CN111321249A - Loop-mediated isothermal amplification detection primer group, kit and method for SARS-CoV-2 - Google Patents
Loop-mediated isothermal amplification detection primer group, kit and method for SARS-CoV-2 Download PDFInfo
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Abstract
The invention provides a loop-mediated isothermal amplification detection primer group, a kit and a method for SARS-CoV-2, belonging to the technical field of medical molecular biology diagnosis. Based on the stability and uniqueness of RdRp, E and N genes in SARS-COV-2, the invention designs three groups of primer groups to carry out amplification detection on the genes, reduces the concentration of the primers, improves the specificity of the reaction and reduces false positive; therefore, the invention provides a detection primer group, a kit and a detection method with high detection speed, high sensitivity and strong specificity, and has good practical application value.
Description
Technical Field
The invention belongs to the technical field of medical molecular biology diagnosis, and particularly relates to a loop-mediated isothermal amplification detection primer group, a kit and a detection method for SARS-CoV-2.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). SARS-COV-2 is a new strain of coronavirus which has never been found in human body before, its gene sequence has 79.5% similarity with SARS coronavirus, and its route into cell is identical with SARS coronavirus, i.e. it passes through ACE2 cell receptor, but SARS-COV-2 is more easily spread than SARS which causes SARS.
Currently, clinical and disease control systems confirm viral infection by detecting SARS-COV-2 nucleic acid or viral nucleic acid sequencing by fluorescence PCR methods. The fluorescence PCR method needs a special nucleic acid amplification instrument and a special detection instrument, and two primers are used during amplification to target two gene segments, so that the accuracy is limited, and false positive or false negative exists. Moreover, due to the limitation of sensitivity, the phenomenon of negative nucleic acid detection of a plurality of patients occurs, so that the patients cannot be diagnosed and treated in time, and viruses are further spread and infected due to missed diagnosis, which is not beneficial to epidemic situation control. The pneumonia epidemic situation caused by the infection of the novel coronavirus (SARS-COV-2) needs a more accurate, rapid and convenient detection method and detection reagent. The loop-mediated isothermal amplification (LAMP) method is a new gene amplification method in recent years, adopts 4-6 primers, targets 6 or more gene segments, has strong specificity and particularly high amplification efficiency, enables amplification products to be visible by naked eyes, has low requirements on instrument equipment, operators and sites, and has the following obvious advantages compared with the common nucleic acid amplification technology, namely 1) the whole amplification process is finished at constant temperature without complex temperature-changing equipment; 2) the amplification efficiency is higher and is 10-100 times of that of the common PCR; 3) The time consumption is less, the general PCR amplification needs 2-3h, and the LAMP amplification can be completed only by 0.5-1 h; 4) the result can be seen visually, and is easy to judge, an electrophoresis or fluorescence detector is not needed, the time is saved, and the pollution is avoided. These advantages of LAMP have led to the widespread interest since the invention, but the inventors found that only the kit for detecting Mycobacterium tuberculosis, Salmonella and Legionella is currently commercialized in the medical field, and there is no report on the method and kit for SARS-COV-2.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primer group, a kit and a detection method for LAMP detection for detecting SARS-COV-2 with high detection speed, high sensitivity and specificity based on the stability and uniqueness of RdRp, E and N genes in SARS-COV-2.
One of the purposes of the invention is to provide a loop-mediated isothermal amplification detection primer group for SARS-CoV-2, wherein the LAMP detection primer group at least comprises a first primer group, a second primer group and a third primer group; wherein, the nucleotide sequence of the first primer group is as follows:
F3:5'-GTTACGATGGTGGCTGTA-3'(SEQ ID NO.1)；
B3:5'-GGCATACTTAAGATTCATTTGAG-3'(SEQ ID NO.2)；
FIP:
5'-AGCCTTACCCCATTTATTAAATGGAGCTAACCAAGTCATCGTCAA-3'(SEQ ID NO.3)；
BIP:5'- AATGAGTTATGAGGATCAAGATGCATTATAGTAGGGATGACATTACGT -3'(SEQ IDNO.4)；
Lp:5'-AAACCAGCTGATTTGTCTAGGTTG-3'(SEQ ID NO.5)。
the nucleotide sequence of the second primer set is as follows:
F3:5'-AGATCACATTGGCACCCG-3'(SEQ ID NO.6)；
B3:5'-CCATTGCCAGCCATTCTAGC-3'(SEQ ID NO.7)；
FIP:5'-TGCTCCCTTCTGCGTAGAAGCCAATGCTGCAATCGTGCTAC-3' (SEQ ID NO.8)；
BIP:5'-GGCGGCAGTCAAGCCTCTTCCCTACTGCTGCCTGGAGTT-3'(SEQ ID NO.9)；
LF:5'-GCAATGTTGTTCCTTGAGGAAGTT-3'(SEQ ID NO.10)；
LB:5'-CGTAGTCGCAACAGTTAAGAAATTC-3'(SEQ ID NO.11)。
the nucleotide sequence of the third primer set is as follows:
F3:5'-AGCTGATGAGTACGAACTT-3'(SEQ ID NO.12)；
B3:5'-TTCAGATTTTTAACACGAGAGT-3'(SEQ ID NO.13)；
FIP:
5'-ACCACGAAAGCAAGAAAAAGAAGTATTCGTTTCGGAAGAGACAG-3' (SEQ ID NO.14)；
BIP:5'-TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTCACGT -3'(SEQ IDNO.15)；
LP:5'-CTGCGCTTCGATTGTGTGCGT-3'(SEQ ID NO.16)；
the second purpose of the invention is to provide the application of the primer group in the detection of SARS-CoV-2 based on the loop-mediated isothermal amplification method.
The present invention also aims to provide a LAMP detection method for detecting SARS-CoV-2, which comprises LAMP amplification using the above primer set.
The invention also aims to provide a LAMP detection kit for SARS-CoV-2, which comprises the primer group, reaction buffer solution, BstDNA polymerase and reverse transcriptase.
Wherein the reaction buffer component comprises Tris-HCl (pH 8.8), KCl, MgSO4、(HN4)2SO4Tween20, betaine and dNTPs.
The kit also comprises a color development liquid, wherein the color development liquid is manganese ion chelate calcein.
The detection kit is also provided with a positive control and a negative control.
The fifth purpose of the invention is to provide the application of the kit in detecting SARS-CoV-2 based on the loop-mediated isothermal amplification method.
The beneficial technical effects of one or more technical schemes are as follows:
1. aiming at 6 fragments in highly conserved and specific RdRp, N and E genes in SARS-COV-2 gene, a primer group which has high specificity and rapid reaction and contains 6 primers is self-designed and optimized. The N gene is most sensitive and has the fastest reaction, the 1copy virus gene can be detected within 20 minutes, and false negative does not exist; the RdRp gene is most specific and has no false positive; the three genes are detected together, so that the sensitivity and the specificity are guaranteed.
2. The steps from RNA to amplification and result interpretation are completed in one reaction tube in one step without prior reverse transcription. Reagents required by each reaction are mixed in advance and placed in a reaction tube, and the reaction can be carried out only by adding sample RNA into the reaction tube during detection, so that pollution and errors caused by multiple sample adding are avoided, the requirement on operators is low, and the detection accuracy and convenience are improved;
3. the reaction solution comprises 2 × reaction buffer solution (200mM Tris-HCl, pH 8.8; 100mM potassium chloride; 100mM ammonium sulfate; 20mM magnesium sulfate and 1% TritonX-100, betaine, dNTP, ultrapure water), enzyme solution (1 unit reverse transcriptase, 100 units BstDNA polymerase), primer group and fluorescent dye, the reaction solution adopts a Tris-HCl buffer system, and is more stable, the concentration of the betaine reaches 32 mu mol/20 mu l, so that the activity of the BstDNA polymerase is better protected, the reaction efficiency is improved, the primer concentration is reduced, the reaction specificity is improved, and false positive is reduced;
4. the dye for result judgment adopts calcein, so that the reaction efficiency is high, the specificity is good, only LAMP reaction can be detected but the conventional PCR reaction is invalid, and false positive caused by weak PCR reaction in nucleic acid amplification is avoided;
4. the dye is added into the reaction liquid in advance, and a reaction tube does not need to be opened after the reaction is finished, so that DNA pollution caused by aerosol formation is avoided, the reaction environment is effectively protected, and false positive in subsequent detection is avoided;
5. the reaction is rapid and can be finished within 30 min; the time range for judging the result is set to be 25-35min, and because false positive can be generated after 40min, the result after 40min is not interpreted, and the accuracy of the detection result is ensured.
Compared with the similar reagent kit, the reagent kit is more convenient, rapid, specific and accurate, and has low requirements on instruments, equipment and operators.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a diagram showing the result of LAMP method visual identification of SARS-COV-2 (RdRp gene) in the example of the present invention, in which the left tube is negative and the right tube is positive;
FIG. 2 is a result chart of specificity (RdRp gene) of the LAMP visual detection kit in the embodiment of the present invention; wherein, 1-2 are positive samples, and 3-8 are negative samples.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present reagent kit for detecting SARS-COV-2 is fluorescence PCR method, and the fluorescence PCR reagent kit for detecting SARS-COV-2 by clinical and disease control system is mainly designed for N gene and ORF1ab (the RdRp gene used by said reagent kit is positioned in ORF1 ab), and some are designed for N gene, ORF1ab (or RdRp) and E gene. In clinical use, the specificity of the N gene is relatively poor, and a plurality of samples only have positive N gene, but have negative ORF1 ab. Besides SARS-COV-2 and SARS and MERS, there are also four kinds of human diseases caused by coronavirus, which are one of the main pathogens of common cold of adults, and the children have high infection rate, mainly upper respiratory tract infection, and generally have little influence on the lower respiratory tract. The N gene has poor specificity to SARS-COV-2, four common coronaviruses may exist in the N gene positive sample, but in order to avoid detection omission, the kit still contains the N gene, and in order to further improve the detection accuracy, the kit can simultaneously detect the E gene and the RdRp gene. Therefore, the invention designs primers aiming at the RdRp, N and E genes of SARS-COV-2, and adopts the loop-mediated isothermal amplification technology to amplify and detect.
In an exemplary embodiment of the invention, a loop-mediated isothermal amplification detection primer set for SARS-CoV-2 is provided, wherein the LAMP detection primer set at least comprises a first primer set, a second primer set and a third primer set; wherein, the first primer group is used for amplifying the RdRp gene, and the nucleotide sequence is as follows:
F3:5'-GTTACGATGGTGGCTGTA-3'(SEQ ID NO.1)；
B3:5'-GGCATACTTAAGATTCATTTGAG-3'(SEQ ID NO.2)；
FIP:5'- AGCCTTACCCCATTTATTAAATGGAGCTAACCAAGTCATCGTCAA-3'(SEQ IDNO.3)；
BIP:5'- AATGAGTTATGAGGATCAAGATGCATTATAGTAGGGATGACATTACGT -3'(SEQ IDNO.4)；
Lp:5'-AAACCAGCTGATTTGTCTAGGTTG-3'(SEQ ID NO.5)。
the second primer group is used for amplifying the N gene, and the nucleotide sequence is as follows:
F3:5'-AGATCACATTGGCACCCG-3'(SEQ ID NO.6)；
B3:5'-CCATTGCCAGCCATTCTAGC-3'(SEQ ID NO.7)；
FIP:5'-TGCTCCCTTCTGCGTAGAAGCCAATGCTGCAATCGTGCTAC-3' (SEQ ID NO.8)；
BIP:5'-GGCGGCAGTCAAGCCTCTTCCCTACTGCTGCCTGGAGTT -3'(SEQ ID NO.9)；
LF:5'-GCAATGTTGTTCCTTGAGGAAGTT-3'(SEQ ID NO.10)；
LB:5'-CGTAGTCGCAACAGTTAAGAAATTC(SEQ ID NO.11)。
the third primer group is used for amplifying the E gene, and the nucleotide sequence is as follows:
F3:5'-AGCTGATGAGTACGAACTT-3'(SEQ ID NO.12)；
B3:5'-TTCAGATTTTTAACACGAGAGT-3'(SEQ ID NO.13)；
FIP:5'- ACCACGAAAGCAAGAAAAAGAAGTATTCGTTTCGGAAGAGACAG-3'(SEQ IDNO.14)；
BIP:5'-TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTCACGT -3'(SEQ IDNO.15)；
LP:5'-CTGCGCTTCGATTGTGTGCGT-3'(SEQ ID NO.16)；
in still another embodiment of the present invention, the primer set is used for detecting SARS-CoV-2 by loop-mediated isothermal amplification.
In still another embodiment of the present invention, there is provided a LAMP detection method for detecting SARS-CoV-2, which comprises performing LAMP amplification using the above-mentioned primer set.
In another embodiment of the present invention, the detection method includes:
s1, configuring an LAMP reaction system, and amplifying a reaction template through an LAMP reaction program;
s2, judging whether the sample contains SARS-CoV-2 by observing the color change of the reaction tube with naked eyes.
Wherein, in the step S1,
the LAMP reaction system comprises a reaction solution and an RNA sample to be detected, wherein the reaction solution comprises the primer group, a reaction buffer solution, Bst DNA polymerase, reverse transcriptase and a color development solution.
Wherein, the color developing solution is manganese ion chelate calcein.
Wherein the RNA template includes, but is not limited to, RNA samples obtained from throat fluid, alveolar lavage fluid, saliva, sputum, blood, urine, and stool;
the LAMP reaction conditions are as follows: reacting at constant temperature of 60-65 ℃ for 20-30 min.
In step S2, if the reaction product is a green turbid liquid by visual observation, SARS-CoV-2 is confirmed to be contained; if the reaction product is an orange transparent liquid, it indicates that SARS-CoV-2 is not contained.
In another embodiment of the present invention, the detection method further comprises setting a positive control and a negative control;
in another embodiment of the present invention, the positive control is a method in which PC RNA is added to the reaction solution; the negative control is that ultrapure water is added into the reaction solution.
In still another embodiment of the present invention, there is provided a LAMP detection kit for SARS-CoV-2, which comprises the above primer set, reaction buffer, BstDNA polymerase and reverse transcriptase.
Wherein the reaction buffer component comprises Tris-HCl (pH 8.8), KCl, MgSO4、(HN4)2SO4Tween20, betaine and dNTPs.
The kit also comprises a color development liquid, wherein the color development liquid is manganese ion chelate calcein.
The detection kit is also provided with a positive control and a negative control.
In still another embodiment of the present invention, the kit is used for detecting SARS-CoV-2 by loop-mediated isothermal amplification.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.
Examples
RNA extraction
RNA used for amplification is extracted by a commercially available qualified extractor and a reagent, and no special requirement exists.
2. Primer design and screening
According to the three gene sequences of RdRp, N and E, Primer Explorer V5 software is respectively utilized to design a plurality of groups of primers, each Primer group comprises a pair of specific inner primers and a pair of specific outer primers, the reaction processes and results of different primers are monitored and screened according to the reaction time and specificity, and the optimal Primer with fast reaction and high specificity is determined. The sequences of the primers screened are shown in Table 1.
TABLE 1 primer sequences
LAMP reaction System
The LAMP reaction system is 12.5. mu.L, 10. mu.L of reaction solution is added during the reaction, and 2.5. mu.L of RNA to be detected is added. Positive and negative controls were set simultaneously.
The positive controls were: during reaction, PC RNA is added into reaction liquid;
negative controls were: during the reaction, ultrapure water was added to the reaction solution.
10 μ L of the reaction solution contained: reaction buffer solution, Bst DNA polymerase, color development solution, an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a loop primer;
wherein, the components and the content of the reaction buffer solution are shown in a table 2;
TABLE 2 reaction buffer composition and content
The active content of Bst DNA polymerase is: 100 units
The active content of the reverse transcriptase is as follows: 1 unit of
The color developing solution is as follows: 0.5nmol of manganese ion chelate calcein;
the content of each primer is shown in Table 3;
TABLE 3 content of each primer in 20. mu.L reaction system
4. Kit set
Each kit comprises a detection tube and a control tube, and at least the detection tube, a positive control and a negative control are arranged, the kit is provided with 8 persons, and comprises 24 detection tubes (8 × 3 rows, 1 row of RdRp, N and E genes respectively), 1 positive control tube and 1 negative control tube.
5. Reaction conditions
Adding 2.5 mu L of RNA to be detected into the reaction solution of the LAMP reaction tube, mixing uniformly, adding PC RNA into a positive control, and adding sterile ultrapure water into a negative control. Placing the LAMP reaction tube in a water bath kettle or a metal bath for amplification, wherein the amplification conditions are as follows: reacting in water bath at 60-65 deg.C for 20-30 min.
6. Determination of results
In the course of the LAMP reaction, pyrophosphate ions, which are a by-product produced as a result of the synthesis of a large amount of DNA, are produced in a concentration proportional to the amount of DNA produced. At the initial stage of the reaction, calcein in the developing solution is combined with manganese ions as a fluorescence quencher and does not fluoresce. The pyrophosphate ions are more easily combined with the manganese ions to release calcein. Free calcein can fluoresce spontaneously, and the fluorescent effect is enhanced in the presence of magnesium ions. And such fluorescence can be observed by the naked eye under natural light. Before the amplification reaction, the reaction solution is light orange, and the reaction solution becomes green after the RNA of the sample to be detected is reversely transcribed and amplified. Therefore, the reaction tube is not required to be opened from the beginning of the reaction to the interpretation of the result, and the DNA pollution and the generation of false positive caused by the formation of aerosol can be effectively avoided.
Therefore, the reaction solution turned green, which is a positive result; the reaction solution was light orange without changing color, which was a negative result. If the reaction time is prolonged to more than 30min, false positive may occur, and the result determination is within 30 minutes.
The kit detects 3 genes, and under the condition that negative and positive controls are normal, the result judgment is as follows:
TABLE 4 combination of genes judged to be positive for SARS-CoV-2
7. Sensitivity detection
And (3) performing serial dilution on the RNA by DEPC water to ensure that the dilution concentration is 10, 20, 40, 80, 100, 200, 400, 800 and 1000 times, simultaneously performing LAMP and fluorescence PCR, detecting the lowest template concentration with positive reactions in the two methods, and calculating the detection limit of the kit. The results show that the fluorescence PCR kit can detect 20 times diluted samples, and the detection limit in the fluorescence PCR instruction is 1000 copies/ml. The N gene in the three genes of the kit can detect a sample diluted by 400 times, which shows that the detection sensitivity is 20 times higher than that of a fluorescent PCR kit, the detection limit is 50copies/ml, the E gene can detect a sample diluted by 80 times, the detection limit is 250copies/ml, RdRp can detect a sample diluted by 40 times, the detection limit is 500copies/ml, the detection limit of the kit is comprehensively evaluated to be 500copies/ml, and the detection sensitivity is 2 times of that of the kit used clinically. Moreover, the sensitivity of the N gene in the kit is very high, which is 20 times of the detection sensitivity of the kit used in clinic, and the positive sample of the N gene needs to be further checked, so that the detection omission can be avoided.
TABLE 5 detection sensitivity of each gene in the kit
8. Specificity detection
Selecting clinical detection positive and negative specimens of the novel coronavirus pneumonia, and positive specimens of influenza A, influenza B and respiratory syncytial virus, extracting RNA, and performing LAMP amplification. As a result, at 35 minutes, only the clinically positive RdRp gene and E gene of the novel coronavirus are positive, and other samples are negative, which indicates that the primer has high specificity.
9. Clinical sample application assay
207 clinically confirmed specimens (21 positive specimens and 186 negative specimens) were detected by using the kit. 20 parts of positive specimen and 187 parts of negative specimen are identified by the kit. The non-verified 1 positive sample was negative again by fluorescence PCR, the reason for analysis was RNA degradation. By adopting the kit for detection, only 30 minutes are needed from the amplification of a sample to the end of result judgment, while the traditional fluorescent PCR needs nearly 3 hours. Compared with the prior art, the kit has the characteristics of rapidness, convenience, accuracy and strong operability.
TABLE 6 results of clinical samples of each gene assay of the kit
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Jinan City central hospital
<120> SARS-CoV-2 loop-mediated isothermal amplification detection primer group, kit and method
<130>
<160>16
<170>PatentIn version 3.3
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Claims (10)
1. The LAMP detection primer group is characterized by at least comprising a first primer group for amplifying the RdRp gene, a second primer group for amplifying the N gene and a third primer group for amplifying the E gene.
2. The loop-mediated isothermal amplification detection primer set according to claim 1, wherein the nucleotide sequence of the first primer set is as follows:
F3:5'-GTTACGATGGTGGCTGTA-3'(SEQ ID NO.1)；
B3:5'-GGCATACTTAAGATTCATTTGAG-3'(SEQ ID NO.2)；
FIP:5'-AGCCTTACCCCATTTATTAAATGGAGCTAACCAAGTCATCGTCAA-3'(SEQ ID NO.3)；
BIP:5'-AATGAGTTATGAGGATCAAGATGCATTATAGTAGGGATGACATTACGT-3'(SEQ ID NO.4)；
Lp:5'-AAACCAGCTGATTTGTCTAGGTTG-3'(SEQ ID NO.5)。
3. the loop-mediated isothermal amplification detection primer set according to claim 1, wherein the second primer set has the following nucleotide sequence:
F3:5'-AGATCACATTGGCACCCG-3'(SEQ ID NO.6)；
B3:5'-CCATTGCCAGCCATTCTAGC-3'(SEQ ID NO.7)；
FIP:5'-TGCTCCCTTCTGCGTAGAAGCCAATGCTGCAATCGTGCTAC-3'(SEQ ID NO.8)；
BIP:5'-GGCGGCAGTCAAGCCTCTTCCCTACTGCTGCCTGGAGTT-3'(SEQ ID NO.9)；
LF:5'-GCAATGTTGTTCCTTGAGGAAGTT-3'(SEQ ID NO.10)；
LB:5'-CGTAGTCGCAACAGTTAAGAAATTC-3'(SEQ ID NO.11)。
4. the loop-mediated isothermal amplification detection primer set according to claim 1, wherein the nucleotide sequence of the third primer set is as follows:
F3:5'-AGCTGATGAGTACGAACTT-3'(SEQ ID NO.12)；
B3:5'-TTCAGATTTTTAACACGAGAGT-3'(SEQ ID NO.13)；
FIP:5'-ACCACGAAAGCAAGAAAAAGAAGTATTCGTTTCGGAAGAGACAG-3'(SEQ ID NO.14)；
BIP:5'-TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTCACGT-3'(SEQ ID NO.15)；
LP:5'-CTGCGCTTCGATTGTGTGCGT-3'(SEQ ID NO.16)。
5. use of the primer set according to any one of claims 1 to 4 for detecting SARS-CoV-2 based on the loop-mediated isothermal amplification method.
6. An LAMP detection method for detecting SARS-CoV-2, which comprises performing LAMP amplification using the primer set according to any one of claims 1 to 4.
7. The LAMP detection method for detecting SARS-CoV-2 according to claim 6, wherein the detection method comprises:
s1, configuring an LAMP reaction system, and amplifying a reaction template through an LAMP reaction program;
s2, judging whether the sample contains SARS-CoV-2 or not by observing the color change of the reaction tube by naked eyes;
preferably, in the step S1,
the LAMP reaction system comprises a reaction solution and an RNA sample to be detected, wherein the reaction solution comprises the primer group, a reaction buffer solution, Bst DNA polymerase, reverse transcriptase and a color development solution;
preferably, the color developing solution is manganese ion chelate calcein.
Preferably, the RNA template includes, but is not limited to, RNA samples obtained from throat fluid, alveolar lavage fluid, saliva, sputum, blood, urine, and stool;
preferably, the LAMP reaction conditions are as follows: reacting at constant temperature of 60-65 ℃ for 20-30 min;
preferably, in step S2, when the reaction product is a green turbid liquid, SARS-CoV-2 is confirmed; if the reaction product is orange transparent liquid, SARS-CoV-2 is not contained;
preferably, the detection method further comprises setting a positive control and a negative control;
8. an LAMP detection kit for SARS-CoV-2, which comprises the primer set according to any one of claims 1 to 4, a reaction buffer, BstDNA polymerase and a reverse transcriptase.
9. The kit of claim 8, wherein the reaction buffer component comprises Tris-HCl, KCl, MgSO4、(HN4)2SO4Tween20, betaine and dNTPs;
preferably, the kit further comprises a color development liquid, wherein the color development liquid is manganese ion chelate calcein;
preferably, the detection kit is also provided with a positive control and a negative control.
10. Use of the kit according to claim 9 for detecting SARS-CoV-2 based on loop-mediated isothermal amplification.
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Application publication date: 20200623 |