the body and is a commonly used method to assess viral replication. HBV-DNA is the most direct, specific, and sensitive indicator of HBV infection. A positive HBV-DNA test indicates HBV replication and infectivity; the higher the HBV-DNA level, the greater the viral replication and the stronger the infectivity. Persistent HBV replication is the fundamental cause of hepatitis B.

[0003] Quantitative DNA testing plays a vital role in diagnosing HBV and assessing the effectiveness of HBV treatment. It can reveal the amount of virus in the body, replication level, infectivity, drug treatment efficacy, and help in developing treatment strategies. It is also the only laboratory test that can help diagnose occult HBV infection and occult chronic HBV.

[0004] Therefore, there is a need in this field for accurate quantitative detection of HBV, especially HBV with low viral load. Summary of the Invention

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

[0006] The first reagent comprises N sets of primers and probes, wherein the N sets of primers and probes are used to amplify and detect at least one portion of at least one of the C region, P region, X region, or S region of HBV;

[0007] The second reagent includes N homologous vectors, wherein each homologous vector includes at least one part of at least one of the C region, P region, X region, or S region of HBV, and the HBV segments in the second reagent correspond one-to-one with the HBV segments 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] Where N and M are both positive integers ≥ 1.

[0011] This invention innovatively introduces a homologous vector to artificially increase the concentration of HBV in the sample, thereby improving the stability and accuracy of the PCR amplification reaction. In particular, it can detect HBV more effectively in samples with low viral load.

[0012] Understandably, the C, P, X, and S regions of HBV refer to the C, P, X, and S genes of HBV, respectively. For example, the X region of HBV (i.e., the X gene) is the smallest open reading frame in the HBV genome, located at 1374-1838 bp, with a total length of 435-462 bp. The X gene sequence is highly conserved in the Hepatoviridae family.

[0013] 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.

[0014] Furthermore, the quantitative vector includes a nucleotide sequence derived from modifications to the HBV segment in the homologous vector, the modifications including 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).

[0015] In some specific embodiments, the quantification vector includes a nucleotide sequence, which is a sequence modified to modify the HBV segment C, P, X, or S region in the homologous vector into a C', P', X', or S' 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 region C of the HBV is shown in SEQ ID NO.4.

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

[0019] Furthermore, the nucleotide sequence of the S region of the HBV is shown in SEQ ID NO.20.

[0020] In some specific implementation schemes, N can be 1, 2, 3, etc.

[0021] For example, when N is 1, the first reagent includes a set of primers and probes that amplify a specific region of the C region of HBV; correspondingly, the second reagent includes a homologous vector that includes the same region as the specific region of the C region of HBV amplified by the first reagent.

[0022] For example, when N is 2, the first reagent includes two sets of primers and probes, which can amplify specific regions of the C and P regions of HBV, respectively; correspondingly, the second reagent includes two homologous vectors, one homologous vector including the same region as the specific region of the C region of HBV amplified by the first reagent, and the other homologous vector including the same region as the specific region of the P region of HBV amplified by the first reagent.

[0023] For example, when N is 3, the first reagent includes 3 sets of primers and probes, which can amplify specific regions of the C, P, and S regions of HBV, respectively; correspondingly, the second reagent includes 3 homologous vectors, one homologous vector including the same region as the specific region of the C region of HBV amplified by the first reagent, one homologous vector including the same region as the specific region of the P region of HBV amplified by the first reagent, and one homologous vector including the same region as the specific region of the S region of HBV amplified by the first reagent.

[0024] In some specific implementation schemes, M can be 1, 2, 3, etc. For example, the values ​​of M in the third and fourth reagents are similar to the values ​​of N in the first and second reagents, and will not be repeated here.

[0025] In some specific implementations, the forward primer for amplifying region C of HBV 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.

[0026] In some specific implementations, the forward primer for amplifying the P region of HBV 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.

[0027] In some specific implementations, the forward primer for amplifying the S region of HBV 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.

[0028] When N=2, the nucleotide sequences of the HBV segment C region, P region or S region in the first reagent and the second reagent are any two of SEQ ID NO.4, SEQ ID NO.8 or SEQ ID NO.20.

[0029] When N=2, the nucleotide sequences of the primers and probes in the first reagent amplify any two of SEQ ID NO.4, 8 or 20.

[0030] When N=3, the nucleotide sequences of the C, P and S regions of HBV in the first and second reagents are SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.20, respectively.

[0031] When N=3, the nucleotide sequences of the primers and probes in the first reagent are as follows: SEQ ID NO.1~SEQ ID NO.3, SEQ ID NO.9~SEQ ID NO.11, and SEQ ID NO.17~SEQ ID NO.19.

[0032] 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.

[0033] 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.

[0034] In some specific implementations, the vector may be a plasmid.

[0035] 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 C region, P region, X region and / or S region, etc.

[0036] In some specific implementations, the backbone plasmid of the homologous plasmid and the quantitative plasmid is the pUC57 plasmid, that is, the sequence of region C is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the A in region C is replaced with T, and the sequence after T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0037] In some specific implementations, the vector may be a recombinant virus comprising the plasmids described above.

[0038] In some specific implementations, the vector may be a liposome comprising the plasmid described above.

[0039] In some specific implementations, the vector may be an artificial chromosome carrying a target segment.

[0040] Furthermore, the quantitative plasmid includes a C' region, the nucleotide sequence of which is as shown in SEQ ID NO.5.

[0041] Furthermore, the quantitative plasmid includes a P' region, the nucleotide sequence of which is as shown in SEQ ID NO.13.

[0042] Furthermore, the quantitative plasmid includes an S' region, the nucleotide sequence of which is as shown in SEQ ID NO.21.

[0043] In some specific implementations, the forward primer for amplifying the C' 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.

[0044] In some specific implementations, the forward primer for amplifying the P' 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.

[0045] In some specific implementations, the forward primer for amplifying the S' 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.

[0046] When M=2, the quantitative carrier includes any two of the nucleotide sequences SEQ ID NO.5, 13 or 21 shown in the C' region, P' region or S' region.

[0047] When M=2, the nucleotide sequences of the primers and probes in the fourth reagent amplify any two of SEQ ID NO.5, SEQ ID NO.13, or SEQ ID NO.21.

[0048] When M=3, the quantitative carrier includes the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.21, which are C', P', or S' regions.

[0049] When M=3, the nucleotide sequences of the primers and probes in the fourth reagent are as follows: SEQ ID NO.6-8, SEQ ID NO.14-SEQ ID NO.16, and SEQ ID NO.22-SEQ ID NO.24.

[0050] Furthermore, the concentration of the homologous carrier is 25% to 66.7% of the minimum quantitative concentration, preferably 30% to 60%.

[0051] Using the concentration of the aforementioned homologous vector allows for more accurate quantification of low-concentration HBV.

[0052] 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.

[0053] 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 differs from the pathogen segment amplified by the first reagent and the HBV segment in the second reagent.

[0054] 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.

[0055] In some specific embodiments, the composition further includes a fifth reagent that amplifies segment Y of the pathogen, the segment Y being different from the nucleotide sequences shown in segments C, P, X, or S of the pathogen.

[0056] In one specific implementation, the modification may be a locked nucleic acid modification.

[0057] In some specific embodiments, the composition further includes replacing the second reagent with a sixth reagent, wherein the sixth reagent comprises a segment of HBV, the HBV segment in the sixth reagent being obtained by modifying the HBV segment included in the second reagent.

[0058] In one specific embodiment, the sixth reagent comprises a segment of HBV as shown in SEQ ID NO. 25. Further, it is obtained by modifying a segment of HBV as shown in SEQ ID NO. 12.

[0059] The seventh reagent includes a probe that can target the HBV segment included in the second reagent but cannot target the HBV segment included in the sixth reagent.

[0060] In one specific implementation, the seventh reagent includes a probe as shown in SEQ ID NO.26.

[0061] In some specific embodiments, the first, fourth, fifth, and seventh reagents in the quantitative composition of the present invention can be modified. Further, the modification can be one or more of locked nucleic acid (LNA) modification, MGB modification, or ZNA (Zip Nucleic Acids) modification.

[0062] In some specific embodiments, the HBV quantification composition of the present invention is used for fluorescent PCR.

[0063] Furthermore, the fluorescent groups of the probes mentioned in this invention are different from each other and do not interfere with each other.

[0064] In this paper, "dissimilar and non-interfering" means that the fluorescent groups used in the probes of the first, fourth, fifth, and seventh 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.

[0065] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0066] Furthermore, the 3' end of the probe also has a quenching group, such as MGB, BHQ1, or BHQ2.

[0067] In one specific embodiment, each component of the HBV quantitative composition of the present invention is contained in a separate package.

[0068] In one specific embodiment, the components of the HBV quantitative composition of the present invention are contained in the same package.

[0069] Furthermore, the components of the HBV quantitative composition of the present invention exist in a mixed form.

[0070] Secondly, the present invention provides the use of the above-mentioned HBV quantitative composition for preparing HBV quantitative kits.

[0071] Thirdly, the present invention provides an HBV quantitative kit comprising the HBV quantitative composition as described above.

[0072] Furthermore, the kit also includes nucleic acid amplification reagents.

[0073] Furthermore, the amplification reagents include dNTPs, PCR buffer, DNA polymerase, and Mg. 2+ At least one of them.

[0074] Furthermore, the kit also includes: nucleic acid release reagent and nucleic acid extraction reagent.

[0075] 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.

[0076] Furthermore, the concentration of the DNA polymerase is 3 U / μL to 15 U / μL, for example, the DNA polymerase can be Taq polymerase.

[0077] In one specific embodiment, the kit of the present invention includes: Taq enzyme, Mg 2+ Mn 2+ dNTPs and PCR buffer.

[0078] 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.

[0079] Fourthly, the present invention provides a method for quantifying HBV, the method comprising the following steps:

[0080] 1) Extract nucleic acid from the sample to be tested;

[0081] 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

[0082] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0083] Furthermore, the quantitative formula is as follows: P1 is the probe for the first reagent, and P2 is the probe for the fourth reagent.

[0084] Furthermore, a method for quantifying HBV for non-diagnostic purposes is provided, the method comprising the following steps:

[0085] 1) Extract nucleic acid from the sample to be tested;

[0086] 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

[0087] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0088] 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

[0089] Figure 1 These are exemplary detection patterns of compositions 1 to 3 of the present invention;

[0090] Figure 2 This is a graph showing the test results of the composition of the present invention.

[0091] Figure 3 This is a graph showing the test results of the comparative composition of the present invention.

[0092] Figure 4 This is an exemplary detection diagram of composition 4 of the present invention;

[0093] Figure 5 This is a graph showing the results of detecting positive HBV samples using composition 4 of the present invention.

[0094] Figure 6 This is a diagram showing the results of detecting a negative HBV sample using composition 4 of the present invention. Detailed Implementation

[0095] 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.

[0096] Measurement of the lowest quantitation concentration (limit of quantitation):

[0097] Using the national standard for HBV, newborn calf serum was diluted to concentrations of 30 IU / mL, 20 IU / mL, 15 IU / mL, 12 IU / mL, 10 IU / mL, 8 IU / mL, and 5 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 quotient (LoQ) was the lowest measurable analyte concentration that could be measured with 100% accuracy. Because primers and probes are part of the system, when replacing primers and probes, the LOQ value of HBV in different embodiment systems needed to be recalculated using the method described above.

[0098] Example 1: The sequence used in this invention

[0099] Some exemplary sequences used in this invention are shown in Table 1 below, where + indicates that the position is a locked nucleic acid modification.

[0100] Table 1

[0101]

[0102]

[0103]

[0104] In Table 1: "+A" means LNA-A; "+T" means LNA-T; "+C" means LNA-C; "+G" means LNA-G.

[0105] Example 2: Quantitative Method for HBV

[0106] Exemplary methods of the present invention are as follows: Figure 1 As shown in the figure. This project used the Sansure Biotech S10012 nucleic acid extraction kit for HBV nucleic acid extraction. Diluted newborn calf serum was used to plot HBV concentration curves or as a sample to test the performance of the internal standard quantitative system.

[0107] Using the national standard for HBV, newborn calf serum was diluted to concentrations of 20 IU / mL, 15 IU / mL, 12 IU / mL, 10 IU / mL, 8 IU / mL, and 5 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 (LOQ) was the lowest measurable analyte concentration that could be measured with 100% accuracy. Based on this system, the LOQ for HBV was calculated to be 12 IU / mL.

[0108] PCR reaction conditions:

[0109] Phyto-Anstart Taq polymerase (5U); Sansure Biotech S08 Buffer; primers / probes synthesized in Bailige and diluted to working concentration of 40 pmol / μL via TE buffer; 1 mol / L Mg 2+ HBV and IC target sequence plasmids were synthesized at Sangon Biotech.

[0110] A single-sample 50 μL amplification system includes: 20.75 μL PCR buffer (S08), 2 μL 100 mmol / L dNTP(T), 0.6 μL 40 pmol / μL PCR forward and reverse primers, 0.1 μL 40 pmol / μL probe, 1.6 μL 5 U Taq DNA polymerase, and 0.4 μL 1 mol / L MgSO₄. 2+ 22.65 μL of purified water.

[0111] PCR amplification program: 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, reaction volume set to 50 μL.

[0112] 10 μL of a 1.00E-7 μg / mL quantitative plasmid or HBV homologous plasmid was added to 400 μL of newborn calf serum. An HBV concentration curve was plotted using diluted Chinese national HBV standard from the newborn calf serum for determination. The average concentration obtained from three independent replicate experiments was set as the HBV determination concentration for 10 μL of the plasmid. In this protocol, 10 μL of the 1.00E-7 μg / mL quantitative plasmid was used for determination in a 400 μL sample system, and the determination concentration was diluted to a usable concentration of 1700 IU / mL using TE buffer. Similarly, 10 μL of the 1.00E-7 μg / mL HBV homologous plasmid was used for determination in a 400 μL sample system, and the determination concentration was diluted to a usable concentration of 1000 IU / mL using TE buffer.

[0113] After completing plasmid determination, add the 1700 IU / mL quantitative plasmid and the corresponding homologous plasmid to 400 μL of the sample to be tested, mix well, and then add 300 μL of extraction solution 1 to the centrifuge tube. Vortex and centrifuge briefly. Add 100 μL of extraction solution 2 to each tube, vortex, and incubate at room temperature for 10 minutes. After brief centrifugation, place the tube on a magnetic separator for 3 minutes. Once the magnetic beads have aggregated on the centrifuge tube wall, aspirate the liquid. Add 600 μL of extraction solution 3 and 200 μL of extraction solution 4 to the centrifuge tube simultaneously, vortex, and centrifuge briefly. Place the centrifuge tube back on the magnetic separator. After approximately 3 minutes, aspirate the liquid again. Elute the magnetic beads with 50 μL of PCR reaction solution and place the tube on the magnetic separator. Transfer the supernatant to a PCR reaction tube, vortex, and centrifuge briefly. Place the tubes in the PCR instrument in sequence, set the running program and relevant parameters, save the file, and run the reaction program.

[0114] For example, the final concentration of the quantitative carrier in the sample is 1700 IU / mL, therefore, according to the formula... Calculate the HBV load in the sample to be tested.

[0115] Example 3: Detection results of test samples of composition 1 of the present invention

[0116] The comparative and example protocols (first reagent: primers and probes shown in SEQ ID NO.1 to SEQ ID NO.3; second reagent: homologous vector including SEQ ID NO.4; third reagent: quantitative vector including SEQ ID NO.5; fourth reagent: primers and probes shown in SEQ ID NO.6 to SEQ ID NO.8) were used to detect diluted Chinese national HBV standard in newborn calf serum. In the comparative example, only 10 μL of quantitative vector was added to the quantitative plasmid with a final concentration of 1700 IU / mL in the sample to be tested. In the example, in addition, homologous plasmid with a final concentration of 5 IU / mL was added.

[0117] The detection results of the two systems on the Chinese national HBV standard diluted at 15 IU / mL newborn calf serum are shown below: Compared with the comparative example, the HBV target sequence amplification curve of the 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 of the comparative example is 91.67% (11 / 12), and the logarithmic deviation of the concentration of 11 detected samples is within ±0.5; the quantitative accuracy of the example is 100% (12 / 12), and the logarithmic deviation of the concentration of 12 detected samples is within ±0.5. The HBV dual-channel dual-plasmid internal standard quantification technology exhibits a lower quantitative lower limit and better detection accuracy for samples with low HBV load.

[0118] Table 2

[0119]

[0120] Example 4: Detection results of test samples of composition 2 of the present invention

[0121] Using the national standard for HBV, newborn calf serum was diluted to concentrations of 20 IU / mL, 18 IU / mL, 16 IU / mL, 15 IU / mL, and 10 IU / mL. The lowest concentration samples were used as test samples. Each test sample was analyzed in 20 replicates, for a total of 60 replicates (3 times each). The accuracy requirement was a logarithmic deviation of within ±0.5%. The lowest quantitation (LOQ) was the lowest measurable analyte concentration that could be measured with 100% accuracy. Based on this system, the LOQ for HBV was calculated to be 16 IU / mL.

[0122] The comparative and example protocols (first reagent: primers and probes shown in SEQ ID NO. 9 to SEQ ID NO. 11; second reagent: homologous vector including SEQ ID NO. 12; third reagent: quantitative vector including SEQ ID NO. 13; fourth reagent: primers and probes shown in SEQ ID NO. 14 to SEQ ID NO. 16) were used to detect diluted Chinese national HBV standard in newborn calf serum. In the comparative example, only 10 μL of quantitative plasmid was added to achieve a final concentration of 4800 IU / mL in the sample to be tested. In the example, an additional homologous plasmid with a final concentration of 8 IU / mL was added.

[0123] The results of the two systems on the detection of the Chinese national HBV standard diluted at 10 IU / mL newborn calf serum are as follows: Figures 2-3 As shown, compared to the comparative example, the amplification curve of the HBV target sequence in the HBV dual-channel dual-plasmid internal standard quantification technology (example) is more concentrated, and the variance of the Ct value is significantly lower than that in the comparative example, indicating more stable detection results. As shown in Table 3, the quantitative accuracy rate of the comparative example is 93.75% (15 / 16), with the logarithmic deviation of the concentration of 15 detected samples within ±0.5; the quantitative accuracy rate of the example is 100% (16 / 16), with the logarithmic deviation of the concentration of 16 detected samples within ±0.4. The HBV dual-channel dual-plasmid internal standard quantification technology exhibits a lower quantitative lower limit and better detection accuracy for samples with low HBV load. The results also show that the system of this invention (…) Figure 2 The curves are more concentrated and have smaller deviations.

[0124] Table 3

[0125]

[0126] *Quantitative inaccuracy; the logarithmic value of the sample being tested deviates from the actual sample concentration by more than ±0.5%.

[0127] The results of two internal standard quantification systems for detecting diluted Chinese national HBV standard at 8 IU / mL newborn calf serum are as follows: Figure 2 As shown, compared to the comparative example, the amplification curve of the HBV target sequence using the dual-channel dual-plasmid internal standard quantification technique (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 4, the quantitative accuracy rate of the comparative example was 81.25% (13 / 16), with the logarithmic deviation of the concentration of 13 detected samples within ±0.5; the quantitative accuracy rate of the example was 100% (16 / 16), with the logarithmic deviation of the concentration of 16 detected samples within ±0.5. The HBV dual-channel dual-plasmid internal standard quantification technique demonstrates a lower quantitative lower limit and better accuracy.

[0128] Table 4

[0129]

[0130] *Quantitative inaccuracy; the logarithmic value of the sample being tested deviates from the actual sample concentration by more than ±0.5%.

[0131] Example 5: Detection results of test samples of composition 2 of the present invention

[0132] The HBV dual-channel dual-plasmid internal standard quantification technique (Example) was used to detect 10 IU / mL diluted Chinese national HBV standard from newborn calf serum. μL of a quantification plasmid with a final concentration of 1700 IU / mL was added to the test sample. Based on this, homologous plasmids with final HBV concentrations of 2 / 3 / 4 / 6 / 8 / 10 / 12 IU / mL were added to each group. In this test system, the lowest quantitation (LOQ) of the HBV single-plasmid internal standard system was approximately 12 IU / mL. As shown in Table 5, the optimal addition amount of HBV homologous plasmid is approximately between 3 IU / mL and 8 IU / mL, which is 25% to 66.7% of the LOQ of the HBV single-plasmid internal standard system.

[0133] Table 5

[0134]

[0135] Example 6: Further optimization of the test samples of composition 2 of the present invention. Test sample results of composition 4.

[0136] In this embodiment, a third probe P3 was designed based on the HBV target region of composition 2 of the present invention. Simultaneously, the target sequence of the corresponding segment of the homologous plasmid of composition 2 of the present invention was modified, specifically by replacing adenine deoxyribonucleotide (A) in the segment corresponding to probe P3 in the homologous plasmid sequence with thymine deoxyribonucleotide (T), and replacing thymine deoxyribonucleotide (T) in the original sequence with adenine deoxyribonucleotide (A). Specific sequence changes are shown in Table 6 below.

[0137] Table 6

[0138]

[0139]

[0140] As shown in the table above, this embodiment modifies a segment of the homologous plasmid to prepare the sixth reagent, and designs a probe sequence for the seventh reagent targeting the corresponding position of the original HBV P segment. This enables both quantitative detection of the HBV target region and qualitative detection of HBV. A schematic diagram of the specific model is shown below. Figure 4 As shown in the image, this is a detailed test result. Figures 5-6 As shown, in HBV-positive samples, the fluorescence signal of the CY5 channel exceeds the threshold line, indicating HBV positivity; in HBV-negative samples, the fluorescence signal of the CY5 channel is below the threshold line, indicating HBV negativity. This optimized scheme, based on the second embodiment of the composition of the present invention, can effectively determine the HBV positivity or negativeness of the sample through the CY5 fluorescence channel.

[0141] In summary, this approach amplifies the HBV P region and a homologous plasmid segment using the same set of primers. A FAM-labeled quantitative probe P1 is used to detect the shared sequence between the HBV P region and the homologous plasmid segment for quantitative analysis. A CY5-labeled probe P3 is used to detect the corresponding sequence of the HBV P region for qualitative analysis of the HBV target sequence.

[0142] Example 7: Detection results of test samples of composition 3 of the present invention

[0143] Using the national standard for HBV, newborn calf serum was diluted to 30 IU / mL, 25 IU / mL, 20 IU / mL, and 15 IU / mL. The lowest concentration of the diluted sample was used as the test sample. 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 the test results within ±0.5%. The lowest quantitation (LOQ) was the lowest measurable concentration of the analyte that could be measured to achieve 100% accuracy. Based on the calculations for this composition system, the LOQ for HBV was 25 IU / mL.

[0144] The comparative and example protocols (first reagent: primers and probes shown in SEQ ID NO.17 to SEQ ID NO.19; second reagent: homologous vector including SEQ ID NO.20; third reagent: quantitative vector including SEQ ID NO.21; fourth reagent: primers and probes shown in SEQ ID NO.22 to SEQ ID NO.24) were used to detect diluted Chinese national HBV standard in newborn calf serum. In the comparative example, only 10 μL of quantitative plasmid was added to achieve a final concentration of 8800 IU / mL in the sample to be tested. In the example, homologous plasmid with a final concentration of 10 IU / mL was added in addition to this.

[0145] The detection results of the two systems on the diluted Chinese national HBV standard at 20 IU / mL newborn calf serum are shown below: Compared with the comparative example, the HBV dual-channel dual-plasmid internal standard quantification technology (example) showed a more concentrated amplification curve of the HBV target sequence, and the variance of the Ct value was significantly lower than that of the comparative example, indicating more stable detection results. As shown in Table 7, the quantitative accuracy of the comparative example was 83.33% (10 / 12), with the logarithmic deviation of the concentration of 10 detected samples within ±0.5; the quantitative accuracy of the example was 100% (12 / 12), with the logarithmic deviation of the concentration of 12 detected samples within ±0.5. The HBV dual-channel dual-plasmid internal standard quantification technology exhibits a lower quantitative lower limit and better detection accuracy for samples with low HBV load.

[0146] Table 7

[0147]

Claims

1. An HBV quantitative composition, comprising: The first reagent comprises N sets of primers and probes, wherein the N sets of primers and probes are used to amplify and detect at least one portion of at least one of the C region, P region, X region or S region of HBV; The second reagent includes N homologous vectors, wherein each homologous vector includes at least one part of at least one of the C region, P region, X region or S region of HBV, and the HBV segments in the second reagent correspond one-to-one with the HBV segments 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 N and M are both positive integers ≥ 1; The quantitative vector comprises a nucleotide sequence, wherein the nucleotide sequence is a sequence in which the HBV segment C, P, X, or S region in the homologous vector is modified into a C', P', X', or S' region. The modification involves 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 third reagent comprises at least one part of at least one of the HBV segment C', P', X', or S' region, and the HBV segment in the third reagent corresponds one-to-one with the HBV segment amplified in the first reagent.

2. The HBV 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 HBV quantitative composition according to claim 1, characterized in that, The nucleotide sequence of region C of the HBV is shown in SEQ ID NO.4; the nucleotide sequence of region P of the HBV is shown in SEQ ID NO.12; or the nucleotide sequence of region S of the HBV is shown in SEQ ID NO.

20.

4. The HBV quantitative composition according to claim 2, characterized in that, The forward primer for amplifying region C of HBV 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 region P of HBV 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 region S of HBV 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.

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5. The HBV quantitative composition according to claim 1, characterized in that, The nucleotide sequence of the C' region is shown in SEQ ID NO.5; the nucleotide sequence of the P' region is shown in SEQ ID NO.13; or the nucleotide sequence of the S' region is shown in SEQ ID NO.

21.

6. The HBV quantitative composition according to claim 5, characterized in that, The forward primer for amplifying the C' 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 P' 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 S' 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.

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7. The HBV quantitative composition according to claim 6, characterized in that, The composition further includes replacing the second reagent with a sixth reagent, wherein the sixth reagent includes a segment of HBV, the HBV segment in the sixth reagent being obtained by modifying the HBV segment included in the second reagent; and further includes a seventh reagent, wherein the seventh reagent includes a probe, the probe in the seventh reagent being capable of targeting the HBV segment included in the second reagent but not the HBV segment included in the sixth reagent.

8. The HBV 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 HBV quantitative kit comprising the HBV quantitative composition as described in any one of claims 1 to 8.

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