Novel nucleic acid detection method established based on Exo III phosphatase activity

Abstract

The invention discloses a novel nucleic acid detection method established based on Exo III phosphatase activity, and belongs to the technical field of biomedical detection. The fluorescent probe is cut by utilizing the phosphatase activity of the Exo III protein, so that a quenching group is released, a fluorescence signal of a fluorophore on the fluorescent probe is increased, after a target sequence in a to-be-detected sample is complementarily combined with the fluorescent probe, the phosphatase activity of the Exo III protein is inhibited, and the target sequence is detected. The fluorescence signal of the fluorophore on the fluorescent probe is weakened; according to the relative intensity of the fluorescence signal, quantitatively detecting the target sequence in the to-be-detected sample; the detection method provided by the invention has the advantages of simple operation, rapid reaction, high sensitivity and good specificity; meanwhile, a detection reagent required in the detection method is easy to obtain, low in price, easy to store and transport, simple to operate and free of special instruments, and one-step detection is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and in particular relates to a novel nucleic acid detection method based on Exo III phosphatase activity. Background Art

[0002] The development of nucleic acid sensing technology is of great significance in the fields of medical diagnosis, food safety and environmental protection. Exo III is a common bacterial exonuclease that is easy to obtain, has high catalytic activity and has low preference for substrate sequence and length.

[0003] According to statistics, since 2010, a large number of literature reports have used Exo III to develop detection methods for nucleic acid and non-nucleic acid targets. These methods all use the exonuclease activity of Exo III. Generally, its cleavage activity for dsDNA is 5-10 times that of ssDNA. In general, the process of detecting nucleic acids based on Exo III is as follows: 1) The hairpin probe template recognizes and hybridizes with the target nucleic acid to form a partial dsDNA structure; 2) Exo III recognizes and degrades the dsDNA part to release a fluorescent signal, and the target nucleic acid is released and participates in the next round of hybridization with the probe template and Exo III cleavage process. The above detection method achieves the purpose of target signal amplification to a certain extent through cyclic amplification of the target. However, this detection method has problems such as non-specific cleavage of ssDNA template and fluorescent probe by Exo III, low amplification efficiency and the generation of false positive signals.

[0004] Therefore, it is necessary to provide a new nucleic acid detection method based on Exo III phosphatase activity to solve the shortcomings of the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a novel nucleic acid detection method based on Exo III phosphatase activity, which is used to solve the problems of non-specific cleavage of ssDNA templates and fluorescent probes by Exo III in existing detection methods, low amplification efficiency and generation of false positive signals.

[0006] In a first aspect, the present invention provides a novel nucleic acid detection method based on Exo III phosphatase activity, comprising the following steps: adding a sample to be tested containing a target sequence to a detection system, mixing and reacting, and then detecting a detectable signal in the system after the reaction to obtain the presence and / or content of the target sequence in the sample to be tested; wherein the detection system comprises a buffer, an Exo III protein, and a fluorescent probe complementary to the target sequence in the sample to be tested; and the fluorescent probe is labeled with a fluorescent group and a quenching group, and the fluorescent signal of the fluorescent group can be quenched by the quenching group.

[0007] In the present invention, the phosphatase activity of the Exo III protein is used to cut the fluorescent probe, thereby releasing the quenching group, resulting in an increase in the fluorescent signal of the fluorescent group on the fluorescent probe. When the target sequence in the sample to be tested is complementary to the fluorescent probe, the phosphatase activity of the Exo III protein is inhibited, resulting in a weakening of the fluorescent signal of the fluorescent group on the fluorescent probe; according to the relative intensity of the fluorescent signal, the target sequence in the sample to be tested is quantitatively detected; the detection method provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity and good specificity.

[0008] In some embodiments, the buffer comprises a HEPES buffer, and the HEPES buffer contains Mn 2+ ions; among them, Mn 2+ The concentration of the ions is 0.05-1 mM, for example, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 1 mM or other values ​​within the range.

[0009] It can be understood that, in the present invention, the buffer can be conventionally selected according to actual use needs. In the present invention, the buffer preferably includes HEPES buffer.

[0010] In some embodiments, the pH value of the buffer is 7.5-8.2, for example, it can be 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2 or other values ​​within this range.

[0011] In some embodiments, the concentration of Exo III protein is 0.01-1 nM, for example, 0.01 nM, 0.03 nM, 0.05 nM, 0.07 nM, 0.09 nM, 1 nM or other values ​​within this range.

[0012] It is understandable that, in the present invention, the Exo III protein can be obtained through commercial routes, or can be expressed using genetically engineered bacteria and obtained after purification.

[0013] In some embodiments, the quencher group is modified at the 3' end of the fluorescent probe; and the fluorescent group is modified at the T base near the 3' end of the fluorescent probe.

[0014] In the present invention, the inventors further discovered that the 3' phosphatase activity of the Exo III protein is used to cut the fluorescent probe, thereby releasing the quenching group at the 3' end of the fluorescent probe, resulting in an increase in the fluorescent signal of the fluorescent group on the fluorescent probe. When the target sequence in the sample to be tested is complementary to the fluorescent probe, the phosphatase activity of the Exo III protein is inhibited, resulting in a weakening of the fluorescent signal of the fluorescent group on the fluorescent probe; based on the relative intensity of the fluorescent signal, the target sequence in the sample to be tested is quantitatively detected.

[0015] In some embodiments, the fluorescent group includes at least one of FAM, CY5, VIC, and ROX; and the quenching group includes BHQ1.

[0016] It is understandable that, in the present invention, the fluorescent group and the quenching group can be conventionally selected according to actual use needs. In the present invention, the fluorescent group preferably includes at least one of FAM, CY5, VIC, and ROX; the quenching group preferably includes BHQ1.

[0017] In some embodiments, reacting after mixing specifically includes: reacting at a temperature of 20-40°C (for example, 20°C, 25°C, 30°C, 35°C, 40°C or other values ​​within the range) for 6-10 minutes, for example, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes or other values ​​within the range.

[0018] In some embodiments, the step of drawing a standard curve is also included, which includes: adding positive standards with different concentration gradients to the detection system respectively, mixing them, reacting them, and detecting detectable signals, and drawing a standard curve according to the detectable signal values ​​corresponding to the positive standards with different concentration gradients; wherein the positive standard includes the target sequence.

[0019] In some embodiments, the target sequence includes at least one of a DNA sequence and an RNA sequence.

[0020] In some embodiments, the detectable signal comprises a fluorescent signal.

[0021] The beneficial effects of the present invention are as follows: different from the prior art, the present invention utilizes the phosphatase activity of the Exo III protein to cut the fluorescent probe, thereby releasing the quenching group, resulting in an increase in the fluorescent signal of the fluorescent group on the fluorescent probe; when the target sequence in the sample to be tested is complementary to the fluorescent probe, the phosphatase activity of the Exo III protein is inhibited, resulting in a weakening of the fluorescent signal of the fluorescent group on the fluorescent probe; according to the relative intensity of the fluorescent signal, the target sequence in the sample to be tested is quantitatively detected; the detection method provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity and good specificity; at the same time, the detection reagent required in the detection method is easy to obtain, low in price, easy to store and transport, and simple to operate, does not require special instruments, and realizes one-step detection, which can meet the needs of high-throughput detection of nucleic acid and non-nucleic acid samples in daily home use and hospitals. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of a novel nucleic acid detection method based on Exo III phosphatase activity in the present invention; Figure 2 This is an SDS-PAGE image of the Exo III protein obtained in Example 1 of the present invention; Figure 3A It is a fluorescence graph of single-stranded DNA 1 of different concentrations (0-20 nM) in the detection system in Example 2 of the present invention; Figure 3B This is a standard curve diagram established after detecting the DNA target in Example 2 of the present invention; Figure 3C This is a standard curve diagram established after detecting the RNA target in Example 2 of the present invention; Figure 3D This is a fluorescence intensity diagram of the detection of multiple target nucleic acids in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0025] See also Figure 1 , which is a schematic diagram of a novel nucleic acid detection method based on Exo III phosphatase activity in the present invention, wherein, for single nucleic acid target detection, the quenching group is modified at the 3' end of the fluorescent probe, and the fluorescent group is modified at the T base near the 3' end of the fluorescent probe; the 3' end of the fluorescent probe is cut under the action of the phosphatase activity of the Exo III protein, thereby releasing the quenching group, resulting in an increase in the fluorescent signal of the fluorescent group on the fluorescent probe, and when the target sequence in the sample to be tested is complementary to the fluorescent probe, the phosphatase activity of the Exo III protein is inhibited, resulting in a weakening of the fluorescent signal of the fluorescent group on the fluorescent probe; according to the relative intensity of the fluorescent signal, the target sequence in the sample to be tested is quantitatively detected; for multiple nucleic acid target detection, it is necessary to design fluorescent probes corresponding to the number of multiple nucleic acid targets, and then perform multiple nucleic acid target detection based on the single nucleic acid target detection method.

[0026] Example 1 Expression and purification of Exo III protein 1.1 Acquisition of the xthA gene The xthA gene sequence (accession number: NC_002695.2) was found in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ); primers were designed based on the xthA gene sequence as shown below: xthA-F:5'-CGCGGATCCATGAAATTTGTCTCTTTTAATATCAATGGC-3' (SEQ ID NO. 1); xthA-R:5'-CCGCTCGAGTTAGCGGCGGAAGGTC-3'(SEQ ID NO.2); The xthA gene fragment was obtained by PCR amplification reaction, wherein the PCR reaction system was as follows: 2×Pfu Mix, 25 μL; xthA-F / xthA-R, 2 μL; E. coli genome, 1 μL; ddH 2O, 20 μL; PCR reaction program is as follows: 95℃, 5 min; 95℃, 0.5 min; 55℃, 0.5 min; 72℃, 1 min; 72℃, 10 min; 30 cycles. After the reaction is completed, the fragment size of the nucleic acid product is analyzed using 1% agarose gel to obtain the xthA gene fragment of the correct size.

[0027] 1.2 Construction of recombinant strains The xthA gene fragment was connected to the expression vector pET28a using a double enzyme digestion system and T4 ligase to obtain the recombinant plasmid pET28a-xthA, which was transformed into E. coli DH5α. After cultivation, a single colony was picked for colony PCR verification, and the plasmid was extracted from the successfully verified strain and sent to Biotech for sequencing. The recombinant plasmid pET28a-xthA that was successfully sequenced was transformed into E. coli BL21. After colony PCR verification, it was sent to Biotech for sequencing to obtain the recombinant strain.

[0028] 1.3 Expression and purification of Exo III protein The recombinant strain in step 1.2 was inoculated into LB medium for culture, and then 10 mL of the bacterial solution was inoculated into 1 L of LB medium containing kanamycin. The culture was carried out at 37 °C and 200 rpm for 3 h until the OD 600 to 0.6; then 80 μL IPTG (0.1 mol / L) was added for induction, and cultured at 16°C and 200 r / min for 18 h; after the culture was completed, the bacterial liquid was collected and centrifuged at 12,000 r / min for 2 min. The resulting precipitate was crushed by high pressure (1,200 bar) and repeated 3-5 times; finally, the supernatant was obtained by high-speed centrifugation (14,000 r / min, 30 min); and the Exo III protein was purified using the Ni-NTA Sefinose Resin Kit.

[0029] The purified Exo III protein was analyzed by SDS-PAGE. Figure 2 shown.

[0030] from Figure 2 As can be seen, the size of the obtained Exo III protein is about 30 kDa, which is consistent with the expected protein size.

[0031] Example 2 A novel nucleic acid detection method based on Exo III phosphatase activity In this example, a novel nucleic acid detection method based on Exo III phosphatase activity was used to detect the target nucleic acid.

[0032] Specifically, firstly prepare a 2+ Ionic HEPES buffer: accurately weigh 0.476g HEPES, 0.4383g NaCl, and 0.0154g DTT powder and dissolve them in deionized water, adjust the pH to 7.8; then weigh 0.0013g MnCl 2 , dilute to 100 mL; store at 4°C until use.

[0033] Then the new nucleic acid detection method was verified. Specifically, 1 μL fluorescent probe 1 (5'-CATACCTCGAGGTCCTC-3'-BHQ1 (SEQ ID NO.3), where 6-FAM is modified at the fifth base T from 3', 10 μM, synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.), 2 μL Exo III protein (1.12 nM), single-stranded DNA 1 (5'-GAGGACCTCGAGGTATG-3' (SEQ ID NO.4)) with different concentrations (0-20 nM) complementary to the fluorescent probe sequence, 10 μL containing Mn 2+ ion HEPES buffer and 11 μL deionized water were mixed, reacted at 30°C for 8 min, and the corresponding fluorescence intensity was detected using a fluorescence microplate reader. The results are as follows Figure 3A shown.

[0034] from Figure 3A It can be seen that as the concentration of the target sequence increases (0-20nM), the fluorescence signal generated by the Exo III reaction system gradually decreases. Therefore, based on this detection method, a relationship between the fluorescence signal and the target sequence concentration can be established to achieve quantitative detection of the nucleic acid sequence in the sample to be tested.

[0035] Finally, the new nucleic acid detection method was used to detect different target nucleic acids.

[0036] Specifically, the DNA target was first detected by using a pipette, specifically: 1 μL of fluorescent probe 1 (5'-CATACCTCGAGGTCCTC-3'-BHQ1 (SEQ ID NO.3), wherein 6-FAM was modified at the fifth base T from 3', 10 μM, synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.), 2 μL of Exo III protein (1.12 nM), single-stranded DNA 1 (5'-GAGGACCTCGAGGTATG-3' (SEQ ID NO.4)) with different concentrations complementary to the fluorescent probe sequence (0, 0.005, 0.001, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 200, 500, 750, 1000, 2500, 5000 nM), 10 μL of Mn-containing2+ ion HEPES buffer and 11 μL deionized water were mixed and reacted at 30°C for 8 min. The corresponding fluorescence intensity was detected using a fluorescence microplate reader. The concentration of the target sequence and the fluorescence signal intensity generated by it were used as X and Y respectively to establish a standard curve. The results are shown in the figure. Figure 3B shown.

[0037] from Figure 3B It can be seen that the standard curve equation is Y=-548.82X+3153.12 (R 2 =0.99), the detection range of this method is 1pM-4nM, and the detection sensitivity is 0.82pM (3σ).

[0038] Then, the RNA target was detected, specifically: 1 μL of fluorescent probe 1 (5'-CATACCTCGAGGTCCTC-3'-BHQ1 (SEQ ID NO.3), wherein 6-FAM was modified at the fifth base T from 3', 10 μM, synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.), 2 μL of Exo III protein (1.12 nM), single-stranded RNA1 (5'-GAGGACCUCGAGGUAUG-3' (SEQ ID NO.5)) with different concentrations complementary to the fluorescent probe sequence (0, 0.0125, 0.025, 0.125, 0.25, 1.25, 2.5, 5, 12.5, 25, 100, 250, 500, 1000 nM), 10 μL of Mn 2+ ion HEPES buffer and 11 μL deionized water were mixed and reacted at 30°C for 8 min. The corresponding fluorescence intensity was detected using a fluorescence microplate reader. The concentration of the target sequence and the fluorescence signal intensity generated by it were used as X and Y respectively to establish a standard curve. The results are shown in the figure. Figure 3B shown.

[0039] from Figure 3C It can be seen that the standard curve equation is Y=-802.80X+3067.91 (R 2 =0.99), the detection range of this method is 0.5pM-1nM, and the detection sensitivity is 0.38pM (3σ).

[0040] Finally, multiple target nucleic acids were detected by synthesizing probes with different sequences and modified with different fluorescent groups at Sangon Biotech (Shanghai) Co., Ltd.: Fluorescent probe 2 (5'-TGATTATTTACCATCCAT-3'-BHQ1 (SEQ ID NO.6), wherein 6-CY5 is modified at the fifth base T from 3'); fluorescent probe 3 (5'-ACTACCTCGAGGACCTC-3'-BHQ1 (SEQ ID NO.7), wherein 6-ROX is modified at the second base T from 3'); fluorescent probe 4 (5'-CCATCTTTACCAGACAGTGTTA-3'-BHQ1 (SEQ ID NO.8), wherein 6-VIC is modified at the fifth base T from 3'); and the corresponding complementary single-stranded DNA 2 (5'-ATGGATGGTAAATAATCA-3' (SEQ ID NO.9)), single-stranded DNA 3 (5'-GAGGTCCTCGAGGTAGT-3' (SEQ ID NO.10) ID NO.10)) and single-chain MiR-141 (5'-UAACACUGUCUGGUAAAGAUGG-3' (SEQ ID NO.11)); The reaction system and conditions are the same as those for the above-mentioned detection of DNA targets. The results are as follows Figure 3D shown.

[0041] from Figure 3D It can be seen that in the single-target mode, the appearance of each target sequence can cause the corresponding signal to be generated, and a single target will not have a cross-effect with other reporter probe sequences. In the dual-target mode, the appearance of the target sequence causes the corresponding detection signal to be generated, and there is no cross-effect between them. When the three target sequences are added to the reaction system at the same time, corresponding detection signals are generated. Compared with reporter probes modified with fluorescent groups such as ROX, VIC, and FAM, the catalytic activity of Exo III on CY5-modified fluorescent probes is slightly weaker, but it can also produce obvious cleavage fluorescence signals.

[0042] The above results show that the novel nucleic acid detection method of the present invention can detect both single nucleic acid targets and multiple nucleic acid targets, and has the advantages of high sensitivity and strong specificity.

[0043] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0044] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A novel nucleic acid detection method based on Exo III phosphatase activity, characterized in that: The steps include: Adding a sample to be tested containing the target sequence to the detection system, mixing and reacting, and then detecting the detectable signal in the system after the reaction to obtain the presence and / or content of the target sequence in the sample to be tested; The detection system includes a buffer, Exo III protein, and a fluorescent probe complementary to the target sequence in the sample to be tested; and the fluorescent probe is labeled with a fluorescent group and a quenching group, and the fluorescent signal of the fluorescent group can be quenched by the quenching group.

2. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The buffer comprises a HEPES buffer, and the HEPES buffer contains Mn 2+ ion; Wherein, the Mn 2+ The concentration of ions is 0.05-1 mM.

3. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The pH value of the buffer solution is 7.5-8.

2.

4. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The concentration of the Exo III protein is 0.01-1 nM.

5. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The quenching group is modified on the 3' end of the fluorescent probe; and the fluorescent group is modified on the T base close to the 3' end of the fluorescent probe.

6. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 5, characterized in that: The fluorescent group includes at least one of FAM, CY5, VIC, and ROX; and the quenching group includes BHQ1.

7. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The step of reacting after mixing specifically includes: reacting for 6-10 minutes at a temperature of 20-40°C.

8. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The method further includes the step of drawing a standard curve, which includes: adding positive standard substances with different concentration gradients to the detection system respectively, mixing them and reacting them, and detecting the detectable signal, and drawing the standard curve according to the detectable signal values ​​corresponding to the positive standard substances with different concentration gradients; Wherein, the positive standard includes the target sequence.

9. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The target sequence includes at least one of a DNA sequence and an RNA sequence.

10. The novel nucleic acid detection method based on Exo III phosphatase activity according to claim 1, characterized in that: The detectable signal comprises a fluorescent signal.

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