HBV 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 HBV quantitative composition, a kit, a method and application. The present invention provides a quantitative composition comprising a homologous vector of at least one portion of at least one of the C region, P region, X region, or S region of the HBV, and a quantitative vector, as well as primers and probes for amplifying the two vectors. The homologous vector is creatively introduced, and the concentration of nucleic acid to be detected is artificially increased, so that the stability and accuracy of PCR amplification reaction are improved. Particularly, in a sample with low virus load, the HBV can be well detected.

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 a HBV quantitative composition, a kit, a method and a use thereof. Background Art

[0002] Hepatitis B virus (Hepatitis B) belongs to the Hepadnaviridae family. The blood of people infected with hepatitis B virus is infectious regardless of whether they are in the latent, acute or chronic stages. The five tests for hepatitis B cannot be used as an indicator to determine whether the virus is replicating. However, DNA testing is sensitive to low levels of HBV virus in the body by amplifying viral nucleic acid, and is a common means of determining viral replication. DNA is the most direct, specific and sensitive indicator of hepatitis B virus infection. A positive HBV-DNA indicates HBV replication and infectiousness. The higher the HBV-DNA, the more the virus replicates and the more infectious it is. The continuous replication of hepatitis B virus is the root cause of hepatitis B disease.

[0003] DNA quantitative testing also plays a very important role in diagnosing HBV and evaluating the effectiveness of HBV treatment. It can be used to understand the number of viruses in the body, the level of replication, the infectivity, the effectiveness of drug treatment, the formulation of treatment strategies, etc. and serve as an evaluation indicator. It is also the only laboratory test indicator that can help diagnose latent HBV infection and latent chronic HBV.

[0004] Therefore, there is a need in the art 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 a HBV quantitative composition, comprising:

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

[0007] A second reagent, comprising N homologous vectors, wherein the homologous vectors respectively comprise at least one portion 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 respectively correspond one-to-one to the HBV segments amplified in the first reagent;

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

[0009] A fourth reagent, comprising 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] Wherein, N and M are positive integers both ≥1.

[0011] The present invention creatively 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, HBV can be better detected in samples with low viral loads.

[0012] It can be understood that the segments C, P, X, and S of HBV refer to the C gene, P gene, X gene, and S gene of HBV, respectively. For example, the X region of HBV (i.e., the X gene) is the smallest open reading frame in the HBV viral genome, located at 1374-1838 bp of the HBV genome, with a total length of 435-462 bp, and the X gene sequence is highly conserved in the Hepadnaviridae family.

[0013] Furthermore, the quantitative vector can be any vector used for quantification 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, which is derived from the modification of the HBV segment in the homologous vector, and the modification includes 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).

[0015] In some specific embodiments, the quantitative vector comprises a nucleotide sequence, which is a sequence of the HBV segment C region, P region, X region, or S region in the homologous vector that is transformed into the C' region, P' region, X' region, or S' 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 HBV segment C region is shown in SEQ ID NO.4.

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

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

[0020] In some specific embodiments, 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 segment C of HBV; accordingly, the second reagent includes a homologous vector that includes the same region as the specific region of segment C of HBV amplified by the first reagent.

[0022] For example, when N is 2, the first reagent includes 2 sets of primers and probes, which can respectively amplify specific regions of segments C and P of HBV; accordingly, the second reagent includes 2 homologous vectors, one homologous vector includes the same region as the specific region of segment C of HBV amplified by the first reagent, and the other homologous vector includes the same region as the specific region of segment P 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 respectively amplify specific regions of segments C, P and S of HBV; accordingly, the second reagent includes 3 homologous vectors, one homologous vector includes the same region as the specific region of segment C of HBV amplified by the first reagent, one homologous vector includes the same region as the specific region of segment P of HBV amplified by the first reagent, and one homologous vector includes the same region as the specific region of segment S of HBV amplified by the first reagent.

[0024] In some specific embodiments, M can be 1, 2, 3, etc. For example, the value of M in the third and fourth reagents is similar to the value of N in the first and second reagents, and will not be repeated.

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

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

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

[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 correspond to amplifying any two of SEQ ID NO. 4, 8 or 20.

[0030] When N=3, the nucleotide sequences of the C region, P region and S region of HBV in the first reagent and the second reagent 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 such as SEQ ID NO.1 to SEQ ID NO.3, SEQ ID NO.9 to SEQ ID NO.11, and SEQ ID NO.17 to SEQ ID NO.19.

[0032] 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.

[0033] 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.

[0034] In some specific embodiments, the vector can be a plasmid.

[0035] Furthermore, the backbone plasmids of the homologous plasmid and the quantitative plasmid are derived from 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 C region, P region, X region and / or S region, etc.

[0036] 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 C region is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the sequence after A in the C region is replaced by T and T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0037] In some specific embodiments, the vector may be a recombinant virus comprising the above-mentioned plasmid.

[0038] In some specific embodiments, the vector can be a liposome comprising the above-mentioned plasmid.

[0039] In some specific embodiments, the vector can be an artificial chromosome carrying the target segment.

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

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

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

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

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

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

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

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

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

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

[0051] Using the above homologous vector concentrations allows for more accurate quantification of low concentrations of HBV.

[0052] 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.

[0053] 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 HBV in the second reagent.

[0054] 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.

[0055] In some specific embodiments, the composition further comprises a fifth reagent, which amplifies segment Y of the pathogen, wherein the segment Y is different from the nucleotide sequence shown in segment C, P, X or S of the pathogen.

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

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

[0058] In a specific embodiment, the sixth agent includes the HBV segment shown in SEQ ID NO. 25. Further, it is obtained by modifying the HBV segment shown in SEQ ID NO. 12.

[0059] and a seventh reagent, wherein the seventh reagent comprises a probe, wherein the probe in the seventh reagent is capable of targeting the segment of HBV included in the second reagent but is not capable of targeting the segment of HBV included in the sixth reagent.

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

[0061] In some specific embodiments, the first reagent, the fourth reagent, the fifth reagent, and the seventh 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) modification, MGB modification, or ZNA (Zip Nucleic Acids) modification.

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

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

[0064] 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, the fifth reagent and the seventh 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.

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

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

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

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

[0070] In a second aspect, the present invention provides use of the above-mentioned HBV quantitative composition for preparing an HBV quantitative kit.

[0071] In a third aspect, the present invention provides an HBV quantitative kit, comprising the HBV quantitative composition as described above.

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

[0073] Furthermore, the amplification reagent includes dNTP, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0074] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0075] 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.

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

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

[0078] Common PCR buffers are composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μL to 200 μL.

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

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

[0081] 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

[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 Among them, P1 is the probe of the first reagent, and P2 is the probe of the fourth reagent.

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

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

[0086] 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

[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 Among them, P1 is the probe of the first reagent, and P2 is the probe of the fourth reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 exemplary detection diagrams of compositions 1 to 3 of the present invention;

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

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

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

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

[0094] Figure 6 This is a graph showing the results of the composition 4 of the present invention detecting a negative HBV sample. DETAILED DESCRIPTION

[0095] 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.

[0096] Measurement of minimum quantitative concentration (limit of quantitation):

[0097] Use HBV national standard, use newborn calf serum to dilute to 30IU / mL, 20IU / mL, 15IU / mL, 12IU / mL, 10IU / mL, 8IU / mL and 5IU / mL concentrations, use the diluted low-concentration sample as the test sample, each test sample is tested 20 times each time, and it is done 3 times in a row, for a total of 60 times of repeated testing. The accuracy requirement is that the logarithmic deviation of the test result is within ±0.5, and the minimum quantitative concentration (LoQ) is the minimum concentration of the test substance that can be measured when the 100% accurate measurement requirement is met. Because the primer probe is part of the system, when the primer probe is replaced, the LOQ value of HBV under the different embodiment systems needs to be recalculated by the above method.

[0098] Example 1: Sequences used in the present invention

[0099] 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.

[0100] Table 1

[0101]

[0102]

[0103]

[0104] In Table 1, “+A” means LNA-A; “+T” means LNA-T; “+C” means LNA-C; and “+G” means LNA-G.

[0105] Example 2: Quantitative method for HBV

[0106] Exemplary methods of the present invention are as follows Figure 1 This project uses the Shengxiang Biotechnology S10012 nucleic acid extraction kit to extract HBV nucleic acid, and the Chinese national HBV standard diluted with newborn calf serum is used to draw the HBV concentration curve or as a sample to be tested to test the performance of the internal standard quantitative system.

[0107] Use HBV national standard and dilute with newborn calf serum to 20IU / mL, 15IU / mL, 12IU / mL, 10IU / mL, 8IU / mL and 5IU / mL. Use the diluted low-concentration samples as test samples. Perform 20 repeated tests for each test sample each time, and do it 3 times in a row, for a total of 60 repeated tests. 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. Calculated under this system, the LOQ of HBV is 12IU / mL.

[0108] PCR reaction conditions:

[0109] Feipeng anstart taq enzyme (5U); Shengxiang Bio S08 Buffer; primers / probes synthesized in Bio-Tech and diluted to a working concentration of 40 pmol / μL by dissolving in TE; 1 mol / L Mg 2+ HBV and IC target sequence plasmids were synthesized at Bioengineering.

[0110] The amplification system for a single person of 50 μL includes: 20.75 μL PCR buffer (S08), 2 μL 100 mmol / L dNTP (T), 0.6 μL 40 pmol / μL PCR upstream and downstream primers, 0.1 μL 40 pmol / μL probe, 1.6 μL 5U Taq DNA polymerase, 0.4 μL 1 mol / L Mg 2+ , 22.65 μL purified water.

[0111] PCR amplification program: 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.

[0112] 10μL 1.00E-7μg / mL quantitative plasmid or HBV homologous plasmid was added to 400μL newborn calf serum, and the HBV concentration curve was drawn by diluting the Chinese national HBV standard with newborn calf serum for determination. The average of the determined concentrations measured by three independent repeated tests was set as the HBV determined concentration at the addition of 10μL of the plasmid. In this scheme, 10μL 1.00E-7μg / mL quantitative plasmid was determined in 400μL of the sample system to be tested, and the determined concentration was diluted to a working concentration of 1700IU / mL by TE, and 10μL 1.00E-7μg / mL HBV homologous plasmid was determined in 400μL of the sample system to be tested, and the determined concentration was diluted to a working concentration of 1000IU / mL by TE.

[0113] After completing the plasmid determination, add the quantitative plasmid with a determination value of 1700IU / mL under the above scheme and the homologous plasmid of the corresponding concentration to 400μL of the sample to be tested. After mixing evenly, add 300μL of extraction solution 1 to the centrifuge tube, shake and mix, and then centrifuge instantly. Add 100μL of extraction solution 2 to each tube, shake and mix, and let stand at room temperature for 10 minutes. After instant centrifugation, place it on the magnetic separator for 3 minutes. When the magnetic beads are gathered on the wall of the centrifuge tube, discard the liquid in the tube, add 600μL of extraction solution 3 and 200μL of extraction solution 4 to the centrifuge tube at the same time, shake and mix, and centrifuge instantly, and then place the centrifuge tube on the magnetic separator again. After about 3 minutes, discard the liquid in the tube again. After 50μL of PCR reaction solution is taken to elute the magnetic beads, place it on the magnetic separator, transfer the separated clear liquid to the PCR reaction tube, shake and mix, and centrifuge instantly. Put it into the PCR instrument in order, set the running program and related parameters, save the file, and run the reaction program.

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

[0115] Example 3: Test results of the test sample of the composition 1 of the present invention

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

[0117] The test results of the two systems for the Chinese national HBV standard diluted with 15 IU / mL newborn calf serum are as follows: Compared with the comparative example, the amplification curve of the HBV target sequence of the HBV dual-channel dual-plasmid internal standard quantitative technology (embodiment) is more concentrated, and the Ct value variance is significantly lower than that of the comparative example, that is, the test results are more stable. 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 embodiment 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 quantitative technology shows a lower quantitative lower limit and better detection accuracy for low HBV load samples.

[0118] Table 2

[0119]

[0120] Example 4: Test results of the test sample of composition 2 of the present invention

[0121] Use HBV national standard and dilute with newborn calf serum to 20IU / mL, 18IU / mL, 16IU / mL, 15IU / mL and 10IU / mL. Use the diluted low-concentration samples as test samples. Perform 20 repeated tests for each test sample each time, and do it three times in a row, for a total of 60 repeated tests. 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. Calculation under this system shows that the LOQ of HBV is 16IU / mL.

[0122] The Chinese national HBV standard diluted with newborn calf serum was detected using the comparative example and the embodiment scheme (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). In the comparative example, only 10 μL of quantitative plasmid was added to make the final concentration of the quantitative vector in the sample to be tested 4800 IU / mL; on this basis, the embodiment additionally added a homologous plasmid with a final concentration of 8 IU / mL.

[0123] The test results of the two systems for the Chinese national HBV standard diluted with 10 IU / mL newborn calf serum are as follows Figures 2-3 As shown, compared with the comparative example, the amplification curve of the HBV target sequence of the HBV 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 3, the quantitative accuracy of the comparative example is 93.75% (15 / 16), and the logarithmic deviation of the concentration of 15 detected samples is within ±0.5; the quantitative accuracy of the embodiment is 100% (16 / 16), and the logarithmic deviation of the concentration of 16 detected samples is within ±0.4. The HBV dual-channel dual-plasmid internal standard quantitative technology shows a lower quantitative lower limit and better detection accuracy for low HBV load samples. It can also be seen from the result diagram that the system of the present invention ( Figure 2 ) has a more concentrated curve and smaller deviation.

[0124] Table 3

[0125]

[0126] *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.

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

[0128] Table 4

[0129]

[0130] *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.

[0131] Example 5. Test results of the test sample of composition 2 of the present invention

[0132] The HBV dual-channel dual-plasmid internal standard quantitative technology (Example) was used to detect the Chinese national HBV standard diluted with 10IU / mL newborn calf serum, and μL of quantitative plasmid with a fixed value of 1700IU / mL was added to the sample to be tested; on this basis, homologous plasmids with a final concentration of 2 / 3 / 4 / 6 / 8 / 10 / 12IU / mL HBV fixed values ​​were added to each group. Under this test system, the minimum quantitative concentration (LOQ) of the HBV single plasmid internal standard system is about 12IU / mL. As shown in Table 5, the optimal addition amount of HBV homologous plasmid is approximately between 3IU / mL and 8IU / mL, that is, 25% to 66.7% of the minimum quantitative concentration (LOQ) of the HBV single plasmid internal standard system.

[0133] Table 5

[0134]

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

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

[0137] Table 6

[0138]

[0139]

[0140] According to the above table, the sixth reagent is prepared by modifying the segment of the homologous plasmid, and the probe sequence of the seventh reagent is designed for the corresponding position of the original HBV segment P segment, thereby achieving quantitative detection of the HBV target area and qualitative detection of HBV. The specific model schematic diagram is shown in FIG. Figure 4 As shown in the figure, the specific test results are shown. Figures 5-6 As shown, in the HBV positive sample, the fluorescence signal of the CY5 channel exceeds the threshold line, and it is judged as HBV positive; in the HBV negative sample, the fluorescence signal of the CY5 channel is lower than the threshold line, and it is judged as HBV negative. This optimization scheme, based on the embodiment 2 of the composition of the present invention, can effectively judge the positivity of HBV in the sample to be tested through the CY5 fluorescence channel.

[0141] In summary, this scheme uses the same set of primers to amplify the HBV segment P segment and the homologous plasmid segment, uses the FAM group-labeled quantitative probe P1 to detect the common sequence of the HBV segment P segment and the homologous plasmid segment for quantitative analysis; uses the CY5 group-labeled P3 probe to detect the corresponding sequence of the HBV segment P segment for qualitative analysis of the HBV target sequence.

[0142] Example 7, test results of the test sample of composition 3 of the present invention

[0143] Use HBV national standard, use newborn calf serum to dilute to 30IU / mL, 25IU / mL, 20IU / mL and 15IU / mL, use the diluted low concentration sample as the test sample, each test sample is tested 20 times each time, and it is done 3 times in a row, for a total of 60 repeated tests. 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. Calculated under this composition system, the LOQ of HBV is 25IU / mL.

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

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

[0146] Table 7

[0147]

Claims

1. A HBV quantitative composition, comprising: A first reagent, comprising N sets of primers and probes, wherein the N sets of primers and probes are respectively used to amplify and detect at least one portion of at least one of the segments C region, P region, X region, or S region of HBV; A second reagent, comprising N homologous vectors, wherein the homologous vectors respectively comprise at least one portion 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 respectively correspond one-to-one to the HBV segments amplified in the first reagent; A third reagent, comprising M quantitative carriers; and A fourth reagent, comprising 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; Wherein, N and M are positive integers both ≥1.

2. The HBV quantitative composition according to claim 1, wherein The quantitative vector includes a nucleotide sequence, which is a sequence modified for the HBV segment in the homologous vector, and the modification includes replacing the adenine deoxyribonucleotide in the homologous vector sequence with a thymine deoxyribonucleotide, and replacing the thymine deoxyribonucleotide in the homologous vector sequence with an adenine deoxyribonucleotide. The quantitative vector in the fourth reagent includes at least one part of at least one of the C' region, P' region, X' region or S' region of the HBV segment, and the HBV segment in the fourth reagent corresponds one-to-one to the HBV segment amplified in the second reagent.

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

20.

5. The HBV quantitative composition according to claim 3, characterized in that: The forward primer for amplifying the HBV segment C 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; the forward primer for amplifying the HBV segment P region 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 HBV segment S 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.

6. The HBV quantitative composition according to claim 2, characterized in that: The nucleotide sequence of the C' region is shown as SEQ ID NO.5; the nucleotide sequence of the P' region is shown as SEQ ID NO.13; or the nucleotide sequence of the S' region is shown as SEQ ID NO.

21.

7. The HBV quantitative composition according to claim 6, characterized in that: The forward primer for amplifying the C' 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 P' 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 S' region is shown as SEQ ID NO.22, the reverse primer is shown as SEQ ID NO.23, and the probe is shown as SEQID NO.

24.

8. The HBV quantitative composition according to claim 7, characterized in that: The composition also includes replacing the second reagent with a sixth reagent, wherein the sixth reagent includes a segment of HBV, and the segment of HBV in the sixth reagent is obtained by modifying the segment of HBV included in the second reagent; and a seventh reagent, wherein the seventh reagent includes a probe, and the probe in the seventh reagent can target the segment of HBV included in the second reagent, but cannot target the segment of HBV included in the sixth reagent.

9. The HBV 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. A HBV quantitative kit comprising the HBV quantitative composition according to any one of claims 1 to 9.

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