Real-time fluorescent quantitative PCR (polymerase chain reaction) based on single-labeled fluorescent probe and application of real-time fluorescent quantitative PCR
Through single-label fluorescent probe technology, combined with DNA molecule I and DNA molecule II for PCR detection, the existing probes have high cost, low specificity and complex design problems in clinical diagnosis, and achieved high sensitivity, Highly specific and economical nucleic acid testing.
Patent Information
- Application Number
- CN202411581693.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-01-10
AI Technical Summary
Existing fluorescent probes have multiple defects in clinical disease diagnosis, such as incomplete quenching of TaqMan probes, high cost, expensive synthesis of MGB probes, complex design of molecular beacon probes, low specificity, and large Most probes require double labeling, which leads to complex and time-consuming synthesis process and is relatively expensive.
Using real-time fluorescence quantitative PCR technology based on single-label fluorescence probes, PCR amplification primers are composed of DNA molecule I (single-stranded DNA with fluorophore modification) and DNA molecule II (normal primers), and fluorophore FAM is used for detection. , simple design, strong specificity, and does not rely on the exocarcinoma of Taq enzymes, reducing synthesis costs.
It realizes nucleic acid detection with high sensitivity, strong specificity, simple design and low cost. It is suitable for clinical disease diagnosis, pathogen detection, gene mutation and genotyping, avoids the defects of traditional probes, and improves detection efficiency and economicality. .
Smart Images
Figure HDA0005123287520000011 
Figure HDA0005123287520000012 
Figure HDA0005123287520000021
Abstract
Description
Technical Field
[0001] The invention belongs to the field of gene detection, and in particular relates to a real-time fluorescence quantitative PCR based on a single-labeled fluorescent probe and an application thereof. Background Art
[0002] Real-time fluorescence quantitative PCR (Quantitative Real-time PCR) is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. At present, fluorescent probe PCR is generally used for detection in clinical disease diagnosis applications. It is based on the principle of fluorescence resonance energy transfer, or FRET. That is to say, when a fluorescent group and a fluorescent quenching group are close to a certain range, fluorescence energy transfer will occur, and the quenching group will absorb the excitation fluorescence of the fluorescent group under the action of excitation light, so that it cannot emit fluorescence. But once the two are separated, the quenching effect disappears. Using the FRET principle, researchers select appropriate fluorescent groups and quenching groups to label nucleic acid probes and primers, and then use the principle that nucleic acid hybridization and nucleic acid hydrolysis can cause the fluorescent group and the quenching group to combine or separate, and establish various real-time fluorescence PCR methods. The fluorescent probes currently used mainly include: TaqMan probes, MGB probes, double hybridization probes, molecular beacon probes, and scorpion probes.
[0003] The two ends of the TaqMan probe are labeled with a reporter group (5' end) and a quencher group (3' end); during PCR annealing, the probe is completely bound to the single-stranded DNA. At this time, the fluorescent signal emitted by the reporter group is absorbed by the quencher group, and no fluorescent signal can be detected. During extension, the 5'-3' exonuclease activity of TaqDNA polymerase hydrolyzes the probe, so that the reporter group and the quencher group are separated, thereby emitting fluorescence. The number of cut fluorescent molecules is proportional to the number of PCR products. Therefore, the purpose of monitoring the amplification amount of PCR products can be achieved by detecting the fluorescence intensity in the PCR reaction system. This method has the characteristics of strong specificity and high sensitivity, and is most widely used in clinical diagnosis in China. At the same time, this method can use different dye-labeled probes to amplify and detect different sequences in the same reaction tube. Compared with the dye method, the disadvantage is that different probes need to be designed and synthesized according to different sequences.
[0004] Based on the TaqMan probe, MGB probes were further developed. Minor groove binder (MGB) probes are also called Eclipse probes. They have a fluorescent group and a quencher group at both ends like hydrolysis probes. However, they also have an MGB near the quencher group. In the absence of a target, the MGB probe randomly coils, bringing the fluorescent group and the quencher group close together, resulting in a lower fluorescence background. MGB binds to the minor groove of the double helix formed by hybridization between the probe and the target gene, which not only promotes the stability and specificity of hybridization between the probe and the target gene, but also greatly increases the Tm of the probe, thereby allowing a wider range of choices for hybridization temperature. This makes MGB probes more advantageous in detecting single nucleotide polymorphisms and quantitatively analyzing methylated alleles.
[0005] Double hybridization probes are also called LightCycler or FRET probes. This technology uses a pair of probes designed to bind to adjacent sequences. They are labeled with a donor fluorescent group and an acceptor fluorescent group, and the excitation light spectra of the two groups overlap to a certain extent. When the two probes bind to the target sequence, the two probes hybridize to the target sequence in a head-to-tail manner. The donor and acceptor dyes are close, FRET occurs, and the donor excites the acceptor to emit another fluorescence. The instrument can detect the fluorescence signal at this time, and the fluorescence intensity is proportional to the amount of DNA synthesis in the PCR reaction. Since two probes are used for target sequence hybridization, and the two different probes must hybridize to the correct target sequence before fluorescence can be detected, the specificity of this method has been greatly improved compared to the traditional single probe detection system.
[0006] Molecular beacon probes (MBP) consist of single-stranded DNA molecules covalently labeled with fluorescent dyes and quenchers at both ends. The two ends of the probe become stems due to complementarity, so that the fluorescent group and the quenching group undergo FRET due to their close distance, quenching the fluorescence; there are 5 or 6 bases near the two ends of the molecular beacon probe that can complement the target sequence to form a hairpin or stem-loop structure. When the probe binds to the target sequence, the stem denatures, separating the fluorescent group and the quenching group, thereby generating fluorescence. Compared with Taqman probes, the hairpin structure of molecular beacon probes requires a certain amount of force to open, and single base mismatches, deletions, or insertion mutations in the target sequence can be detected, so it has better specificity and is usually used to identify point mutations. However, the design of molecular beacon probes is complex, and the overall probe annealing temperature and the annealing temperature of the loop region must be considered.
[0007] Scorpion probes consist of probes, primers, and PCR terminators. The probe part is similar to molecular beacons, with a fluorescent group at the 5' end, a quenching group at the 3' end, and a stem-loop structure. Unlike molecular beacons, scorpion probes carry a specific primer that can bind to the corresponding target nucleic acid, polymerize and extend under the action of Taq DNA polymerase to obtain an amplified product, and the probe part can hybridize and bind with the complementary sequence in the specific product at the extension end. Therefore, if amplification exists, the scorpion probe can continuously hybridize with the amplicon during the continuous denaturation and renaturation process, the stem-loop structure opens, the fluorescent group is no longer quenched and is emitted, and the fluorescence intensity is proportional to the content of the amplified product. Since the tail of the scorpion probe and the amplicon are on the same DNA chain, mutual hybridization is an intramolecular interaction. The advantages are rapid reaction, stronger fluorescence signal, and better discrimination and specificity. It has been used for pathogen nucleic acid detection, allele discrimination, SNP and mutation detection.
[0008] In summary, the existing probes still have many defects. For example, it is difficult to completely quench the TaqMan probe, and the background is high. The hydrolysis of the probe depends on the exoenzyme activity of Taq, so the quantification is affected by the enzyme performance and reagent quality. The synthesis price of the MGB probe is several times that of the ordinary TaqMan probe, which greatly increases the detection cost. In addition, the MGB probe is a patented technology of LIFE Company and is subject to many restrictions in its promotion and use. The MBP structure is relatively complex, and there are many factors that affect it. It is not easy to design. When there is a mismatch between the target sequence and the probe, the hybridization efficiency of the two is very low. In addition, all of the above probes require double labeling, and the synthesis process is complex and time-consuming, and the cost is high. Summary of the invention
[0009] The invention provides a real-time fluorescence quantitative PCR based on a single-labeled fluorescent probe and application thereof.
[0010] The present invention provides a DNA molecule combination, which consists of DNA molecule I (single fluorescent labeling probe) and DNA molecule II (common primer);
[0011] DNA molecule I is a single-stranded DNA molecule modified with a fluorescent group, wherein the fluorescent group modification is located at a specific nucleotide, and the specific nucleotide is the nucleotide T closest to the 3' end of the single-stranded DNA molecule;
[0012] DNA molecule II is a single-stranded DNA molecule (unmodified);
[0013] The DNA molecule I and the DNA molecule II form a PCR amplification primer pair, and the target sequence thereof is 50-80 bp;
[0014] The fluorescent group is the fluorescent group FAM.
[0015] Specifically, the DNA molecule I consists of 15-30 nucleotides.
[0016] Specifically, the DNA molecule I consists of 18-20 nucleotides.
[0017] Specifically, the DNA molecule I consists of 19 nucleotides;
[0018] The DNA molecule II consists of 15-30 nucleotides.
[0019] Specifically, the DNA molecule II consists of 17-20 nucleotides.
[0020] In the DNA molecule I, the specific nucleotide is not located at the first position from the 3' end of the DNA molecule.
[0021] The Tm value of DNA molecule I is 50-60°C, and the Tm value of DNA molecule II is 50-60°C.
[0022] The difference in Tm values between DNA molecule I and DNA molecule II is less than or equal to 3°C.
[0023] In DNA molecule I, the specific nucleotide is located at the Nth position from the 3' end of the DNA molecule; N is 2 or 3 or 4 or 5 or 6 or 7.
[0024] Specifically, the target sequence is 56-77 bp.
[0025] In the DNA molecule combination, the molar ratio of DNA molecule I to DNA molecule II is 1:1.
[0026] As an example, the DNA molecule combination is a DNA molecule combination for detecting SARS-CoV-2.
[0027] As an example, the DNA molecule I is SARS2-Nfam.
[0028] As an example, the DNA molecule II is SARS2-NR1, SARS2-NR2, SARS2-NR3 or SARS2-NR4.
[0029] SARS2-Nfam is a single-stranded DNA molecule modified with a fluorescent group. The nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 1, and the fluorescent group FAM is modified on the second nucleotide from the 3' end.
[0030] SARS2-NR1 is a single-stranded DNA molecule, as shown in SEQ ID NO: 2.
[0031] SARS2-NR2 is a single-stranded DNA molecule, as shown in SEQ ID NO: 3.
[0032] SARS2-NR3 is a single-stranded DNA molecule, as shown in SEQ ID NO:4.
[0033] SARS2-NR4 is a single-stranded DNA molecule, as shown in SEQ ID NO:5.
[0034] As an example, the DNA molecule combination is a DNA molecule combination for detecting Helicobacter pylori.
[0035] As an example, the DNA molecule I is Hp-Ffam.
[0036] As an example, the DNA molecule II is Hp-R1 or Hp-R2.
[0037] Hp-Ffam is a single-stranded DNA molecule modified with a fluorescent group. The nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 9, and the fluorescent group FAM is modified on the second nucleotide from the 3' end.
[0038] Hp-R1 is a single-stranded DNA molecule, as shown in SEQ ID NO:10.
[0039] Hp-R2 is a single-stranded DNA molecule, as shown in SEQ ID NO:11.
[0040] The present invention also protects a reagent or a kit, which contains any of the above-mentioned DNA molecule combinations.
[0041] The present invention also protects the use of any of the above-mentioned DNA molecule combinations in the preparation of nucleic acid detection products.
[0042] As an example, the nucleic acid detection product is a product for detecting SARS-CoV-2 nucleic acid.
[0043] As an example, the nucleic acid detection product is a product for detecting Helicobacter pylori nucleic acid.
[0044] The present invention also protects the use of any of the above-mentioned DNA molecule combinations in nucleic acid detection.
[0045] As an example, the nucleic acid detection is to detect the nucleic acid of SARS-CoV-2.
[0046] As an example, the nucleic acid detection is the detection of Helicobacter pylori nucleic acid.
[0047] The present invention also provides a nucleic acid detection method, comprising the following steps: using a test DNA as a template, using any of the above-mentioned DNA molecule combinations to perform real-time fluorescence quantitative PCR, and judging whether the test DNA contains the target sequence of the DNA molecule combination according to the amplification curve.
[0048] As an example, the nucleic acid detection is the detection of Helicobacter pylori nucleic acid.
[0049] As an example, the reaction procedure of real-time fluorescence quantitative PCR is: 50°C for 2 min; 95°C for 3 min; 95°C denaturation for 30 s, 52°C annealing for 30 s, 40 cycles. During annealing, the fluorescence signal of the FAM channel is collected.
[0050] The present invention also provides a nucleic acid detection method, comprising the following steps: using a test RNA as a template, using any of the above-mentioned DNA molecule combinations to perform real-time fluorescence quantitative reverse transcription PCR, and judging whether the test RNA contains the target sequence of the DNA molecule combination according to the amplification curve.
[0051] As an example, the nucleic acid detection is to detect the nucleic acid of SARS-CoV-2.
[0052] As an example, the reaction procedure of real-time fluorescence quantitative reverse transcription PCR is: 42°C for 20 min, 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 52°C for 30 s, 40 cycles; collecting FAM channel fluorescence signals during annealing.
[0053] In any of the above methods, the annealing temperature of PCR is 52-60°C.
[0054] In any of the above methods, the annealing temperature of PCR is 52°C.
[0055] Any of the above products is a reagent or a kit.
[0056] Any of the above nucleic acids is DNA or RNA.
[0057] The principle schematic diagram of the method provided by the present invention is shown in Figure 1 . The fluorescent group FAM has a function similar to SYBR Green. The free single fluorescent labeled probe is in a randomly folded state, and the fluorescent group FAM is in a quenched state. When the test nucleic acid molecule contains a target sequence, a primer pair consisting of a single fluorescent labeled probe and a common primer is used for PCR amplification to obtain a PCR amplification product, in which the fluorescent group FAM emits a strong fluorescent signal. When performing melting curve analysis, as the temperature rises, the double chain opens, the FAM fluorescence is greatly weakened, and a single peak graph can be obtained.
[0058] The nucleic acid detection can be a short-fragment nucleic acid detection. The nucleic acid detection is particularly suitable for special sample materials such as complex, trace or degraded. The nucleic acid detection can be a nucleic acid detection based on the following methods: real-time fluorescence PCR, gene chip, membrane hybridization.
[0059] The DNA molecule combination provided by the present invention has the advantages of simple design, high sensitivity, strong specificity, simple and fast, low synthesis cost, etc., and does not rely on the exolytic activity of Taq enzyme. The present invention can be used for: clinical disease medical diagnosis, pathogen detection, gene mutation, genotyping or life science experimental research. BRIEF DESCRIPTION OF THE DRAWINGS
[0060] Figure 1 The schematic diagram of the principle of the method provided by the present invention.
[0061] Figure 2 This is the result diagram of Example 1.
[0062] Figure 3 This is the result diagram of Example 2.
[0063] Figure 4 This is the result diagram of Example 3. DETAILED DESCRIPTION
[0064] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0065] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. Each single-labeled fluorescent probe, each common primer, primer NF, primer NR and probe NP are prepared by Jiangsu Saisuofei Biotechnology Co., Ltd.
[0066] Example 1: Detection of SARS-CoV-2 RNA by real-time fluorescence quantitative PCR based on single-labeled fluorescent probe
[0067] 1. Design and prepare single-labeled fluorescent probes and common primers
[0068] Single-labeled fluorescent probes (SARS2-NFam) and common primers (SARS2-NR1, SARS2-NR2, SARS2-NR3, and SARS2-NR4) were designed based on the conserved sequences in the SARS-CoV-2 genome (NCBI Reference Sequence: NC_045512.2, 18-JUL-2020). SARS2-Nfam is a single-stranded DNA molecule modified with a fluorescent group, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 1, and the fluorescent group FAM is modified on the second nucleotide (bold T) from the 3' end. SARS2-NR1 is a single-stranded DNA molecule, as shown in SEQ ID NO: 2. SARS2-NR2 is a single-stranded DNA molecule, as shown in SEQ ID NO: 3. SARS2-NR3 is a single-stranded DNA molecule, as shown in SEQ ID NO: 4. SARS2-NR4 is a single-stranded DNA molecule, as shown in SEQ ID NO: 5.
[0069] SARS2-NFam (SEQ ID NO: 1): 5'-CAACAATCCATGAGCAGTG-3'.
[0070] SARS2-NR1 (SEQ ID NO: 2): 5'-GTGTGGTCTGCATGAGTTTA-3'.
[0071] SARS2-NR2 (SEQ ID NO: 3): 5'-TGCCTTGTTGTGCTCTGC-3'.
[0072] SARS2-NR3 (SEQ ID NO: 4): 5'-ATAGCCCATCTGCCTTGT-3'.
[0073] SARS2-NR4 (SEQ ID NO: 5): 5'-TAAACGGAAAAGCGAAAC-3'.
[0074] In the whole genome of SARS-CoV-2, the target sequence for PCR amplification using SARS2-NFam and SARS2-NR1 is 56bp, the target sequence for PCR amplification using SARS2-NFam and SARS2-NR2 is 62bp, the target sequence for PCR amplification using SARS2-NFam and SARS2-NR3 is 72bp, and the target sequence for PCR amplification using SARS2-NFam and SARS2-NR4 is 97bp.
[0075] 2. Prepare the reaction system and carry out the reaction
[0076] Template solution: RNA solution obtained by RNA extraction from throat swabs of confirmed SARS-CoV-2 positive patients, with an RNA concentration of 1×10 7 copies / mL.
[0077] Reaction system composition (25 μL): 2×One Step RT-PCR Buffer III 12.5 μL, TaKaRa Ex Taq HS 0.5 μL, PrimeScript RT Enzyme Mix II 0.5 μL, single-labeled fluorescent probe solution, common primer solution, template solution 2 μL, the balance is RNase Free dH 2 O. The concentration of the single-labeled fluorescent probe in the reaction system was 0.2 μmol / L, and the concentration of the common primer was 0.2 μmol / L. Two biological replicates were set.
[0078] The active ingredient provided by the single-labeled fluorescent probe solution is SARS2-NFam. The active ingredient provided by the common primer solution is SARS2-NR1, SARS2-NR2, SARS2-NR3 or SARS2-NR4. One-Step RT-PCR Kit One StepPrimeScript TM RT-PCR Kit: Takara product, catalog number RR064A. The kit provides the following components: 2×One Step RT-PCR BufferⅢ, TaKaRa Ex Taq HS (5U / μl), PrimeScript RT Enzyme MixⅡ and RNase Free dH 2 O.
[0079] The reaction was run on a CFX96 fluorescence quantitative PCR instrument.
[0080] Reaction procedure: reverse transcription at 42℃ for 20min, pre-denaturation at 95℃ for 2min; denaturation at 95℃ for 10s, annealing at 52℃ for 30s, 40 cycles; collect FAM channel fluorescence signal during annealing. In melting curve analysis, the temperature was raised from 45℃ to 90℃ at a rate of 0.5℃ / s, and the FAM channel fluorescence signal was collected every 5s.
[0081] 3. Results Analysis
[0082] Amplification curves are shown in Figure 2 A. The fluorescence signals generated by the single-labeled fluorescent probe and the common primers at different positions were similar in intensity within the amplified fragment of 56-72 bp, and the fluorescence signal was significantly weakened (the Ct value was significantly increased) when the amplified length was 97 bp.
[0083] Melting curve Figure 2B. The melting curves all showed a single peak.
[0084] The results show that the method provided by the present invention has good specificity, and the size of the amplified fragment using the single-labeled fluorescent probe and common primers is more suitable within the range of 56-72 bp.
[0085] Twenty template solutions from different SARS-CoV-2 positive patients were repeatedly validated according to the above steps, and the results were consistent.
[0086] Example 2: Comparison of the effects of real-time fluorescence quantitative PCR based on single-labeled fluorescent probe and TaqMan probe in detecting SARS-CoV-2 RNA
[0087] 1. Design and prepare TaqMan primer probe sets
[0088] Primers NF, primers NR and probes NP for real-time fluorescence quantitative PCR based on TaqMan probes were designed based on the conserved sequences in the SARS-CoV-2 genome (NCBI Reference Sequence: NC_045512.2, 18-JUL-2020). Primer NF is shown in SEQ ID NO: 6, and primer NR is shown in SEQ ID NO: 7, both of which are single-stranded DNA molecules. Probe NP is a modified single-stranded DNA molecule, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 8, with a fluorescent group FAM modified at the 5' end and a quenching group BHQ1 modified at the 3' end.
[0089] NF (SEQ ID NO: 6): 5'-TTTGCTGAATAAGCATATTGACGC-3'.
[0090] NR (SEQ ID NO: 7): 5'-CTGTTTCTTCTGTCTCTGCGG-3'.
[0091] NP (SEQ ID NO: 8): 5'-AAGRCTKATGAAACTCAAGCC-3'.
[0092] In SEQ ID NO: 8, R represents A or G, and K represents G or T.
[0093] 2. Real-time fluorescence quantitative PCR using TaqMan primer probe set
[0094] Template solution: RNA solution obtained by RNA extraction from throat swabs of confirmed SARS-CoV-2 positive patients, with an RNA concentration of 1×10 7 copies / mL.
[0095] Reaction system composition (25 μL): 2×One Step RT-PCR Buffer III 12.5 μL, TaKaRa Ex Taq HS 0.5 μL, PrimeScript RT Enzyme Mix II 0.5 μL, primer NF solution, primer NR solution, probe NP solution, template solution 2 μL, the balance is RNase Free dH 2 O. The concentrations of primer NF, primer NR and probe NP in the reaction system were all 0.2 μmol / L. An equal volume of RNase Free dH 2 O was used as a blank control instead of the template solution.
[0096] The reaction procedure is the same as that in step 2 of Example 1.
[0097] 3. Design and preparation of single-labeled fluorescent probes and common primers
[0098] A single-labeled fluorescent probe (SARS2-NFam) and a common primer (SARS2-NR1) were designed based on the conserved sequence in the SARS-CoV-2 genome (NCBI Reference Sequence: NC_045512.2, 18-JUL-2020). SARS2-Nfam is a single-stranded DNA molecule modified with a fluorescent group. The nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 1, and the fluorescent group FAM is modified on the second nucleotide (bold T) from the 3' end. SARS2-NR1 is a single-stranded DNA molecule, as shown in SEQ ID NO: 2.
[0099] SARS2-NFam (SEQ ID NO: 1): 5'-CAACAATCCATGAGCAGTG-3'.
[0100] SARS2-NR1 (SEQ ID NO: 2): 5'-GTGTGGTCTGCATGAGTTTA-3'.
[0101] 4. Real-time fluorescence quantitative PCR using single-labeled fluorescent probes and common primers
[0102] The composition of the reaction system is the same as that of the reaction system in step 2 of Example 1. The active ingredient provided by the single-labeled fluorescent probe solution is SARS2-NFam. The active ingredient provided by the common primer solution is SARS2-NR1.
[0103] The reaction procedure is the same as that in step 2 of Example 1.
[0104] 5. Results Analysis
[0105] Amplification curves are shown in Figure 3 A (the arrow marks the amplification curve). The effects of real-time fluorescence quantitative PCR based on single-labeled fluorescent probe and TaqMan probe in detecting SARS-CoV-2 RNA are similar, and the fluorescence curve of single-labeled fluorescent probe is slightly better than that of TaqMan probe.
[0106] Melting curve Figure 3 B (the arrow indicates the melting curve). The amplification product of the single-labeled fluorescent probe shows a single peak, while the TaqMan probe does not show a peak due to cleavage.
[0107] The results showed that real-time fluorescence quantitative PCR based on single-labeled fluorescent probes has good detection performance, which is slightly better than the classic TaMan probe method. It can also be combined with melting curves to achieve specific monitoring. In the future, multi-throughput detection can be achieved through different melting curves.
[0108] Twenty template solutions from different SARS-CoV-2 positive patients were repeatedly validated according to the above steps, and the results were consistent.
[0109] Example 3: Real-time fluorescence quantitative PCR detection of Hp DNA based on single-labeled fluorescent probe
[0110] 1. Design and prepare single-labeled fluorescent probes and common primers
[0111] A single-labeled fluorescent probe (Hp-Ffam) and common primers (Hp-R1 and Hp-R2) were designed based on the conserved sequence in the Helicobacter pylori 23S ribosomal RNA gene (NCBI Reference Sequence: NR_076155.1, 03-FEB-2015). Hp-Ffam is a single-stranded DNA molecule modified with a fluorescent group, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 9, and the fluorescent group FAM is modified on the second nucleotide (bold T) from the 3' end. Hp-R1 is a single-stranded DNA molecule, as shown in SEQ ID NO: 10. Hp-R2 is a single-stranded DNA molecule, as shown in SEQ ID NO: 11.
[0112] Hp-Ffam (SEQ ID NO: 9): 5'-TGAATGGCGTAACGAGATG-3'.
[0113] Hp-R1 (SEQ ID NO: 10): 5'-AGGAATTTTCACCTCCACTA-3'.
[0114] Hp-R2 (SEQ ID NO: 11): 5'-GTAGGAGGAATTTTCACCTC-3'.
[0115] In the Helicobacter pylori 23S ribosomal RNA gene, the target sequence for PCR amplification using Hp-Ffam and Hp-R1 is 72 bp, and the target sequence for PCR amplification using Hp-Ffam and Hp-R2 is 77 bp.
[0116] 2. Prepare the reaction system and carry out the reaction
[0117] Template solution: DNA solution obtained by extracting DNA from the saliva of patients with confirmed Helicobacter pylori positive, with a DNA concentration of 1×10 9 copies / mL.
[0118] Reaction system composition (25 μL): 5×AK Taq buffer (with Mg 2+ )5μL, AK Taq Master PCRMix (25×)1μL, single-labeled fluorescent probe solution, common primer solution, template solution 2μL, the balance is ddH 2 O. The concentration of the single-labeled fluorescent probe in the reaction system was 0.2 μmol / L, and the concentration of the common primer was 0.2 μmol / L. Four biological replicates were set. An equal volume of RNase Free dH 2 O was used as a blank control instead of the template solution.
[0119] The active ingredient provided by the single-label fluorescent probe solution is Hp-Ffam. The active ingredient provided by the common primer solution is Hp-R1 or Hp-R2. AK Taq Master PCR Mix Kit: Feipeng Biotechnology Co., Ltd., product catalog number is MD099M. The components provided by the AKTaq Master PCR Mix Kit are 5×AK Taq buffer (with Mg 2+ ) and AK Taq Master PCR Mix (25×).
[0120] Reaction program: 50℃2min; 95℃3min; 95℃denaturation 30s, 52℃annealing 30s, 40 cycles. FAM channel fluorescence signal was collected during annealing. During melting curve analysis, the temperature was increased from 45℃ to 90℃ at a rate of 0.5℃ / s, and the FAM channel fluorescence signal was collected every 5s.
[0121] 3. Results Analysis
[0122] Amplification curves are shown in Figure 4A (the arrow marks the amplification curve). The fluorescence signals generated by the single-labeled fluorescent probe and the common primers at different positions are both strong, and the fluorescence signal of the amplified fragment of 77 bp is stronger than the fluorescence signal of the amplified fragment of 72 bp.
[0123] Melting curve Figure 4 B (the arrows indicate the melting curve). The amplified products all show a single peak.
[0124] The results showed that the PCR system with single-labeled fluorescent probe was also suitable for Hp DNA detection.
[0125] Twenty template solutions from different Helicobacter pylori-positive patients were repeatedly validated according to the above steps, and the results were consistent.
[0126] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
Claims
1. DNA molecule combination, consisting of DNA molecule I and DNA molecule II; DNA molecule I is a single-stranded DNA molecule modified with a fluorescent group, wherein the fluorescent group modification is located at a specific nucleotide, and the specific nucleotide is the nucleotide T closest to the 3' end of the single-stranded DNA molecule; DNA molecule II is a single-stranded DNA molecule; The DNA molecule I and the DNA molecule II form a PCR amplification primer pair, and the target sequence thereof is 50-80 bp; The fluorescent group is the fluorescent group FAM.
2. The DNA molecule combination according to claim 1, characterized in that: The DNA molecule I consists of 15-30 nucleotides; The DNA molecule II consists of 15-30 nucleotides.
3. The DNA molecule combination according to claim 1 or 2, characterized in that: In the DNA molecule I, the specific nucleotide is located at the Nth position from the 3' end of the DNA molecule; N is 2 or 3 or 4 or 5 or 6 or 7.
4. The DNA molecule combination according to any one of claims 1 to 3, characterized in that: The molar ratio of the DNA molecule I to the DNA molecule II is 1:
1.
5. A reagent or kit comprising the DNA molecule combination according to any one of claims 1 to 4.
6. Use of the DNA molecule combination described in any one of claims 1 to 4 in the preparation of nucleic acid detection products.
7. Use of the DNA molecule combination according to any one of claims 1 to 4 in nucleic acid detection.
8. A method for nucleic acid detection, comprising the following steps: using a test DNA as a template, performing real-time fluorescence quantitative PCR using the DNA molecule combination described in any one of claims 1 to 4, and judging whether the test DNA contains the target sequence of the DNA molecule combination based on the amplification curve.
9. A nucleic acid detection method, comprising the following steps: using a test RNA as a template, performing real-time fluorescence quantitative reverse transcription PCR using the DNA molecule combination described in any one of claims 1 to 4, and judging whether the test RNA contains the target sequence of the DNA molecule combination based on the amplification curve.