A quantitative composition, kit, method and use

Abstract

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 pathogen detection. The present invention provides a quantitative composition comprising segments X1 to X2 of the pathogen. N The present invention utilizes a homologous vector and a quantitative vector, as well as primers and probes for amplifying both vectors. The invention creatively incorporates a homologous vector to artificially increase the concentration of the nucleic acid being tested, thereby improving the stability and accuracy of the PCR amplification reaction. This allows for better detection in low-load pathogen samples.

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 testing plays a very important role in diagnosing pathogen infection and evaluating the treatment effect after infection. Through quantitative detection technology, we can 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 their incubation, acute, or chronic stages. Most other tests cannot be used as indicators of pathogen replication. However, nucleic acid testing, which amplifies pathogen nucleic acids, is highly sensitive to low levels of pathogens in the body and is a commonly used method for determining 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:

[0006] 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 ;

[0007] The second reagent includes N homologous vectors, wherein the homologous vectors include 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;

[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] This invention creatively introduces homologous vectors to artificially increase the concentration of pathogens in samples, thereby improving the stability and accuracy of PCR amplification reactions. This allows for more accurate detection in low-load pathogen samples. Furthermore, the introduction of two or more homologous vectors further facilitates the quantification of pathogens with high mutation rates (such as RNA viruses), thereby preventing false positives.

[0012] Furthermore, N≥M.

[0013] 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.

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

[0015] For example, when N is 1, the first reagent includes a set of primers and probes that amplify segment X1 of the pathogen; correspondingly, the second reagent includes a homologous vector that includes segment X1 of the pathogen.

[0016] For example, when N is 2, the first reagent includes 2 sets of primers and probes, which can amplify segments X1 and X2 of the pathogen respectively; 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.

[0017] For example, when N is 3, the first reagent includes 3 sets of primers and probes, which can amplify segments X1, X2 and X3 of the pathogen respectively; 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.

[0018] 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.

[0019] 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 here.

[0020] 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. These regions are highly conserved, and 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.

[0021] 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, that is, 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.

[0022] Furthermore, the pathogens may be bacteria, fungi, viruses, and parasites. Examples include bacteria such as Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi, and Salmonella paratyphi; viruses such as Enterovirus 71, Astrovirus, Norovirus, Rotavirus, and Enteric Adenovirus; parasites such as pathogenic Echinococcus multilocularis and Echinococcus granulosus; and fungi such as Aspergillus flavus.

[0023] 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.

[0024] Furthermore, the pathogens may be the same or different.

[0025] 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.

[0026] Furthermore, the quantitative vector can be any vector used in the prior art for quantitative analysis, 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.

[0027] Furthermore, the quantitative vector can be directed against the pathogen segment X in the homologous vector. N The nucleotide sequence of the gene was 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).

[0028] This design aims to ensure that the amplification efficiency of the target segment of the quantitative vector is similar to that of the homologous vector, effectively simplifying the process of calculating the concentration of the target segment of the homologous vector based on the quantitative vector value.

[0029] 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.

[0030] In the present invention, the term "set" refers to at least one upstream primer, at least one downstream primer, and at least one probe that match each other for detecting a single target. Specifically, for example, it may be one upstream primer, one downstream primer, and one probe that match each other for detecting a single target; it may also be two upstream primers, one downstream primer, and one probe that match each other for detecting a single target; it may also be one upstream primer, two downstream primers, and one probe that match each other for detecting a single target; it may also be one upstream primer, one downstream primer, and two probes that match each other for detecting a single target, etc.

[0031] In some specific embodiments, the vector can be a plasmid.

[0032] 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 X1, X2 and / or X3, etc.

[0033] 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 in X1 is replaced by T and T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0034] In some specific embodiments, the vector may be a recombinant virus comprising the above-mentioned plasmid.

[0035] In some specific embodiments, the vector can be a liposome comprising the above-mentioned plasmid.

[0036] In some specific embodiments, the vector can be an artificial chromosome carrying the target segment.

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

[0038] Using the above concentrations of homologous vectors allows for more accurate quantification of low concentrations of pathogens.

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

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

[0041] By introducing the fifth reagent, a qualitative operation can be performed on the sample, that is, it can be determined whether the pathogen exists in the sample.

[0042] 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 All different.

[0043] 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.

[0044] Furthermore, the composition also includes a sixth reagent for replacing 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.

[0045] and a seventh reagent, wherein the seventh reagent comprises a probe, wherein the probe in the seventh reagent is capable of targeting the segment of the pathogen included in the first reagent but cannot target the segment of the pathogen included in the sixth reagent.

[0046] By introducing the sixth reagent and the seventh reagent, it is also possible to perform a qualitative operation on the sample, that is, it is possible to determine whether the pathogen is present in the sample.

[0047] 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.

[0048] Furthermore, the modification may be one or more of locked nucleic acid (LNA) modification, MGB modification, or ZNA (Zip Nucleic Acids) modification.

[0049] In some specific embodiments, the quantitative composition of the present invention is used for fluorescent PCR.

[0050] 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.

[0051] In this context, "different and non-interfering" means that the fluorescent groups used in the probes of the first, fourth, fifth, and seventh reagents in the composition are different and do not interfere with each other's detection, that is, they can be detected using different channels. For example, ATTO 425, Quasar 705, FAM, HEX, ROX, CY5, and CY5.5 can be used. These groups have different absorbance values ​​and can be used in different channels, thus preventing interference.

[0052] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0053] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, BHQ1 or BHQ2.

[0054] In a specific embodiment, each component of the quantitative composition of the present invention is present in a separate package.

[0055] In a particular embodiment, the components of the quantitative composition of the present invention are present in the same package.

[0056] Furthermore, the components of the quantitative composition of the present invention are present in a mixed form.

[0057] In a specific embodiment, the present invention provides a quantitative composition for quantifying HBV, comprising:

[0058] The first reagent includes a set of primers and probes having 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 HBV segment X1, and the nucleotide sequence thereof is shown in SEQ ID NO. 4;

[0059] A second reagent includes a homologous vector, wherein the homologous vector includes a nucleotide sequence as shown in SEQ ID NO. 4;

[0060] A third reagent, comprising a quantitative carrier; and

[0061] The fourth reagent includes a set of primers and a probe, wherein the primers and the probe are used to amplify and detect the segments of the quantitative carrier, respectively.

[0062] Furthermore, the nucleotide sequence of the segment of the quantitative vector is shown in SEQ ID NO.5.

[0063] 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.

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

[0065] In a specific embodiment, the present invention provides a quantitative composition for quantifying HCV, comprising:

[0066] 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 shown in SEQ ID NO. 12 and SEQ ID NO. 52;

[0067] 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;

[0068] A third reagent, comprising two quantitative carriers; and

[0069] 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 carrier respectively.

[0070] Furthermore, the nucleotide sequences of the segments of the quantitative vector are shown in SEQ ID NO.13 and SEQ ID NO.53.

[0071] Furthermore, the nucleotide sequences of the primers and probes of the fourth reagent are shown as SEQ ID NO.14 to SEQ ID NO.16 and SEQ ID NO.54 to SEQ ID NO.56.

[0072] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration, preferably 30% to 60%.

[0073] Furthermore, the composition further comprises a fifth reagent, 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.

[0074] In a specific embodiment, the present invention provides a quantitative composition for quantifying HIV, comprising:

[0075] 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 HIV segment X 1~ X3, the nucleotide sequence of which is shown in SEQ ID NO. 26 to SEQ ID NO. 28;

[0076] 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;

[0077] A third reagent comprising 1 or 3 quantitative carriers; and

[0078] The fourth reagent includes three sets of primers and probes, which are used to amplify and detect segments of the quantitative carrier, respectively.

[0079] Furthermore, the nucleotide sequence of the segment of the quantitative vector is shown as SEQ ID NO.29 to SEQ ID NO.31.

[0080] 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.

[0081] Furthermore, the concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration, preferably 30% to 60%.

[0082] Furthermore, the composition further comprises a fifth reagent, 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.

[0083] In some specific embodiments, the third reagent may include three quantitative carriers. Preferably, the three quantitative carriers are mixed in a ratio of 1:1:1, that is, mixed in equal amounts.

[0084] 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.

[0085] In a second aspect, the present invention provides use of the quantitative composition for preparing a quantitative kit.

[0086] In a third aspect, the present invention provides a quantitative kit comprising the quantitative composition described above.

[0087] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0088] Furthermore, the amplification reagents include dNTP, PCR buffer, DNA polymerase, reverse transcriptase, and Mg 2+ At least one of .

[0089] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0090] Furthermore, the concentration of the DNA polymerase is 3 U / μL to 15 U / μL, for example, the DNA polymerase may be Taq enzyme.

[0091] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ 、Mn 2+ , dNTPs, and PCR buffer.

[0092] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, RT enzyme, Mg 2+ 、Mn 2+ , dNTPs, and PCR buffer.

[0093] 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.

[0094] In a fourth aspect, the present invention provides a quantitative method comprising the following steps:

[0095] 1) Extract nucleic acid from the sample to be tested;

[0096] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention or the kit of the present invention as described above; and

[0097] 3) Quantify the nucleic acid in the sample based on the carrier concentration and CT value.

[0098] Furthermore, the quantitative formula is:

[0099] , 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.

[0100] Furthermore, the present invention provides a quantitative method for non-diagnostic purposes, the method comprising the following steps:

[0101] 1) Extract nucleic acid from the sample to be tested;

[0102] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention or the kit of the present invention as described above; and

[0103] 3) Quantify the nucleic acid in the sample based on the carrier concentration and CT value.

[0104] Furthermore, the quantitative formula is:

[0105] , wherein P1 is the probe of the first reagent and P2 is the probe of the fourth reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 Schematic diagram of single-carrier quantitative mode;

[0107] Figure 2 This is a schematic diagram of the dual-carrier quantitative model of the present invention Figure 1 ;

[0108] Figure 3 This is a schematic diagram of the dual-carrier quantitative model of the present invention Figure 2 ;

[0109] Figure 4 This is a schematic diagram of the dual-carrier quantitative model of the present invention Figure 3 ;

[0110] Figure 5 This is a schematic diagram of the dual-carrier quantitative model of the present invention Figure 4 ;

[0111] Figure 6 This is a graph showing the test results of the HBV quantitative composition 1 of the present invention;

[0112] Figure 7 This is a graph showing the results of the sample detection using the quantitative composition for the HBV comparative example of the present invention;

[0113] Figure 8 This is a graph showing the results of the HBV quantitative composition 2 of the present invention detecting a positive HBV sample;

[0114] Figure 9 This is a graph showing the results of the HBV quantitative composition 2 of the present invention detecting a negative HBV sample;

[0115] Figure 10 This is a graph showing the results of the HCV quantitative composition 2 of the present invention detecting a positive HBV sample;

[0116] Figure 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

[0117] 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 for illustrating the present invention, rather than for limiting the present invention.

[0118] Measurement of the minimum quantitative concentration (limit of quantitation):

[0119] Using pathogen standards and serial dilutions of standards using newborn calf serum, the diluted low-concentration samples were used as test samples. Each test sample was tested 20 times, three times in a row, for a total of 60 replicates. The accuracy requirement was a logarithmic deviation within ±0.5 of the test result. The minimum quantification concentration (LOQ) was the lowest measurable concentration of the analyte that met the 100% accuracy requirement.

[0120] Taking HBV as an example, the HBV 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 this was repeated 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. The lowest quantitative concentration (LOQ) is the lowest analyte concentration that can be measured when the measurement requirement is 100% accurate. Through the above method, it was finally determined that the LOQ of HBV under this patented PCR system is 12 IU / mL.

[0121] 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 shown as follows Figure 5 shown.

[0122] Example 1: Sequences used in the present invention

[0123] Table 1

[0124]

[0125]

[0126]

[0127] The fluorescent group of the probe of the first HBV reagent is FAM; the fluorescent group of the probe of the fourth HBV reagent is HEX.

[0128] The fluorescent group of the probe of the first HCV reagent is FAM; the fluorescent group of the probe of the fourth HCV reagent is HEX; and the fluorescent group of the probe of the fifth HCV reagent is CY5.

[0129] 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.

[0130] Example 2: Quantification of pathogens

[0131] This project uses Shengxiang Bio's S10012 (HBV), S10013 (HCV) or S1001 (HIV) nucleic acid extraction kits to extract pathogen nucleic acids. Newborn calf serum is diluted with Chinese national pathogen standards to draw pathogen concentration curves or as test samples to test the performance of the internal standard quantitative system.

[0132] PCR reaction conditions:

[0133] HBV:

[0134] Feipeng Anstart Taq enzyme (5U); Shengxiang Bio S08 Buffer; primers / probes synthesized at Bio-Tech and diluted to a working concentration of 40 pmol / μL in TE; 1 mol / L Mg 2+ Pathogen and IC target sequence plasmids were synthesized at Bioengineering.

[0135] The amplification system for a single person (50 μL) includes: 20.75 μL PCR buffer (S08), 2 μL 100 mmol / L dNTP (T), 0.6 μL 40 pmol / μL PCR upstream and downstream primers, 0.1 μL 40 pmol / μL probe, 1.6 μL 5U Taq DNA polymerase, 0.4 μL 1 mol / L Mg 2+ , 22.65 μL purified water.

[0136] PCR amplification program: 95°C for 8 min, 1 cycle; 95°C for 15 s, 57°C for 30 s, 45 cycles, with the fluorescence signal read once after each cycle; 25°C for 10 s, 1 cycle, and the reaction system was set to 50 μL.

[0137] HCV:

[0138] Kangde NRT enzyme (5U); Kangde Taq DNA polymerase (5U); Shengxiang Biotechnology S15 Buffer; primers / probes 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 synthesized at Bio-Tech.

[0139] 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.

[0140] PCR amplification program: 1 cycle at 95°C for 1 min; 1 cycle at 60°C for 30 min; 1 cycle at 95°C for 8 min; 45 cycles at 95°C for 15 s and 57°C for 30 s, with fluorescence readings taken after each cycle; 1 cycle at 25°C for 10 s. HCV target 1 was set to the FAM channel, the quantitative internal standard IC was set to the HEX channel, and the HCV target 2 was set to the CY5 channel. The reaction volume was set to 50 μL.

[0141] HIV:

[0142] 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 in TE; 1 mol / L Mg 2+ HIV-1 and IC target sequence plasmids were synthesized at Bioengineering.

[0143] The amplification system for a single person (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 Mg2+ , 25.54 μL purified water.

[0144] 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, with the fluorescence signal read once after each cycle; 25°C for 10 s, 1 cycle, and the reaction system was set to 50 μL.

[0145] Add 10 μL of 1.00E-7 μg / mL quantitative vector or pathogen homologous vector to 400 μL of newborn calf serum. Draw a pathogen concentration curve using the Chinese national pathogen standard diluted in newborn calf serum for determination. The mean of the determined concentrations measured in three independent replicates is set as the pathogen determined concentration for the plasmid.

[0146] For example, after plasmid titration, 10 μL of 1700 IU / mL quantitative vector and 10 μL of the corresponding homologous vector were added to 400 μL of the sample to be tested. After thorough mixing, pathogen nucleic acid (HBV) was extracted using the Shengxiang Biotechnology S10012 Nucleic Acid Extraction Kit. 300 μL of Extraction Solution 1 was added to a centrifuge tube, vortexed to mix, and then centrifuged briefly. 100 μL of Extraction Solution 2 was added to each tube, vortexed to mix, and allowed to stand at room temperature for 10 minutes. After brief centrifugation, the tubes were placed on a magnetic separator for 3 minutes. Once the magnetic beads aggregated on the tube walls, the liquid was discarded by aspiration. 600 μL of Extraction Solution 3 and 200 μL of Extraction Solution 4 were added to the tubes simultaneously. After vortexing and brief centrifugation, the tubes were placed back on the magnetic separator. After approximately 3 minutes, the liquid was again aspirated. Pipette 50 μL of PCR reaction solution to elute the magnetic beads and place on a magnetic separator. Transfer the supernatant to a PCR reaction tube, vortex to mix, and centrifuge briefly. Place the tubes in the PCR instrument in the correct order, set the program and relevant parameters, save the file, and run the reaction.

[0147] 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.

[0148] Example 3: Detection results of HBV samples tested with the HBV quantitative composition 1 of the present invention

[0149] Using the HBV national standard, diluted with newborn calf serum to 20 IU / mL, 18 IU / mL, 16 IU / mL, 15 IU / mL, and 10 IU / mL, the diluted low-concentration samples were used as test samples. Each test sample was tested three times with 20 replicates each time, for a total of 60 replicates. The accuracy requirement was a logarithmic deviation within ±0.5 of the test result. The lowest quantification concentration (LOQ) was the lowest measurable analyte concentration that met the 100% accuracy requirement. Calculated using this system, the LOQ for HBV was 16 IU / mL.

[0150] The comparative example and the example 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 in newborn calf serum. In the comparative example, only 10 μL of a quantitative plasmid with a total concentration of 1700 IU / mL was added; on this basis, the example additionally added a homologous plasmid with a final concentration of 5 IU / mL.

[0151] 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 7 As shown, compared with the control 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 control example, that is, the detection result is more stable. As shown in Table 2, the quantitative accuracy of the control 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.

[0152] Table 2

[0153]

[0154] *Quantitation is inaccurate, and the logarithm of the sample to be tested deviates by more than ±0.5 compared to the actual sample concentration.

[0155] 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 7As shown, compared to the comparative example, the HBV dual-channel dual-plasmid internal standard quantification technique (Example) showed a more concentrated amplification curve for the HBV target sequence, and the Ct value variance was significantly lower than that of the comparative example, indicating more stable detection results. As shown in Table 3, the quantitative accuracy of the comparative example was 81.25% (13 / 16), with the logarithmic deviation of the concentration of 13 detected samples within ±0.5. The quantitative accuracy of the Example was 100% (16 / 16), with the logarithmic deviation of the concentration of 16 detected samples within ±0.5. The HBV dual-channel dual-plasmid internal standard quantification technique demonstrated a lower quantification threshold and better accuracy.

[0156] Table 3

[0157]

[0158] *Quantitation is inaccurate, and the logarithm of the sample to be tested deviates by more than ±0.5 compared to the actual sample concentration.

[0159] Example 4: Detection results of HBV samples tested with the HBV quantitative composition 1 of the present invention

[0160] The HBV dual-channel, dual-plasmid internal standard quantitative technique (Example) was used to detect the Chinese National HBV Standard diluted in 10 IU / mL newborn calf serum. A quantitative plasmid with a fixed concentration of 1700 IU / mL was added to the test sample. Furthermore, homologous plasmids with final concentrations of 2, 3, 4, 6, 8, 10, and 12 IU / mL of HBV were added to each group. Under this test system, the minimum quantification (LOQ) of the HBV single-plasmid internal standard system was approximately 12 IU / mL. As shown in Table 4, the optimal addition amount of the HBV homologous plasmid ranged from approximately 3 IU / mL to 8 IU / mL, representing 25% to 66.7% of the LOQ of the HBV single-plasmid internal standard system.

[0161] Table 4

[0162]

[0163] Example 5: Detection results of HCV samples tested with the HCV quantitative composition of the present invention

[0164] The HCV national standard was 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 three times with 20 replicates each time, for a total of 60 replicates. The accuracy requirement was a logarithmic deviation within ±0.5 of the test result. The lowest quantification concentration (LOQ) was the lowest measurable analyte concentration that met the 100% accuracy requirement. Using this method, the LOQ for HCV was determined to be 18 IU / mL using this combined PCR system.

[0165] The comparative example and the example scheme (sequences shown as SEQ ID NO.9 to SEQ ID NO.16 and SEQ ID NO.49 to SEQ ID NO.56, N=2) were used to detect the Chinese national HCV standard diluted in newborn calf serum. In the comparative example, only 10 μL of the quantitative plasmid was added to make the final concentration of the quantitative vector in the test sample 2500 IU / mL; on this basis, the example additionally added the homologous plasmid to a final concentration of 15 IU / mL.

[0166] Compared to the control group, the two systems tested the Chinese National HCV Standard diluted in 15 IU / mL newborn calf serum. The HCV dual-channel, dual-plasmid internal standard quantitation technique (Example) showed more concentrated amplification curves for the HCV target sequence, and the Ct value variance was significantly lower than that of the control group, indicating more stable detection results. As shown in Table 5, the quantitative accuracy of the control group was 88% (14 / 16), with the logarithmic deviation of the concentration within ±0.5 for all 14 detected samples. The quantitative accuracy of the Example was 100% (16 / 16), with the logarithmic deviation of the concentration within ±0.4 for all 16 detected samples. The HCV dual-channel, dual-plasmid internal standard quantitation technique demonstrated a lower quantitation threshold and improved detection accuracy for samples with low HCV loads.

[0167] Table 5

[0168]

[0169] *Quantitation is inaccurate, and the logarithm of the sample to be tested deviates by more than ±0.5 compared to the actual sample concentration.

[0170] Example 7: Detection results of HIV samples tested with the HIV quantitative composition of the present invention

[0171] Using the national HIV standard, 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 served as test samples. Each test sample was tested three times, with 20 replicates performed each time, for a total of 60 replicates. The accuracy requirement was a logarithmic deviation within ±0.5 of the test result. The lowest quantification concentration (LOQ) was the lowest measurable analyte concentration that met the 100% accuracy requirement. Using this method, the LOQ for HIV was determined to be 30 IU / mL for this combined PCR system.

[0172] The comparative example and the example 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. In the comparative example, only 10 μL of the quantitative plasmid was added to make the final concentration of the quantitative vector in the test sample 600 IU / mL; on this basis, the example additionally added the homologous plasmid to a final concentration of 12 IU / mL.

[0173] Compared to the control group, the HIV dual-channel dual-plasmid internal standard quantitation technique (Example) demonstrated more concentrated amplification curves for the HIV target sequence using both systems, with significantly lower Ct value variance than the control group, indicating more stable detection results. As shown in Table 6, the control group achieved a quantitation accuracy of 78.57% (13 / 16), with the logarithmic deviation of the concentration within ±0.5 for all 13 detected samples. The Example achieved a quantitation accuracy of 100% (16 / 16), with the logarithmic deviation within ±0.4 for all 16 detected samples. The HIV dual-channel dual-plasmid internal standard quantitation technique demonstrated a lower threshold for quantitation and improved detection accuracy for samples with low HIV loads.

[0174] Table 6

[0175]

[0176] *Quantitation is inaccurate, and the logarithm of the sample to be tested deviates by more than ±0.5 compared to the actual sample concentration.

[0177] Example 8: Detection results of HBV samples tested with the HBV quantitative composition 2 of the present invention

[0178] In this example, a third probe, P3, was designed based on the HBV target region of the HBV quantitative composition of the present invention. Simultaneously, the target sequence of the corresponding segment of the homologous plasmid of the HBV quantitative composition of the present invention was modified. Specifically, the adenine deoxyribonucleotide (A) in the segment corresponding to the P3 probe in the homologous plasmid sequence was replaced with a thymine deoxyribonucleotide (T), and the thymine deoxyribonucleotide (T) in the original sequence was replaced with an adenine deoxyribonucleotide (A). The specific sequence changes are shown in Table 7 below.

[0179] Table 7

[0180]

[0181] According to the above table, this embodiment transforms the homologous plasmid segment to prepare the sixth reagent, and designs the probe sequence of the seventh reagent for the corresponding position of the original HBV segment P segment, thereby achieving quantitative detection of the HBV target region and qualitative detection of HBV at the same time. The specific model 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 HBV-positive samples, the fluorescence signal of the CY5 channel exceeds the threshold line, indicating HBV positive; in HBV-negative samples, the fluorescence signal of the CY5 channel falls below the threshold line, indicating HBV negative. This optimized solution, based on the embodiment of the composition of the present invention, can effectively determine the HBV positive or negative of the test sample through the CY5 fluorescence channel.

[0182] In summary, this scheme uses the same set of primers to amplify the HBV segment P segment and the segment of the homologous plasmid, uses the FAM group-labeled quantitative probe P1 to detect the common sequence of the HBV segment P segment and the segment of the homologous plasmid 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.

[0183] Example 9: Detection results of the HCV quantitative composition 2 of the present invention in testing HBV samples

[0184] 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: Figure 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 tested does not exist in the sample, the red curve does not rise. Figure 11 As shown, it is shown that the addition of the fifth reagent can effectively determine the positive or negative 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 includes N homologous vectors, wherein the homologous vectors include 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 groups of primers and probes, wherein the M groups of primers and probes are used to amplify and detect segments of the quantitative vector, respectively; Wherein, N is any positive integer from 1 to 10, and / or M is any positive integer from 1 to 10; Wherein, the quantitative vector includes the pathogen segments X1~X1 in the homologous vector. N The modified nucleotide sequence replaces adenine deoxyribonucleotides in the homologous vector sequence with thymine deoxyribonucleotides, and replaces thymine deoxyribonucleotides in the homologous vector sequence with adenine deoxyribonucleotides; The concentration of the homologous vector is 25% to 66.7% of the minimum quantitative concentration.

2. 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.

3. 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.

4. 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 having 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 HBV segment X1, and the nucleotide sequence thereof is shown in SEQ ID NO. 4; A second reagent includes a homologous vector, wherein the homologous vector includes a nucleotide sequence as shown in SEQ ID NO. 4; A third reagent, comprising 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 with nucleotide sequences as shown in SEQ ID NOs. 9 to 11 and SEQ ID NOs. 49 to 51, and the primers and probes are used to amplify and detect HCV segments X1 and X2, and the nucleotide sequences thereof are shown in SEQ ID NOs. 12 and 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 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.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, and 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, the primers and probes being used to amplify and detect 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.

5. 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.

6. Use of the quantitative composition according to any one of claims 1 to 5 for preparing a quantitative kit.

7. A quantitative kit comprising the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit further comprises at least one of a nucleic acid amplification reagent, a nucleic acid release reagent, or a nucleic acid extraction reagent.

9. A quantitative method for non-diagnostic purposes, 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 5 or the kit according to claim 7 or 8; and 3) Quantify the nucleic acid in the sample based on the carrier concentration and CT value.

10. The method according to claim 9, characterized in that The quantitative formula is: , where P1 is the probe of the first reagent, P2 is the probe of the fourth reagent, and ΔCT(P1−P2) is the difference between the Ct value of the fluorescent channel where the probe of the first reagent is located and the Ct value of the fluorescent channel where the probe of the fourth reagent is located.

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