CN116463461A - Novel freeze-dried coronavirus detection kit based on visual RT-LAMP - Google Patents
Novel freeze-dried coronavirus detection kit based on visual RT-LAMP Download PDFInfo
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Abstract
The invention provides a novel RT-LAMP primer group for coronavirus detection and a freeze-drying type novel coronavirus detection kit based on visual RT-LAMP, wherein the primer group comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; the nucleotide sequences of the inner primers FIP and BIP are shown in SEQ ID NO. 1-2; the nucleotide sequences of the outer primers F3 and B3 are shown in SEQ ID NO. 3-4; the nucleotide sequences of the loop primers LF and LB are shown in SEQ ID NO. 5-6. The kit comprises RT-LAMP reaction liquid, positive control, negative control and double solution. The kit is used for detecting the novel coronavirus and has high specificity and sensitivity.
Description
Technical Field
The invention relates to a freeze-dried novel coronavirus detection kit based on a visual RT-LAMP, and belongs to the technical field of detection of novel coronavirus nucleic acid.
Background
Coronaviruses are a large virus family and are known to cause relatively serious diseases such as common cold, middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus (2019-nCoV) is a novel strain of coronavirus that has never been found in humans before, and is known as the 7 th coronavirus that can infect humans.
The novel coronavirus is a positive-strand RNA virus with an envelope, and is mainly characterized by human transmission, rapid propagation speed, high transmission rate and rapid variation, and clinical diagnosis difficulty is increased due to combined infection with other respiratory viruses. Common signs of a person infected with coronavirus are common respiratory symptoms, fever, cough, shortness of breath, dyspnea, and the like. In more severe cases, infection with a novel coronavirus can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.
According to the research of the diagnosis guidelines and detection technology of novel coronaviruses at home and abroad, the detection method mainly comprises pathogen separation culture detection, nucleic acid amplification detection, sequencing, immunological detection and the like.
Reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification, RT-LAMP) is a detection technology for rapidly and efficiently amplifying RNA by using high-temperature resistant reverse transcriptase, strand displacement DNA polymerase and 4-6 primers designed for 6-8 regions of a target gene at a constant temperature of 60-65 ℃. Colorimetric RT-LAMP (cRT-LAMP) refers to a nucleic acid amplification technology in which a visual dye is added into a system before a reaction, and a rapid interpretation reaction result is realized by observing the color change difference before and after the RT-LAMP reaction. cRT-LAMP can realize rapid detection of samples, and provides a method and means for rapid screening and treatment of primary intimate contact persons and suspected cases.
However, the specificity and sensitivity of the detection of the reagent for detecting novel coronavirus cRT-LAMP in the prior art are not ideal.
Disclosure of Invention
The invention provides a freeze-dried novel coronavirus detection kit based on a visual RT-LAMP, which can effectively solve the problems.
The invention is realized in the following way:
in a first aspect, the invention provides a novel coronavirus detection RT-LAMP primer group, which comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; the nucleotide sequences of the inner primers FIP and BIP are shown in SEQ ID NO. 1-2; the nucleotide sequences of the outer primers F3 and B3 are shown in SEQ ID NO. 3-4; the nucleotide sequences of the loop primers LF and LB are shown in SEQ ID NO. 5-6.
As a further improvement, the molar ratio of the inner primer, the outer primer and the loop primer is 8:1:2.
in a second aspect, the invention provides a novel coronavirus detection RT-LAMP reaction solution, which comprises the primer group, RT-LAMP Buffer and enzyme Mix; the enzyme Mix comprises Bst DNA polymerase, gao Wenni-resistant transcriptase and RNase inhibitor.
As a further improvement, the RT-LAMP Buffer includes (NH 4 ) 2 SO 4 、MgSO 4 KCl, DTT, dNTPs, phenol red.
As a further improvement, a lyoprotectant is also included, comprising 6wt% sucrose, 2wt% sorbitol, 6wt% peg20000, 3wt% pvp k30, the remainder being DEPC water.
In a third aspect, the invention provides a freeze-drying method of novel coronavirus detection RT-LAMP reaction liquid, the novel coronavirus detection RT-LAMP reaction liquid is subpackaged and then freeze-dried, and freeze-drying curve parameters of the freeze-drying are as follows:
pre-freezing: -50 ℃ for 2 hours;
sublimation drying: -40 ℃,0.5h, vacuum degree 10Pa; -40 ℃,10h, and 10Pa vacuum degree;
and (5) analysis and drying: -30 ℃,0.5h, vacuum degree 10Pa; -20 ℃,0.5h, vacuum degree 10Pa; -10 ℃,0.5h, vacuum degree 10Pa;0 ℃ for 0.5h, and the vacuum degree is 10Pa;10 ℃ for 2 hours, and the vacuum degree is 10Pa;20 ℃,4h and 10Pa vacuum degree.
In a fourth aspect, the invention provides a novel lyophilized coronavirus detection kit based on visual RT-LAMP, which comprises the RT-LAMP reaction solution, positive control, negative control and double solution.
As a further improvement, the positive control is a novel coronavirus N gene fragment plasmid.
In a fifth aspect, the present invention provides a method for using the above-mentioned novel lyophilized coronavirus detection kit based on visual RT-LAMP, adding a reconstitution solution into an RT-LAMP reaction solution to reconstitute a lyophilized reagent, then sequentially adding a nucleic acid to be tested after treatment, a negative control and a positive control, mixing uniformly, performing RT-LAMP reaction amplification, observing color change of a liquid in a reaction tube after the reaction is finished, and determining a result according to the color change.
As a further improvement, the reaction temperature of the RT-LAMP reaction is 63-65 ℃ and the reaction time is 28-32min.
The beneficial effects of the invention are as follows:
the design of the primer group is a technical key, 6 specific amplification primers are designed according to the N gene sequence of the novel coronavirus, the primers comprise inner primers FIP and BIP, outer primers F3 and B3 and loop primers LF and LB, the primer group can only specifically identify and amplify the nucleic acid of the novel coronavirus (2019-nCoV), can not amplify the nucleic acid of other coronaviruses, can not amplify the nucleic acid of other respiratory viruses such as influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, human metapneumovirus and the like, and has strong specificity for detecting the novel coronavirus (2019-nCoV).
The freeze-dried novel coronavirus detection kit based on the visual RT-LAMP has high sensitivity, and the amplification efficiency can be obviously improved by adding a pair of loop primers and an RNase inhibitor into a reaction system, and the detection LOD is 10copies/rxn.
The novel freeze-dried coronavirus detection kit based on the visual RT-LAMP adds the phenol red indicator in the reaction system, and can directly achieve the effect of judging visual results according to the color change before and after the reaction.
The invention provides a freeze-drying protective agent and a freeze-drying curve of a colorimetric RT-LAMP, which can enable a kit to be stored for 6 months under normal temperature conditions and not depend on low temperature conditions for storage and cold chain transportation, so that the freeze-drying kit can effectively ensure the performance of the kit and is convenient and fast to store, transport and use.
The freeze-dried novel coronavirus detection kit based on the visual RT-LAMP is used for detecting novel coronaviruses, has the advantages of rapidness, high efficiency, simplicity and convenience in operation, good practicability and the like, does not need expensive special instruments, and is low in cost. Therefore, the kit is suitable for field detection and provides convenience for effectively diagnosing novel coronaviruses on the national border.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of an RT-LAMP reaction tube after lyophilization, containing a lyophilization reagent, according to an embodiment of the present invention.
Fig. 2 is a negative result diagram of a freeze-dried novel coronavirus detection kit based on visual RT-LAMP for detecting novel coronaviruses, which is provided by the embodiment of the invention.
Fig. 3 is a positive result diagram of the use of the freeze-dried novel coronavirus detection kit based on the visual RT-LAMP in detecting novel coronaviruses.
Fig. 4 is a graph showing the results of calculating LOD using SPSS in a sensitivity experiment for detecting novel coronaviruses using a freeze-dried type novel coronavirus detection kit based on visual RT-LAMP provided by the embodiment of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
The invention provides a novel RT-LAMP primer group for coronavirus detection, which comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; the nucleotide sequences of the inner primers FIP and BIP are shown in SEQ ID NO. 1-2; the nucleotide sequences of the outer primers F3 and B3 are shown in SEQ ID NO. 3-4; the nucleotide sequences of the loop primers LF and LB are shown in SEQ ID NO. 5-6. The primer group designs 6 specific amplification primers according to the characteristic site (GenBank: NC-045512.2 (543-741)) of the N gene sequence of the novel coronavirus, and can only specifically identify and amplify the nucleic acid of the novel coronavirus (2019-nCoV), so that the detection specificity is high.
As a further improvement, the molar ratio of the inner primer, the outer primer and the loop primer is 8:1:2, for example, each 25. Mu.L of RT-LAMP reaction solution contains 1.6. Mu. Mol/L of inner primers FIP and BIP, 0.4. Mu. Mol/L of loop primers LF and LB, and 0.2. Mu. Mol/L of outer primers F3 and B3. The amplification efficiency and sensitivity are optimal at this ratio.
In a second aspect, the invention provides a novel coronavirus detection RT-LAMP reaction solution, which comprises the primer group, RT-LAMP Buffer and enzyme Mix. The enzyme Mix comprises Bst DNA polymerase, gao Wenni-resistant transcriptase and RNase inhibitor, and the proportion of each enzyme in the enzyme Mix is Bst DNA polymerase 8U and Gao Wenni-resistant transcriptase 200U, RNA enzyme inhibitor 50U. The high temperature resistant reverse transcriptase is a reverse transcriptase cloned and expressed by a gene recombination technology, partial structural domains of reverse transcriptase genes of different species are modified into chimeras by a genetic engineering means, the RNaseH activity is completely lost, and the high temperature resistant reverse transcriptase has good heat stability and the activity range is 37-65 ℃. The RNase inhibitor can inhibit the activities of RNase A and B, C enzyme with high efficiency, has no influence on the activities of Bst DNA polymerase and reverse transcriptase, and can obviously improve the amplification efficiency and detection sensitivity by adding the RNase inhibitor.
As a further improvement, the RT-LAMP Buffer includes (NH 4 ) 2 SO 4 MgSO4, KCl, DTT, dNTPs, phenol red. Wherein the concentration of each reagent is (NH) 4 ) 2 SO 4 ：100mM，MgSO 4 :80mM, KCl:100mM, DTT:120mM, dNTPs:14mM, phenol red: 0.5mM. A phenol red indicator is added in the RT-LAMP reaction system, the color of the phenol red indicator changes from red to yellow, and compared with the traditional Hydroxy Naphthol Blue (HNB), the color of the phenol red indicator changes from violet to sky blue. Therefore, compared with the phenol red indicator, the color change is more obvious, and the interpretation of the detection result is more facilitated.
As a further improvement, a lyoprotectant is also included, comprising 6wt% sucrose, 2wt% sorbitol, 6wt% peg20000, 3wt% pvp k30, the remainder being DEPC water. The sucrose can protect protein in the freeze-drying and water-losing process, and is used as a framework support to shape a freeze-drying reagent; sorbitol as a cryoprotectant and excipient; PEG20000 and PVP K30 are mainly used as skeleton supports to shape freeze-dried reagents, and promote RT-LAMP reaction.
In a third aspect, the invention provides a freeze-drying method of novel coronavirus detection RT-LAMP reaction liquid, and the novel coronavirus detection RT-LAMP reaction liquid is subpackaged and then freeze-dried.
The vacuum freeze drying technology is a drying technology which freezes wet materials or solution into solid state at a lower temperature (-10 ℃ to minus 50 ℃), then sublimates moisture in the solid state into gas state without liquid state under the condition of vacuum (1.3 to 13 Pa), and finally dehydrates the materials. The freeze-drying protective agent is added into the RT-LAMP system to carry out vacuum freeze-drying, short for freeze-drying, and the aim is to remove free water in substances and part of bound water adsorbed in solid lattice gaps in a low-temperature and vacuum environment. Freeze-drying is generally divided into three steps: prefreezing, sublimation drying, and desorption drying. In the first drying step, not all the material is dried at the same time, but there is an interface that always moves inward from the dry outer layer boundary. In the second drying step, a small percentage of non-frozen or partially bound water left over from the first drying step is removed and reduced to an optimal value for product storage.
The freeze-drying curve parameters of the freeze-drying are as follows:
pre-freezing: -50 ℃ for 2 hours;
sublimation drying: -40 ℃,0.5h, vacuum degree 10Pa; -40 ℃,10h, and 10Pa vacuum degree;
and (5) analysis and drying: -30 ℃,0.5h, vacuum degree 10Pa; -20 ℃,0.5h, vacuum degree 10Pa; -10 ℃,0.5h, vacuum degree 10Pa;0 ℃ for 0.5h, and the vacuum degree is 10Pa;10 ℃ for 2 hours, and the vacuum degree is 10Pa;20 ℃,4h and 10Pa vacuum degree.
Since conventional liquid version reagents are dependent on low temperature conditions for storage and cold chain transport, lyophilized reagents can be stored and transported at ambient temperature. The freeze-drying protective agent and the freeze-drying curve of the colorimetric method RT-LAMP can obtain a freeze-drying reagent with complete appearance and water content of less than 5%, and provide convenience for preserving, transporting and using the kit while ensuring the stability and performance of the reagent.
In a fourth aspect, the invention provides a novel lyophilized coronavirus detection kit based on visual RT-LAMP, which comprises the RT-LAMP reaction solution, positive control, negative control and double solution.
As a further improvement, the positive control is a novel coronavirus N gene fragment plasmid.
In a fifth aspect, the present invention provides a method for using the above-mentioned novel lyophilized coronavirus detection kit based on visual RT-LAMP, adding a reconstitution solution into an RT-LAMP reaction solution to reconstitute a lyophilized reagent, then sequentially adding a nucleic acid to be tested after treatment, a negative control and a positive control, mixing uniformly, performing RT-LAMP reaction amplification, observing color change of a liquid in a reaction tube after the reaction is finished, and determining a result according to the color change. The pH change range of the phenol red indicator is 6.8 (yellow) to 8.4 (red), and pyrophosphate and hydrogen ions generated in the RT-LAMP reaction are released as byproducts, so that the pH gradually changes to be acidic, and the color before and after the reaction is changed, thereby achieving the effect of judging the visual result.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is divided into three stages.
Stage 1 is a reverse transcription stage, in which the RNA template is reverse transcribed into cDNA complementary thereto using a thermostable reverse transcriptase.
Stage 2 is the initiation of amplification, and when any one of the primers base-pair extends to the complementary site of the double-stranded DNA, the other strand is dissociated into single strands. The F2 sequence of the upstream inner primer FIP is first bound to the template F2c and extended forward to initiate strand displacement synthesis by the action of DNA polymerase having strand displacement activity. The outer primer F3 binds to the template F3c and extends, displacing the complete FIP-ligated complementary single strand. F1c on FIP and F1 on the single strand are complementary structures, and self base pairing forms a circular structure. With the strand as a template, the downstream primer BIP and the downstream primer B3 sequentially start synthesis similar to FIP and F3 to form a single strand with a dumbbell structure. Rapidly taking the F1 segment at the 3' -end as a starting point and taking the F1 segment as a template, and performing DNA synthesis and extension to form a stem-loop structure. This structure is the starting structure for the LAMP gene amplification cycle.
The 3 rd stage is an amplification cycle stage, and FIP is combined with the F2c region of the stem loop by taking the stem loop structure as a template. The strand displacement synthesis is started, and a circular structure is formed on the dissociated single-stranded nucleic acid. Rapidly taking the B1 segment at the 3' end as a starting point, taking the B1 segment as a template, performing DNA synthesis extension and strand displacement to form 2 pieces of DNA with new stem loop structures with different lengths. B2 on the BIP primer hybridizes to it, a new round of amplification is initiated, and the product DNA length is doubled. 2 cyclic primers LF and LB are added into the reaction system, and are combined with a stem-loop structure to start strand displacement synthesis, so that the cycle is repeated. The final product of amplification is a mixture of DNA of different length with different numbers of stem-loop structures. And the product DNA is an alternating inverted repeat of the amplified target sequence.
As a further improvement, the reaction temperature of the RT-LAMP reaction is 63-65 ℃ and the reaction time is 28-32min.
EXAMPLE 1 preparation of RT-LAMP reaction solution
1) Colorimetric RT-LAMP reaction solutions were prepared as follows in Table 1:
TABLE 1
High temperature resistant reverse transcriptase: guangzhou Meiger Biotechnology Co., ltd (goods number: A003S); rnase inhibitors: ebolac Biotechnology Inc. (cat# RK 21401).
The above-mentioned inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB were all artificially synthesized DNA fragments, which were synthesized by the division of Biotechnology (Shanghai) and purified by HPLC. The base sequences are respectively as follows:
inner primer FIP:
5’-TGCCAGCCATTCTAGCAGGAGATCACGTAGTCGCAACAGTTC-3’
(SEQ ID NO:1)
inner primer BIP:
5’-GCGGTGATGCTGCTCTTGCTTTTGTTGGCCTTTACCAGACA-3’(SEQ ID NO:2)
outer primer F3:5'-AGCCTCTTCTCGTTCCTCA-3' (SEQ ID NO: 3)
Outer primer B3:5'-AGTGACAGTTTGGCCTTGTT-3' (SEQ ID NO: 4)
Loop primer LF:5'-GTTCCCCTACTGCTGCCT-3' (SEQ ID NO: 5)
Loop primer LB:5'-GCTGCTTGACAGATTGAACCAG-3' (SEQ ID NO: 6)
2) And (3) subpackaging the RT-LAMP reaction liquid into eight-connecting tubes by using an eight-connecting-row pipettor, wherein the subpackaging volume is 20 mu L, and the subpackaging is finished and the RT-LAMP reaction liquid is transferred into a freeze dryer for freeze drying.
3) Lyophilization was performed by setting lyophilization curve parameters as in table 2 below.
TABLE 2
4) And after the freeze-drying is finished, taking out the eight-connecting tube, and covering the eight-connecting tube with an eight-connecting tube cover. 3 strips/bag are put into an aluminum foil bag, a bag of drier is put into the aluminum foil bag, and a sealing machine is used for sealing.
5) Positive control preparation and split charging: the plasmid containing the novel coronavirus (2019-nCoV) N gene fragment was diluted to 1000 copies/. Mu.L and then split-packed into 1.5mL centrifuge tubes at a loading of 150. Mu.L/tube.
6) Split charging of negative control: DEPC water was dispensed in a 1.5mL centrifuge tube at a loading rate of 150. Mu.L/tube.
7) Split charging of compound solution: DEPC water was dispensed in a 1.5mL centrifuge tube at 600. Mu.L/tube. Example 2 use of a Freeze-dried novel coronavirus detection kit based on visual RT-LAMP
1) The kit components are shown in table 3:
TABLE 3 Table 3
Novel coronavirus (2019-nCoV) N gene fragment plasmid: the novel coronavirus (2019-nCoV) N gene fragment sequence was constructed on a PGEM-T easy vector and synthesized by general biosystems (Anhui) Inc.
2) Taking out N samples according to the number of the samples to be tested (N=the number of the samples to be tested+negative control+positive control)
RT-LAMP reaction tube.
3) And respectively adding 20 mu L of a re-solution into the RT-LAMP reaction tube to re-dissolve the freeze-drying reagent, sequentially adding 5 mu L of treated sample nucleic acid to be tested, negative control and positive control, tightly covering a tube cover, and carrying out instantaneous centrifugation for 5 seconds after vortex mixing.
4) The amplification is carried out by using a PCR instrument, a water bath kettle or a constant-temperature metal bath, the reaction temperature is 65 ℃, and the reaction time is 30min. After the reaction was completed, the color change of the liquid in the reaction tube was observed.
5) And (3) result judgment:
positive results: the color of the liquid in the reaction tube is yellow;
negative results: the liquid in the reaction tube was red in color.
Example 3 specificity experiments
a) Sample: local human coronavirus (HKU 1, OC43, NL63, 229E), SARS coronavirus, MERS coronavirus, influenza A virus (H1N 1, H7N 9), influenza B virus (Victoria),
Yamagata), respiratory syncytial virus (A, B type), adenovirus (types 1, 2, 3, 4, 5, 7, 55), parainfluenza virus (types 1, 2, 3), nucleic acid DNA/RNA of human metapneumovirus, novel coronavirus nucleic acid RNA as positive control, DEPC water as negative control. Novel coronavirus nucleic acid standard was purchased from China national institute of metrology, under the number GBW (E) 091089.
b) And (3) re-dissolving: 20. Mu.L of the reconstituted solution was added to the RT-LAMP reaction tube to reconstitute the lyophilized reagents.
c) Sample adding: 5 μl of each of the above samples was added to the RT-LAMP reaction tube, each sample was repeated 2 times, then the tube cap was closed, and the mixture was vortexed and homogenized and then centrifuged instantaneously for 5 seconds.
d) Amplification: amplification was performed using a trigeminal 960PCR apparatus at 65℃for 30min. e) The results of the specificity experiments are shown in Table 4:
TABLE 4 Table 4
Conclusion: the experimental result shows that the detection result of the novel coronavirus nucleic acid is positive, and the detection result of the local human coronavirus (HKU 1, OC43, NL63, 229E), SARS coronavirus, MERS coronavirus, influenza A virus (H1N 1, H7N 9), influenza B virus (Victoria, yamagata), respiratory syncytial virus (A, B type), adenovirus (1, 2, 3, 4, 5, 7, 55 type), parainfluenza virus (1, 2, 3 type) and human metapneumovirus nucleic acid is negative, which indicates that the detection specificity of the kit is good and the kit is not cross-interfered by other viruses.
Example 4 sensitivity experiment
a) Sample: diluting the novel coronavirus nucleic acid standard to 10 copies/. Mu.L, 4 copies/. Mu.L,
2 copies/. Mu.L, 1 copies/. Mu.L, DEPC water as negative control (NTC).
b) And (3) re-dissolving: 20. Mu.L of the reconstituted solution was added to the RT-LAMP reaction tube to reconstitute the lyophilized reagents.
c) Sample adding: 5 μl of each of the above samples was added to the RT-LAMP reaction tube, and each sample was repeated 20 times, then the tube cap was closed, and immediately centrifuged for 5 seconds after vortex mixing.
d) Amplification: amplification was performed using a trigeminal 960PCR apparatus at 65℃for 30min. e) The sensitivity test results are shown in table 5:
TABLE 5
The LOD was calculated using SPSS as shown in fig. 4.
Conclusion: the cell count and residual are recorded and an estimate after 0.95 is found in the confidence limit table, taking an integer. According to the calculation result, when α=95%, lod=2 copies/. Mu.l, i.e. 10copies/rxn of the kit.
Example 5 real-time stability experiment
And (3) preserving the kit under normal temperature conditions, and sampling and detecting the performance of the kit in the 0 th month, the 1 st month, the 2 nd month, the 3 th month, the 4 th month, the 5 th month and the 6 th month respectively.
a) Sample: diluting the novel coronavirus nucleic acid standard to 100 copies/. Mu.L, 10 copies/. Mu.L,
4 copies/. Mu.L, 2 copies/. Mu.L, DEPC water as negative control and the novel crown N gene fragment plasmid as positive control.
b) And (3) re-dissolving: 20. Mu.L of the reconstituted solution was added to the RT-LAMP reaction tube to reconstitute the lyophilized reagents.
c) Sample adding: 5 μl of each of the above samples was added to the RT-LAMP reaction tube, each sample was repeated 2 times, then the tube cap was closed, and the mixture was vortexed and homogenized and then centrifuged instantaneously for 5 seconds.
d) Amplification: amplification was performed using a trigeminal 960PCR apparatus at 65℃for 30min. e) The results of the real-time stability experiments are shown in Table 6:
TABLE 6
Conclusion: experimental results show that the performance of the detection kit meets the requirements after the kit is stored for 6 months under the normal temperature condition. Therefore, the storage conditions and the validity period of the kit are specified as follows: the kit is preserved at normal temperature and has a valid period of 6 months.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The novel RT-LAMP primer group for coronavirus detection is characterized by comprising inner primers FIP and BIP, outer primers F3 and B3 and loop primers LF and LB; the nucleotide sequences of the inner primers FIP and BIP are shown in SEQ ID NO. 1-2; the nucleotide sequences of the outer primers F3 and B3 are shown in SEQ ID NO. 3-4; the nucleotide sequences of the loop primers LF and LB are shown in SEQ ID NO. 5-6.
2. The novel coronavirus detection RT-LAMP primer set of claim 1, wherein the molar ratio of the inner primer, the outer primer, and the loop primer is 8:1:2.
3. a novel coronavirus detection RT-LAMP reaction solution, which is characterized by comprising the primer group, RT-LAMP Buffer and enzyme Mix as set forth in claim 1 or 2; the enzyme Mix comprises Bst DNA polymerase, gao Wenni-resistant transcriptase and RNase inhibitor.
4. The novel coronavirus detection RT-LAMP reaction solution according to claim 3, wherein the RT-LAMP Buffer comprises (NH 4 ) 2 SO 4 、MgSO 4 KCl, DTT, dNTPs, phenol red.
5. The novel coronavirus detection RT-LAMP reaction solution of claim 3, further comprising a lyoprotectant comprising 6wt% sucrose, 2wt% sorbitol, 6wt% peg20000, 3wt% pvp k30, the remainder being DEPC water.
6. The method for freeze-drying the novel coronavirus detection RT-LAMP reaction solution according to claim 5, wherein the novel coronavirus detection RT-LAMP reaction solution is subjected to sub-packaging and then freeze-drying, and freeze-drying curve parameters are as follows:
pre-freezing: -50 ℃ for 2 hours;
sublimation drying: -40 ℃,0.5h, vacuum degree 10Pa; -40 ℃,10h, and 10Pa vacuum degree;
and (5) analysis and drying: -30 ℃,0.5h, vacuum degree 10Pa; -20 ℃,0.5h, vacuum degree 10Pa; -10 ℃,0.5h, vacuum degree 10Pa;0 ℃ for 0.5h, and the vacuum degree is 10Pa;10 ℃ for 2 hours, and the vacuum degree is 10Pa;20 ℃,4h and 10Pa vacuum degree.
7. A novel freeze-dried coronavirus detection kit based on visual RT-LAMP, which is characterized by comprising RT-LAMP reaction solution, positive control, negative control and double solution according to any one of claims 3 to 5.
8. The visual RT-LAMP based freeze-dried novel coronavirus detection kit of claim 7, wherein said positive control is a novel coronavirus N gene fragment plasmid.
9. The method for using the novel freeze-dried coronavirus detection kit based on the visual RT-LAMP, which is disclosed in claim 7 or 8, is characterized in that a re-solution is added into RT-LAMP reaction liquid to re-dissolve freeze-dried reagents, then treated sample nucleic acid to be detected, negative control and positive control are sequentially added, uniformly mixed, amplified by RT-LAMP reaction, after the reaction is finished, the color change of liquid in a reaction tube is observed, and the result is judged according to the color change.
10. The method of claim 9, wherein the reaction temperature of the RT-LAMP reaction is 63-65 ℃ and the reaction time is 28-32min.
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