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 a quantitative composition, a kit, a method and application of pathogens. The present invention provides a quantitative composition comprising a homologous vector of segments X1-XN of said pathogen, and a quantitative vector, as well as primers and probes for amplifying both 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 low-load pathogen sample, the pathogen 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 quantitative composition, a kit, a method and a use thereof for pathogens. Background Art

[0002] Quantitative nucleic acid detection plays a very important role in diagnosing pathogen infection and evaluating the treatment effect after infection. Quantitative detection technology can be used to understand the number of pathogens in the body, replication level, infectivity, drug treatment effect, formulation of treatment strategies, etc., and use it as an evaluation indicator. It is also the only laboratory test indicator that can help diagnose latent infection and latent chronic infection (such as HBV, HCV, and HIV, etc.).

[0003] Many pathogens are contagious during the incubation period, acute period or chronic period. Most other tests cannot be used as indicators of whether the pathogen replicates, but nucleic acid testing is very sensitive to low levels of pathogens in the body by amplifying pathogen nucleic acid, and is a common method for judging pathogen replication.

[0004] Therefore, there is a need in the art for accurate quantitative detection of pathogens, especially low-load pathogens, that is, ultra-high sensitive quantitative detection. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a quantitative composition, comprising: The first reagent includes N sets of primers and probes, wherein the N sets of primers and probes are used to amplify and detect N segments X1 to X2 of the pathogen, respectively. N ; The second reagent comprises N homologous vectors, wherein the homologous vectors comprise segments of the pathogen, and the segments of the pathogen are respectively identical to the segments X1 to X2 of the pathogen in the first reagent. N One to one correspondence; 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.

[0006] The present invention creatively introduces homologous vectors to artificially increase the concentration of pathogens in the sample, thereby improving the stability and accuracy of the PCR amplification reaction. In particular, in low-load pathogen samples, more accurate detection is possible. At the same time, if two or more homologous vectors are introduced, it will be more helpful to quantify pathogens with many mutations (such as RNA viruses), thereby preventing misdetection.

[0007] Furthermore, N≥M.

[0008] Furthermore, N is any positive integer from 1 to 100, any positive integer from 1 to 50, any positive integer from 1 to 20, or any positive integer from 1 to 10.

[0009] In some specific embodiments, N can be 1, 2, 3, 4, 5, 6, etc.

[0010] For example, when N is 1, the first reagent includes 1 set of primers and probes, which amplify segment X1 of the pathogen; accordingly, the second reagent includes 1 homologous vector, including segment X1 of the pathogen.

[0011] For example, when N is 2, the first reagent includes 2 sets of primers and probes, which can respectively amplify segments X1 and X2 of the pathogen; accordingly, the second reagent includes 2 homologous vectors, one homologous vector includes segment X1 of the pathogen, and one homologous vector includes segment X2 of the pathogen.

[0012] For example, when N is 3, the first reagent includes 3 sets of primers and probes, which can respectively amplify segments X1, X2 and X3 of the pathogen; accordingly, the second reagent includes 3 homologous vectors, one homologous vector includes segment X1 of the pathogen, one homologous vector includes segment X2 of the pathogen, and one homologous vector includes segment X3 of the pathogen.

[0013] Furthermore, M is any positive integer from 1 to 100, any positive integer from 1 to 50, any positive integer from 1 to 20, or any positive integer from 1 to 10.

[0014] In some specific embodiments, M can be 1, 2, 3, 4, 5, 6, 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.

[0015] Furthermore, the X1~X N It is determined for different pathogen target areas; further, the pathogen target area can be a conserved region of the pathogen, for example, the C, S, P, and X regions of HBV, which are highly conserved. There is one target area, that is, N=1, which means that quantification can be completed; when N=2 or above, it can naturally meet the quantification requirements.

[0016] Furthermore, the pathogen target region can be a non-conserved region of the pathogen, for example, the POL, GAG, and LTR regions of HIV. These regions are highly variable, and there are at least 3 target regions, namely N=3, which can ensure that quantification is completed without missing detection. When N>3, it can naturally meet the quantitative requirements. Admittedly, when N=1 or 2, it may miss detection, but it still does not affect its ability to more accurately quantitatively detect pathogens compared to existing technologies.

[0017] Furthermore, the pathogen may be bacteria, fungi, viruses and parasites. For example, bacteria: Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi; viruses: Enterovirus 71, astrovirus, norovirus, rotavirus, enteroadenovirus; parasites: pathogenic Echinococcus multilocularis and Echinococcus granulosus; fungi: Aspergillus flavus, etc.

[0018] Furthermore, the nucleic acid of the pathogen may be DNA or RNA. For example, the pathogen may be a DNA virus or an RNA virus. For example, the pathogen of the present invention may be hepatitis B virus, hepatitis C virus, human immunodeficiency virus, etc.

[0019] Furthermore, the pathogens may be the same or different.

[0020] Furthermore, the pathogens are the same, that is, the segments of the pathogen in the first reagent and / or the second reagent belong to different segments of the same pathogen.

[0021] 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.

[0022] Furthermore, the quantitative vector may be directed against the pathogen segment X in the homologous vector. N The nucleotide sequence of the gene is modified by replacing the adenine deoxyribonucleotide (A) in the original sequence with thymine deoxyribonucleotide (T), and replacing the thymine deoxyribonucleotide (T) in the original sequence with adenine deoxyribonucleotide (A).

[0023] 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.

[0024] In the present invention, the term "vector" refers to all substances in the art that can carry nucleic acid and can be used in the process of nucleic acid amplification. For example, common vectors include plasmids, recombinant viruses containing plasmids (lentivirus, adenovirus, pseudovirus, etc.), liposomes containing plasmids, artificial chromosomes, etc.

[0025] 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.

[0026] In some specific embodiments, the vector can be a plasmid.

[0027] 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 X1, X2 and / or X3, etc.

[0028] In some specific embodiments, the backbone plasmid of the homologous plasmid and the quantitative plasmid is a pUC57 plasmid, that is, the sequence of X1 is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the sequence after A of X1 is replaced by T and T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0029] In some specific embodiments, the vector may be a recombinant virus comprising the above-mentioned plasmid.

[0030] In some specific embodiments, the vector can be a liposome comprising the above-mentioned plasmid.

[0031] In some specific embodiments, the vector can be an artificial chromosome carrying the target segment.

[0032] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration, preferably, 30% to 60%.

[0033] Using the above concentrations of homologous vectors allows for more accurate quantification of low concentrations of pathogens.

[0034] The term "lowest quantitative concentration" refers to the limit of quantitation (LOQ), which refers to the ability to stably and accurately test samples with known specific concentration values, that is, the lowest concentration of the analyte that can be measured when the preset accuracy requirements are met.

[0035] 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 the pathogen in the second reagent.

[0036] By introducing the fifth reagent, it is possible to perform a qualitative operation on the sample, that is, it is possible to determine whether the pathogen exists in the sample.

[0037] In some specific embodiments, the composition further comprises a fifth reagent, which amplifies segment Y of the pathogen, and the segment Y is amplified by segments X1 to X2 of the pathogen. N Are different.

[0038] Furthermore, the first reagent further comprises at least one additional second probe, the second probe targeting the segments X1 to X2 of the homologous vector. N and mutating the bases of a portion of the segment targeted by the probe on the homologous vector so that the probe cannot target the pathogen.

[0039] Furthermore, the composition also includes a sixth reagent, which is used to replace the second reagent, wherein the sixth reagent includes a segment of a pathogen, and the segment of the pathogen in the sixth reagent is obtained by modifying the segment of the pathogen included in the second reagent.

[0040] and a seventh reagent, wherein the seventh reagent comprises a probe, wherein the probe in the seventh reagent is capable of targeting a segment of the pathogen included in the first reagent but is not capable of targeting a segment of the pathogen included in the sixth reagent.

[0041] By introducing the sixth reagent and the seventh reagent, it is also possible to perform qualitative operations on the sample, that is, it is possible to determine whether the pathogen exists in the sample.

[0042] 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.

[0043] Furthermore, the modification may be one or more of locked nucleic acid (LNA) modification, MGB modification, or ZNA (Zip Nucleic Acids) modification.

[0044] In some specific embodiments, the quantitative composition of the present invention is used for fluorescent PCR.

[0045] Furthermore, the fluorescent groups of the probes in the composition of the present invention are different from each other and do not interfere with each other.

[0046] 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 in the composition 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.

[0047] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0048] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, BHQ1 or BHQ2.

[0049] In a specific embodiment, each component of the quantitative composition of the present invention is present in a separate package.

[0050] In a particular embodiment, the components of the quantitative composition of the invention are present in the same package.

[0051] Furthermore, the components of the quantitative composition of the present invention are present in a mixed form.

[0052] In a specific embodiment, the present invention provides a quantitative composition for quantifying HBV, comprising: The first reagent includes a set of primers and probes with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.3, wherein the primers and probes are used to amplify and detect the segment X1 of HBV, and the nucleotide sequence thereof is shown in SEQ ID NO.4; A second reagent, comprising a homologous vector, wherein the homologous vector comprises a nucleotide sequence as shown in SEQ ID NO.4; A third reagent includes a quantitative carrier; and The fourth reagent includes a set of primers and a probe, wherein the primers and the probe are respectively used to amplify and detect the segments of the quantitative carrier.

[0053] Furthermore, the nucleotide sequence of the segment of the quantitative vector is shown in SEQ ID NO.5.

[0054] Furthermore, the nucleotide sequences of the primers and probes of the fourth reagent are shown in SEQ ID NO.6 to SEQ ID NO.8.

[0055] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration, preferably, 30% to 60%.

[0056] In a specific embodiment, the present invention provides a quantitative composition for quantifying HCV, comprising: The first reagent includes two sets of primers and probes with nucleotide sequences as shown in SEQ ID NO.9 to SEQ ID NO.11 and SEQ ID NO.49 to SEQ ID NO.51, and the primers and probes are used to amplify and detect HCV segments X1 and X2, and the nucleotide sequences thereof are as shown in SEQ ID NO.12 and SEQ ID NO.52; A second reagent, comprising two homologous vectors, wherein the homologous vectors comprise nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.52; A third reagent, comprising two quantitative carriers; and The fourth reagent includes two sets of primers and probes, and the primers and probes are used to amplify and detect the segments of the quantitative vector respectively.

[0057] Furthermore, the nucleotide sequences of the segments of the quantitative vector are shown in SEQ ID NO.13 and SEQ ID NO.53.

[0058] Furthermore, the nucleotide sequences of the primers and probes of the fourth reagent are shown in SEQ ID NO.14 to SEQ ID NO.16 and SEQ ID NO.54 to SEQ ID NO.56.

[0059] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration, preferably, 30% to 60%.

[0060] Furthermore, the composition further comprises a fifth reagent, and the nucleotide sequences of the primers and probes of the fifth reagent are shown in SEQ ID NO.41 to SEQ ID NO.43. The amplification target nucleotide sequence of the fifth reagent is shown in SEQ ID NO.44.

[0061] In a specific embodiment, the present invention provides a quantitative composition for quantifying HIV, comprising: The first reagent includes three sets of primers and probes with nucleotide sequences as shown in SEQ ID NO.17 to SEQ ID NO.25, and the primers and probes are used to amplify and detect segment X of HIV. 1~ X3, the nucleotide sequence of which is shown in SEQ ID NO.26 to SEQ ID NO.28; The second reagent includes three homologous vectors, each of which includes a nucleotide sequence as shown in SEQ ID NO.26 to SEQ ID NO.28; A third reagent comprising 1 or 3 quantitative carriers; and The fourth reagent includes three sets of primers and probes, and the primers and probes are used to amplify and detect the segments of the quantitative vector respectively.

[0062] Furthermore, the nucleotide sequence of the segment of the quantitative vector is shown in SEQ ID NO.29 to SEQ ID NO.31.

[0063] Furthermore, the nucleotide sequences of the primers and probes of the fourth reagent are shown in SEQ ID NO.32 to SEQ ID NO.40.

[0064] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration, preferably, 30% to 60%.

[0065] Furthermore, the composition further comprises a fifth reagent, and the nucleotide sequences of the primers and probes of the fifth reagent are shown in SEQ ID NO.45 to SEQ ID NO.47. The amplification target nucleotide sequence of the fifth reagent is shown in SEQ ID NO.48.

[0066] In some specific embodiments, the third reagent may include 3 quantitative carriers. Preferably, the 3 quantitative carriers are mixed in a ratio of 1:1:1, that is, mixed in equal amounts.

[0067] In some specific embodiments, the third reagent may include one quantitative vector, that is, the three segments of the quantitative vector are integrated into one vector, preferably, integrated into one segment at a copy ratio of 1:1:1.

[0068] In a second aspect, the present invention provides use of the quantitative composition for preparing a quantitative kit.

[0069] In a third aspect, the present invention provides a quantitative kit, comprising the quantitative composition as described above.

[0070] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0071] Furthermore, the amplification reagents include dNTPs, PCR buffer, DNA polymerase, reverse transcriptase, and Mg 2+ At least one of .

[0072] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0073] Furthermore, the concentration of the DNA polymerase is 3 U / μL to 15 U / μL, for example, the DNA polymerase may be Taq enzyme.

[0074] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , Mn 2+ , dNTPs, and PCR buffer.

[0075] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, RT enzyme, Mg 2+ , Mn 2+ , dNTPs, and PCR buffer.

[0076] 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~200 µL.

[0077] In a fourth aspect, the present invention provides a quantitative method, the method comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 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 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0078] Furthermore, the quantitative formula is: , wherein P1 is the probe of the first reagent, and P2 is the probe of the fourth reagent. Further, C 病原体 represents the concentration of the pathogen to be tested, C 定量载体 represents the concentration of the quantitative carrier, C 同源载体 represents the concentration of homologous vector.

[0079] Furthermore, the present invention provides a quantitative method for non-diagnostic purposes, the method comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 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 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0080] Furthermore, the quantitative formula is: , wherein P1 is the probe of the first reagent and P2 is the probe of the fourth reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 It is a schematic diagram of the single carrier quantitative mode; Figure 2 Schematic diagram of the dual-carrier quantitative model of the present invention Figure 1 ; Figure 3 Schematic diagram of the dual-carrier quantitative model of the present invention Figure 2 ; Figure 4 Schematic diagram of the dual-carrier quantitative model of the present invention Figure 3 ; Figure 5 Schematic diagram of the dual-carrier quantitative model of the present invention Figure 4 ; Figure 6 This is a sample test result diagram of the HBV quantitative composition 1 of the present invention; Figure 7 This is a graph showing the results of the sample detection of the HBV comparative quantitative composition of the present invention; Figure 8 This is a graph showing the results of the HBV quantitative composition 2 of the present invention detecting a positive HBV sample; Fig. 9 This is a graph showing the results of the HBV quantitative composition 2 of the present invention detecting a negative HBV sample; Fig.10 This is a graph showing the results of the HCV quantitative composition 2 of the present invention detecting a positive HBV sample; Fig.11 This is a graph showing the results of the HCV quantitative composition 2 of the present invention detecting a negative HBV sample. DETAILED DESCRIPTION

[0082] 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.

[0083] Measurement of the minimum quantitative concentration (limit of quantitation): Using pathogen standards, using newborn calf serum to dilute the standards, using the diluted low-concentration samples as the test samples, each test sample was tested 20 times each time, and repeated 3 times in a row, for a total of 60 times. The accuracy requirement is that the logarithmic deviation of the test result is within ±0.5, and the lowest quantitative concentration (LOQ) is the lowest concentration of the test substance that can be measured when the 100% accurate measurement requirement is met.

[0084] Taking HBV as an example, HBV national standard was used, and newborn calf serum was used to dilute to 20 IU / mL, 15 IU / mL, 12 IU / mL, 10 IU / mL, 8 IU / mL and 5 IU / mL. The diluted low-concentration samples were used as test samples, and each test sample was tested 20 times each time, for 3 consecutive times, 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. Through the above method, it was finally determined that the LOQ of HBV was 12 IU / mL under this patented PCR system.

[0085] The conventional quantitative model of the prior art is as follows Figure 1 As shown, the present invention creatively introduces homologous vectors, and its quantitative pattern is as follows Figure 2 It is also possible to add additional primer and probe sets, such as Figure 3 When two or more homologous vectors are introduced, the exemplary quantitative pattern is as follows Figure 4 At the same time, the probe of the seventh reagent can be added to the first reagent, and the homologous plasmid of the second reagent can be adaptively modified to obtain the sixth reagent to replace the second reagent, so that the qualitative and quantitative detection of pathogens can be achieved at the same time. The exemplary mode is as follows Figure 5 shown.

[0086] Example 1: Sequences used in the present invention Table 1

[0087]

[0088]

[0089] Among them, the fluorescent group of the probe of the first reagent of HBV is FAM; the fluorescent group of the probe of the fourth reagent of HBV is HEX.

[0090] The fluorescent group of the probe of the first reagent of HCV is FAM; the fluorescent group of the probe of the fourth reagent of HCV is HEX; and the fluorescent group of the probe of the fifth reagent of HCV is CY5.

[0091] Among them, the fluorescent group of the probe of the first HIV reagent is FAM; the fluorescent group of the probe of the fourth HIV reagent is HEX; and the fluorescent group of the probe of the fifth HIV reagent is CY5.

[0092] Example 2: Quantification of pathogens This project uses Shengxiang Bio's S10012 (HBV), S10013 (HCV) or S1001 (HIV) nucleic acid extraction kits to extract pathogen nucleic acids, and Chinese national pathogen standards diluted with newborn calf serum are used to draw pathogen concentration curves or as test samples to test the performance of the internal standard quantitative system.

[0093] PCR reaction conditions: HBV: 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+ ; The pathogen and IC target sequence plasmids were synthesized at Bioengineering.

[0094] The amplification system for a single person (50 μL) includes: 20.75 μL PCR buffer (S08), 2 μL 100mmol / 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.

[0095] 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.

[0096] HCV: Kangde NRT enzyme (5U); Kangde Taq DNA polymerase (5U); Shengxiang Biotechnology S15 Buffer; primers / probes were synthesized at Bio-Tech and diluted to a working concentration of 40 pmol / μL by dissolving in TE; 1 mol / L Mg2+; HCV and IC target sequence plasmids were synthesized at Bio-Tech.

[0097] The amplification system for a single person (50 μL) includes: 17 μL PCR buffer (S15), 0.8 μL 100 mmol / L dNTP (T), 0.4 μL 40 pmol / μL PCR upstream and downstream primers, 0.125 μL 40 pmol / μL probe, 1.5 μL 5U Taq DNA polymerase, 0.5 μL 5U NRT enzyme, 0.4 μL 1 mol / L Mg 2+ , 27.025 μL purified water.

[0098] 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.

[0099] HIV: Baorui MMLV RT enzyme (5U); Kande Taq DNA polymerase (5U); Shengxiang Bio S15 Buffer; primers / probes synthesized in Bio-Tech and diluted to a working concentration of 40 pmol / μL by dissolving in TE; 1 mol / L Mg 2+ ; HIV-1 and IC target sequence plasmids were synthesized at Bioengineering.

[0100] The amplification system for a single person of 50 μL includes: 17 μL PCR buffer (S15), 1 μL 100 mmol / L dNTP (T), 0.24 μL 40 pmol / μL PCR upstream and downstream primers, 0.1 μL 40 pmol / μL probe, 1.5 μL 5U Taq DNA polymerase, 0.5 μL 5U MMLV RT enzyme, 0.4 μL 1 mol / L Mg 2+ , 25.54 μL purified water.

[0101] PCR amplification program: 50°C for 30 min, 1 cycle; 95°C for 8 min, 1 cycle; 95°C for 15 s, 57°C for 30 s, 45 cycles, reading the fluorescence signal once after each cycle; 25°C for 10 s, 1 cycle, and the reaction system was set to 50 μL.

[0102] Add 10 μL of 1.00E-7 μg / mL quantitative vector or pathogen homologous vector to 400 μL of newborn calf serum, and draw a pathogen concentration curve using the Chinese national pathogen standard diluted with newborn calf serum for determination. The average of the determined concentrations measured by three independent repeated experiments is set as the pathogen determined concentration of the plasmid.

[0103] Exemplarily, after completing the plasmid determination, add 10 μL of 1700 IU / mL quantitative vector and 10 μL of homologous vector of corresponding concentration to 400 μL of the sample to be tested, mix evenly, and use the Shengxiang Bio S10012 nucleic acid extraction kit to extract pathogen nucleic acid (HBV). Add 300 μL of extraction solution 1 to the centrifuge tube, shake and mix, and then centrifuge instantly. Add 100 μL 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 a magnetic separator for 3 minutes. When the magnetic beads are aggregated on the wall of the centrifuge tube, discard the liquid in the tube, add 600 μL of extraction solution 3 and 200 μL 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. Pipette 50 μL PCR reaction solution to elute the magnetic beads and place them on a magnetic separator. Transfer the separated clear solution to a 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.

[0104] The final concentration of the quantitative carrier in the sample to be tested is 1700 IU / mL, so according to the formula Calculate the pathogen load in the sample to be tested.

[0105] Example 3: Detection results of HBV samples tested with the HBV quantitative composition 1 of the present invention Using the HBV national standard, newborn calf serum was used to dilute to 20 IU / mL, 18 IU / mL, 16 IU / mL, 15IU / mL and 10 IU / mL. The diluted low-concentration samples were used as the test samples. Each test sample was tested 20 times each time, and it was repeated 3 times in a row, for a total of 60 times. The accuracy requirement is that the logarithmic deviation of the test result is within ±0.5, and the lowest quantitative concentration (LOQ) is the lowest concentration of the analyte that can be measured when the 100% accurate measurement requirement is met. Calculation under this system shows that the LOQ of HBV is 16 IU / mL.

[0106] The comparative example and the embodiment scheme (sequences shown in SEQ ID NO.1 to SEQ ID NO.8, N=1) were used to detect the Chinese national HBV standard diluted with newborn calf serum. The comparative example only added 10 μL of a quantitative plasmid with a total fixed value of 1700 IU / mL; on this basis, the embodiment additionally added a homologous plasmid with a final fixed value of 5 IU / mL.

[0107] The results of the two systems for the Chinese national HBV standard diluted with 10 IU / mL newborn calf serum are as follows Figures 6 and 7As 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 2, 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 6 ) has a more concentrated curve and smaller deviation.

[0108] Table 2

[0109] *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.

[0110] 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 7 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 3, 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. The HBV dual-channel dual-plasmid internal standard quantitative technology shows a lower quantitative lower limit and better accuracy.

[0111] Table 3

[0112] *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.

[0113] Example 4: Detection results of HBV samples tested with the HBV quantitative composition 1 of the present invention The HBV dual-channel dual-plasmid internal standard quantitative technology (Example) was used to detect the Chinese national HBV standard diluted with 10 IU / mL newborn calf serum, and a quantitative plasmid with a fixed value of 1700 IU / 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 / 12 IU / 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 12 IU / mL. As shown in Table 4, the optimal addition amount of the HBV homologous plasmid is approximately between 3IU / mL and 8 IU / mL, that is, 25%~66.7% of the minimum quantitative concentration (LOQ) of the HBV single plasmid internal standard system.

[0114] Table 4

[0115] Example 5: Detection results of HCV samples tested with the HCV quantitative composition of the present invention The HCV national standard was used and diluted with newborn calf serum to 20 IU / mL, 15 IU / mL, 12 IU / mL, 10 IU / mL, 8 IU / mL and 5 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 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 analyte that can be measured when the 100% accurate measurement requirement is met. Through the above method, it was finally determined that the LOQ of HCV was 18 IU / mL under this combined PCR system.

[0116] The comparative example and the embodiment scheme (sequences shown in SEQ ID NO.9 to SEQ ID NO.16 and SEQ ID NO.49 to SEQID NO.56, N=2) were used to detect the Chinese national HCV standard diluted with newborn calf serum. The comparative example only added 10 μL of a quantitative plasmid so that the final concentration of the quantitative vector in the sample to be tested was 2500 IU / mL; on this basis, the embodiment additionally added a homologous plasmid with a final concentration of 15 IU / mL.

[0117] The detection results of the Chinese national HCV standard diluted with 15 IU / mL newborn calf serum by the two systems showed that the amplification curve of the HCV target sequence of the HCV dual-channel dual-plasmid internal standard quantitative technology (embodiment) was more concentrated than that of the comparative example, and the Ct value variance was significantly lower than that of the comparative example, that is, the detection results were more stable. As shown in Table 5, the quantitative accuracy of the comparative example was 88% (14 / 16), and the logarithmic deviation of the concentration of 14 detected samples was within ±0.5; the quantitative accuracy of the embodiment was 100% (16 / 16), and the logarithmic deviation of the concentration of 16 detected samples was within ±0.4. The HCV dual-channel dual-plasmid internal standard quantitative technology showed a lower quantitative limit and better detection accuracy for low HCV load samples.

[0118] Table 5

[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 7: Detection results of HIV samples tested with the HIV quantitative composition of the present invention HIV national standard was used, and newborn calf serum was used to dilute to 20 IU / mL, 15 IU / mL, 12 IU / mL, 10 IU / mL, 8 IU / mL and 5 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 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. Through the above method, it was finally determined that the LOQ of HIV was 30 IU / mL under this combined PCR system.

[0121] The comparative example and the embodiment scheme (sequences shown in SEQ ID NO.17 to SEQ ID NO.40, N=3) were used to detect the Chinese national HIV standard diluted in newborn calf serum. The comparative example only added 10 μL of a quantitative plasmid so that the final concentration of the quantitative vector in the sample to be tested was 600 IU / mL; on this basis, the embodiment additionally added a homologous plasmid with a final concentration of 12 IU / mL.

[0122] The test results of the two systems for the Chinese National HIV Standard diluted with 12 IU / mL newborn calf serum showed that compared with the control example, the amplification curve of the HIV target sequence of the HIV dual-channel dual-plasmid internal standard quantitative technology (implementation) was more concentrated, and the Ct value variance was significantly lower than that of the control example, that is, the test results were more stable. As shown in Table 6, the quantitative accuracy of the control example was 78.57% (13 / 16), and the logarithmic deviation of the concentration of 13 detected samples was within ±0.5; the quantitative accuracy of the embodiment was 100% (16 / 16), and the logarithmic deviation of the concentration of 16 detected samples was within ±0.4. The HIV dual-channel dual-plasmid internal standard quantitative technology showed a lower quantitative limit and better detection accuracy for low HIV load samples.

[0123] Table 6

[0124] *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.

[0125] Example 8: Detection results of HBV samples tested with the HBV quantitative composition 2 of the present invention In this embodiment, a third probe P3 is designed based on the target region of HBV of the HBV quantitative composition of the present invention, and the target sequence of the corresponding segment of the homologous plasmid of the HBV quantitative composition 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 7 below.

[0126] Table 7

[0127] 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 5 As shown in the figure, the specific test results are as follows: Figures 8 and 9 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 of the composition of the present invention, can effectively judge the positive and negative of the HBV sample to be tested through the CY5 fluorescence channel.

[0128] 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.

[0129] Example 9: Detection results of the HCV quantitative composition 2 of the present invention in testing HBV samples On the basis of HCV quantitative composition 1, a fifth reagent was further introduced: primers and probes shown in SEQ ID NOs. 41 to 43, and the same test was performed. The test results of the Chinese national HCV standard diluted with 15 IU / mL newborn calf serum were as follows: Fig.10 As shown in the figure, the red curve shows that the qualitative detection of the present invention avoids the possibility of false positives in the sample. When the HCV nucleic acid to be detected does not exist in the sample, the red curve does not rise. Fig.11 As shown, it is shown that the addition of the fifth reagent can effectively determine the positive and negative nature of HCV nucleic acid in the test sample.

Claims

1. A quantitative composition comprising: The first reagent includes N sets of primers and probes, wherein the N sets of primers and probes are used to amplify and detect N segments X1 to X2 of the pathogen, respectively. N ; The second reagent comprises N homologous vectors, wherein the homologous vectors comprise segments of the pathogen, and the segments of the pathogen are respectively identical to the segments X1 to X2 of the pathogen in the first reagent. N One to one correspondence; 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 quantitative composition according to claim 1, characterized in that The N is any positive integer from 1 to 10, and / or the M is any positive integer from 1 to 10.

3. The quantitative composition according to claim 1, characterized in that The quantitative vector includes segments X1 to X2 in the homologous vector. N The modified nucleotide sequence replaces adenine deoxyribonucleotides in homologous vector sequences with thymine deoxyribonucleotides, and replaces thymine deoxyribonucleotides in homologous vector sequences with adenine deoxyribonucleotides.

4. The quantitative composition according to claim 1, characterized in that The composition also includes a fifth reagent, which includes primers and probes, wherein the primers and probes in the fifth reagent are used to amplify and detect segments of pathogens, wherein the segments of pathogens amplified by the fifth reagent are different from the segments of pathogens amplified by the first reagent and the segments of pathogens in the second reagent.

5. The quantitative composition according to claim 1, characterized in that The composition further includes a sixth agent and a seventh agent; The sixth reagent is used to replace the second reagent, wherein the sixth reagent includes a segment of a pathogen, and the segment of the pathogen in the sixth reagent is obtained by modifying the segment of the pathogen included in the second reagent; The seventh reagent includes a probe, and the probe in the seventh reagent can target the segment of the pathogen included in the first reagent, but cannot target the segment of the pathogen included in the sixth reagent.

6. The quantitative composition according to claim 1, characterized in that The quantitative composition includes any one of the following compositions: 1) A quantitative composition for quantifying HBV, comprising: The first reagent includes a set of primers and probes with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.3, wherein the primers and probes are used to amplify and detect the segment X1 of HBV, and the nucleotide sequence thereof is shown in SEQ ID NO.4; A second reagent, comprising a homologous vector, wherein the homologous vector comprises a nucleotide sequence as shown in SEQ ID NO.4; A third reagent includes a quantitative carrier; and A fourth reagent, comprising a set of primers and a probe, wherein the primers and the probe are respectively used to amplify and detect a segment of the quantitative carrier; The nucleotide sequences of the primers and probes of the fourth reagent are shown in SEQ ID NO.6 to SEQ ID NO.8; 2) A quantitative composition for quantifying HCV, comprising: The first reagent includes two sets of primers and probes whose nucleotide sequences are shown in SEQ ID NO.9 to 11 and 49 to SEQ ID NO.51, and the primers and probes are used to amplify and detect HCV segments X1 and X2, whose nucleotide sequences are shown in SEQ ID NO.12 and SEQ ID NO.52; A second reagent, comprising two homologous vectors, wherein the homologous vectors comprise nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.52; A third reagent, comprising two quantitative carriers; and A fourth reagent, comprising two sets of primers and probes, wherein the primers and probes are used to amplify and detect the segments of the quantitative vector, respectively; The nucleotide sequences of the primers and probes of the fourth reagent are shown in SEQ ID NO.14 to SEQ ID NO.16 and SEQ ID NO.54 to SEQ ID NO.56; 3) A quantitative composition for quantifying HIV, comprising: The first reagent includes three sets of primers and probes with nucleotide sequences as shown in SEQ ID NO.17 to SEQ ID NO.25, wherein the primers and probes are used to amplify and detect HIV segment X, 1~ X3, the nucleotide sequence of which is shown in SEQ ID NO.26 to SEQ ID NO.28; The second reagent includes three homologous vectors, each of which includes a nucleotide sequence as shown in SEQ ID NO.26 to SEQ ID NO.28; A third reagent, comprising three quantitative carriers; and A fourth reagent, comprising three sets of primers and probes, wherein the primers and probes are used to amplify and detect the segments of the quantitative vector respectively; The nucleotide sequences of the primers and probes of the fourth reagent are shown in SEQ ID NO.32 to SEQ ID NO.

40.

7. The quantitative composition according to claim 1, characterized in that The vector is one or more of a plasmid, a recombinant virus containing a plasmid, a liposome containing a plasmid, or an artificial chromosome.

8. The quantitative composition according to any one of claims 1 to 7, characterized in that The concentration of the homologous vector is 25% to 66.7% of the lowest quantitative concentration.

9. Use of the quantitative composition according to any one of claims 1 to 8 for preparing a quantitative kit.

10. A quantitative kit comprising the composition according to any one of claims 1 to 8.

11. The kit according to claim 10, characterized in that The kit also includes at least one of a nucleic acid amplification reagent, a nucleic acid release reagent, or a nucleic acid extraction reagent.

12. A quantitative method for non-diagnostic purposes, the method comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 8 or the kit according to claim 10 or 11; and 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

13. The method according to claim 12, characterized in that The quantitative formula is: , wherein P1 is the probe of the first reagent and P2 is the probe of the fourth reagent.

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