CN112795629A - Method for improving gene detection sensitivity and speed - Google Patents
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Abstract
The invention relates to a method for improving the sensitivity and speed of nucleic acid detection, which can obviously improve the sensitivity and speed of gene detection no matter aiming at the detection of DNA or RNA by increasing the number of primers in a peripheral or internal structure when the polymerase without exo activity is used for nucleic acid synthesis. By using the technology, the advantages of nucleic acid detection can be further exerted, and the technology has great significance in various application scenes.
Description
[ technical field ]
The present invention relates to the field of molecular biology, and more particularly, to methods for gene detection.
[ background art ]
The invention of multiple aggregation chain reaction (PCR) technology has been developed for 20 years, during which the technology is continuously developed, and the real-time quantitative PCR technology appeared in recent years realizes the leap of PCR from qualitative to quantitative, and it becomes an important tool in molecular biology research with the advantages of strong specificity, high sensitivity, good repeatability, accurate quantification, fast speed, full-closed reaction, etc.
Since the 9O's of the last century, several new nucleic acid amplification methods emerged: nucleic acid isothermal amplification (NAS-BA), self-sequencing replication (3 SR), Strand Displacement Amplification (SDA), and the like. Noto-mi et al, developed a novel isothermal nucleic acid amplification method in 2000, namely, loop-mediated isothermal amplification (LAMP), which is characterized in that 4 specific primers are designed for 6 regions of a target gene, and a strand displacement DNA polymerase (Bst DNA polymerase) is used for heat preservation for dozens of minutes under an isothermal condition (about 60-65 ℃) to complete the nucleic acid amplification reaction without the processes of thermal denaturation of a template, long-time temperature circulation, complicated electrophoresis, ultraviolet observation and the like. LAMP is a novel DNA amplification method, has the characteristics of simplicity, rapidness and strong specificity, and has the possibility of replacing a PCR method.
Although the PCR detection system and the isothermal amplification system are the most sensitive detection means, the situation of insufficient sensitivity still occurs in actual work. In addition, the speed of detection is also large
[ summary of the invention ]
In a nucleic acid amplification reaction by a DNA polymerase having no exo activity, if the number of primers is increased at the periphery of the primers or inside the structure, since the polymerase has no exo activity, the product of each amplification is retained, and the process of polymerization is a strand displacement process. The latter primer will remove double chain from the new chain synthesized by the former primer to form a naked single chain, and will combine with the new primer more easily to continue gene amplification. In addition, because the primers are positioned differently, they do not interfere with each other and bind to the matching region, and the chance of annealing to form a double strand is significantly increased. Therefore, the product of nucleic acid amplification will be greatly increased, and the speed and sensitivity of nucleic acid synthesis will be greatly improved finally.
The LAMP method relies on the loop-mediated gene synthesis with the participation of a large polymerase Bst segment with a strand displacement function, only needs one reaction temperature, and utilizes a hairpin structure formed between primers to carry out continuous amplification. By using the invention, the number of primers is increased at the periphery of the primers and in the loop structure, so that the nucleic acid synthesis speed of the LAMP method can be increased. Through experimental comparison, the speed and sensitivity of nucleic acid synthesis are remarkably improved under the condition of adding the primer.
In addition, the present invention is also applicable to the reverse transcription process of reverse transcription quantitative PCR. Primers are added at the periphery of the PCR primers, reverse transcriptase without RNase H activity is selected, or strand displacement polymerase with a reverse transcription function is used, so that the template can be multiplied in the reverse transcription process, and the sensitivity and the speed of subsequent PCR detection are improved.
Drawings
FIG. 1 is a graph of a first experimental effect of the present invention;
FIG. 2 is a diagram of a second experimental effect of the present invention;
FIG. 3 is a third experimental effect graph of the present invention;
FIG. 4 is a graph showing the effect of a fourth experiment according to the present invention;
FIG. 5 is a diagram illustrating a fifth experimental effect of the present invention;
FIG. 6 is a graph showing the effect of a sixth experiment according to the present invention;
fig. 7 is a diagram showing the effect of the seventh experiment of the present invention.
[ detailed description of the invention ]
The method of the present invention for increasing the sensitivity and speed of nucleic acid synthesis is illustrated by the following examples.
Materials and methods
Positive plasmids and primers were synthesized by Shanghai Czeri Bio, Bst DNA polymerase from NEB, and Betaine and HNB from Sigma.
The synthetic plasmid sequence was as follows:
TAATTGTGTTAAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACCGGAAGCCAATA TGGATCAAGAATCCTTTGGTGGTGCATCGTGTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAG GATTTTGTGACTTAAAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTA AAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGC TTCAGTCAGCTGATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCAC AGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCT AAAAACTAATTGTTGTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTTGTAGTTAAGAG ACACACTTTCTCTAACTAC
the primer sequences were synthesized as follows:
FIP:CGGTACAGACTGTGTTTTTACCTACAACTTGTGCTAATGAC
BIP:TCTGCGGTATGTGGAAAGGTTATAAACGATTGTGCATCAGC
LB:GCTGTAGTTGTGATCAACTCC
LF:AGTGTAAAACCCACAG
LF2：GCGAACCCATGCTTCAGTC
F3:GGTGCATCGTGTTGTCTGTAC
F4:TGCCGTTGCCACATAGATC
F5 CCTAAAGGATTTTGTGACTTAAAAGG
B3:CAAAAGCCCTGTATACGAC
B4:CAGCTACTTTATCATTGTAG
B5：CATCAGTACTAGTGCCTGTGC
the LAMP reaction system is as follows: peripheral primers F3, F4, F5, B3, B4, B5 each 10pmol, the remaining primers 40pmol, 1mmol dNTP, 1mmol Betaine, 6mmol MgSO4, 10u BST DNA polymerase, 1mM HNB. After the addition, 50ul of paraffin was added, the lid was closed, and the vessel was placed in a water bath.
HNB is a metal ion color indicator and can be used for indicating the existence of amplification reaction in LAMP experiments. The basic principle is that a large amount of pyrophosphate is released in the isothermal gene amplification process, magnesium pyrophosphate precipitate is produced by pyrophosphate and magnesium ions, the magnesium ions in the solution are reduced, and the HNB color is changed from purple red to sky blue. This color change is visible to the naked eye and the effect of the photograph is weaker due to the difference in light and image, and the illustration is provided only for reference.
Result is one
As shown in FIG. 1, the conventional LAMP assay method is adopted, the primers are F3+ B3+ FIP + BIP + LF + LB, and the reaction is carried out in a 62-degree water bath for 30 minutes. The amount of plasmid copy added from left to right was about: 1000,200, 40, 8, 0. As a result, the plasmid reactions of 1000 and 200 copies were positive, and the others were negative.
As shown in FIG. 2, the conventional LAMP assay method was adopted, and the primers were F3+ B3+ F4+ B4+ FIP + BIP + LF + LB, and were applied in a 62 ℃ water bath for 30 minutes. The amount of plasmid copy added from left to right was about: 1000,200, 40, 8, 0. As a result, 1000,200 and 40 copies of the plasmid reaction were positive, and the remainder were negative
As shown in FIG. 3, the conventional LAMP assay method was adopted, and the primers were F3+ B3+ F4+ B4+ F5+ B5FIP + BIP + LF + LB, and were subjected to 62-degree water bath for 30 minutes. The amount of plasmid copy added from left to right was about: 1000,200, 40, 8, 0. As a result, the plasmid reactions of 1000,200, 40 and 8 copies were positive, and 0 copy (negative control) was negative.
Combining the three diagrams above, the following conclusions can be drawn: in the LAMP experiment, the detection sensitivity can be increased by increasing the number of primers.
Result two
The same number of about 1000 copy plasmid templates were added to each tube using the usual LAMP protocol, and the primers added from left to right were: f3+ B3+ F4+ B4+ F5+ B5FIP + BIP + LF + LB + LF2, F3+ B3+ F4+ B4+ F5+ B5FIP + BIP + LF + LB, F3+ B3+ F4+ B4+ FIP + BIP + LF + LB, F3+ B3+ FIP + BIP + LF + LB, the 5 th tube is a negative control without adding a plasmid template, and the 62-degree water bath is adopted.
FIG. 4 reaction time is 20 minutes, the fourth tube with the largest number of primers has shown a positive result, the third tube is nearly positive, and the rest are negative.
FIG. 5 the reaction time is 25 minutes, the fourth and third tubes have shown a positive result, and the remainder are negative.
FIG. 6 reaction time was 30 minutes, second, third, and fourth tubes showed positive results, and the remainder were negative.
FIG. 7 shows that the reaction time is 40 minutes, the first four reaction tubes show positive results, and the negative control is normal.
Combining the above four diagrams, the following conclusions can be drawn: in the LAMP experiment, increasing the number of primers can increase the detection speed.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
[ second embodiment ]
The following examples illustrate the methods of the present invention for increasing the sensitivity and speed of nucleic acid synthesis in the field of RNA virus detection.
Materials and methods
Primers were synthesized by Shanghai Czeri Bio, Bst3.0 DNA polymerase from NEB, Betaine and HNB from Sigma.
The sequence of armored virus donated by Xiamen Zhishi Biotech is as follows:
TGGTGCATCGTGTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTAA AAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGTCTGTA CCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATG CACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGA TGTCGTATACAGGGCTTTTGACATCTACAATGATAAAGTAGCTGGTTTTGCTAA
the primer sequences were synthesized as follows:
forward primer (F): CCCTGTGGGTTTTACACTTAA 13342-
Reverse primer (R): ACGATTGTGCATCAGCTGA 13460-
Fluorescent probe (P): 5' -FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ 1-3'
Forward primer (F2): GTACAAATACCTACAACTTGTGC
Forward primer (F3): GCCACATAGATCATCCAAATCC
Reverse primer (R2): CTGCACTTACACCGCAAACC
Reverse primer (R3): TGTGCCGCACGGTGTAAGAC
The one-step RT-QPCR reaction system is as follows: each of primers F, F2, F3, B, B2, B3 and probe was 10pmol, 0.5mmol dNTP, 2mmol MgSO4, 5U BST3.0 DNA polymerase, 5U Taq polymerase. The armored RNA was added separately in 1000/200/40/8/1.6 copies. The control group used only primer F and primer R, the remaining components were the same.
Setting real-time amplification conditions: the first step is 50 ℃ for 20 minutes, and 1 cycle is carried out; a second step, the temperature is 95 ℃ for 10 minutes, and a cycle is performed; and the third step is 95 ℃ for 15 seconds, 60 ℃ for 3 seconds and 35 cycles, and the fluorescence signals are collected.
As a result:
without the addition of the peripheral primers, 200 copies could be detected with sensitivity. After adding the primers, 1.6 copies can be detected, and the sensitivity is improved by 125 times.
When the peripheral primers are not added, 32 cycles are needed for 200 copies, and after the peripheral primers are added, only 26 cycles are needed for 200 copies, so that the whole reaction speed is accelerated, and the detection time is shortened greatly.
In summary, the following conclusions can be drawn: in one-step RT-QPCR experiments, increasing the number of peripheral primers can increase the sensitivity and speed of detection.
The two embodiments described above are merely illustrative of the principles and effects of the present invention, and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
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Claims (5)
1. A method for improving the sensitivity and speed of nucleic acid detection features that when the polymerase without exo activity is used to synthesize nucleic acid, the number of primers in peripheral or internal structure is increased, so obviously improving the sensitivity and speed of gene detection.
2. A method relating to increased sensitivity and speed of nucleic acid detection according to claim 1, wherein: the polymerase used for the synthesis cannot have exo-activity.
3. A method relating to increased sensitivity and speed of nucleic acid detection according to claim 1, wherein: the number of peripheral primers, or intra-loop primers, at the target site is meant to increase the number of primers at different positions.
4. A method relating to increased sensitivity and speed of nucleic acid detection according to claim 1, wherein: the nucleic acid synthesis includes DNA synthesis depending on a DNA sequence as a template, and also includes DNA synthesis depending on RNA as a template.
5. A method relating to increased sensitivity and speed of nucleic acid detection according to claim 1, wherein: the primer at the periphery of the target site needs to be as close to the target site as possible, and the primer positioned outside the target gene is 0-3000bp away from the target gene.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104334745A (en) * | 2012-05-14 | 2015-02-04 | P·瓦尔霍 | A combined technique of PCR and loop-mediated isothermal amplification for nucleic acid detection |
| WO2017212904A1 (en) * | 2016-06-06 | 2017-12-14 | 国立大学法人 宮崎大学 | Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined |
| CN108431235A (en) * | 2015-12-22 | 2018-08-21 | 索灵股份公司 | The method of the fluoroscopic examination of the isothermal loop mediated amplification (LAMP) of target nucleic acid, oligonucleotides and its kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104334745A (en) * | 2012-05-14 | 2015-02-04 | P·瓦尔霍 | A combined technique of PCR and loop-mediated isothermal amplification for nucleic acid detection |
| CN108431235A (en) * | 2015-12-22 | 2018-08-21 | 索灵股份公司 | The method of the fluoroscopic examination of the isothermal loop mediated amplification (LAMP) of target nucleic acid, oligonucleotides and its kit |
| WO2017212904A1 (en) * | 2016-06-06 | 2017-12-14 | 国立大学法人 宮崎大学 | Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined |
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