A universal one-step multiplex PCR reaction system, a nucleic acid detection kit and a PCR detection method

Abstract

The application relates to the field of PCR technology, and particularly relates to a universal one-step multiplex PCR reaction system, a nucleic acid detection kit and a PCR detection method. The PCR reaction system comprises a PCR buffer reagent and an enzyme reaction reagent; the PCR buffer reagent contains tetramethylammonium chloride and Brij35; the enzyme reaction reagent comprises an enzyme storage solution and PCR enzyme, and the enzyme storage solution contains Brij35 and tris(2-carboxyethyl) phosphine. The PCR buffer system provided by the application can be prepared into a reaction system suitable for multiplex fluorescence PCR detection and multiplex RT-PCR detection by using relatively simple components, and the overall reaction sensitivity and specificity are good in cooperation with an efficient automatic magnetic bead nucleic acid extraction method; the sensitivity of the detection target can be as low as 100 copies / mL, the sensitivity of part of the detection targets can be as low as 5 copies / mL, and the method has obvious advantages over the currently disclosed methods.

Description

Technical Field

[0001] This invention relates to the field of PCR technology, specifically to a universal one-step multiplex PCR reaction system, a nucleic acid detection kit, and a PCR detection method. Background Technology

[0002] Currently, the main methods used for nucleic acid detection include: conventional PCR, real-time quantitative PCR, nucleic acid sequencing, nucleic acid mass spectrometry, and gene chips. Among these, real-time quantitative PCR is the closest to the present invention.

[0003] Real-time quantitative PCR (qPCR) is a nucleic acid amplification and detection technique that combines polymerase chain reaction (PCR) with real-time fluorescence detection. Based on the characteristics of the fluorescent dyes, qPCR is further divided into two types: a non-specific dye method represented by SYBR Green I dye, and a specific probe method represented by TaqMan probes. The former has lower application costs, but the detection results are easily affected by non-specific amplification and it is difficult to achieve multiplexing. The latter has slightly higher application costs, higher detection sensitivity and specificity, and can achieve single-tube multiplexing, and is currently widely used for nucleic acid detection in fields such as microbial pathogens and genetic diseases.

[0004] Among similar patents, Chinese invention patent CN108998506B discloses a one-step real-time fluorescent RT-PCR reaction buffer, its reaction system, and PCR method; Chinese invention patent CN113999897B discloses an RT-PCR reaction system, RT-PCR method, and applications, introducing an RT-PCR reaction system suitable for one-step RNA detection; Chinese invention patent CN102465120B discloses a fluorescent quantitative PCR reaction solution and fluorescent quantitative PCR method; and Chinese invention patent CN110804652B discloses additives, kits, and reaction methods for rapid DNA detection in real-time quantitative PCR, introducing PCR reaction systems for DNA detection and optimizing the corresponding PCR enhancers. However, none of these patents address the issue of simultaneously detecting DNA and RNA, nor do they propose optimizations for multiplex PCR.

[0005] Current PCR detection technologies employ complex reaction systems, requiring the addition of multiple PCR enhancers to improve amplification efficiency. Furthermore, most PCR reaction systems are not compatible with multiplex PCR amplification and cannot simultaneously achieve efficient amplification of both DNA and RNA. The poor compatibility of fluorescent PCR reaction systems may be due to inadequate buffer group ratios, failing to simultaneously accommodate the reactivity of reverse transcriptase and Taq enzymes. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a universal one-step multiplex PCR reaction system, nucleic acid detection kit, and PCR detection method. Addressing the issues of complex PCR reaction systems in existing technologies and their inability to be compatible with multiplex fluorescent PCR reactions to achieve efficient amplification of all targets, this invention optimizes the multiplex PCR reaction system. This achieves the technical effect of being applicable to the detection of DNA and RNA in ordinary PCR systems, as well as to the detection of single / multiple DNA, single / multiple RNA, and multiplex DNA / RNA in quantitative fluorescent PCR systems.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is to provide a universal one-step multiplex PCR reaction system, the PCR reaction system comprising PCR buffer reagent and enzyme reaction reagent; The PCR buffer reagent contains tetramethylammonium chloride and Brij35, wherein the concentration of tetramethylammonium chloride is 2-20 mM and the volume percentage of Brij35 is 0.5-10%. The enzyme reaction reagent includes an enzyme preservation solution and a PCR enzyme. The enzyme preservation solution contains Brij35 and tris(2-carboxyethyl)phosphine (TCEP), and the volume percentage of Brij35 is 0.5-5%, and the concentration of tris(2-carboxyethyl)phosphine is 0.1-10 mM.

[0008] The volume percentages (%) mentioned above refer to v / v (volume / volume).

[0009] Furthermore, the PCR buffer reagent contains 5-15 mM tetramethylammonium chloride and 1-5% Brij35 by volume; the enzyme preservation solution contains 1-2% Brij35 by volume and 1-5 mM tris(2-carboxyethyl)phosphine.

[0010] Furthermore, the PCR buffer reagent also includes Tris-HCl, ammonium sulfate, and magnesium chloride.

[0011] Furthermore, in the PCR buffer reagent, the concentration of Tris-HCl is 20-100 mM and the pH is 8.5-9.0; preferably, the concentration is 40-70 mM and the pH is preferably 8.5-8.8.

[0012] Furthermore, in the PCR buffer reagent, the concentration of ammonium sulfate is 10-50 mM; preferably 20-40 mM.

[0013] Furthermore, in the PCR buffer reagent, the concentration of magnesium chloride is 2.5 ~ 6 mM; preferably 3 ~ 5 mM.

[0014] Furthermore, the PCR buffer reagent also includes dNTPs (deoxynucleotide triphosphates), and the dNTPs include dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate) and / or dUTP (deoxyuridine triphosphate). Further, the concentration of the dNTPs in the PCR buffer is 0.1 ~ 1.2 mM, preferably 0.2 ~ 0.8 mM.

[0015] Furthermore, the volume ratio of dATP:dCTP:dGTP:(dTTP and / or dUTP) is 1:1:1:1.

[0016] Furthermore, the ratio of dTTP to dUTP is 1:(1~5), with a preferred ratio of 1:(2~4).

[0017] Furthermore, the PCR buffer reagent comprises 40-70 mM Tris-HCl, 20-40 mM ammonium sulfate, 3-5 mM magnesium chloride, 5-15 mM tetramethylammonium chloride, 1%-5% Brij35, and 0.2-0.8 mM dNTPs.

[0018] Furthermore, the enzyme preservation solution also includes Tris-HCl, potassium chloride, ethylenediaminetetraacetic acid (EDTA), and glycerol.

[0019] Furthermore, in the enzyme preservation solution, the concentration of Tris-HCl is 10-100 mM and the pH is 7.0-8.5; preferably, the concentration is 20-50 mM and the pH is preferably 7.5-8.0.

[0020] Furthermore, in the enzyme reaction reagent, the concentration of potassium chloride is 10-150 mM; preferably 20-70 mM.

[0021] Furthermore, in the enzyme reaction reagent, the concentration of ethylenediaminetetraacetic acid is 0.05 ~ 5 mM; preferably 0.1 ~ 1 mM.

[0022] Furthermore, in the enzyme reaction reagent, the volume percentage of glycerol is 30% to 70%; preferably 40% to 60%.

[0023] Furthermore, the enzyme preservation solution comprises 20-50 mM Tris-HCl, 20-70 mM potassium chloride, 0.1-1 mM ethylenediaminetetraacetic acid, 1%-2% Brij35, 1-5 mM TCEP, and 40%-60% glycerol.

[0024] Furthermore, the PCR reaction system includes enzyme reaction reagent A or enzyme reaction reagent B.

[0025] Furthermore, the PCR enzyme in enzyme reaction reagent A includes Taq enzyme and UNG enzyme; the PCR enzyme in enzyme reaction reagent B includes Taq enzyme, UNG enzyme, reverse transcriptase and RNA protectant.

[0026] Furthermore, in enzyme reaction reagent A or enzyme reaction reagent B, the concentration range of the Taq enzyme is 1 ~ 10 U / test; preferably 5 ~ 10 U / test.

[0027] Furthermore, in the enzyme reaction reagent A / enzyme reaction reagent B, the concentration range of the UNG enzyme is 0.1 ~ 5 U / test; preferably 0.5 ~ 2 U / test.

[0028] Furthermore, in the enzyme reaction reagent B, the concentration range of the reverse transcriptase is 10 ~ 400 U / test; preferably 50 ~ 200 U / test.

[0029] Furthermore, in the enzyme reaction reagent B, the concentration of the RNA protectant ranges from 5 to 50 U / test; preferably from 10 to 40 U / test.

[0030] The Taq enzyme is a hot-start Taq enzyme with 5'-3' DNA polymerase and 5'-3' exonuclease activities; the UNG enzyme is a thermosensitive UNG enzyme suitable for both conventional PCR and RT-PCR reaction systems, accommodating both DNA and RNA detection; the reverse transcriptase is a genetically modified MMLV-derived reverse transcriptase used in RT-PCR reaction systems requiring RNA detection, using RNA as a template to reverse transcribe and synthesize cDNA. The enzyme's reaction temperature range is 42℃~50℃; the RNA protectant is an RNase inhibitor used in RT-PCR reaction systems requiring RNA detection, protecting the RNA template from RNase degradation.

[0031] A second aspect of the present invention is to provide a nucleic acid detection kit, wherein the product is a kit comprising the PCR reaction system described in any one of the first aspects of the present invention, as well as specific primers and TaqMan fluorescent probes, for multiplex fluorescent PCR detection.

[0032] Furthermore, the fluorescent probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

[0033] Furthermore, the fluorescent reporter group is selected from at least one of FAM, HEX, VIC, JOE, TET, Cy3, TAMRA, ROX, TexasRed, Cy5, Cy5.5, Quasar 705, or Cy7. Furthermore, the fluorescence quenching group is selected from at least one of BHQ1, BHQ2, or BHQ3.

[0034] Furthermore, the kit includes a multiplex fluorescent PCR detection kit for detecting blood pathogens, a multiplex fluorescent RT-PCR detection kit for detecting respiratory pathogens, and a multiplex fluorescent PCR detection kit for detecting sexually transmitted pathogens.

[0035] Furthermore, the aforementioned multiplex fluorescent PCR detection kit for detecting blood pathogens, using the above-described PCR reaction system and adding specific primers and probes, can simultaneously detect multiple DNA / RNA targets, including hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, human immunodeficiency virus (HIV-1+HIV-2) RNA, and internal control RNA. The specific probes for detecting HBV DNA, HCV RNA, HIV RNA, and internal control RNA are labeled with different fluorescent groups, such as FAM, VIC, TAMRA, and Cy5.

[0036] Furthermore, the aforementioned multiplex fluorescent RT-PCR detection kit for detecting respiratory pathogens, using the above-described PCR reaction system and adding specific primers and probes, can simultaneously detect multiple RNA targets: SARS-CoV-2 RNA, influenza A virus (FluA) RNA, influenza B virus (FluB) RNA, respiratory syncytial virus (RSV) RNA, and internal control RNA. The specific probes for detecting SARS-CoV-2 RNA, FluA RNA, FluB RNA, RSV RNA, and internal control RNA are labeled with different fluorescent groups, such as FAM, VIC, ROX, Cy5, and Cy5.5.

[0037] Furthermore, the multiplex fluorescent PCR detection kit for detecting sexually transmitted pathogens, using the aforementioned PCR reaction system and adding specific primers and probes, can simultaneously detect multiple DNA targets: herpes simplex virus (HSV-1+HSV-2) DNA, Chlamydia trachomatis (CT) DNA, Neisseria gonorrhoeae (NG) DNA, Ureaplasma urealyticum (UU) DNA, and internal control DNA. The specific probes for detecting herpes simplex virus DNA, Chlamydia trachomatis DNA, Neisseria gonorrhoeae DNA, Ureaplasma urealyticum DNA, and internal control DNA are labeled with different fluorescent groups, such as FAM, HEX, ROX, Cy5, and Cy5.5.

[0038] A third aspect of the present invention is to provide a one-step multiplex PCR detection method, the method comprising performing a reaction using a PCR reaction system as described in any one of the first aspects of the present invention, or performing a reaction using a kit as described in any one of the second aspects of the present invention.

[0039] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: (1) Tetramethylammonium chloride is an organic amine that can improve the amplification efficiency of PCR reaction by reducing the non-specific binding of primers and probes to nucleic acid templates; Brij35 is a nonionic surfactant that can enhance the stability of the PCR reaction system by changing the surface tension of the liquid and improving the compatibility of the components. Both can be used as PCR additives suitable for this invention and can synergistically improve the amplification efficiency when added to the PCR reaction system.

[0040] (2) Brij35 is a nonionic surfactant that can improve the compatibility of the components of the enzyme preservation solution by changing the surface tension of the liquid; TCEP is a non-thiol reducing agent that can improve the stability of biological enzymes during long-term preservation by protecting the active groups of the enzyme from oxidation; glycerol is a stable biocompatible solvent that can provide a stable preservation environment for biological enzymes. Among them, Brij35 and TCEP are enzyme protectants suitable for the present invention. When added to the enzyme preservation solution at the same time, they can synergistically protect the reactivity of the enzyme and improve the preservation stability.

[0041] (3) The PCR buffer system provided by the present invention has simple components and can be prepared into a reaction system suitable for multiplex fluorescent PCR and multiplex RT-PCR detection. Combined with an efficient automated magnetic bead nucleic acid extraction method, the overall reaction sensitivity and specificity are good. The detection target sensitivity can be as low as 100 copies / mL, and some targets can be as low as 5 copies / mL, which has obvious advantages over the currently disclosed methods. Attached Figure Description

[0042] Figure 1This is a process of TaqMan real-time quantitative PCR in one embodiment of the present invention; Figure 2 This is an embodiment of the present invention showing the results of sensitivity analysis for PCR detection of blood pathogens, including amplification curves of HBV sample (A), HCV sample (B), HIV-1 sample (C), HIV-2 sample (D), and internal standard (E). Figure 3 This is an embodiment of the present invention showing the specific test results of PCR detection of blood pathogens, including amplification curves of HBV DNA (A), HCV RNA (B), HIV RNA (C), and internal standard RNA (D). Figure 4 This is an embodiment of the present invention showing the results of sensitivity analysis for PCR detection of respiratory pathogens, including amplification curves of SARS-CoV-2 sample (A), FluA sample (B), FluB sample (C), RSV sample (D), and internal standard (E); Figure 5 This is an embodiment of the present invention showing the specific test results of PCR detection of respiratory pathogens, including amplification curves of SARS-CoV-2 RNA (A), FluA RNA (B), FluB RNA (C), RSV RNA (D), and internal standard (E); Figure 6 This is an embodiment of the present invention showing the sensitivity analysis results of PCR detection of sexually transmitted pathogens, including amplification curves of HSV-1 sample (A), HSV-2 sample (B), CT sample (C), NG sample (D), UU sample (E) and internal standard (F); Figure 7 This is an embodiment of the present invention showing the specific test results of PCR detection of sexually transmitted pathogens, including amplification curves of HSV-1 DNA (A), CT DNA (B), NG DNA (C), UU DNA (D), and internal standard DNA (E). Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify the source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are mass percentages or volume percentages. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0044] This invention proposes a PCR buffer reagent comprising 40-70 mM Tris-HCl, 20-40 mM ammonium sulfate, 3-5 mM magnesium chloride, 5-15 mM tetramethylammonium chloride, and 1%-5% Brij35, as well as 0.2-0.8 mM dNTPs.

[0045] Tris-HCl is a general-purpose biochemical buffer solution used to provide a stable reaction environment for biological enzyme reactions; ammonium sulfate is an inorganic salt used to provide a salt ion environment for enzyme reactions; magnesium chloride provides the Mg... 2+ It is an essential ion for the activation of Taq enzyme activity, and an appropriate concentration of Mg... 2+ This ensures high reactivity of the Taq enzyme; tetramethylammonium chloride, an organic amine, reduces non-specific binding of primers and probes to nucleic acid templates, improving PCR amplification efficiency; Brij35, a nonionic surfactant, alters the surface tension of the liquid, improving the compatibility of components in the PCR reaction system and thus enhancing its stability. Tetramethylammonium chloride and Brij35 are suitable PCR additives for this invention; their simultaneous addition to the PCR reaction system synergistically improves amplification efficiency.

[0046] Organic amines similar to tetramethylammonium chloride include benzyltrimethylammonium chloride, benzyltriethylammonium chloride, spermidine, spermine, and formamide. Nonionic surfactants similar to Brij35 include Tween 20, Triton X-100, NP-40, and IGEPA C-630. This scheme only uses Brij35 and the tetramethylammonium chloride phase as examples; other similar substances and substances with similar effects are within the scope of protection of this scheme.

[0047] Among them, dNTPs include dATP, dCTP, dGTP, dTTP and / or dUTP. dUTP is used in combination with UNG enzyme to eliminate contamination of PCR products.

[0048] The present invention also proposes a PCR reaction system, including the above-mentioned PCR buffer reagent, and enzyme reaction reagent A and enzyme reaction reagent B, wherein enzyme reaction reagent A and enzyme reaction reagent B have the same enzyme preservation solution, which includes 20-50 mM Tris-HCl, 20-70 mM potassium chloride, 0.1-1 mM ethylenediaminetetraacetic acid, 1%-2% Brij35, 1-5 mM TCEP and 40%-60% glycerol.

[0049] Tris-HCl is a general-purpose biochemical buffer solution used to provide a stable storage environment for biological enzyme reactions; potassium chloride is an inorganic salt that provides a low-concentration salt ion environment for the storage of biological enzymes; ethylenediaminetetraacetic acid (EDTA) is an ion chelating agent used to chelate metal ions in the solution, preventing the activity of biological enzymes from being activated by metal ions during storage; Brij35 is a nonionic surfactant used to change the surface tension of the liquid and improve the compatibility of the components of the enzyme storage solution; TCEP is a non-thiol reducing agent used to protect the active groups of the enzyme from oxidation and improve the stability of biological enzymes during long-term storage; glycerol is a stable biocompatible solvent used to provide a stable storage environment for biological enzymes. Brij35 and TCEP are enzyme protectants suitable for this invention. When both are added to the enzyme storage solution simultaneously, they can synergistically protect the reactivity of the enzyme and improve the storage stability of the biological enzyme.

[0050] Compared to traditional thiol reducing agents (β-mercaptoethanol (BME), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH / GSSG)), TCEP has no obvious irritating odor, is not easily oxidized by air, and has good water solubility, which can more stably protect enzyme activity. TCEP is usually prepared as a TCEP-HCl solution before being used in the preparation of other reagents. Non-thiol reducing agents similar to TCEP include THPP (tris(3-hydroxypropyl)phosphine), TEPP (triethylphosphine), and TBUP (tributylphosphine). Other similar substances and substances with similar effects are all within the scope of protection of this scheme.

[0051] Enzyme Reaction Reagent A includes enzyme preservation solution, Taq enzyme, and UNG enzyme, and is used for PCR reaction systems that detect only DNA; specifically, it includes 5 ~ 10 U / test Taq enzyme and 0.5 ~ 2 U / test UNG enzyme.

[0052] Enzyme Reaction Reagent B includes enzyme preservation solution, Taq enzyme, UNG enzyme, reverse transcriptase, and RNA protectant, and is used for PCR reaction systems that simultaneously detect DNA / RNA or detect RNA only; specifically, it includes 5 ~ 10 U / test Taq enzyme, 0.5 ~ 2 U / test UNG enzyme, 50 ~ 200 U / test reverse transcriptase, and 10 ~ 40 U / test RNA protectant.

[0053] The Taq polymerase is a hot-start Taq polymerase with both 5'-3' DNA polymerase and 5'-3' exonuclease activities. It releases its activity upon activation at 93°C or above for 30 seconds to 10 minutes. Once activated, the enzyme initiates 5'-3' DNA strand synthesis in PCR cycles. When the DNA strand extends to the template region that specifically binds to the TaqMan probe, the 5'-3' exonuclease hydrolyzes the TaqMan probe, releasing a fluorescent group, which then produces a fluorescent signal under excitation light. Taq polymerase can be modified chemically, with antibody modification or ligand modification. The modification method determines the activation time; the thermal activation times for the three modification methods are 5–10 minutes, 30 seconds to 1 minute, and 30 seconds to 2 minutes, respectively.

[0054] The UNG enzyme is a thermosensitive UNG enzyme, suitable for both conventional PCR and RT-PCR reaction systems, and can detect both DNA and RNA.

[0055] The reverse transcriptase is a genetically modified MMLV-derived reverse transcriptase used in RT-PCR reaction systems that require RNA detection. It uses RNA as a template to reverse transcribe and synthesize cDNA. The reaction temperature range of this enzyme is 42℃~50℃.

[0056] The RNA protectant is a ribonuclease inhibitor used in RT-PCR reaction systems that require RNA detection, and can protect the RNA template from RNase degradation.

[0057] This invention also proposes a product comprising the above-described PCR reaction system, wherein the product is a kit, and the product further comprises primers and a fluorescent probe, wherein the fluorescent probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end. The fluorescent reporter groups include, but are not limited to, FAM, HEX, VIC, JOE, TET, Cy3, TAMRA, ROX, TexasRed, Cy5, Cy5.5, Quasar 705, or Cy7; the fluorescent quencher groups include, but are not limited to, BHQ1, BHQ2, or BHQ3.

[0058] The PCR reaction system and PCR kit described in this invention mainly utilize... Figure 1 The TaqMan real-time quantitative PCR method was used for detection, and the specific steps are as follows: 1. Taq enzyme activation Hot-start Taq polymerase is activated at high temperatures and exhibits both 5'-3' polymerase and 5'-3' exonuclease activities.

[0059] 2. PCR cycling (1) Transsexualism The DNA template is destranded at high temperature to form single-stranded DNA.

[0060] (2) Annealing The specific primers and probes bind to the complementary sequences of the single-stranded DNA template. At this time, the 5' fluorescent group and the 3' quencher group of the TaqMan probe are close to each other. The fluorescence signal generated by the fluorescent group is absorbed by the quencher group, and the fluorescence signal cannot be detected by the fluorescence PCR instrument.

[0061] (3) Extension Using dNTPs as raw materials, Taq polymerase utilizes its 5'-3' polymerase activity to extend from the 3' end of the primer and synthesize a new DNA strand complementary to the template.

[0062] (4) Probe hydrolysis When the newly synthesized DNA strand extends to the probe binding region, synthesis is inhibited. The 5'-3' exonuclease activity of Taq polymerase hydrolyzes the probe, allowing the DNA strand to continue extending. After the TaqMan probe is hydrolyzed, the 5' fluorescent group is far from the 3' quencher group, and the fluorescence signal generated by the fluorescent group cannot be absorbed by the quencher group, thus it can be detected by a fluorescence PCR instrument. After each PCR amplification cycle, a fluorescence signal value positively correlated with the amount of PCR product synthesized is generated. The fluorescence PCR instrument detects the amount of PCR product synthesized by acquiring the fluorescence signal in real time.

[0063] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0064] Example 1 This embodiment relates to a multiplex fluorescent PCR detection kit for detecting blood pathogens according to the present invention, which can simultaneously achieve single-tube multiplex detection of DNA and RNA.

[0065] (a) PCR detection The PCR reaction system in this embodiment includes PCR buffer, enzyme reaction solution B, specific primers, and fluorescently labeled TaqMan probes. The specific probes for detecting HBV DNA, HCV RNA, HIV RNA, and internal control RNA are labeled with different fluorescent groups, thereby enabling single-tube multiplex detection.

[0066] Specifically, the PCR reaction system is 60 μl / test (20 μl / test of PCR reaction solution + 40 μl / test of nucleic acid template), wherein the PCR reaction solution contains 15 μl / test of PCR reaction buffer, 3 μl / test of primer and probe mixture, and 2 μl / test of enzyme reaction solution B.

[0067] The composition and concentration of the PCR reaction buffer are as follows: 50 mM Tris-HCl (pH=8.7), 20 mM ammonium sulfate, 3 mM magnesium chloride, 5 mM tetramethylammonium chloride, 1% Brij35, 0.12 mM dATP, 0.12 mM dCTP, 0.12 mM dGTP, 0.04 mM dTTP, and 0.08 mM dUTP.

[0068] The composition and concentration of enzyme reaction solution B are as follows: 20 mM Tris-HCl (pH=7.5), 20 mM potassium chloride, 0.5 mM EDTA, 1% Brij35, 1 mM TCEP, 5 U / test Taq enzyme, 100 U / test reverse transcriptase, 40 U / test RNA protectant, 2 U / test UNG enzyme, and 50% glycerol.

[0069] The specific primer and probe sequences are shown in the table below: Table 1: Specific primer sequences for PCR detection of blood pathogens The reaction system used MS2 RNA fragment as an internal standard IC assay to monitor the entire process of nucleic acid extraction and PCR amplification.

[0070] In this kit, the Cy5 channel is selected to detect HBV DNA, the TAMRA channel to detect HCV RNA, the FAM channel to detect HIV RNA, and the VIC channel to detect internal control RNA, enabling multiplex detection in a single tube.

[0071] The PCR reaction conditions are as follows: Step1: 50℃ 20 min, 1 cycle; Step2: 94℃10 min, 1 cycle; Step 3: 94℃ for 10 s, 55℃ for 15 s, 65℃ for 30 s, 45 cycles (fluorescence signal is collected at 65℃).

[0072] (II) Evaluation of PCR test results (1) Sensitivity analysis WHO HBV DNA, WHO HCV RNA, WHO HIV-1 RNA, and WHO HIV-2 RNA international standards were diluted to analytically sensitive concentrations of 9.7 copies / mL, 9.5 copies / mL, and 5.5 copies / mL, respectively. Nucleic acid was extracted from the samples using a high-efficiency automated magnetic bead method. The kits provided in this invention were then used on an ABI 7500 real-time quantitative PCR instrument. Each assay was performed in 20 replicates. The detection rate for each assay was ≥95%. Specific results are shown in Table 2. Amplification curves for HBV, HCV, HIV-1, HIV-2 samples, and the internal standard are shown in Table 2. Figure 2 As shown.

[0073] Table 2: Sensitivity analysis results of PCR detection of blood pathogens Using WHO international standards as analytical samples, this kit extracts pathogen nucleic acids from plasma samples using a high-efficiency automated magnetic bead method. Detection using this kit on an ABI 7500 real-time quantitative PCR instrument showed analytical sensitivities of 9.7 copies / mL, 9.5 copies / mL, and 5.5 copies / mL for HBV DNA, HCV RNA, and HIV RNA, respectively. When used for blood screening, its detection capabilities far exceed industry standards (75 copies / mL for HBV DNA, 135 copies / mL for HCV RNA, and 30 copies / mL for HIV RNA), effectively reducing the risk of missed detections and improving transfusion safety.

[0074] (2) Specificity test Using negative plasma as the test sample, nucleic acids were extracted from the sample using a high-efficiency automated magnetic bead method. The kit provided by this invention was then used for testing. A total of 300 samples were tested, and all results were negative (HBV DNA, HCV RNA, and HIV RNA were all non-reactive). This indicates that this embodiment has good specificity for multiplex PCR detection of blood pathogen DNA / RNA, and its amplification curves are shown below. Figure 3 As shown.

[0075] Example 2 This embodiment relates to the multiplex fluorescent RT-PCR detection kit of the present invention for detecting respiratory pathogens, which enables single-tube multiplex detection of RNA.

[0076] (a) PCR detection The PCR reaction system in this embodiment includes PCR buffer, enzyme reaction solution B, specific primers, and fluorescently labeled TaqMan probes. The specific probes for detecting SARS-CoV-2 RNA, FluA RNA, FluB RNA, RSV RNA, and internal control RNA are labeled with different fluorescent groups, thereby enabling single-tube multiplex detection.

[0077] Specifically, the PCR reaction system is 50 μl / test (25 μl / test of PCR reaction solution + 25 μl / test of nucleic acid template), wherein the PCR reaction solution contains 20 μl / test of PCR reaction buffer, 3 μl / test of primer and probe mixture, and 2 μl / test of enzyme reaction solution.

[0078] The composition and concentration of the PCR reaction buffer are as follows: 50 mM Tris-HCl (pH 8.5), 20 mM ammonium sulfate, 4 mM magnesium chloride, 10 mM tetramethylammonium chloride, 1.5% Brij35, 0.12 mM dATP, 0.12 mM dCTP, 0.12 mM dGTP, 0.04 mM dTTP, and 0.08 mM dUTP.

[0079] The composition and concentration of enzyme reaction solution B are as follows: 50 mM Tris-HCl (pH 7.8), 20 mM potassium chloride, 1 mM EDTA, 1% Brij35, 2 mM TCEP (tris(2-carboxyethyl)phosphine), 5 U / test Taq enzyme, 200 U / test reverse transcriptase, 20 U / test RNA protectant, 2 U / test UNG enzyme, and 50% glycerol.

[0080] The specific primer and probe sequences are shown in the table below: Table 3: Specific primer sequences for RT-PCR detection of respiratory pathogens The reaction system uses RNA transcribed from the RPP30 gene as an internal standard IC assay to monitor the entire process of nucleic acid extraction and PCR amplification.

[0081] In this kit, the FAM channel is selected to detect SARS-CoV-2 RNA, the ROX channel to detect FluA RNA, the Cy5 channel to detect FluB RNA, the Cy5.5 channel to detect RSV RNA, and the VIC channel to detect internal control RNA, enabling multiplex detection in a single tube.

[0082] The PCR reaction conditions are as follows: Step1: 50℃ 20 min, 1 cycle; Step2: 94℃ 2 min, 1 cycle; Step 3: 94℃ for 10 s, 60℃ for 45 s, 45 cycles (fluorescence signal is collected at 60℃).

[0083] (II) Evaluation of PCR test results (1) Sensitivity analysis ATCC-derived SARS-CoV-2 reference virus strains, FluA reference virus strains, FluB reference virus strains, and RSV reference virus strains were diluted to analytically sensitive concentrations of 25 copies / mL, 15 copies / mL, 140 copies / mL, and 140 copies / mL, respectively. Nucleic acid was extracted from the samples using a high-efficiency automated magnetic bead method. The samples were then tested using the kit provided in this invention on a Bio-Rad CFX 96 real-time quantitative PCR instrument. Each assay was performed in 20 replicates, and the detection rate for each assay was ≥95%. Specific results are shown in Table 4. The amplification curves of the SARS-CoV-2 reference virus strain, FluA reference virus strain, FluB reference virus strain, RSV reference virus strain, and internal standard are shown in Table 4. Figure 4 As shown.

[0084] Table 4: Sensitivity analysis results of PCR detection of respiratory pathogens Using the ATCC reference virus strain as the analysis sample, a high-efficiency automated magnetic bead method was used to extract pathogen nucleic acids from nasal / pharyngeal swab samples. The kit, when used on a Bio-Rad CFX 96 real-time quantitative PCR instrument, achieved analytical sensitivities of 25 copies / mL, 15 copies / mL, 140 copies / mL, and 140 copies / mL for SARS-CoV-2 RNA, respectively. This detection capability far exceeds the industry standard (industry standard: SARS-CoV-2 RNA analytical sensitivity 3703 copies / mL). When used for respiratory pathogen screening, it can effectively improve the detection rate of respiratory pathogens.

[0085] (2) Specificity test Using negative pharyngeal swabs as test samples, nucleic acids were extracted from the samples using a high-efficiency automated magnetic bead method. The samples were then tested using the kit provided in this invention. A total of 100 samples were tested, and all results were negative (SARS-CoV-2 RNA, FluA RNA, FluB RNA, and RSV RNA were all non-reactive). This indicates that this embodiment has good specificity for multiplex PCR detection of respiratory pathogen RNA, and its amplification curves are shown below. Figure 5 As shown.

[0086] Example 3 This embodiment relates to the multiplex fluorescent PCR detection kit of the present invention for detecting sexually transmitted pathogens, which enables single-tube multiplex detection of DNA.

[0087] (a) PCR detection The PCR reaction system in this embodiment includes PCR buffer, enzyme reaction solution A, specific primers, and fluorescently labeled TaqMan probes. The specific probes for detecting HSV DNA, CT DNA, NG DNA, UU DNA, and internal standard DNA are labeled with different fluorescent groups, thereby enabling single-tube multiplex detection.

[0088] Specifically, the PCR reaction system is 50 μl / test (25 μl / test of PCR reaction solution + 25 μl / test of nucleic acid template), wherein the PCR reaction solution contains 20 μl / test of PCR reaction buffer, 3 μl / test of primer and probe mixture, and 2 μl / test of enzyme reaction solution A.

[0089] The composition and concentration of the PCR reaction buffer are as follows: 40 mM Tris-HCl (pH 8.7), 30 mM ammonium sulfate, 3.5 mM magnesium chloride, 5 mM tetramethylammonium chloride, 1% Brij35, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.04 mM dTTP, and 0.16 mM dUTP.

[0090] The composition and concentration of enzyme reaction solution A are as follows: 50 mM Tris-HCl (pH 7.5), 20 mM potassium chloride, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 2% Brij35, 2 mM TCEP (tris(2-carboxyethyl)phosphine), 10 U / test Taq enzyme, 1 U / test UNG enzyme, and 50% glycerol.

[0091] The specific primer and probe sequences are shown in the table below: Table 5: Specific primer sequences for PCR detection of sexually transmitted pathogens The reaction system uses human genomic β-actin gene DNA as an internal standard IC assay to monitor the entire process of nucleic acid extraction and PCR amplification.

[0092] In this kit, the FAM channel is selected to detect HSV DNA, the ROX channel to detect CT DNA, the Cy5 channel to detect NG DNA, the Cy5.5 channel to detect UU DNA, and the HEX channel to detect internal standard DNA, enabling multiplex detection in a single tube.

[0093] The PCR reaction conditions are as follows: Step1: 50℃ 5 min, 1 cycle; Step2: 94℃ 2 min, 1 cycle; Step 3: 94℃ for 10 s, 60℃ for 30 s, 45 cycles (fluorescence signal is collected at 60℃).

[0094] (II) Evaluation of PCR test results (1) Sensitivity analysis WHO HSV-1 international standard, WHO HSV-2 international standard, ATCC-derived CT reference virus strain, NG reference virus strain, and UU reference virus strain were diluted to analytical sensitivity concentrations of 50 copies / mL, 50 copies / mL, 50 copies / mL, 100 copies / mL, and 100 copies / mL, respectively. Nucleic acid was extracted from the samples using a high-efficiency automated magnetic bead method. The kit provided in this invention was used on a SLAN-96S real-time quantitative PCR instrument for testing. Each test was performed in 20 replicates, and the detection rate for each test item was ≥95%. Specific results are shown in Table 6. The amplification curves of HSV-1 reference virus strain, HSV-2 reference virus strain, CT reference virus strain, NG reference virus strain, UU reference virus strain, and internal standard are shown in Table 6. Figure 6 As shown.

[0095] Table 6: Sensitivity Analysis Results of PCR Detection for Sexually Transmitted Pathogens Using WHO international standards or ATCC reference virus strains as analytical samples, pathogen nucleic acids were extracted from the samples using a high-efficiency automated magnetic bead method. The kit was used to detect HSV DNA, CTDNA, NG DNA, and UU DNA on the SLAN-96S real-time quantitative PCR instrument. The analytical sensitivities reached 50 copies / mL, 50 copies / mL, 100 copies / mL, and 100 copies / mL, respectively.

[0096] Currently, similar kits are generally multiplex fluorescent PCR detection kits that only detect HSV (type 1+2) nucleic acid, or multiplex fluorescent PCR detection kits that only detect Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) nucleic acid. Several similar kits with the best detection performance were selected for a comparison of analytical sensitivity; the results are shown in the table below.

[0097] Table 7: Comparison of analytical sensitivity among similar kits Note: " / " in the table indicates that the kit does not test the corresponding item.

[0098] As shown in the table above, the analytical sensitivity of similar kits ranges from 100 copies / mL to 1000 copies / mL. The analytical sensitivity of each detection item in this example is comparable to or better than that of similar kits, and can effectively improve the detection rate of sexually transmitted pathogens when used for screening.

[0099] (2) Specificity test Using negative genital swabs as test samples, nucleic acids were extracted from the samples using a high-efficiency automated magnetic bead method. The samples were then tested using the kit provided in this invention. A total of 100 samples were tested, and all results were negative (HSV DNA, CTDNA, NG DNA, and UU DNA were all non-reactive). This indicates that this embodiment has good specificity for multiplex PCR detection of sexually transmitted pathogen DNA, and its amplification curves are shown below. Figure 7 As shown.

[0100] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A universal one-step multiplex PCR reaction system, characterized in that, The PCR reaction system includes PCR buffer reagents and enzyme reaction reagents; The PCR buffer reagent contains tetramethylammonium chloride and Brij35, wherein the concentration of tetramethylammonium chloride is 2-20 mM and the volume percentage of Brij35 is 0.5-10%. The enzyme reaction reagent includes an enzyme preservation solution and a PCR enzyme. The enzyme preservation solution contains Brij35 and tris(2-carboxyethyl)phosphine, and the volume percentage of Brij35 is 0.5-5%, and the concentration of tris(2-carboxyethyl)phosphine is 0.1-10 mM.

2. The PCR reaction system according to claim 1, characterized in that, The PCR buffer reagent also includes Tris-HCl, ammonium sulfate, and magnesium chloride; In the PCR buffer reagent, the concentration of Tris-HCl is 20 ~ 100 mM, and the pH is 8.5 ~ 9.0; And / or, the concentration of the ammonium sulfate is 10 to 50 mM; And / or, the concentration of the magnesium chloride is 2.5 ~ 6 mM.

3. The PCR reaction system according to claim 1, characterized in that, The PCR buffer reagent also includes dNTPs, and the dNTPs include dATP, dCTP, dGTP, dTTP and / or dUTP; The concentration of the dNTPs in the PCR buffer is 0.1 ~ 1.2 mM.

4. The PCR reaction system according to claim 1, characterized in that, The enzyme reaction reagent includes enzyme reaction reagent A or enzyme reaction reagent B; The PCR enzymes in enzyme reaction reagent A include Taq enzyme and UNG enzyme; The PCR enzymes in the enzyme reaction reagent B include Taq enzyme, UNG enzyme, reverse transcriptase, and RNA protectant.

5. The PCR reaction system according to claim 1, characterized in that, The enzyme preservation solution also includes Tris-HCl, potassium chloride, ethylenediaminetetraacetic acid and glycerol; In the enzyme preservation solution, the concentration of Tris-HCl is 10 ~ 100 mM, and the pH is 7.0 ~ 8.5; And / or, the concentration of potassium chloride is 10 ~ 150 mM; And / or, the concentration of the ethylenediaminetetraacetic acid is 0.05 ~ 5 mM; And / or, the glycerol has a volume percentage of 30 to 70%.

6. The PCR reaction system according to claim 4, characterized in that, In enzyme reaction reagent A or enzyme reaction reagent B, the concentration range of the Taq enzyme is 1 ~ 10 U / test; And / or, in enzyme reaction reagent A or enzyme reaction reagent B, the concentration of the UNG enzyme is in the range of 0.1 ~ 5 U / test; And / or, in the enzyme reaction reagent B, the concentration range of the reverse transcriptase is 10 ~ 400 U / test; And / or, in enzyme reaction reagent B, the concentration of the RNA protectant ranges from 5 to 50 U / test.

7. A nucleic acid detection kit, characterized in that, The PCR reaction system comprising any one of claims 1 to 6, as well as specific primers and TaqMan fluorescent probes, is used for multiplex fluorescent PCR detection.

8. The reagent kit according to claim 7, characterized in that, The fluorescent probe has a fluorescent reporter group labeled at its 5' end and a fluorescent quencher group labeled at its 3' end, wherein the fluorescent reporter group is selected from at least one of FAM, HEX, VIC, JOE, TET, Cy3, TAMRA, ROX, Texas Red, Cy5, Cy5.5, Quasar 705 or Cy7.

9. The reagent kit according to claim 7, characterized in that, The fluorescence quenching group is selected from at least one of BHQ1, BHQ2 or BHQ3.

10. A one-step multiplex PCR detection method, characterized in that, The method includes performing the reaction using the PCR reaction system according to any one of claims 1 to 6, or performing the reaction using the kit according to any one of claims 7 to 9.

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