The present disclosure claims the priority to the Chinese patent application filed on Dec. 29, 2018 with the Chinese Patent Office with the filing number 201811643407.0, and entitled “Structure and Application of Double-stranded Oligonucleotide Nucleic Acid Probe”, which is incorporated by reference herein in entirety.
The present disclosure relates to a tool for biological detection technology, in particular, a double-stranded oligonucleotide nucleic acid probe, a method of using the same, and application of the same in gene fluorescence qualitative and quantitative analysis, medical diagnosis and other life science researches, belonging to the technical field of gene detection.
Currently, the fluorescence probe method is usually used in molecular diagnosis of infectious diseases and the like, individualized companion diagnosis, and other disease diagnosis applications. The fluorescence probe method relies on fluorescent resonance energy transfer (FRET) to realize detection, including TaqMan probes, molecular beacons, Scorpion probes, and the like. The method only detects a specific amplification product, and therefore the specificity is strong. Currently, the most widely used is the TaqMan probe technology, and this technology mainly utilizes 5′ exonuclease activity of Taq enzyme. Firstly, a probe capable of hybridizing to a PCR product is synthesized, 5′ end of the probe labels a fluorescent molecule, 3′ end labels a corresponding fluorescent quenching molecule, and quenching molecule at 3′ end can absorb fluorescence emitted by the fluorescent molecule at 5′ end. Under normal conditions, the probe theoretically does not emit fluorescence, but when there is a PCR product in a solution, the probe is bound with the PCR product, to activate the 5′ end exonuclease activity of the Taq enzyme, and cleave the probe into a mononucleotide, at the same time, the fluorescent group labeled on the probe is free, as a result, fluorescence is emitted and the number of cleaved fluorescent molecules is proportional to the number of PCR products, therefore, the concentration of an initial template can be calculated from the fluorescence signal intensity in a PCR reaction liquid. TaqMan probe technology has many drawbacks despite its wide application.
The present disclosure aims at providing a new structure of double-stranded oligonucleotide nucleic acid probe, a method of using the same, and use of the same in gene fluorescence qualitative and quantitative analysis, medical diagnosis, life science researches and other fields, to solve at least one problem existing in the prior art.
The basic principle of the present disclosure is as follows.
As shown in the schematic diagram of qualitative and quantitative analysis of a double-stranded oligonucleotide nucleic acid probe in
The objective of the present disclosure is achieved as follows: a double-stranded oligonucleotide nucleic acid probe, consisting of two completely or partially base-complementary oligonucleotide chains, is prepared, wherein for the two probes, an end of each oligonucleotide chain may be linked to a fluorescent group or a corresponding fluorescent quenching group, and the two oligonucleotide probe chains may be both hybridized and bound with a partial fragment of a target DNA or RNA nucleic acid sequence to be detected according to the base pairing principle. Each probe of the double-stranded probe independently consists of 6-50 oligonucleotides, and preferably, a long-strand probe in the double-stranded probe consists of 25-30 nucleotides, and a short-strand probe consists of 15-25 nucleotides. The number of fluorescent molecules and quenching molecules linked to the probe may be 1-5, generally being 1 in consideration of cost and synthesis convenience. The fluorescent group may be one or more selected from the group consisting of FAM, HEX, TET, ROX, CY3, CYS, VIC, JOE, SIMA, Alexa Fluor 488, TexasRed or Quasar 670, and the quenching group linked to the other end of this chain may be one or more selected from the group consisting of TAMRA, Dabcyl, BHQ-1, BHQ-2, BHQ-3, MGB or Eclipse.
The present disclosure discloses a method for using a double-stranded oligonucleotide nucleic acid probe in gene detection, which includes the following steps:
(1) preparing a double-stranded oligonucleotide nucleic acid probe;
(2) designing and synthesizing a pair of upstream and downstream primers according to a gene sequence to be detected, wherein Tm values of the primers are lower than a Tm value of a long-strand probe, the primers do not overlap with the probe and are adjacent to two ends of the probe, and are 1-150 nucleotides from the two ends of the probe;
(3) adding a template to a reaction mixture containing the probe, the primers, a PCR buffer solution, magnesium ions or manganese ions, dNTPs, and a Taq DNA polymerase to perform regular PCR, and amplification for 25-60 cycles, wherein the nucleic acid as a template preferably has a length of 60-500 bases, more preferably 70-150 bases, and fluorescence values are recorded in annealing or extension of each cycle;
(4) performing regression analysis on the number of cycles of threshold fluorescence with a logarithm of an initial concentration of the template, preparing a standard curve, and performing quantitative analysis on a concentration of the gene to be detected, wherein the threshold fluorescence refers to the fluorescence intensity of the gene to be detected that is 2 times a background fluorescence variation coefficient.
According to the above detection steps, the double-stranded oligonucleotide nucleic acid probe of the present disclosure can be widely applied to gene fluorescence qualitative and quantitative analysis, medical diagnosis and other gene detection fields, and in particular, it has greater advantages in the simultaneous detection and typing of multiple genes, and high-sensitivity detection of genes.
In the double-stranded oligonucleotide nucleic acid probe of the present disclosure, the double-stranded probes are fluorescent probes and quenching probes to each other, the fluorescent group and the quenching group are closer to each other, the quenching is more thorough, and the fluorescence background is greatly reduced; the double-stranded oligonucleotide nucleic acid probe does not completely rely on exonuclease activity, and may label two or more fluorescent molecules, and the two probes both bind to the template, thus improving the detection sensitivity. In summary, the double-stranded oligonucleotide nucleic acid probe technology has a greater promotion and application value.
Specific embodiments of the present disclosure are further described in detail with reference to the accompanying drawings.
In order to further illustrate the technique and use of the double-stranded oligonucleotide nucleic acid probe, description is made with reference to the following examples, and the following examples are intended to illustrate rather than limit the present disclosure in any way.
1. Design of Primers and Probes for HBV Detection
According to the qualitative and quantitative analysis principle of double-stranded oligonucleotide nucleic acid probe, and in accordance with DNA sequence of a target molecule HBV to be detected, primers F and R, a long-strand oligonucleotide probe P1, a short-strand oligonucleotide probe P2, a Taqman probe P3, and a mutant base-containing oligonucleotide probe P4 were designed and synthesized. See Table 1 for the primer and probe sequences.
(1) Primers
The upstream primer F, having 21 nucleotides in total, was 17 nucleotides away from the long-strand oligonucleotide probe, and 22 nucleotides away from the short-strand oligonucleotide probe.
The downstream primer R, having 21 nucleotides in total, was 27 nucleotides away from the long-strand oligonucleotide probe, and 32 nucleotides away from the short-strand oligonucleotide probe.
(2) Probes
The long-strand oligonucleotide probe P1 was complementary to a minus strand of the target sequence, and consisted of 27 nucleotides, in which a 5′ end had a fluorescein molecule, and a 3′ end had a quenching molecule; the short-strand oligonucleotide probe P2 was complementary to a plus strand of the target sequence, and consisted of 17 nucleotides, in which a 5′ end had a fluorescein molecule, a 3′ end had a quenching molecule, the short-strand oligonucleotide probe P2 was 5 bases away from the probe P1 in position, and 10 bases shorter than the P1; the Taqman probe P3 was complementary to the minus strand of the target sequence, and consisted of 25 nucleotides, wherein a 5′ end had a fluorescein molecule, and a 3′ end had a quenching molecule; the mutant base-containing probe P4 had a mutant base which was incompletely reversely complementary to the plus strand of the target sequence; and the mutant base-containing probe P4 consisted of 27 nucleotides, wherein a 5′ end had a fluorescein molecule, and a 3′ end had a quenching molecule.
2. PCR detection
(1) PCR Detection by the Double-Stranded Oligonucleotide Nucleic Acid Probe
A PCR reaction system was formulated, including: 10× PCR buffer solution 4 μL, dNTPs 0.2 mmol/L, the upstream and downstream primers each 0.55 μmol/L, Taq DNA polymerase 2.5 U, a long-strand oligonucleotide probe 0.275 μmol/L, a short-strand oligonucleotide probe 0.330 μmol/L, and an HBV template 20 μL extracted with a nucleic acid extraction kit, a total reaction volume being 40 μL;
Reaction condition: 50° C., 2 min; 94° C., 2 min; 94° C., 15 s, 55 ° C., 45 s, 45 cycles in total, and collecting fluorescence during annealing.
(2) Fluorescent PCR Detection by the Double-Stranded Oligonucleotide Nucleic Acid Probe That Was Incompletely Reversely Complementary
A PCR reaction system was formulated, including: 10× PCR buffer solution 4 μL, dNTPs 0.2 mmol/L, the upstream and downstream primers each 0.55 μmol/L, Taq DNA polymerase 2.5 U, a long-strand oligonucleotide probe 0.275 μmol/L, an incompletely-reversely-complementary oligonucleotide probe 0.330 μmol/L, and an HBV template 20 μL extracted with a nucleic acid extraction kit, a total reaction volume being 40 μL;
Reaction condition: 50° C., 2 min; 94° C., 2 min; 94° C., 15 s, 55° C., 45 s, 45 cycles in total, and collecting fluorescence during annealing.
(3) Fluorescent PCR Detection by the Taqman Probe
A PCR reaction system was formulated, including: 10× PCR buffer solution 4 μL, dNTPs 0.2 mmol/L, the upstream and downstream primers each 0.55 μmol/L, Taq DNA polymerase 2.5 U, Taqman probe 0.275 μmol/L, and an HBV template 20 μL extracted with a nucleic acid extraction kit, a total reaction volume being 40 μL;
Reaction condition: 50° C., 2 min; 94° C., 2 min; 94° C., 15 s, 55° C., 45 s, 45 cycles in total, and collecting fluorescence during annealing.
3. Specificity of the Double-Stranded Oligonucleotide Nucleic Acid Probe
The primers were F and R, the probes were the double-stranded oligonucleotide nucleic acid probe (P1/P2) and the Taqman probe (P3), respectively, the templates were 105 IU/mL hepatitis B virus (HBV), 105 IU/mL hepatitis C virus (HCV), 105 IU/mL hepatitis A virus (HAV), 105 IU/mL human cytomegalovirus (CMV), 105 IU/mL herpes simplex virus type I (HSV-1), and herpes simplex virus type II (HSV-2), respectively, and ddH2O was a negative control. The PCR detection was performed according to operations in step 2.
Experiment results are as shown in
4. Quenching Efficiency of the Double-Stranded Oligonucleotide Nucleic Acid Probe
The primers were F and R, the probes were the double-stranded oligonucleotide nucleic acid probe (P1/P2) and Taqman probe (P3), respectively, and ddH2O was a template. The amplification was performed for 15 cycles according to the operations in step 2, and the quenching efficiencies of the double-stranded oligonucleotide nucleic acid probe and the Taqman probe were detected.
As shown in
5. Detection Range and Sensitivity of the Double-Stranded Oligonucleotide Nucleic Acid Probe (1) Linear Range Detection
The primers were F and R, the probes were the double-stranded oligonucleotide nucleic acid probe (P1/P2) and the Taqman probe (P3), respectively, and the template was an HBV nucleic acid quantitative detection standard substance at a concentration of 109 IU/mL, and was diluted to 109 IU/mL-10 IU/mL in 10-fold gradient. Amplification was performed according to the operations in step 2, to detect the quantitative range and sensitivity of the double-stranded oligonucleotide nucleic acid probe and the Taqman probe.
As shown in
(2) Sensitivity Detection
The primers were F and R, the probes were the double-stranded oligonucleotide nucleic acid probe (P1/P2), a double-stranded oligonucleotide nucleic acid probe (P1/P4), and the Taqman probe (P3), respectively, the template was an HBV nucleic acid quantitative detection standard substance at a concentration of 10 IU/mL. Detection was repeated 8 times. The amplification was performed according to the operations in step 2, to detect the lowest detection limits of the double-stranded oligonucleotide nucleic acid probe and the Taqman probe.
As shown in
It thus indicates that the double-stranded oligonucleotide nucleic acid probe may perform an accurate quantitative detection on samples at a concentration in the range of 109 IU/mL-10 IU/mL, and provide a reference to the detection results for samples at a concentration of 10 IU/mL. The Taqman probe may perform an accurate quantitative detection on samples at a concentration in the range of 109 IU/mL-102 IU/mL.
6. Quantitative Detection Analysis for HBV Clinical Sample
The primers were F and R, the probes were the double-stranded oligonucleotide nucleic acid probe (P1/P2) and the Taqman probe (P3), respectively, and the templates were 18 cases of HBV DNA positive sera with a fixed value. Amplification was performed according to the operations in step 2.
Results are as shown in
In the above, in
1. Design of primers and probes for 2C19*2 gene detection
According to the qualitative and quantitative analysis principle of double-stranded oligonucleotide nucleic acid probe, and in accordance with DNA sequence of a target molecule 2C19*2 gene to be detected, upstream and downstream primers, a mutant chain oligonucleotide probe, and a wild chain oligonucleotide probe were designed and synthesized. See Table 6 for the primer and probe sequences.
2. PCR Detection
(1) A PCR reaction system was formulated, including: 10×Buffer solution 2.5 μL, the upstream and downstream primers each 1.2 μL (10 μM), THE double-stranded oligonucleotide probe 0.6 μL (10 μM), Mg2+2.5 μL, 50×ST enzyme 0.5 μL (BIORI, China), and a human DNA template 3 μL extracted with a nucleic acid extraction kit, a total reaction volume being 25 μL.
Reaction condition: 50° C., 2 min; 95° C., 5 min; 95° C., 20 s, 60° C., 45 s, 40 cycles in total, and collecting fluorescence during annealing.
(2) Templates: 2C19*2 gene mutant type NA, wild type G/G, and heterozygous type G/A were each 12 cases, ddH2O was a negative control. PCR detection was performed according to the operations in step (1).
3. Experiment Results
As shown in Table 6 and
From the above results, it can be seen that the double-stranded probe can detect genotype of mononucleotide polymorphism sites, and accurately detect the 2C19*2 gene mutant type NA, wild type G/G, heterozygous type G/A, and negative samples. For the mutant type samples, the mutant chain probe labeled with the FAM fluorescent group increases in the fluorescence signal intensity with the increase of the number of cycles, and the wild chain probe has no change; for the wild type samples, the wild chain probe labeled with the HEX fluorescent group increases in the fluorescence signal intensity with the increase of the number of cycles, and the mutant chain probe has no change; and for the heterozygous type samples, the fluorescence signal intensity of both increases with the increase of the number of cycles, and there is no change in the negatives.
The double-stranded oligonucleotide nucleic acid probe provided in the present disclosure renders more thorough fluorescence quenching, greatly reduces the fluorescence background, and does not completely rely on exonuclease activity; besides, the double-stranded oligonucleotide nucleic acid probe may label two or more fluorescent molecules, thus improving the detection sensitivity and having a greater promotion and application value.
Number | Date | Country | Kind |
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201811643407.0 | Dec 2018 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2019/077879 | 3/12/2019 | WO | 00 |