HCV 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 HCV 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 at least one of the 5 'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, or 3' UTR region of said HCV, and a quantitative vector, as well as primers and probes for amplifying both vectors. The homologous vector is creatively introduced, and the concentration of the sample to be detected is artificially increased, so that the stability and accuracy of PCR amplification reaction are improved. Particularly, in a sample with low virus load, the HCV 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 an HCV quantitative composition, a kit, a method and a use thereof. Background Art

[0002] Hepatitis C virus (Hepatitis C) belongs to the Hepadnaviridae family. The blood of people infected with hepatitis C virus is infectious regardless of whether they are in the latent, acute or chronic stages. DNA testing is sensitive to low levels of HCV virus in the body by amplifying viral nucleic acid, and is a common method for determining viral replication. DNA is the most direct, specific and sensitive indicator of hepatitis C virus infection. A positive HCV-DNA indicates HCV replication and infectiousness. The higher the HCV-DNA, the more viral replication and the stronger the infectiousness. The continuous replication of hepatitis C virus is the root cause of hepatitis C.

[0003] Quantitative DNA testing also plays a very important role in diagnosing HCV and evaluating the effectiveness of HCV 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 HCV infection and latent chronic HCV.

[0004] Therefore, there is a need in the art for accurate quantitative detection of HCV, particularly, HCV at low viral loads. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides an HCV 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 5'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, or 3'UTR region of HCV;

[0007] A second reagent, comprising N homologous vectors, wherein the homologous vectors respectively comprise at least one portion of at least one of the 5'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, or 3'UTR region of HCV segments, and the segments of HCV in the second reagent respectively correspond one-to-one to the segments of HCV 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 both positive integers ≥1.

[0011] The present invention creatively introduces a homologous vector to artificially increase the concentration of HCV in the sample, thereby improving the stability and accuracy of the PCR amplification reaction. In particular, HCV can be better detected in samples with low viral loads.

[0012] Furthermore, the quantitative vector can be any vector used for quantitative analysis in the prior art, as long as it does not interfere with the amplification of the corresponding pathogen in the PCR system. For example, it can be a vector containing GAPDH, RNASE gene fragments, etc.

[0013] Furthermore, the quantitative vector includes a nucleotide sequence, and the nucleotide sequence is modified for the HCV 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). The quantitative vector includes at least one part of at least one of the 5'UTR' region, CORE' region, E1' region, E2' region, P7' region, NS2' region, NS3' region, NS4A' region, NS4B' region, NS5A' region, NS5' region, or 3'UTR' region of the HCV segment, and the segment of the quantitative vector HCV corresponds one-to-one to the segment of the HCV amplified in the homologous vector.

[0014] In some specific embodiments, the quantitative vector includes a nucleotide sequence, which is a sequence of the 5'UTR region, CORE region, or NS3 region of the HCV segment in the homologous vector that is transformed into a 5'UTR' region, CORE' region, or NS3' region, and the transformation 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] 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.

[0016] Furthermore, the nucleotide sequence of the 5'UTR region of the HCV segment is shown as SEQ ID NO.4.

[0017] Furthermore, the nucleotide sequence of the CORE region of the HCV segment is shown as SEQ ID NO.12.

[0018] Furthermore, the nucleotide sequence of the NS3 region of the HCV segment is shown in SEQ ID NO.20.

[0019] In some specific embodiments, N can be 1, 2, 3, etc.

[0020] For example, when N is 1, the first reagent includes a set of primers and probes that amplify a specific region of the 5'UTR region of the HCV segment; accordingly, the second reagent includes a homologous vector including the same region as the specific region of the 5'UTR region of the HCV segment amplified by the first reagent.

[0021] For example, when N is 2, the first reagent includes 2 sets of primers and probes, which can respectively amplify specific regions of the 5'UTR region and the CORE region of the HCV segment; accordingly, the second reagent includes 2 homologous vectors, one homologous vector includes the same region as the specific region of the 5'UTR region of the HCV segment amplified by the first reagent, and the other homologous vector includes the same region as the specific region of the CORE region of the HCV segment amplified by the first reagent.

[0022] For example, when N is 3, the first reagent includes 3 sets of primers and probes, which can respectively amplify specific regions of the 5'UTR region, CORE region and NS3 region of HCV segments; accordingly, the second reagent includes 3 homologous vectors, one homologous vector includes the same region as the specific region of the 5'UTR region of HCV segment amplified by the first reagent, one homologous vector includes the same region as the specific region of the CORE region of HCV segment amplified by the first reagent, and one homologous vector includes the same region as the specific region of the NS3 region of HCV segment amplified by the first reagent.

[0023] 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.

[0024] In some specific embodiments, the forward primer for amplifying the 5'UTR region of the HCV segment is shown as SEQ ID NO.1, the reverse primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO.3.

[0025] In some specific embodiments, the forward primer for amplifying the CORE region of HCV 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.

[0026] In some specific embodiments, the forward primer for amplifying the NS3 region of HCV 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.

[0027] When N=2, the nucleotide sequence of the 5'UTR region, CORE region, or NS3 region of the HCV segment in the first reagent and the second reagent is any two of SEQ ID NO.4, SEQ ID NO.8, or SEQ ID NO.20.

[0028] 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, SEQ ID NO.8 or SEQ ID NO.20.

[0029] When N=3, the nucleotide sequences of the 5'UTR region, CORE region and NS3 region of HCV in the first reagent and the second reagent are SEQ ID NO. 4, SEQ ID NO. 8 and SEQ ID NO. 20, respectively.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] In the present invention, the term "5'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, 3'UTR region" refers to at least one nucleotide sequence in the 5' untranslated region, CORE gene, E1 gene, E2 gene, P7 gene, NS2 gene, NS3 gene, NS4A gene, NS4B gene, NS5A gene, NS5 gene, and 3' untranslated region of HIV. In some specific embodiments, the vector can be a plasmid.

[0034] Furthermore, the backbone plasmids of the homologous plasmid and the quantitative plasmid can be any plasmid in the prior art, for example, pUC57 plasmid, pBluescript II SK+ plasmid, pUC-SP plasmid, etc., which are used to carry target segments, such as 5'UTR region, CORE region and / or NS3 region, etc.

[0035] 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 5'UTR region is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the sequence after A in the 5'UTR region is replaced by T and T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0036] In some specific embodiments, the vector may be a recombinant virus comprising the above-mentioned plasmid.

[0037] In some specific embodiments, the vector can be a liposome comprising the above-mentioned plasmid.

[0038] In some specific embodiments, the vector can be an artificial chromosome carrying the target segment.

[0039] Furthermore, the quantitative plasmid includes a 5'UTR' region, and the nucleotide sequence of the 5'UTR' region is as shown in SEQ ID NO.5.

[0040] Furthermore, the quantitative plasmid includes a CORE' region, and the nucleotide sequence of the CORE' region is as shown in SEQ ID NO.13.

[0041] Furthermore, the quantitative plasmid includes an NS3' region, and the nucleotide sequence of the NS3' region is as shown in SEQ ID NO.21.

[0042] In some specific embodiments, the forward primer for amplifying the 5'UTR' 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.

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

[0044] In some specific embodiments, the forward primer for amplifying the NS3' 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.

[0045] When M=2, the quantitative vector includes any two of the nucleotide sequences SEQ ID NO.5, SEQ ID NO.13, or SEQ ID NO.21 shown in the 5'UTR' region, the CORE' region, or the NS3' region.

[0046] When M=2, the nucleotide sequences of the primers and probe in the fourth reagent correspond to amplify any two of SEQ ID NO.5, SEQ ID NO.13, or SEQ ID NO.21.

[0047] 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 5'UTR' region, the CORE' region, or the NS3' region.

[0048] When M=3, the nucleotide sequences of the primers and probes in the fourth reagent are as follows: SEQ ID NO.6 to SEQ ID NO.8, SEQ ID NO.14 to SEQ ID NO.16, and SEQ ID NO.22 to SEQ ID NO.24.

[0049] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the lowest quantitative concentration, preferably, 30% to 60%.

[0050] Using the above concentrations of homologous vectors allows for more accurate quantification of low concentrations of HCV.

[0051] The term "minimum 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.

[0052] Further, the composition also includes a fifth reagent, the fifth reagent includes 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. Further, 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 HCV in the second reagent.

[0053] 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.

[0054] In some specific embodiments, the composition further comprises a fifth reagent, which amplifies the segment NS5B region of the pathogen, wherein the segment NS5B region is different from the nucleotide sequence shown in the segment 5'UTR region, CORE region, or NS3 region of the pathogen.

[0055] In some specific embodiments, the nucleotide sequence of the NS5B region is shown as SEQ ID NO.25 or SEQ ID NO.29.

[0056] In some specific embodiments, the forward primer for amplifying the NS5B region is shown as SEQ ID NO.26, the reverse primer is shown as SEQ ID NO.27, and the probe is shown as SEQ ID NO.28.

[0057] In some specific embodiments, the forward primer for amplifying the NS5B region is shown as SEQ ID NO.30, the reverse primer is shown as SEQ ID NO.31, and the probe is shown as SEQ ID NO.32.

[0058] In some specific embodiments, the forward primer for amplifying the NS5B region is shown as SEQ ID NO.33, the reverse primer is shown as SEQ ID NO.34, and the probe is shown as SEQ ID NO.35.

[0059] In some specific embodiments, the first reagent, the fourth reagent and the fifth reagent in the quantitative composition of the present invention can be modified. Further, the modification can be one or more of locked nucleic acid (LNA) modification, MGB modification, or ZNA (Zip Nucleic Acids) modification.

[0060] In a specific embodiment, the modification may be a locked nucleic acid modification.

[0061] In some specific embodiments, the HCV quantitative composition of the present invention is used for fluorescent PCR.

[0062] Furthermore, the fluorescent groups of the probes mentioned in the present invention are different from each other and do not interfere with each other.

[0063] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in the probes of the first reagent, the fourth reagent and the fifth reagent are different from each other and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO 425, Quasar705, FAM, HEX, ROX, CNS5B region 5 and CNS5B region 5.5 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.

[0064] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0065] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, BHQ1 or BHQ2.

[0066] In a specific embodiment, each component of the HCV quantitative composition of the present invention is present in a separate package.

[0067] In a specific embodiment, the components of the HCV quantitative composition of the present invention are present in the same package.

[0068] Furthermore, the components of the HCV quantitative composition of the present invention are present in a mixed form.

[0069] In a second aspect, the present invention provides use of the above HCV quantitative composition for preparing an HCV quantitative kit.

[0070] In a third aspect, the present invention provides an HCV quantitative kit, comprising the HCV quantitative composition as described above.

[0071] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0072] Furthermore, the amplification reagent includes dNTP, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0073] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0074] 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.

[0075] Furthermore, the concentration of the DNA polymerase is 3U / μL to 15U / μL, for example, the DNA polymerase may be Taq enzyme.

[0076] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , Mn 2+ , dNTPs, and PCR buffer.

[0077] 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.

[0078] In a fourth aspect, the present invention provides a method for quantifying HCV, the method comprising the following steps:

[0079] 1) Extracting nucleic acid from the sample to be tested;

[0080] 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

[0081] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0082] Furthermore, the quantitative formula is Among them, P1 is the probe of the first reagent, and P2 is the probe of the fourth reagent.

[0083] Further, a method for quantifying HCV for non-diagnostic purposes is provided, the method comprising the following steps:

[0084] 1) Extracting nucleic acid from the sample to be tested;

[0085] 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

[0086] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0087] 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

[0088] Figures 1-2 is an exemplary detection diagram of the composition of the present invention;

[0089] Figure 3 This is a graph showing the test results of a sample of a comparative example composition of the present invention;

[0090] Figure 4 The results of the sample test for the exemplary composition of the present invention are shown in FIG.

[0091] Figure 5This is a graph showing the results of testing a negative sample using an exemplary composition of the present invention. DETAILED DESCRIPTION

[0092] 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.

[0093] In the present invention, the 5'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, or 3'UTR region of HCV are conservative regions. Those skilled in the art can select the above-mentioned regions according to the logic of the present invention to design the quantitative composition of the present invention, and the effects of the present invention can be achieved.

[0094] Measurement of minimum quantitative concentration (limit of quantitation):

[0095] Use HCV national standard, use newborn calf serum to dilute to 30IU / mL, 25IU / mL, 20IU / mL, 15IU / mL, 12IU / mL, 10IU / mL, 8IU / mL and 5IU / mL concentration gradient, 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 lowest quantitative concentration (LOQ) is the lowest concentration of the analyte that can be measured when the 100% accurate measurement requirement is met.

[0096] Example 1: Sequences used in the present invention

[0097] 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.

[0098] Table 1

[0099]

[0100]

[0101] In Table 1, “+A” means LNA-A; “+T” means LNA-T; “+C” means LNA-C; and “+G” means LNA-G.

[0102] Among them, the fluorescent group of the probe of the first reagent is FAM; the fluorescent group of the probe of the fourth reagent is HEX; and the fluorescent group of the probe of the fifth reagent is CY5.

[0103] Example 2: Quantitative method for HCV

[0104] Use HCV national standard, use newborn calf serum to dilute to 25IU / mL, 20IU / mL, 18IU / mL, 15IU / mL, 12IU / 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 times of repeated testing. The accuracy requirement is that the logarithmic deviation of the test result is within ± 0.5, and the lowest quantitative concentration (LOQ) is the lowest concentration of the test substance that can be measured when the 100% accurate measurement requirement is met. Through the above method, it is determined that the LOQ of HCV is 18IU / mL under this sequence combination PCR system.

[0105] The method of the present invention is as follows Figures 1-2 This project uses Shengxiang Biotechnology S10013 nucleic acid extraction kit to extract HCV nucleic acid, and Chinese national HCV standard diluted with newborn calf serum is used to draw HCV concentration curve or as a sample to be tested to test the performance of the internal standard quantitative system.

[0106] PCR reaction conditions:

[0107] Kangde NRT enzyme (5U); Kangde Taq DNA polymerase (5U); Shengxiang Bio S15 Buffer; Primers / probes synthesized in Bio-Tech and diluted to a working concentration of 40pmol / μL by dissolving in TE; 1mol / L Mg 2+ ; HCV and IC target sequence plasmids were synthesized at Bioengineering.

[0108] The amplification system for a single person of 50 μL includes: 17 μL PCR buffer (S15), 0.8 μL 100mmol / L dNTP (T), 0.4 μL 40pmol / μL PCR upstream and downstream primers, 0.125 μL 40pmol / μL probe, 1.5 μL 5UTaq DNA polymerase, 0.5 μL 5U NRT enzyme, 0.4 μL 1mol / L Mg 2+ , 27.025 μL purified water.

[0109] PCR amplification program: 95℃1min, 1 cycle; 60℃30min, 1 cycle; 95℃8min, 1 cycle; 95℃15s, 57℃30s, 45 cycles, reading the fluorescence signal once after each cycle; 25℃10s, 1 cycle. HCV target 1 was set as FAM channel, quantitative internal standard IC was set as HEX channel, HCV target 2 was set as CY5 channel, and the reaction system was set as 50μL.

[0110] 10 μL of 1.00E-7 μg / mL quantitative plasmid or HCV homologous plasmid was added to 300 μL of newborn calf serum, and the HCV concentration curve was drawn by diluting the Chinese national HCV standard with newborn calf serum for determination, and the mean of the determined concentration measured by three independent repeated tests was set as the HCV determined concentration at the addition amount of 10 μL of the plasmid. In this scheme, illustratively, 10 μL of 1.00E-7 μg / mL quantitative plasmid was determined in 300 μL of the sample system to be tested, and the determined concentration was diluted to a working concentration of 2500 IU / mL by TE, and 10 μL of 1.00E-7 μg / mL HCV homologous plasmid was determined in 300 μL of the sample system to be tested, and the determined concentration was diluted to a working concentration of 1000 IU / mL by TE.

[0111] After completing the plasmid determination, add the quantitative plasmid with a final concentration of 2500IU / mL in the sample to be tested and the homologous plasmid of the corresponding concentration to 300μL of the sample to be tested, mix evenly, add 600μL 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 aggregated 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. Place them in the PCR instrument in order, set the running program and related parameters, save the file, and run the reaction program.

[0112] For example, the final concentration of the quantitative plasmid in the sample to be tested is 2500 IU / mL, so according to the formula Calculate the HCV load in the sample to be tested.

[0113] Example 3: Test results of the test sample of the composition 1 of the present invention

[0114] The comparative example and the embodiment scheme (the first reagent: the primers and probes shown in SEQ ID NO.1-3, 9-11; the second reagent: the homologous vector including SEQ ID NO.4 and SEQ ID NO.12; the third reagent: the quantitative vector including SEQ ID NO.5 and SEQ ID NO.13; the fourth reagent: the primers and probes shown in SEQ ID NO.6-SEQ ID NO.8, SEQ ID NO.14-SEQ ID NO.16; the fifth reagent: the primers and probes shown in SEQ ID NO.26-SEQ ID NO.28) were used to detect the Chinese national HCV standard diluted with newborn calf serum. The comparative example only added 10 μL of the quantitative plasmid with a total fixed value of 2500 IU / mL; on this basis, the embodiment additionally added the homologous plasmid with a final fixed concentration of 8 IU / mL.

[0115] The results of the two systems for the Chinese national HCV standard diluted with 15 IU / mL newborn calf serum are as follows Figures 3-4 As shown, compared with the comparative example, the amplification curve of the HCV target sequence of the HCV 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. At the same time, the red curve in the figure shows that the qualitative detection of the present invention avoids the possibility of false positive samples. For example, Figure 5 As shown, when the HCV nucleic acid to be tested does not exist in the sample, the red curve does not form a line, indicating that the addition of the fifth reagent can effectively determine the positive and negative nature of the HCV nucleic acid in the test sample.

[0116] As shown in Table 2, the quantitative accuracy of the comparative example is 88% (14 / 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 HCV dual-channel dual-plasmid internal standard quantitative technology shows a lower quantitative limit and better detection accuracy for low HCV load samples. It can also be seen from the result graph that the system of the present invention ( Figure 4 ) has a more concentrated curve and smaller deviation.

[0117] Table 2

[0118]

[0119] *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.

[0120] Example 4: Test results of the test sample of the composition 1 of the present invention

[0121] HCV national standard was used, and newborn calf serum was used to dilute to 20 IU / mL, 18 IU / mL, 15 IU / mL, 12 IU / mL, and 10 IU / mL. The diluted low-concentration samples were used as test samples. Each test sample was tested 20 times each time, and it was tested 3 times in a row, for a total of 60 times of repeated tests. The accuracy requirement was that the logarithmic deviation of the test result was within ± 0.5, and the lowest quantitative concentration (LOQ) was the lowest concentration of the test substance that could be measured when the 100% accurate measurement requirement was met. Through the above method, it was determined that the LOQ of HCV was 18 IU / mL under the PCR system of the present invention.

[0122] The HCV dual-channel dual-plasmid internal standard quantitative technology (Example) was used to detect the Chinese national HCV standard diluted with 15IU / mL newborn calf serum, and μL of quantitative plasmid with a fixed value of 2500IU / mL was added to the sample to be tested; on this basis, homologous plasmids with a final concentration of 4 / 5 / 8 / 10 / 12 / 14IU / mL HCV fixed values ​​were added to each group. Under this test system, the minimum quantitative concentration (LOQ) of the HCV single plasmid internal standard system is about 18IU / mL. As shown in Table 3, the optimal addition amount of the HCV homologous plasmid is approximately between 5IU / mL and 12IU / mL, that is, 25% to 66.7% of the minimum quantitative concentration (LOQ) of the HCV single plasmid internal standard system.

[0123] Table 3

[0124]

[0125] *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.

[0126] Example 5. Test results of the test sample of composition 2 of the present invention

[0127] Use HCV national standard, use newborn calf serum to dilute to 40IU / mL, 30IU / mL, 25IU / mL, 20IU / mL, 10IU / 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 times of repeated testing. The accuracy requirement is that the logarithmic deviation of the test result is within ± 0.5, and the lowest quantitative concentration (LOQ) is the lowest concentration of the test substance that can be measured when the 100% accurate measurement requirement is met. Through the above method, it is determined that the LOQ of HCV is 30IU / mL under this sequence combination PCR system.

[0128] The Chinese national HCV 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 the quantitative vector, i.e., the quantitative plasmid with a fixed final concentration of 5500 IU / mL in the sample to be tested was added; on this basis, the embodiment additionally added a homologous plasmid with a fixed final concentration of 12 IU / mL.

[0129] The test results of the two systems for the Chinese national HCV standard diluted with 25 IU / mL newborn calf serum are as follows: Compared with the comparative example, the amplification curve of the HCV target sequence of the HCV 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 4, 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 HCV dual-channel dual-plasmid internal standard quantitative technology shows a lower quantitative lower limit and better detection accuracy for low HCV load samples.

[0130] Table 4

[0131]

[0132]

[0133] Example 6, test results of the test sample of composition 3 of the present invention

[0134] Use HCV national standard, use newborn calf serum to dilute to 40IU / mL, 30IU / mL, 25IU / mL, 20IU / mL, 10IU / 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 times of repeated testing. The accuracy requirement is that the logarithmic deviation of the test result is within ± 0.5, and the lowest quantitative concentration (LOQ) is the lowest concentration of the test substance that can be measured when the 100% accurate measurement requirement is met. Through the above method, it is determined that the LOQ of HCV is 30IU / mL under this sequence combination PCR system.

[0135] The Chinese national HCV standard diluted in 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 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 the quantitative vector, i.e., the quantitative plasmid with a fixed final concentration of 3000 IU / mL in the sample to be tested was added; on this basis, the embodiment additionally added a homologous plasmid with a fixed final concentration of 12 IU / mL.

[0136] The test results of the two systems for the Chinese national HCV standard diluted with 25 IU / mL newborn calf serum are as follows: Compared with the comparative example, the amplification curve of the HCV target sequence of the HCV 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 5, 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 HCV dual-channel dual-plasmid internal standard quantitative technology shows a lower quantitative lower limit and better detection accuracy for low HCV load samples.

[0137] Table 5

[0138]

[0139]

Claims

1. A HCV 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 5'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, or 3'UTR region of HCV; A second reagent, comprising N homologous vectors, wherein the homologous vectors respectively comprise at least one portion of at least one of the 5'UTR region, CORE region, E1 region, E2 region, P7 region, NS2 region, NS3 region, NS4A region, NS4B region, NS5A region, NS5 region, or 3'UTR region of HCV segments, and the segments of HCV in the second reagent respectively correspond one-to-one to the segments of HCV 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 both positive integers ≥1.

2. The HCV quantitative composition according to claim 1, wherein The quantitative vector includes a nucleotide sequence, which is a sequence modified for the HCV 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 5'UTR' region, CORE' region, E1' region, E2' region, P7' region, NS2' region, NS3' region, NS4A' region, NS4B' region, NS5A' region, NS5' region, or 3'UTR' region of the HCV segment, and the HCV segment in the fourth reagent corresponds one-to-one to the HCV segment amplified in the second reagent.

3. The HCV 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 HCV quantitative composition according to claim 1, characterized in that The nucleotide sequence of the 5'UTR region of the HCV segment is shown as SEQ ID NO.4; the nucleotide sequence of the CORE region of the HCV segment is shown as SEQ ID NO.12; or the nucleotide sequence of the NS3 region of the HCV segment is shown as SEQ ID NO.

20.

5. The HCV quantitative composition according to claim 3, characterized in that The forward primer for amplifying the 5'UTR region of the HCV segment is shown as SEQ ID NO.1, the reverse primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO.3; the forward primer for amplifying the CORE region of the HCV segment is shown as SEQ ID NO.9, the reverse primer is shown as SEQ ID NO.10, and the probe is shown as SEQ ID NO.11; or the forward primer for amplifying the NS3 region of the HCV segment is shown as SEQ ID NO.17, the reverse primer is shown as SEQ ID NO.18, and the probe is shown as SEQ ID NO.

19.

6. The HCV quantitative composition according to claim 2, characterized in that The nucleotide sequence of the 5'UTR' region is shown as SEQ ID NO.5; the nucleotide sequence of the CORE' region is shown as SEQ ID NO.13; and the nucleotide sequence of the NS3' region is shown as SEQ ID NO.

21.

7. The HCV quantitative composition according to claim 6, characterized in that The forward primer for amplifying the 5'UTR' 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 CORE' 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 NS3' region is shown as SEQ ID NO.22, the reverse primer is shown as SEQ ID NO.23, and the probe is shown as SEQ ID NO.

24.

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

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

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

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