CN112795630A - A method for rapid detection of loop-mediated isothermal amplification nucleic acid products using magnetic bead probes - Google Patents
A method for rapid detection of loop-mediated isothermal amplification nucleic acid products using magnetic bead probes Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 82
- 238000007397 LAMP assay Methods 0.000 title claims abstract description 52
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- 102000053602 DNA Human genes 0.000 claims description 4
- 238000011901 isothermal amplification Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
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- 238000010839 reverse transcription Methods 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 abstract description 3
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- 238000007885 magnetic separation Methods 0.000 abstract description 2
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- 230000003321 amplification Effects 0.000 abstract 1
- 230000005540 biological transmission Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 239000013615 primer Substances 0.000 description 21
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- 239000003298 DNA probe Substances 0.000 description 9
- 241000711573 Coronaviridae Species 0.000 description 7
- 241000710771 Tick-borne encephalitis virus Species 0.000 description 5
- 241000710959 Venezuelan equine encephalitis virus Species 0.000 description 5
- 241000150230 Crimean-Congo hemorrhagic fever orthonairovirus Species 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
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- 241001678559 COVID-19 virus Species 0.000 description 2
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Abstract
本发明提供了一种利用磁珠探针快速检测环介导等温扩增核酸产物的方法，可用于与核酸鉴定相关的诸多领域，其根据环介导等温扩增产物的单链环状区域设计一段与其互补的磁珠探针，将其与待检核酸混合并进行环介导等温扩增反应，磁珠探针识别并结合扩增产物，磁分离后在紫外灯透射下可直接观察荧光，判断检测样品是否为阳性。本发明大大降低了原有环介导等温扩增最大缺陷即假阳性率高的缺点，同时该方法稳定准确，特异性强，操作简便快捷，整个检测过程不需要昂贵的精密设备，降低成本，具有广阔的市场前景。
The present invention provides a method for rapidly detecting loop-mediated isothermal amplification nucleic acid products using magnetic bead probes, which can be used in many fields related to nucleic acid identification. A magnetic bead probe complementary to it is mixed with the nucleic acid to be detected and subjected to a loop-mediated isothermal amplification reaction. The magnetic bead probe recognizes and binds to the amplification product. After magnetic separation, the fluorescence can be directly observed under the transmission of ultraviolet light. Determine whether the test sample is positive. The invention greatly reduces the defect of the original loop-mediated isothermal amplification, that is, the high false positive rate. At the same time, the method is stable and accurate, has strong specificity, is simple and quick to operate, does not require expensive precision equipment in the entire detection process, and reduces costs. Has broad market prospects.
Description
Technical Field
The invention belongs to the field of nucleic acid detection in biotechnology, and particularly relates to a method for rapidly detecting a loop-mediated isothermal amplification nucleic acid product by using a magnetic bead probe.
Background
At present, the detection of pathogen DNA and RNA mainly depends on PCR technology and qPCR technology, and although the PCR and qPCR can detect nucleic acid rapidly and sensitively, the use of the PCR and qPCR technology is greatly limited due to the requirement of expensive equipment, especially in basic medical institutions. Loop-mediated isothermal amplification (LAMP) has attracted attention in recent years because of its constant temperature, simple equipment and easy operation. LAMP requires 4-6 primers, and can amplify a small amount of nucleic acid to millions of parts after 30-60 min. Compared with the conventional PCR, the LAMP sensitivity is higher, 10 to 100 times higher than that of the conventional PCR, and sometimes even single-copy nucleic acid can be amplified by LAMP. The temperature condition required in the LAMP process is stable, and repeated temperature change is not required, so that the required time is short. Most importantly, no complex and expensive instrument and equipment are needed in the LAMP process, and the requirements of primary level and on-site pathogen detection work are met. However, LAMP is prone to produce false positive results, mainly because LAMP product aerosol is prone to environmental pollution and excessive DNA primers in the system may form non-specific complementarity between primers to produce false positive results.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting a loop-mediated isothermal amplification nucleic acid product by using a magnetic bead probe, and aims to solve the problems of high false positive rate and inaccurate detection result when a specific nucleic acid sequence is detected by loop-mediated isothermal amplification.
The purpose of the invention is realized by the following technical scheme:
a method for rapidly detecting a nucleic acid product of loop-mediated isothermal amplification by using a magnetic bead probe comprises the following steps:
s1, designing a loop-mediated isothermal amplification primer of a sample to be detected and preparing a magnetic bead probe specifically combined with the sample to be detected;
s2, premixing the loop-mediated isothermal amplification primer, a buffer solution and DNA polymerase, adding a sample to be detected, heating for reaction to obtain a loop-mediated isothermal amplification product;
s3, adding the magnetic bead probe into the loop-mediated isothermal amplification product, and incubating;
s4, washing the magnetic beads by the isothermal amplification solution, adding a double-stranded DNA dye, and incubating;
and S5, observing the detection signal, and judging whether the sample to be detected is positive.
As a more preferable technical scheme, the magnetic bead probe is a complementary single-stranded probe designed according to a single-stranded annular structure of the loop-mediated isothermal amplification product, and the probe is modified on the surface of the magnetic bead and is used for combining the loop-mediated isothermal amplification product. A plurality of single-chain circular DNA structures exist in a stem-loop sample structure and a multi-loop cauliflower sample structure in the LAMP product, a complementary single-chain DNA probe is designed according to the single-chain circular structure, the DNA probe is combined with magnetic beads to form a magnetic bead probe, and whether target nucleic acid exists in a sample or not is judged through fluorescence.
In a more preferred embodiment of the present invention, the magnetic beads in the magnetic bead probes are, but not limited to, magnetic beads having superparamagnetism.
As a more preferred technical scheme of the invention, the sample to be detected is a gene sample from a pathogen.
As a more preferred technical scheme of the invention, the pathogen gene sample is a pathogen DNA sample or an RNA reverse transcription sample from the pathogen.
As a more preferred embodiment of the present invention, the detection signal in step S5 is a fluorescence signal obtained under an ultraviolet lamp.
As a more preferred technical scheme, the step 5 further comprises the step of setting a negative control group, wherein the negative control group is a sample to be detected without a target nucleic acid sequence, if fluorescence exists or is stronger, the result is judged to be positive, and if no fluorescence exists, the result is judged to be negative.
The invention also provides a method for rapidly detecting the loop-mediated isothermal amplification nucleic acid product by using the magnetic bead probe, which is applied to detecting a target nucleic acid sequence.
The beneficial effects are as follows:
the invention designs the detection method by the characteristics of the magnetic bead probe, greatly reduces the maximum defect of the prior loop-mediated isothermal amplification, namely the defect of high false positive rate, has the advantages of stability, accuracy, strong specificity and simple and quick operation, does not need expensive precise equipment in the whole detection process, and reduces the cost.
The invention can be used in a plurality of fields related to nucleic acid identification and has wide market prospect.
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FIG. 1 is a schematic diagram of the present invention for rapid detection of nucleic acid products by loop-mediated isothermal amplification using magnetic bead probes;
FIG. 2 shows the results of detecting CMV nucleic acid in example 1;
FIG. 3 shows the results of detection of FMD nucleic acid in example 2;
FIG. 4 shows the results of detection of TBEV nucleic acid in example 3;
FIG. 5 shows the results of detecting VEE nucleic acid in example 4;
FIG. 6 shows the results of detection of CCHFV nucleic acid in example 5;
FIG. 7 shows the results of detecting the nucleic acid of the novel coronavirus 0 gene in example 6;
FIG. 8 shows the results of example 7 for detecting the N gene nucleic acid of the novel coronavirus.
Detailed Description
The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the description of the embodiment is only for illustrating and explaining the present invention and is not for limiting the present invention described in detail in the claims. Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional methods, and test materials used therein are available from commercial companies, unless otherwise specified.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the examples of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
Example 1 detection of Cytomegalovirus (CMV)
Step one, selecting a cytomegalovirus gene as a target sequence, wherein the sequence is as follows (5 '-3'): ATGGAGGTCTTGGTGGCCGGCGTGACAGACACGGCGTATGGCGTCCGCGGTTCGGGCACTAGTTCGCCCACGCTGGCAATGACCTCACGCAGCCTATCGGTGTCGCTGTACTCACAGTAAAAGTAGCTGCGCTGCCCGAAAACGTTGACGCAGATACTGTAGCCGTGTTCTGTGGCCCCGAAGAAACGCAACACGTTCCCCGAAGGCACCAGATGCTGACGATAGCGCGGCGACACGTTTTCGGGCGAGTCGAAGAAGAGCACGGCGTCCGTCTGATCGTAGGTGTGAAAACGAATAGGTCCCACCACGCGACCCA
Step two, the loop-mediated isothermal amplification primer is prepared byhttp://primerexplorer.jp/e/Design software design, primer sequence as follows (5 '-3'):
CMV-F3:CTCGCCCGAAAACGTGTC,
CMV-B3:CGCTGGCAATGACCTCAC,
CMV-FIP:CTGTGGCCCCGAAGAAACGCCGCGCTATCGTCAGCATC,
CMV-BIP:TCTGCGTCAACGTTTTCGGGCCCTATCGGTGTCGCTGTACT
step three, the DNA probe sequence is as follows (5 '-3')
CMV-bio-probe：biotin-TTTTTT-CTTTTACTGT；
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20;
isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Step four, preparing a magnetic bead probe:
1. placing the magnetic bead bottle on an endangered vortex oscillator for 20s, oscillating the resuspended magnetic beads, taking 20 mu l of the magnetic beads to a new centrifugal tube, placing the centrifugal tube on a magnetic frame, and removing supernatant after 1 min;
2. washing the magnetic beads twice with 1ml of buffer I;
3. adding 2 μ l of bio-probe into 500 μ l of buffer solution I, adding the washed magnetic beads, fully oscillating the resuspended magnetic beads, placing the centrifuge tube on a rotary mixer, and rotationally mixing at room temperature for 30 min;
4. magnetic separation, transferring the supernatant to a new centrifuge tube, and washing the magnetic beads with a buffer solution I for three times;
5. adding 100 mul of isothermal amplification solution, and resuspending magnetic beads;
step five, loop-mediated isothermal amplification
1. And mixing FIP and BIP 8 μ l each, F3 and B3 1 μ l each, and double distilled water 82 μ l each to obtain primer mixture.
2. Mixing a loop-mediated isothermal amplification primer mixture, a buffer solution, DNA polymerase, double distilled water and a sample to be detected, wherein the volume ratio is 5: 12.5: 1: 4.5: and 2, replacing the negative control group with double distilled water of 2 mu l without adding the sample to be detected, reacting at the constant temperature of 60-65 ℃ for 30-60min, and reacting at 80 ℃ for 5 min.
Step six, detecting nucleic acid by double-stranded DNA dye
1. Adding 10 mu l of magnetic bead probe into the loop-mediated isothermal amplification product, and incubating for 1h at 55 DEG C
2. The beads were washed 3 times with isothermal amplification solution, 10. mu.l of double stranded DNA dye was added and incubated for 10 min.
3. Under ultraviolet lamp for observation
The detection result is shown in fig. 2, the negative control group has no fluorescence, the fluorescence intensity of the sample group is stronger, and the cytomegalovirus in the detected sample can be judged.
Example 2 detection of foot-and-mouth disease Virus (FMD)
Step one, selecting a foot-and-mouth disease virus gene as a target sequence, wherein the sequence is as follows (5 '-3'): ACTATGAGGGAGTTGAGCTGGATACTTACACCATGATCTCCTACGGAGACGACATTGTGGTGGCAAGTGATTATGATCTGGACTTTGAGGCTCTCAAGCCCCATTTCAAATCTTTGGGCCAAACCATCACTCCAGCAGACAAAAGTGACAAGGGCTTCGTCCTTGGTCACTCCATAACTGATGTCACCTTCCTCAAAAGACACTTCCACATGGATTACGGAACTGGGTTTTACAAACCTGTGATGGCTTCGAAGACTCTCGAAGCTATCCTCTCCTTTGCACGCCGTGGGACCATACAGGAGAAGTTGATCTCCGTGGCAGGACTCGCAGTCCACTCTGGACCTGACGAGTACCAGCGTCTCTTCGAGCCCTTCCAGGGCCTCTTTGAGATTCCAAGCTACAGATCACTTTACCTGCGTTGGGTGACGGCCGTGTG
Step two, designing the loop-mediated isothermal amplification primers by http:// primer explorer. jp/e/design software, wherein the primer sequences are as follows (5 '-3'):
FMD-F3:ACATTGTGGTGGCAAGTGA
FMD-B3:CGAAGCCATCACAGGTTTGT
FMD-FIP:TGCTGGAGTGATGGTTTGGCC-TCTGGACTTTGAGGCTCTCA
FMD-BIP:GACAAGGGCTTCGTCCTTGGT-CCCAGTTCCGTAATCCATGT
step three, the DNA probe sequence is as follows (5 '-3')
FMD-bio-probe：biotin-TTTTTTTTTTTTTTTT-CACTCCATAACTGATGTCACCTTC。
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20;
isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Reference example 1 Steps four to six
The detection result is shown in FIG. 3, the negative control group has no fluorescence, the sample group has strong fluorescence intensity, and the foot-and-mouth disease virus in the detected sample can be judged.
Example 3 detection of tick-borne encephalitis Virus (TBEV)
Step one, selecting tick-borne encephalitis virus genes as target sequences, wherein the sequences are as follows (5 '-3'): GCTGTGGACACGGAACGAATGGAGCTCCGCTGTGGCGAGGGCCTGGTCGTGTGGAGAGAGGTCTCCGAATGGTATGACAACTATGCCTACTACCCGGAGACACCGGGGGCCCTTGCATCAGCCATAAAGGAGACATTTGAGGAGGGAAGCTGTGGCGTAGTCCCCCAGAACAGGCTCGAGATGGCCATGTGGAGAAGCTCGGTCACAGAGCTGAATCTGGCTCTGGCGGAAGGGGAGGCAAATCTCACAGTGGTGGTGGACAAGTTTGACCCCACTGACTACCGAGGTGGTGTCCCTGGTTTACTGAAAAAAGGAAAGGACATAAA
Step two, the loop-mediated isothermal amplification primer is prepared byhttp://primerexplorer.jp/e/Design software design, primer sequence as follows (5 '-3'):
TBEV-F3:GAGAGAGGTCTCCGAATGGT
TBEV-B3:GGGTCAAACTTGTCCACCAC
TBEV-FIP:TACGCCACAGCTTCCCTCCTC-GCCTACTACCCGGAGACAC
TBEV-BIP:GCTCGAGATGGCCATGTGGA-GTGAGATTTGCCTCCCCTTC
step three, the DNA probe sequence is as follows (5 '-3')
TBEV-bio-probe:biotin-TTTTTTTTTTTTTTTT-GAAGCTCGGTCACAGAGCTGAA；
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20;
isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Step four to six, step four to six of reference example 1
The detection result is shown in figure 4, the negative control group has no fluorescence, the sample group has strong fluorescence intensity, and the tick-borne encephalitis virus in the detection sample can be judged.
Example 4 detection of Venezuelan equine encephalitis Virus (VEE)
Step one, selecting a Venezuelan equine encephalitis virus gene as a target sequence, wherein the sequence is as follows (5 '-3'): ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAG
Step two, the loop-mediated isothermal amplification primer is prepared byhttp://primerexplorer.jp/e/Design software design, primer sequence as follows (5 '-3'):
VEE-F3:TCAGTCCAGGCCTGTATGG
VEE-B3:CAGTTTTTGCGCGTCGTC
VEE-FIP:ACCCTCTCCCCGTTCAATGTGT-CCTTCAGGCTATGCTGCTAC
VEE-BIP:TCCCGTGTGCACGTATGTGC-CGCACTGACATCTGTTGCC
step three, the DNA probe sequence is as follows (5 '-3')
VEE-bio-probe:biotin-TTTTTTTTTTTTTTTT-CAGCTACATTGTGTGACCAAATG；
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20.
Isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Reference example 1 Steps four to six
As shown in FIG. 5, the detection result shows that the negative control group has no fluorescence, the fluorescence intensity of the sample group is strong, and the venezuelan equine encephalitis virus in the detected sample can be judged.
Example 5 detection of Crimean-Congo hemorrhagic fever Virus (CCHFV)
Step one, selecting a gene of the Climean-Congo hemorrhagic fever virus as a target sequence, wherein the sequence is as follows (5 '-3'): GCAAGTGCTGACAGCATGATAACAAACCTTCTCAAGCATATTGCTAAAGCACAGGAGCTTTACAAAAACTCGTCTGCTCTCCGTGCACAGGGTGCACAGATTGATACCGTCTTTAGCTCGTACTACTGGCTCTACAAGGCCGGCGTGACTCCAGATACTTTCCCGACTGTCTCACAGTTCCTTTTTGAGTTAGGGAAGCAGCCAAGGGGTACCAAGAAAATGAAGAAGGCACTCTTGAGCACCCCAATGAAGTGGGGGAAGAAGCTTTATGAGCTTTTCGCCGATGATTCTTTCCAGCAGAACAGGATCTACATGCACCCTGCTGTGCTGACAGCTGGCA
Step two, the loop-mediated isothermal amplification primer is prepared byhttp://primerexplorer.jp/e/Design software design, primer sequence as follows (5 '-3'):
CCHFV-F3:AACTCGTCTGCTCTCCGT
CCHFV-B3:TTCTTCCCCCACTTCATTGG
CCHFV-FIP:GTCACGCCGGCCTTGTAGAGGGGTGCACAGATTGATACCG
CCHFV-BIP:TCCCGACTGTCTCACAGTTCCTGCTCAAGAGTGCCTTCTTCA
step three, the DNA probe sequence is as follows (5 '-3')
CCHFV-bio-probe:biotin-TTTTTTTTTTTTTTTT–TTTTGAGTTAGGGAAGCAGCC；
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20;
isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Reference example 1 Steps four to six
The detection result is shown in fig. 6, the negative control group has no fluorescence, the fluorescence intensity of the sample group is strong, and the crimean-congo hemorrhagic fever virus in the detection sample can be judged.
Example 6 detection of New coronavirus (Orf1ab)
Step one, selecting a new coronavirus Orf1ab gene as a target sequence, wherein the sequence is as follows (5 '-3'): AAATACCTGGTGTATACGTTGTCTTTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTTGATGTACTGAAGTCAGAGGACGCGCAGGGAATGGATAATCTTGCCTGCGAAGATCTAAAACCAGTCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAAGAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGAATTATCTAG
Step two, the loop-mediated isothermal amplification primer is prepared byhttp://primerexplorer.jp/e/Design software design, primer sequence as follows (5 '-3'):
F3:CGTTGTCTTTGGAGCACAA
B3:TCTCCTACAACTTCGGTAGT
FIP:TTATCCATTCCCTGCGCGTC-ACCAGTTGAAACATCAAATTCG
BIP:TCTTGCCTGCGAAGATCTAAAAC-CATTACACTCAAGAACGTCTT
step three, the DNA probe sequence is as follows (5 '-3')
bio-probe:biotin-TTTTTTTTTTTTTTTT-CAGTCTCTGAAGAAGTAGTGGAAAA；
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20;
isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Reference example 1 Steps four to six
The detection result is shown in fig. 7, the negative control group has no fluorescence, the fluorescence intensity of the sample group is strong, and the new coronavirus in the detection sample can be judged.
Example 7 detection of New coronavirus (nCoV N)
Step one, selecting a new coronavirus nCoV N gene as a target sequence, wherein the sequence is as follows (5 '-3'): CCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACAT
Step two, the loop-mediated isothermal amplification primer is prepared byhttp://primerexplorer.jp/e/Design software design, primer sequence as follows (5 '-3'):
F3:TGGCTACTACCGAAGAGCT
B3:TGCAGCATTGTTAGCAGGAT
FIP:TCTGGCCCAGTTCCTAGGTAGT-GACGAATTCGTGGTGGTGA
BIP:AGACGGCATCATATGGGTTGCA-GCGGGTGCCAATGTGATC
step three, the DNA probe sequence is as follows (5 '-3')
bio-probe:biotin-TTTTTTTTTTTTTTTT-TGGACTGAGATCTTTCATTTTACCG；
Buffer I: 10mM Tris-HC1(pH7.5),1mM EDTA,1M NaC1, 0.01% -0.1% Tween-20;
isothermal amplification solution: 20mM tris-hcl, 10mM ammonium sulphate, 50mM potassium chloride, 2mM magnesium sulphate, 0.1% tween20, ph 8.8.
Reference example 1 Steps four to six
The detection result is shown in fig. 8, the negative control group has no fluorescence, the fluorescence intensity of the sample group is strong, and the new coronavirus in the detection sample can be judged.
As can be seen from FIGS. 2 to 8, the method provided by the present invention can be used for detecting specific nucleic acid sequences, and the detection method is truly effective.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (9)
1. A method for rapidly detecting a nucleic acid product of loop-mediated isothermal amplification by using a magnetic bead probe is characterized by comprising the following steps of:
s1, designing a loop-mediated isothermal amplification primer of a sample to be detected and preparing a magnetic bead probe specifically combined with the sample to be detected;
s2, premixing the loop-mediated isothermal amplification primer, a buffer solution and DNA polymerase, adding a sample to be detected, heating for reaction to obtain a loop-mediated isothermal amplification product;
s3, adding the magnetic bead probe into the loop-mediated isothermal amplification product, and incubating;
s4, washing the magnetic beads by the isothermal amplification solution, adding a double-stranded DNA dye, and incubating;
s5, observing if fluorescence exists, and judging whether the sample to be detected contains a target nucleic acid sequence. The virus was judged to be positive, and if there was no fluorescence, the virus was judged to be negative.
2. The method of claim 1, wherein the magnetic bead probe is used for rapid detection of the loop-mediated isothermal amplification nucleic acid product, and the method comprises: the magnetic bead probe is obtained by modifying a single-stranded probe which is designed and complemented according to a single-stranded annular structure of a loop-mediated isothermal amplification product on the surface of a magnetic bead.
3. The method of claim 1, wherein the magnetic bead probe is used for rapid detection of the loop-mediated isothermal amplification nucleic acid product, and the method comprises: the single-chain probe is modified on the surface of magnetic beads by a biotin-streptavidin system.
4. The method of claim 1, wherein the magnetic bead probe is used for rapid detection of the loop-mediated isothermal amplification nucleic acid product, and the method comprises: the magnetic beads in the magnetic bead probes are, but not limited to, magnetic beads with superparamagnetism.
5. The method of claim 1, wherein the magnetic bead probe is used for rapid detection of the loop-mediated isothermal amplification nucleic acid product, and the method comprises: the sample to be detected is a gene sample from a pathogen.
6. The method of claim 5, wherein the nucleic acid products of the loop-mediated isothermal amplification are rapidly detected using magnetic bead probes, and the method comprises: the pathogen gene sample is a pathogen DNA sample or an RNA reverse transcription sample from the pathogen.
7. The method of claim 1, wherein the magnetic bead probe is used for rapid detection of the loop-mediated isothermal amplification nucleic acid product, and the method comprises: the detection signal in step S5 is a fluorescence signal obtained under an ultraviolet lamp.
8. The method of claim 1, wherein the magnetic bead probe is used for rapid detection of the loop-mediated isothermal amplification nucleic acid product, and the method comprises: and step 5, setting a negative control group, wherein the negative control group is a sample to be detected without a target nucleic acid sequence, if fluorescence exists or the fluorescence is strong, the result is judged to be positive, and if no fluorescence exists, the result is judged to be negative.
9. Use of the method for rapid detection of loop-mediated isothermal amplification nucleic acid products according to any of claims 1 to 9 with magnetic bead probes for detection of target nucleic acid sequences.
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