Quantitative composition, kit, method and use

Abstract

Provided are a quantitative composition, a kit, a method, and a use. More specifically, provided are a pathogen quantitative composition, a kit, a method, and a use. Provided is a quantitative composition comprising homologous vectors of segments X1-XN of the pathogen, quantitative vectors, and primers and probes for amplifying the two types of vectors. A homologous vector is introduced into the quantitative composition to artificially increase the concentration of nucleic acids to be tested, thereby improving the stability and accuracy of a PCR amplification reaction. In particular, pathogens can be better detected in low-load pathogen samples.

Description

A quantitative composition, kit, method and use Technical Field

[0001] This disclosure pertains to the field of molecular biology detection, specifically relating to a quantitative composition, kit, method, and use; more specifically, relating to a quantitative composition, kit, method, and use for a pathogen. Background Technology

[0002] Nucleic acid quantitative detection plays a vital role in confirming pathogen infection and assessing the effectiveness of treatment after infection. Quantitative detection technology can reveal the quantity, replication level, infectivity, drug treatment efficacy, and treatment strategy of pathogens in the body, and serve as an assessment indicator. It is also the only laboratory test indicator that can help confirm occult infections and occult chronic infections (such as HBV, HCV, and HIV).

[0003] Many pathogens are infectious during their incubation, acute, or chronic phases. Most other tests cannot serve as indicators of whether a pathogen is replicating, but nucleic acid testing, by amplifying the pathogen's nucleic acid, is highly sensitive to low levels of the pathogen in the body and is a commonly used method for determining pathogen replication.

[0004] Therefore, there is a need in this field for accurate quantitative detection of pathogens, especially low-virulence pathogens, i.e., ultra-sensitive quantitative detection. Summary of the Invention

[0005] In view of the above, in a first aspect, this disclosure provides a quantitative composition comprising:

[0006] The first reagent comprises N sets of primers and probes, wherein the N sets of primers and probes are used to amplify and detect N segments X1 to X2 of the pathogen, respectively. N ;

[0007] The second reagent comprises N homologous vectors, wherein each homologous vector includes a segment of the pathogen, and the segment of the pathogen is respectively associated with segment X1 to X2 of the pathogen in the first reagent. N One-to-one correspondence;

[0008] The third reagent includes M quantitative carriers; and

[0009] The fourth reagent includes M sets of primers and probes, wherein the M sets of primers and probes are used to amplify and detect segments of the quantitative vector, respectively.

[0010] Where N and M are both positive integers ≥ 1.

[0011] Secondly, this disclosure provides the use of the above-described quantitative composition in the preparation of quantitative reagent kits.

[0012] Thirdly, this disclosure provides a quantitative kit comprising the quantitative composition described above.

[0013] Fourthly, this disclosure provides a quantitative method, the method comprising the following steps:

[0014] 1) Extract nucleic acid from the sample to be tested;

[0015] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit described above; and

[0016] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0017] The above technical solution achieves the following technical effects:

[0018] This disclosure creatively introduces homologous vectors to artificially increase the concentration of pathogens in samples, thereby improving the stability and accuracy of PCR amplification reactions. In particular, it enables more accurate detection in samples with low pathogen loads. Furthermore, introducing two or more homologous vectors is more helpful in quantifying pathogens with numerous mutations (such as RNA viruses), thus preventing false detections. Detailed Implementation

[0019] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0020] In this disclosure, the term "vector" refers to any substance in the art capable of loading nucleic acids and used in the process of nucleic acid amplification. Common vectors include plasmids, recombinant viruses containing plasmids (lentiviruses, adenoviruses, pseudoviruses, etc.), liposomes containing plasmids, artificial chromosomes, etc.

[0021] In this disclosure, the term "homologous vector" refers to a vector (optionally plasmid, recombinant virus, liposome, or artificial chromosome, etc.) containing a nucleotide sequence that corresponds one-to-one with the target region of the pathogen to be tested.

[0022] In this disclosure, the term "quantitative vector" refers to a vector used for quantitative PCR calibration, which can be obtained by modifying the pathogen segment of a homologous vector (AT / TA base substitution), has a similar amplification efficiency to the homologous vector, and is equipped with dedicated primers and probes for detection. The target load can be calculated from its concentration and CT value.

[0023] In this disclosure, the term "set" refers to at least one upstream primer, at least one downstream primer, and at least one probe that are mutually matched to detect a target. Specifically, for example, it could be one upstream primer, one downstream primer, and one probe that are mutually matched to detect a target; it could also be two upstream primers, one downstream primer, and one probe that are mutually matched to detect a target; it could also be one upstream primer, two downstream primers, and one probe that are mutually matched to detect a target; or it could be one upstream primer, one downstream primer, and two probes that are mutually matched to detect a target, etc.

[0024] The term "lowest quantitation concentration" refers to the limit of quantitation (LOQ), which is the ability to stably and accurately test samples with known specific concentration values. In other words, it is the lowest concentration of the analyte that can be measured while meeting the preset accuracy requirements.

[0025] In this article, "dissimilar 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 will not affect each other's detection; that is, different channels can be used for detection. For example, ATTO 425, Quasar 705, FAM, HEX, ROX, CY5, and CY5.5 can be used. These groups have different absorbance values, allowing for the selection of different channels and thus preventing mutual interference.

[0026] In this article, the term "nucleic acid amplification reagent" refers to the core reagent combination that supports real-time quantitative PCR, which includes at least dNTPs, PCR buffer, DNA polymerase (such as Taq polymerase), reverse transcriptase, and Mg2+. 2+ These conditions provide the necessary conditions for nucleic acid amplification.

[0027] In this article, the term "nucleic acid release reagent" refers to a reagent used to lyse a sample (such as serum, tissue, etc.) that can destroy the structure of sample cells or pathogens, releasing nucleic acids from the cells or pathogens into the reaction system in preparation for subsequent extraction or amplification.

[0028] In this article, the term "nucleic acid extraction reagent" refers to reagents used to separate and purify nucleic acids in samples. These reagents can remove interfering substances such as proteins and impurities from the samples, obtain high-purity nucleic acids, and ensure the accuracy of PCR detection.

[0029] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0030] In some specific embodiments, a quantitative composition is provided, comprising:

[0031] The first reagent comprises 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 ;

[0032] The second reagent comprises N homologous vectors, wherein each homologous vector includes a segment of the pathogen, and the segment of the pathogen is respectively associated with segment X1 to X2 of the pathogen in the first reagent. N One-to-one correspondence;

[0033] The third reagent includes M quantitative carriers; and

[0034] The fourth reagent includes M sets of primers and probes, wherein the M sets of primers and probes are used to amplify and detect segments of the quantitative vector, respectively.

[0035] Where N and M are both positive integers ≥ 1.

[0036] Furthermore, N≥M.

[0037] 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.

[0038] In some specific implementation schemes, N can be 1, 2, 3, 4, 5, 6, etc.

[0039] 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.

[0040] For example, when N is 2, the first reagent includes two sets of primers and probes, which can amplify the pathogen segments X1 and X2 respectively; correspondingly, the second reagent includes two homologous vectors, one homologous vector including the pathogen segment X1 and the other homologous vector including the pathogen segment X2.

[0041] For example, when N is 3, the first reagent includes 3 sets of primers and probes, which can amplify the pathogen segments X1, X2 and X3 respectively; correspondingly, the second reagent includes 3 homologous vectors, one homologous vector including the pathogen segment X1, one homologous vector including the pathogen segment X2, and one homologous vector including the pathogen segment X3.

[0042] 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.

[0043] In some specific implementation schemes, 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 mentioned above. The situation for the first and second reagents is similar and will not be repeated here.

[0044] Furthermore, the X1 to X N It is determined for different pathogen target regions; furthermore, the pathogen target region can be a conserved region of the pathogen, such as the Core region (C region), Surface region (S region), Polymerase region (P region), and X gene region (X region) of HBV. These regions are highly conserved, and there is only one target region, that is, N=1, which can complete the quantification; when N=2 or more, it can naturally also meet the quantification requirements.

[0045] Furthermore, the pathogen target region can be a non-conserved region of the pathogen, such as the Polymerase region (POL region), Group-specific Antigen region (GAG region), and Long Terminal Repeat region (LTR region) of HIV. These regions are highly variable, and there are at least three target regions, i.e., N=3, which can ensure quantification without missed detection. When N>3, it can naturally also meet the quantification requirements. When N=1 or 2, there may be missed detections, but it still does not affect its ability to quantify pathogens more accurately than existing technologies.

[0046] Furthermore, the pathogen can be bacteria, fungi, viruses, and parasites. For example, bacteria include, but are not limited to: Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi, etc.; viruses include, but are not limited to: Enterovirus 71, Astrovirus, Norovirus, Rotavirus, Enteric Adenovirus, etc.; parasites include, but are not limited to: Echinococcus multilocularis and Echinococcus granulosus; fungi include, but are not limited to: Aspergillus flavus, etc.

[0047] Furthermore, the nucleic acid of the pathogen can be DNA or RNA. For example, the pathogen can be a DNA virus or an RNA virus. For example, the pathogens disclosed herein can be hepatitis B virus, hepatitis C virus, human immunodeficiency virus, etc.

[0048] Furthermore, the pathogens may be the same or different.

[0049] Furthermore, the pathogens are the same, meaning that the segments of the pathogens in the first reagent and / or the second reagent belong to different segments of the same pathogen.

[0050] Furthermore, the quantification vector can be any vector used in the prior art for quantification, provided that its amplification efficiency is similar to or consistent with that of the target or homologous vector. For example, it can be a vector containing GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), RNASE (Ribonuclease) gene fragments, etc.

[0051] Furthermore, the quantitative vector can be targeted at pathogen segment X in the homologous vector. N The nucleotide sequence was modified by replacing adenine deoxyribonucleotide (A) with thymine deoxyribonucleotide (T) in the original sequence, and replacing thymine deoxyribonucleotide (T) with adenine deoxyribonucleotide (A) in the original sequence.

[0052] The purpose of this design is to ensure that the amplification efficiency of the target region of the quantitative vector is similar to that of the homologous vector, effectively simplifying the process of calculating the target region concentration of the homologous vector based on the quantitative vector.

[0053] In some specific implementations, the vector may be a plasmid.

[0054] Furthermore, the backbone plasmids of the homologous plasmids and quantitative plasmids are derived from any plasmids in the prior art, for example, such as 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.

[0055] In some specific implementations, the backbone plasmid of the homologous plasmid and the quantitative plasmid is the pUC57 plasmid, that is, the sequence of X1 is synthesized and integrated into the pUC57 plasmid to form a homologous plasmid; the A of X1 is replaced with T, and the sequence after T replaces A is synthesized and integrated into the pUC57 plasmid to form a quantitative plasmid.

[0056] In some specific implementations, the vector may be a recombinant virus comprising the plasmids described above.

[0057] In some specific implementations, the vector may be a liposome comprising the plasmid described above.

[0058] In some specific implementations, the vector may be an artificial chromosome carrying a target segment.

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

[0060] Using the concentration of the aforementioned homologous vectors allows for more accurate quantification of low-concentration pathogens.

[0061] Furthermore, the composition further includes a fifth reagent comprising primers and probes, wherein the primers and probes in the fifth reagent are used to amplify and detect segments of the pathogen. Even further, the pathogen segment amplified by the fifth reagent differs from the pathogen segment amplified by the first reagent and the pathogen segment in the second reagent.

[0062] By introducing a fifth reagent, it is possible to perform qualitative analysis on the sample, that is, to determine whether the pathogen is present in the sample.

[0063] In some specific embodiments, the composition further includes a fifth reagent that amplifies segment Y of the pathogen, segment Y being analogous to segments X1 to X2 of the pathogen. N They are all different.

[0064] Furthermore, the first reagent also includes at least one additional second probe, which targets segments X1 to X of the homologous vector. N At least a portion of at least one of the pathogens, and mutating a portion of the bases of the segment targeted by the probe on the homologous vector, such that the probe cannot target the pathogen.

[0065] Furthermore, the composition further includes a sixth reagent for replacing the second reagent, wherein the sixth reagent comprises a segment of a pathogen, the segment of the pathogen in the sixth reagent being obtained by modifying the segment of the pathogen included in the second reagent.

[0066] Furthermore, the composition further includes a seventh reagent, wherein the seventh reagent includes a probe capable of targeting segments of pathogens included in the first reagent, but not segments of pathogens included in the sixth reagent.

[0067] By introducing the sixth and seventh reagents, it is also possible to perform qualitative operations on the sample, that is, to determine whether the pathogen is present in the sample.

[0068] In some specific embodiments, the first, fourth, fifth, and seventh reagents in the quantitative composition of this disclosure may be modified.

[0069] Furthermore, the modification can be one or more of locked nucleic acid (LNA) modification, MGB (Minor Groove Binder) modification, or ZNA (Zip Nucleic Acids) modification.

[0070] In some specific embodiments, the quantitative compositions disclosed herein are used for fluorescent PCR.

[0071] Furthermore, the fluorescent groups of the probes in this disclosed composition are different from each other and do not interfere with each other.

[0072] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0073] Furthermore, the 3' end of the probe also has a quenching group, such as MGB, BHQ1, or BHQ2.

[0074] In one specific embodiment, each component of the quantitative composition disclosed herein is contained in a separate package.

[0075] In one specific embodiment, the components of the quantitative composition disclosed herein are contained in the same package.

[0076] Furthermore, the components of the quantitative composition disclosed herein exist in a mixed form.

[0077] In one specific embodiment, this disclosure provides a quantitative composition for quantifying HBV, comprising:

[0078] The first reagent comprises 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 HBV segment X1, and the nucleotide sequence is shown in SEQ ID NO.4;

[0079] The second reagent includes a homology vector comprising a nucleotide sequence as shown in SEQ ID NO.4;

[0080] The third reagent includes a quantitative carrier; and

[0081] The fourth reagent includes a set of primers and probes, which are used to amplify and detect segments of the quantitative vector, respectively.

[0082] Furthermore, the nucleotide sequence of the quantification vector segment is shown in SEQ ID NO.5.

[0083] 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.

[0084] Furthermore, the concentration of the homologous carrier is 25% to 66.7% of the minimum quantitative concentration, preferably 30% to 60%.

[0085] In one specific embodiment, this disclosure provides a quantitative composition for quantifying HCV, comprising:

[0086] The first reagent comprises 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, wherein the primers and probes are used to amplify and detect HCV segments X1 and X2, and their nucleotide sequences are shown in SEQ ID NO. 12 and SEQ ID NO. 52.

[0087] The second reagent comprises two homologous vectors, wherein the homologous vectors comprise nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.52;

[0088] The third reagent includes two quantitative carriers; and

[0089] The fourth reagent includes two sets of primers and probes, which are used to amplify and detect segments of the quantitative vector, respectively.

[0090] Furthermore, the nucleotide sequences of the segments of the quantitative vector are shown in SEQ ID NO.13 and SEQ ID NO.53.

[0091] Furthermore, the nucleotide sequences of the two sets of 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.

[0092] Furthermore, the concentration of the homologous carrier is 25% to 66.7% of the minimum quantitative concentration, preferably 30% to 60%.

[0093] Furthermore, the composition further includes a fifth reagent, the nucleotide sequences of the primers and probes of which 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.

[0094] In one specific embodiment, this disclosure provides a quantitative composition for quantifying HIV, comprising:

[0095] The first reagent comprises three sets of primers and probes with nucleotide sequences as shown in SEQ ID NO.17 to SEQ ID NO.25, which are used to amplify and detect HIV segment X. 1～ X3, whose nucleotide sequence is shown in SEQ ID NO.26~SEQ ID NO.28;

[0096] The second reagent comprises three homologous vectors, each containing a nucleotide sequence as shown in SEQ ID NO.26 to SEQ ID NO.28.

[0097] The third reagent includes one or three quantitative carriers; and

[0098] The fourth reagent comprises three sets of primers and probes, which are used to amplify and detect segments of the quantitative vector, respectively.

[0099] Furthermore, the nucleotide sequences of the segments of the quantitative vector are shown in SEQ ID NO.29 to SEQ ID NO.31.

[0100] Furthermore, the nucleotide sequences of the three sets of primers and probes of the fourth reagent are shown in SEQ ID NO.32 to SEQ ID NO.40.

[0101] Furthermore, the concentration of the homologous carrier is 25% to 66.7% of the minimum quantitative concentration, preferably 30% to 60%.

[0102] Furthermore, the composition further includes a fifth reagent, the nucleotide sequences of the primers and probes of which 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.

[0103] In some specific implementations, the third reagent may include three quantitative carriers. Preferably, the three quantitative carriers are mixed in a 1:1:1 ratio (molar ratio), that is, they are mixed in equal amounts.

[0104] In some specific implementations, the third reagent may include a quantitative carrier, that is, the three segments of the quantitative carrier are integrated into one carrier, preferably, integrated into one segment at a copy ratio of 1:1:1.

[0105] Secondly, this disclosure provides the use of the above-described quantitative composition in the preparation of quantitative reagent kits.

[0106] Thirdly, this disclosure provides a quantitative kit comprising the quantitative composition described above.

[0107] Furthermore, the kit also includes nucleic acid amplification reagents.

[0108] Furthermore, the amplification reagents include dNTPs, PCR buffer, DNA polymerase, reverse transcriptase, and Mg. 2+ At least one of them.

[0109] Furthermore, the kit also includes: nucleic acid release reagent and nucleic acid extraction reagent.

[0110] Furthermore, the concentration of the DNA polymerase is 3 U / μL to 15 U / μL, for example, the DNA polymerase can be Taq polymerase.

[0111] In one specific implementation, the kit disclosed herein includes: Taq enzyme, Mg 2+ Mn 2+ dNTPs and PCR buffer.

[0112] In one specific implementation, the kit disclosed herein includes: Taq enzyme, RT enzyme, and Mg... 2+ Mn 2+ dNTPs and PCR buffer.

[0113] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume in a single PCR reaction tube is generally 20 μL to 200 μL.

[0114] Fourthly, this disclosure provides a quantitative method, the method comprising the following steps:

[0115] 1) Extract nucleic acid from the sample to be tested;

[0116] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit described above; and

[0117] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0118] Furthermore, the quantitative formula is as follows: Where C represents concentration, C 病原体 This refers to the concentration of the pathogen, C. 定量载体 This refers to the concentration of the quantitative vector. P1 is the probe of the first reagent, P2 is the probe of the fourth reagent, and -ΔCT(P1-P2) represents the difference between the Ct value of the qPCR amplification curve corresponding to the probe of the first reagent (P1 - target) and the Ct value of the qPCR amplification curve corresponding to the probe of the fourth reagent (P4 - internal standard); C 同源载体 This refers to the concentration of the homologous vector.

[0119] Furthermore, this disclosure provides a quantitative method for non-diagnostic purposes, the method comprising the following steps:

[0120] 1) Extract nucleic acid from the sample to be tested;

[0121] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit described above; and

[0122] 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

[0123] Furthermore, the quantitative formula is as follows: P1 is the probe for the first reagent, and P2 is the probe for the fourth reagent. Attached Figure Description

[0124] Figure 1 is a schematic diagram of the single-carrier quantitative mode;

[0125] Figure 2 is a schematic diagram of the dual-carrier quantitative mode of this disclosure;

[0126] Figure 3 is a schematic diagram of the dual-carrier quantitative mode of this disclosure;

[0127] Figure 4 is a schematic diagram of the dual-carrier quantitative mode of this disclosure.

[0128] Figure 5 is a schematic diagram of the dual-carrier quantitative mode of this disclosure;

[0129] Figure 6 shows the results of the detection of HBV quantitative composition 1 disclosed in this invention.

[0130] Figure 7 shows the results of the HBV comparative quantitative composition detection in this disclosure.

[0131] Figure 8 shows the results of detecting positive HBV samples using the HBV quantitative composition 2 disclosed in this invention.

[0132] Figure 9 shows the results of detecting negative HBV samples using the HBV quantitative composition 2 disclosed in this invention.

[0133] Figure 10 shows the results of detecting positive HCV samples using the HCV quantitative composition 2 disclosed in this invention.

[0134] Figure 11 shows the results of detecting negative HCV samples using the HCV quantitative composition 2 disclosed in this invention.

[0135] Detailed Implementation

[0136] The present disclosure will be further elaborated below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present disclosure clearer. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present disclosure.

[0137] Measurement of the lowest quantitation concentration (limit of quantitation):

[0138] Pathogen standards were used, and newborn calf serum was used to serially dilute the pathogen standards. The diluted low-concentration samples were used as test samples. Each test sample was tested 20 times in a row, for a total of 60 repeated tests. The accuracy requirement was that the logarithmic deviation of the test results was within ±0.5. The lowest quantitation (LOQ) was the lowest measurable concentration of the analyte that could be measured to meet the requirement of 100% accuracy.

[0139] Using HBV as an example, the national standard for HBV was used, and newborn calf serum was diluted to concentrations of 20 IU / mL, 15 IU / mL, 12 IU / mL, 10 IU / mL, 8 IU / mL, and 5 IU / mL. The lowest concentration samples were used as test samples, and 20 replicates were performed for each sample, for a total of 60 replicates (3 times each). The accuracy requirement was a logarithmic deviation of within ±0.5%. The lowest quantitation level (LOQ) was the lowest measurable concentration of the analyte required to achieve 100% accuracy. Using the above method, the LOQ for HBV in this patented PCR system was ultimately determined to be 12 IU / mL.

[0140] The conventional quantitative model in existing technologies is shown in Figure 1. This disclosure innovatively introduces a homologous vector, and its quantitative model is shown in Figure 2. Additional primer and probe sets can also be added, as shown in Figure 3. When two or more homologous vectors are introduced, an exemplary quantitative model is shown in Figure 4. Simultaneously, a probe of a seventh reagent can be added to the first reagent, and the homologous plasmid of the second reagent can be adaptively modified to obtain a sixth reagent to replace the second reagent, thereby enabling simultaneous qualitative and quantitative analysis of pathogens, as shown in Figure 5.

[0141] Example 1: Sequences used in this disclosure

[0142] Table 1

[0143] The probe for the first HBV reagent has a FAM fluorescent group, and the probe for the fourth HBV reagent has a HEX fluorescent group.

[0144] The probe of the first HCV reagent has a fluorescent group of FAM; the probe of the fourth HCV reagent has a fluorescent group of HEX; and the probe of the fifth HCV reagent has a fluorescent group of CY5.

[0145] The probe for the first HIV reagent has a fluorescent group of FAM; the probe for the fourth HIV reagent has a fluorescent group of HEX; and the probe for the fifth HIV reagent has a fluorescent group of CY5.

[0146] Example 2: Quantitative Methods for Pathogens

[0147] This project uses Sansure Biotech's S10012 (HBV), S10013 (HCV), or S1001 (HIV) nucleic acid extraction kits to extract pathogen nucleic acids. Diluted newborn calf serum was used to plot pathogen concentration curves or as a sample to test the performance of the internal standard quantification system.

[0148] PCR reaction conditions:

[0149] HBV:

[0150] Phyto-Anstart Taq polymerase (5U); Sansure Biotech S08 Buffer; primers / probes synthesized in Bailige and diluted to working concentration of 40 pmol / μL via TE buffer; 1 mol / L Mg 2+ Pathogen and IC target sequence plasmid synthesis was carried out at Sangon Biotech.

[0151] A single-sample 50 μL amplification system includes: 20.75 μL PCR buffer (S08), 2 μL 100 mmol / L dNTP(T), 0.6 μL 40 pmol / μL PCR forward and reverse primers, 0.1 μL 40 pmol / μL probe, 1.6 μL 5 U Taq DNA polymerase, and 0.4 μL 1 mol / L MgSO₄. 2+ 22.65 μL of purified water.

[0152] PCR amplification program: 95℃ for 8 min, 1 cycle; 95℃ for 15 s, 57℃ for 30 s, 45 cycles, with fluorescence signal read once after each cycle; 25℃ for 10 s, 1 cycle, reaction volume set to 50 μL.

[0153] HCV:

[0154] KND NRT enzyme (5U); KND Taq DNA polymerase (5U); Sansure Biotech S15 Buffer; primers / probes synthesized at Bailige and diluted to working concentration of 40pmol / μL via TE buffer; 1mol / L Mg2+; HCV and IC target sequence plasmids synthesized at Sangon Biotech.

[0155] A single-sample 50 μL amplification system includes: 17 μL PCR buffer (S15), 0.8 μL 100 mmol / L dNTP(T), 0.4 μL 40 pmol / μL PCR forward and reverse primers, 0.125 μL 40 pmol / μL probe, 1.5 μL 5 U Taq DNA polymerase, 0.5 μL 5 U NRT enzyme, and 0.4 μL 1 mol / L MgSO₄. 2+ 27.025 μL of purified water.

[0156] PCR amplification program: 95℃ for 1 min, 1 cycle; 60℃ for 30 min, 1 cycle; 95℃ for 8 min, 1 cycle; 95℃ for 15 s, 57℃ for 30 s, 45 cycles, with fluorescence signal read once after each cycle; 25℃ for 10 s, 1 cycle. HCV target 1 was set to the FAM channel, the quantitative internal standard IC was set to the HEX channel, and HCV target 2 was set to the CY5 channel. The reaction volume was 50 μL.

[0157] HIV:

[0158] Baorui MMLV RT enzyme (5U); Kangde Taq DNA polymerase (5U); Sansure Biotech S15 Buffer; primers / probes synthesized in Bailige and diluted to working concentration of 40 pmol / μL via TE buffer; 1 mol / L Mg 2+ HIV-1 and IC target sequence plasmids were synthesized at Sangon Biotech.

[0159] A single-sample 50 μL amplification system includes: 17 μL PCR buffer (S15), 1 μL 100 mmol / L dNTP(T), 0.24 μL 40 pmol / μL PCR forward and reverse primers, 0.1 μL 40 pmol / μL probe, 1.5 μL 5 U Taq DNA polymerase, 0.5 μL 5 U MMLV RT enzyme, and 0.4 μL 1 mol / L MgSO4. 2+ 25.54 μL of purified water.

[0160] PCR amplification program: 50℃ for 30 min, 1 cycle; 95℃ for 8 min, 1 cycle; 95℃ for 15 s, 57℃ for 30 s, 45 cycles, with fluorescence signal read once after each cycle; 25℃ for 10 s, 1 cycle, with a reaction volume of 50 μL.

[0161] Add 10 μL of 1.00E-7 μg / mL quantitative vector or pathogen homologous vector to 400 μL of newborn calf serum. Plot a pathogen concentration curve using Chinese national pathogen standards diluted with newborn calf serum to determine the concentration. The average concentration obtained from three independent replicate experiments is set as the pathogen concentration of the plasmid.

[0162] For example, after plasmid determination, 10 μL of 1700 IU / mL quantitative vector and 10 μL of homologous vector at the corresponding concentration are added to 400 μL of the sample to be tested. After mixing thoroughly, pathogen nucleic acid (HBV) is extracted using the Sansure Biotech S10012 nucleic acid extraction kit. 300 μL of extraction solution 1 is added to a centrifuge tube, vortexed, and then briefly centrifuged. 100 μL of extraction solution 2 is added to each tube, vortexed, and incubated at room temperature for 10 minutes. After brief centrifugation, the tube is placed on a magnetic separator for 3 minutes. Once the magnetic beads have aggregated on the centrifuge tube wall, the liquid is aspirated. 600 μL of extraction solution 3 and 200 μL of extraction solution 4 are added to the centrifuge tube simultaneously, vortexed, and briefly centrifuged. The centrifuge tube is then placed on the magnetic separator again. After approximately 3 minutes, the liquid is aspirated again. After eluting the magnetic beads with 50 μL of PCR reaction solution, place the solution on a magnetic separator. Transfer the supernatant to a PCR reaction tube, vortex to mix, and centrifuge briefly. Place the tubes into the PCR instrument in sequence, set the running program and relevant parameters, save the file, and run the reaction program.

[0163] The final concentration of the quantitative carrier in the sample was set at 1700 IU / mL. Therefore, according to the formula... Calculate the pathogen load in the sample to be tested.

[0164] Example 3: Detection results of HBV samples tested using the HBV quantitative composition 1 disclosed herein.

[0165] Using the national standard for HBV, newborn calf serum was diluted to concentrations of 20 IU / mL, 18 IU / mL, 16 IU / mL, 15 IU / mL, and 10 IU / mL. The lowest concentration samples were used as test samples. Each test sample was analyzed in 20 replicates, for a total of 60 replicates (3 times each). The accuracy requirement was a logarithmic deviation of within ±0.5%. The lowest quantitation (LOQ) was the lowest measurable analyte concentration that could be measured with 100% accuracy. Based on this system, the LOQ for HBV was calculated to be 16 IU / mL.

[0166] The comparative and example schemes (sequences shown in SEQ ID NO.1 to SEQ ID NO.8, N=1) were used to detect diluted Chinese national HBV standard in newborn calf serum. The comparative example only added 10 μL of a quantitative plasmid with a total set value of 1700 IU / mL; the example added an additional homologous plasmid with a final concentration of 5 IU / mL.

[0167] The detection results of the two systems on the Chinese national HBV standard diluted with 10 IU / mL newborn calf serum are shown in Figures 6 and 7. Compared with the comparative example, the amplification curve of the HBV target sequence of the HBV dual-channel dual-plasmid internal standard quantification technology (detection by composition 1 in Example 1) in Figure 6 is more concentrated, and the variance of the Ct value is significantly lower than that of the comparative example (Figure 7), indicating that the detection results are 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 example 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 quantification technology shows a lower quantitative lower limit and better detection accuracy for samples with low HBV load. It can also be seen from the results figures that the curve of the system disclosed in this disclosure (Figure 6) is more concentrated and has smaller deviation.

[0168] Table 2

[0169] *Quantitative inaccuracy; the logarithmic value of the sample being tested deviates from the actual sample concentration by more than ±0.5%.

[0170] Figure 7 shows the detection results of the two internal standard quantification systems on the diluted Chinese national HBV standard at 8 IU / mL newborn calf serum. Compared with the comparative example, the amplification curve of the HBV target sequence using the HBV dual-channel dual-plasmid internal standard quantification technology (example) was more concentrated, and the variance of the Ct value was significantly lower than that of the comparative example, indicating more stable detection results. As shown in Table 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 technology exhibits a lower quantitative lower limit and better accuracy.

[0171] Table 3

[0172] *Quantitative inaccuracy; the logarithmic value of the sample being tested deviates from the actual sample concentration by more than ±0.5%.

[0173] Example 4: Detection results of HBV samples tested using the HBV quantitative composition 1 disclosed herein.

[0174] The HBV dual-channel dual-plasmid internal standard quantification technique (Example) was used to detect 10 IU / mL diluted Chinese national HBV standard from newborn calf serum. A quantification plasmid with a final concentration of 1700 IU / mL was added to the test sample. Based on this, homologous plasmids with final HBV concentrations of 2 / 3 / 4 / 6 / 8 / 10 / 12 IU / mL were added to each group. In this test system, the lowest quantitation (LOQ) of the HBV single-plasmid internal standard system was approximately 12 IU / mL. As shown in Table 4, the optimal addition amount of HBV homologous plasmid is approximately between 3 IU / mL and 8 IU / mL, which is 25% to 66.7% of the LOQ of the HBV single-plasmid internal standard system.

Claims

1. A quantitative composition comprising: The first reagent comprises N sets of primers and probes, wherein the N sets of primers and probes are respectively used for amplifying and detecting N segments X1-X N ; a second reagent comprising N homologous vectors, wherein the homologous vectors comprise segments of the pathogen that respectively correspond to segments X1~X N one-to-one correspondence; The third reagent includes M quantitative carriers; and The fourth reagent includes M sets of primers and probes, wherein the M sets of primers and probes are used to amplify and detect segments of the quantitative vector, respectively. Where N and M are both positive integers ≥ 1.

2. The quantitative composition according to claim 1, characterized in that, N is any positive integer from 1 to 10, and / or M is any positive integer from 1 to 10.

3. The quantitative composition according to claim 1 or 2, characterized in that, The quantitative vector includes a target for pathogen segments X1 to X2 in the homologous vector. N The modified nucleotide sequence replaces adenine deoxyribonucleotides in the homology vector sequence with thymine deoxyribonucleotides, and replaces thymine deoxyribonucleotides in the homology vector sequence with adenine deoxyribonucleotides.

4. The quantitative composition according to any one of claims 1-3, characterized in that, The composition further includes a fifth reagent comprising primers and probes, wherein the primers and probes in the fifth reagent are used to amplify and detect segments of a pathogen, wherein 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.

5. The quantitative composition according to any one of claims 1-4, characterized in that, The composition further includes a sixth reagent and a seventh reagent; The sixth reagent is used to replace the second reagent, and the sixth reagent includes a segment of the pathogen, which is obtained by modifying the segment of the pathogen included in the second reagent. The seventh reagent includes a probe that can target segments of pathogens included in the first reagent, but cannot target segments of pathogens included in the sixth reagent.

6. The quantitative composition according to any one of claims 1-5, characterized in that, The quantitative composition includes any one of the following compositions: 1) A quantitative composition for quantifying HBV, comprising: The first reagent comprises 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 HBV segment X1, and the nucleotide sequence is shown in SEQ ID NO.4; The second reagent includes a homology vector comprising a nucleotide sequence as shown in SEQ ID NO.4; The third reagent includes a quantitative carrier; and The fourth reagent includes a set of primers and probes, which are 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.6 to SEQ ID NO.8; 2) A quantitative composition for quantifying HCV, comprising: The first reagent comprises two sets of primers and probes with nucleotide sequences as shown in SEQ ID NO. 9–11 and SEQ ID NO. 49–51, wherein the primers and probes are used to amplify and detect HCV segments X1 and X2, and their nucleotide sequences are shown in SEQ ID NO. 12 and SEQ ID NO.

52. The second reagent comprises two homologous vectors, wherein the homologous vectors comprise nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.52; The third reagent includes two quantitative carriers; and The fourth reagent includes two sets of primers and probes, which are 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.14~SEQ ID NO.16 and SEQ ID NO.54~SEQ ID NO.56; 3) A quantitative composition for quantifying HIV, comprising: The first reagent comprises 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, whose nucleotide sequence is shown in SEQ ID NO.26~SEQ ID NO.28; The second reagent comprises three homologous vectors, each containing a nucleotide sequence as shown in SEQ ID NO.26 to SEQ ID NO.

28. The third reagent includes three quantitative carriers; and The fourth reagent includes three sets of primers and probes, which are 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.

7. The quantitative composition according to any one of claims 1-6, characterized in that, The vector is one or more of the following: plasmid, recombinant virus containing plasmid, liposome containing plasmid, or artificial chromosome.

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

9. The quantitative composition according to any one of claims 1 to 8, characterized in that, The concentration of the homologous carrier is 30% to 60% of the minimum quantitative concentration.

10. The quantitative composition according to any one of claims 1 to 9, characterized in that, The pathogens include bacteria, fungi, viruses, and parasites.

11. Use of the quantitative composition according to any one of claims 1 to 10 for the preparation of a quantitative reagent kit.

12. A quantitative reagent kit comprising the composition as described in any one of claims 1 to 10.

13. The reagent kit according to claim 12, characterized in that, The kit also includes at least one of nucleic acid amplification reagent, nucleic acid release reagent, or nucleic acid extraction reagent.

14. A quantitative method, the method comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition as described in any one of claims 1 to 10 or the kit as described in claim 12 or 13; and 3) Quantify the nucleic acid in the sample based on the concentration of the carrier and the CT value.

15. The method according to claim 14, characterized in that, The quantitative formula is as follows: P1 is the probe for the first reagent, and P2 is the probe for the fourth reagent.

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