CN114317834A - Kit and method for detecting new coronavirus - Google Patents

Abstract

The invention relates to the field of biological detection, in particular to a kit and a detection method for detecting new coronavirus. The kit contains a nucleic acid amplification reagent, and the nucleic acid amplification reagent contains an isothermal amplification primer of a nucleotide sequence shown as SEQ ID NO 1-SEQ ID NO 4; or isothermal amplification primers containing nucleotide sequences shown as SEQ ID NO 9-SEQ ID NO 12. The invention develops a new coronavirus detection reagent capable of rapidly detecting in real time on site, can improve the detection efficiency and provides an effective technical means for prevention and control of new coronavirus.

Description

Kit and method for detecting new coronavirus

Technical Field

The invention relates to the field of biological detection, in particular to a kit and a detection method for detecting new coronavirus.

Background

The novel Coronavirus (SARS-CoV-2) causes the large spread of epidemic situation in the global scope, the strong infectivity and high lethality rate thereof provide great challenges for global epidemic situation prevention and control work, and the development of accurate and efficient detection method is especially important for effectively controlling the development of the epidemic situation. Current detection methods include pathogenic, molecular biological, and immunological detection.

The etiology detection is that the pathogene is directly detected by utilizing the technologies such as a virus isolation culture method or electron microscope observation and the like, can provide direct evidence for determining the pathogene in the early stage of disease outbreak, and is one of the traditional methods for diagnosing virus infection. However, compared with other detection methods, the pathogenic detection technology requires harsh experimental environment, complex operation and long time consumption, and cannot meet the virus detection requirement under large-scale outbreak of epidemic situations.

The immunological detection method is to detect specific antigens or antibodies of viruses in blood, and the diagnosis and treatment protocol for coronavirus pneumonia (trial eighth edition) uses detection of specific antibodies of new coronavirus, detection of nucleic acid, and sequencing of viral genes as evidence of diagnosis. Compared with nucleic acid detection, the immunological detection is easy in sample collection, simple in detection operation and easy in realization of high flux, can detect the change condition of a specific antibody in a patient body, finds the crowd generating immunity and has important significance for disease prevention and treatment and vaccine research and development. At present, the immunological detection method mainly comprises a lateral flow immunochromatography technology, an enzyme-linked immunosorbent assay (ELISA), a chemiluminescence immunoassay and the like. The lateral flow immunochromatography technology combines the chromatographic analysis and the immunoreaction principle, takes colloidal gold, carbon nano particles and the like as immune labeled probes, and carries out rapid detection on the new coronavirus antigen protein in the sample. ELISA is a common immunoassay method combining antigen-antibody reaction and enzyme science, and utilizes the specific combination of antigen and antibody to perform qualitative and quantitative detection of immunoreaction. Chemiluminescence immunoassay is a novel immunoassay technology, and has the specificity of immunoreaction and the sensitivity of chemiluminescence immunoassay. The immunological detection method has the advantages of short detection time, high accuracy and simple and convenient operation, but because the immunoreaction of a human body generates a detectable antibody only after the body is infected for 1 week, the immunological detection has a detection window period and is mainly used as a means for auxiliary diagnosis.

The molecular biological detection is to detect the virus RNA generated by replication after a new coronavirus infects an organism, and is different from IgM and IgG antibodies generated by an immune system, and the virus RNA generated by replication can be detected earlier, so the current most main diagnostic method of the new coronavirus is virus nucleic acid detection, and mainly comprises whole genome sequencing, real-time fluorescence quantitative PCR (RT-PCR), CRISPR (short-palindrome), reverse transcription loop-mediated isothermal amplification technology (RT-LAMP) and the like, wherein the real-time fluorescence quantitative PCR (RT-PCR) method is still the gold standard for detecting the new coronavirus. The whole genome sequencing technology can comprehensively reflect genetic information of pathogens and has high accuracy and sensitivity, but instruments and equipment of the technology are expensive, the sequencing data interpretation threshold is high, and the technology is difficult to apply to large-scale clinical detection. RT-PCR diagnosis takes short time, a plurality of detection samples, simple and convenient result judgment and low detection cost, and is the most common means for detecting the new coronavirus at present, but the method needs a professional laboratory and operators, and the false positive can be caused by aerosol pollution due to insufficient laboratory conditions or improper operation. RT-LAMP can amplify a nucleic acid template specifically, efficiently and quickly under isothermal conditions, although the primer design of the method is relatively complex, the sensitivity of the detection result is high, visual judgment of the detection result can be realized visually, and the method has very wide development potential.

In addition, the new coronavirus is a single-stranded positive-strand RNA virus, and the genome thereof is mutated through various mechanisms during the replication process, thereby causing the generation of highly pathogenic and more transmissible variant strains, and the World Health Organization (WHO) classifies the variant strains into VOCs and "variant of interest (VOI)" according to differences in the transmissibility, the pathogenicity, and the like of the variant strains. The VOI satisfies the change in viral phenotype, or the amino acid variation causes or potentially causes the change in viral phenotype, as compared to the early reference strain. At present, 5 VOI variant strains are reported, wherein the Delta new crown virus variant strain has been spread to 96 countries/regions in the world, has the characteristics of strong transmission capacity, infection latency period, strong pathogenicity, quick disease development and the like, and gradually replaces other VOCs to become a global epidemic strain with the strongest harm.

Aiming at the characteristic of strong variability of the new coronavirus, it is necessary to develop a new coronavirus detection method capable of detecting various variants. At present, the nucleic acid detection method mainly aims at designing primers and probes in an open reading frame OFR1 region and a nucleocapsid protein region of a new coronavirus, and the continuously mutated virus can cause the failure of nucleic acid detection. A recent research shows that by comparing genome samples of 31421 strains of new coronavirus, ORF7b region of the new coronavirus has the lowest mutation rate, so that the new coronavirus is more suitable for being used as a target point to develop a new coronavirus detection reagent.

The microfluidic Chip technology is a novel technology which integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes into a micron-scale Chip to automatically complete the whole analysis process. Aiming at the problems of low intelligent degree of virus detection and single or less detection and analysis target objects in complex food matrixes, a microfluidic chip method is adopted, chip manufacturing and processing technologies are optimized, a nucleic acid extraction-isothermal amplification-hybridization integrated system is developed, and a chip and peripheral on-line detector and reading device identification matching technology is researched, so that rapid high-precision high-sensitivity instant diagnosis (POCT) of virus genes is realized, and automation of a virus detection process and informatization of detection results are achieved. The micro-fluidic chip detection has the characteristics of integration, automation and the like, can be used for simultaneously detecting a plurality of samples and a plurality of targets, and has the characteristics of high efficiency, sensitivity, accuracy and automation. The detection system is small and portable and is easy to carry. And all channels on the chip tray are completely isolated, so that cross contamination can be effectively avoided.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The primary object of the present invention is to provide a kit for detecting a novel coronavirus.

The second object of the present invention is to provide a method for detecting a novel coronavirus.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the invention provides a kit for detecting a new coronavirus, which is characterized by comprising a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent contains an isothermal amplification primer of a nucleotide sequence shown as SEQ ID NO 1-SEQ ID NO 4; or the nucleic acid amplification reagent contains isothermal amplification primers of nucleotide sequences shown as SEQ ID NO. 9-SEQ ID NO. 12.

Optionally, the concentration of SEQ ID NO 1 and SEQ ID NO 2 is 0.1-0.4. mu.M, preferably 0.1. mu.M; the concentration of SEQ ID NO 3 and SEQ ID NO 4 is 1.0. mu.M to 1.6. mu.M, preferably 1.2. mu.M;

the concentration of SEQ ID NO 9 and 10 is 0.1. mu.M-0.4. mu.M, preferably 0.1. mu.M; the concentration of SEQ ID NO 11 and SEQ ID NO 12 is 1.0. mu.M to 1.6. mu.M, preferably 1.2. mu.M.

Optionally, the kit further comprises a negative control and a positive control, the positive control is a new coronavirus ORF7 pseudovirus plasmid, and the negative control is deionized water.

Optionally, the kit further comprises a microfluidic chip.

Optionally, the sample used in the kit is a nucleic acid sample obtained by extracting an extract of a nasal swab or a pharyngeal swab to be detected with an RNA extraction kit.

Optionally, the nucleic acid amplification reagent further comprises a fluorescent dye and a nucleic acid amplification enzyme including a reverse enzyme, and the nucleic acid amplification enzyme is used for hot start of the multi-purpose isothermal amplification enzyme;

preferably, the content of the fluorescent dye is 50 μ L;

more preferably, the nucleic acid amplification enzyme including a reverse enzyme is contained in an amount of 1.25mL of the hot-start multi-purpose isothermal amplification enzyme.

Optionally, the isothermal amplification temperature of the kit is 63-67 ℃, preferably 65 ℃; the isothermal amplification time of the kit is 40-80 minutes, preferably 60 minutes.

The invention also provides a method for detecting the new coronavirus by adopting the kit, which detects by isothermal amplification, wherein the temperature of the isothermal amplification is 63-67 ℃, and preferably 65 ℃; the isothermal amplification time is 40-80 minutes, preferably 60 minutes.

Optionally, the reaction system of isothermal amplification is:

the nucleic acid amplification enzyme including the invertase hot-start multi-purpose isothermal amplification enzyme 12.5 mu L;

2 mu L of template;

0.5 mu L of fluorescent dye;

0.01. mu.L of each of SEQ ID NO 1 and SEQ ID NO 2 at a concentration of 0.1. mu.M or 0.01. mu.L of each of SEQ ID NO 9 and SEQ ID NO 10 at a concentration of 0.1. mu.M; 0.12. mu.L of each of SEQ ID NO 3 and SEQ ID NO 4 at a concentration of 1.2. mu.M or 0.01. mu.L of each of SEQ ID NO 11 and SEQ ID NO 12 at a concentration of 0.12. mu.M;

make up to 20 μ L with deionized water.

The invention has at least the following beneficial effects:

the invention provides a novel coronavirus detection reagent capable of being rapidly detected on site in real time. The detection efficiency can be improved, the detection difficulty is greatly reduced, and an effective technical means is provided for prevention and control of the new coronavirus.

The kit of the invention can detect the gene fragment with the minimum copy number of 10 copies/. mu.L.

Drawings

FIG. 1 is a diagram showing the result of the LAMP isothermal amplification primer screening experiment;

FIG. 2 is a diagram showing the experimental results of temperature optimization of LAMP isothermal amplification reaction;

FIG. 3 is a graph showing the result of the experiment for optimizing the concentration of the primers in the LAMP isothermal amplification reaction;

FIG. 4 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention;

FIG. 5 shows the experimental results of the detection with the microfluidic chip according to the embodiment of the present invention;

fig. 6 is a result of an experiment for detecting the minimum detection amount using the microfluidic chip according to the embodiment of the present invention.

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 also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides a kit for detecting a new coronavirus, which comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent contains an isothermal amplification primer of a nucleotide sequence shown as SEQ ID NO 1-SEQ ID NO 4; or the nucleic acid amplification reagent contains isothermal amplification primers of the nucleotide sequences shown as SEQ ID NO. 9-SEQ ID NO. 12. The isothermal amplification primer obtained by a large number of screening experiments in the embodiment of the invention has the technical advantages of high amplification efficiency, good amplification specificity, small cycle number required by amplification and the like.

Specifically, the sequences of the primers are shown in Table 1.

TABLE 1

In the embodiment of the invention, the concentration of the primer plays an important role in the LAMP amplification result, the target fragment cannot be effectively amplified by using too low primer concentration, the amplification of the product can be inhibited by using too high primer concentration, the amplification efficiency can be effectively improved by using proper primer concentration, and the nonspecific amplification can be reduced, so that the dosage of the primer is systematically optimized. The method specifically comprises the following steps: the concentration of SEQ ID NO 1 and SEQ ID NO 2 is 0.1. mu.M to 0.4. mu.M, preferably 0.1. mu.M to 0.2. mu.M, more preferably 0.1. mu.M; the concentration of SEQ ID NO 3 and SEQ ID NO 4 is 1.0. mu.M to 1.6. mu.M, preferably 1.2. mu.M to 1.4. mu.M, more preferably 1.2. mu.M. The concentration of SEQ ID NO 9 and 10 is 0.1. mu.M to 0.4. mu.M, preferably 0.1. mu.M to 0.2. mu.M, and more preferably 0.1. mu.M; the concentration of SEQ ID NO 11 and SEQ ID NO 12 is 1.0. mu.M to 1.6. mu.M, preferably 1.2. mu.M to 1.4. mu.M, more preferably 1.2. mu.M. By further controlling the concentration of the primer, the optimal amplification efficiency can be obtained, the detection sensitivity is improved, and the detection time is shortened.

The kit of the embodiment of the invention also contains a negative control product and a positive control product, wherein the positive control product is a pseudoviral plasmid in the ORF7 region of the new coronavirus, for example, a positive plasmid pCMV-ORF7 obtained by cloning the ORF7 region sequence into a pCMV vector, and the negative control product is deionized water.

The kit of the embodiment of the invention also comprises a microfluidic chip. The micro-fluidic chip detection has the characteristics of integration, automation and the like, can be used for detecting a plurality of samples at the same time, and has the characteristics of high efficiency, sensitivity, accuracy and automation.

In the kit of the embodiment of the present invention, the sample is an extract from a nasal swab or a pharyngeal swab, and the nucleic acid is extracted by using the viral nucleic acid extraction kit.

In the kit of the embodiment of the present invention, the nucleic acid amplification reagent further contains a fluorescent dye and a nucleic acid amplification enzyme (WarmStart Multi-purple RT-LAMP 2X Master Mix (with UDG));

preferably, the content of the fluorescent dye is 50. mu.L, and 100 reactions can be performed.

The content of nucleic acid amplification enzyme WarmStart Multi-plasmid RT-LAMP 2X Master Mix (with UDG) was 1.25 mL.

In the kit of the embodiment of the invention, the isothermal amplification temperature of the kit is 63-67 ℃, preferably 65 ℃; the isothermal amplification time of the kit is 40-80 minutes, preferably 60 minutes.

The embodiment of the invention also relates to a method for detecting the new coronavirus by adopting the kit, which detects by isothermal amplification, wherein the temperature of the isothermal amplification is 63-67 ℃, and preferably 65 ℃; the isothermal amplification time is 40 to 80 minutes, preferably 60 minutes.

The reaction system of isothermal amplification is as follows:

12.5. mu.L of nucleic acid amplification enzyme WarmStart Multi-plasmid RT-LAMP 2X Master Mix (with UDG);

2 mu L of template;

0.5 mu L of fluorescent dye;

0.01. mu.L of each of SEQ ID NO 1 and SEQ ID NO 2 at a concentration of 0.1. mu.M or 0.01. mu.L of each of SEQ ID NO 9 and SEQ ID NO 10 at a concentration of 0.1. mu.M; 0.12. mu.L of each of SEQ ID NO 3 and SEQ ID NO 4 at a concentration of 1.2. mu.M or 0.01. mu.L of each of SEQ ID NO 11 and SEQ ID NO 12 at a concentration of 0.12. mu.M;

make up to 20 μ L with deionized water.

The following further explains and explains the embodiments:

sources of experimental materials and instruments:

an experimental instrument: MA2000PLUS microfluidic isothermal amplification nucleic acid analyzer, 8-channel microfluidic chip disc, purchased from Shanghai Suo-Ching organism;

experimental materials: nucleic acid amplification enzymes WarmStart Multi-Purpose RT-LAMP 2 × Master Mix (with UDG), Fluorescent dye (50 ×) were purchased from NEB (NEW ENGLAND Biolabs), pCMV-ORF7 plasmid was purchased from Zhongkaydig (Tianjin) science and technology development Co., Ltd, and 32-channel microfluidic chip trays containing pre-embedded primer mixtures were purchased from Shanghai Suzhonghai Chi organisms. Other reagents were all commercially available.

Example 1

A kit for detecting a novel coronavirus, the composition of which is shown in table 2:

TABLE 2

The use method of the kit comprises the following steps:

1. sample treatment:

mixing 2 μ L of nucleic acid extract of nasal swab or throat swab with 12.5 μ L of nucleic acid amplification enzyme and 0.5 μ L of fluorescent dye reagent, mixing, and adding into sample application region of microfluidic chip. Simultaneously, 2 mu L of positive control reagent and negative control reagent are mixed with nucleic acid amplification reagent and fluorescent dye, the mixture is added into a sample adding area of the chip tray after being uniformly mixed, the chip tray is packaged by a sealing film, and air bubbles are removed by scraper treatment.

2. And (3) detection:

and fixing the packaged chip disc on a microfluidic isothermal amplification accounting analyzer, setting a reaction program to be 65 ℃ for 60 minutes, and setting a detection hole, a positive control hole and a negative control hole according to the sample adding sequence. And clicking a starting response menu to detect after the setting is finished, and analyzing after the detection is finished. The detection result is valid if the positive control hole is amplified and the negative control hole is not amplified.

Experimental example 1: primer screening assays

Isothermal amplification primers were designed according to the ORF7b region with the lowest mutation rate on the new coronavirus, and the design primers are shown in Table 3:

TABLE 3

The primers were tested using common Taq enzyme, and the reaction system is shown in table 4:

TABLE 4

Composition (I)	Volume (μ L)
		TaKaRa Ex Taq(5U/μl)	0.2
10×Ex Taq Buffer(20mM Mg2+plus)	2
		dNTP(2.5mM each)	1.6
Form panel	1
		Primer 1 (10. mu.M)	1
Primer 2 (10. mu.M)	1
		H2O	13.2

Reaction procedure: 10s at 98 ℃, 15s at 60 ℃, 40s at 72 ℃, 40 cycles at 72 ℃ for 5min, and infinity at 4 ℃. Wherein, primer 1 and primer 2 represent a pair of upstream and downstream primers, including each pair of F3 and B3 primers or FIP and BIP primers.

The experimental results obtained are shown in FIG. 1. As can be seen from FIG. 1, the primer combination of ORF7-1 and ORF7-3 can clone clear LAMP amplification bands for subsequent condition optimization experiments.

Experimental example 2:

screening of temperature conditions for isothermal amplification experiments

Set 4 gradients: 60 ℃, 63 ℃, 65 ℃ and 67 ℃. The amplification is carried out by preparing a reaction solution according to the primer combination determined in the experimental example 1 and an LAMP reaction system, wherein the LAMP isothermal amplification reaction system is shown in Table 5, and the reaction time is 60 min.

Table 5: LAMP isothermal amplification reaction system

Composition (I)	Volume of
		WarmStart Multi-Purpose RT-LAMP 2X Master Mix(with UDG)	12.5μL
Fluorescent dye(50×)	0.5μL
		Form panel	2μL
SEQ ID NO:3(40μM)	0.8μL
		SEQ ID NO:4(40μM)	0.8μL
SEQ ID NO:1(10μM)	0.4μL
		SEQ ID NO:2(10μM)	0.4μL
Supplement H2O to	20μL

The amplification products were identified by agarose gel electrophoresis, and the amplification results are shown in FIG. 2. As shown by the results of agarose gel electrophoresis in FIG. 2, the reaction at 65 ℃ was satisfactory.

Experimental example 3: and (4) screening the concentration of the primers in the isothermal amplification experiment.

Setting 4 primer concentration gradients, so that the final concentrations in the reaction system are as follows in sequence: the concentrations of SEQ ID NO 3 and SEQ ID NO 4 are 1.0 muM, 1.2 muM, 1.4 muM and 1.6 muM in sequence; the concentrations of SEQ ID NO 1 and SEQ ID NO 2 are 0.1. mu.M, 0.2. mu.M, 0.3. mu.M and 0.4. mu.M in sequence, the reaction temperature is 65 ℃, the reaction time is 60min, and corresponding reaction liquids are prepared for LAMP amplification, and the amplification system is as shown in Table 4. The products were identified by agarose gel electrophoresis and the results are shown in FIG. 3.

As shown in FIG. 3, the agarose results showed that SEQ ID NO 3 and SEQ ID NO 4 were 1.2. mu.M; the results of SEQ ID NO 1 and SEQ ID NO 2 are preferably 0.1. mu.M.

The same screening experiment was carried out for SEQ ID NO 9-12, with SEQ ID NO 11, SEQ ID NO 12 being 1.2. mu.M; the results of 0.1. mu.M for SEQ ID NO 9 and 10 are preferable.

Experimental example 4:

the reaction system is realized on a microfluidic chip. Chip As shown in FIG. 4, 1. mu.L of premixed primers containing the final concentrations of SEQ ID NO 3, SEQ ID NO 4 of 1.2. mu.M, SEQ ID NO 1, and SEQ ID NO 2 of 0.1. mu.M were added to A1 and A2 wells.

The chip was encapsulated by embedding the primers in the reaction zone, and 1. mu.L of deionized water was added to well A3 and A4. Then, the positive plasmid and the reaction system are mixed and added into the sample addition area, and the standard plasmid and the negative sample are detected, so that the experimental result is shown in fig. 5.

As can be seen from FIG. 5, the standard plasmid produced positive amplification, and deionized water did not produce positive amplification, indicating that the isothermal amplification detection method for the ORF7 region of the novel coronavirus was successfully established.

Experimental example 5:

and (3) carrying out gradient dilution on the standard plasmid in the OFR7 area, and detecting by using the isothermal amplification detection method based on the microfluidic chip. Positive plasmids were diluted to a final concentration of 10⁷、10⁶、10⁵、10⁴、10³、10²、10¹、10⁰copying/L, mixing with a reaction reagent, adding the mixture into an A-G reaction hole on a chip disc in which an LAMP detection primer is embedded in advance to perform isothermal amplification reaction, reacting at 65 ℃ for 60min, and observing the result.

As a result of the experiment, as shown in FIG. 6, it was confirmed that the gene fragment of the lowest copy number/L could be detected.

Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.

Sequence listing

<110> institute of environmental and operational medicine of military medical research institute of military science institute

<120> kit and detection method for detecting new coronavirus

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgtgttagag gtacaacagt ac 22

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cctcttgtct gatgaacagt 20

<210> 3

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgcaaatttg ttatcagcta gaggagaacc ttgctcttct gga 43

<210> 4

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctgacttgct ttagcactca atttgtgaaa ctgatctggc acg 43

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcgctacttg tgagcttta 19

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatctggcac gtaactgat 19

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtatgttcca gaagagcaag gtttaccaag agtgtgttag aggt 44

<210> 8

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agctgataac aaatttgcac tgacgtttta cgccgtcagg ac 42

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tgtgttagag gtacaacagt ac 22

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggtgaaactg atctggca 18

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcagctagag gatgaaatgg tgatttaaaa gaaccttgct cttct 45

<210> 12

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

taacaaattt gcactgactt gcttgtaact gatagacgtg ttttacg 47

Claims (10)

1. A kit for detecting a new coronavirus is characterized by comprising a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent contains an isothermal amplification primer of a nucleotide sequence shown as SEQ ID NO 1-SEQ ID NO 4;

or the nucleic acid amplification reagent contains isothermal amplification primers of nucleotide sequences shown as SEQ ID NO. 9-SEQ ID NO. 12.

2. The kit according to claim 1, wherein the concentration of SEQ ID NO 1, 2 is 0.1. mu.M to 0.4. mu.M, preferably 0.1. mu.M; the concentration of SEQ ID NO 3 and SEQ ID NO 4 is 1.0. mu.M to 1.6. mu.M, preferably 1.2. mu.M;

the concentration of SEQ ID NO 9 and 10 is 0.1. mu.M-0.4. mu.M, preferably 0.1. mu.M; the concentration of SEQ ID NO 11 and SEQ ID NO 12 is 1.0. mu.M to 1.6. mu.M, preferably 1.2. mu.M.

3. The kit according to claim 1, further comprising a negative control and a positive control, wherein the positive control is a new coronavirus ORF7 pseudovirus plasmid, and the negative control is deionized water.

4. The kit of claim 1, further comprising a microfluidic chip.

5. The kit according to claim 1, wherein the sample used in the kit is a nucleic acid sample obtained by subjecting an extract from a nasal swab or a pharyngeal swab to be detected to an RNA extraction kit.

6. The kit according to claim 1, wherein the nucleic acid amplification reagent further comprises a fluorescent dye and a nucleic acid amplification enzyme including a reverse transcriptase hot-start multi-purpose isothermal amplification enzyme.

7. The kit according to claim 6, wherein the content of the fluorescent dye is 50 μ L; preferably, the nucleic acid amplification enzyme including a reverse enzyme is contained in an amount of 1.25mL of the hot-start multi-purpose isothermal amplification enzyme.

8. The kit according to claim 1, characterized in that the isothermal amplification temperature of the kit is 63 ℃ to 67 ℃, preferably 65 ℃; the isothermal amplification time of the kit is 40-80 minutes, preferably 60 minutes.

9. A method for detecting a new coronavirus by using the kit of any one of claims 1 to 8, wherein the detection is carried out by isothermal amplification at a temperature of 63 ℃ to 67 ℃, preferably 65 ℃; the isothermal amplification time is 40-80 minutes, preferably 60 minutes.

10. The method of claim 9, wherein the reaction system of isothermal amplification is:

the nucleic acid amplification enzyme including the invertase hot-start multi-purpose isothermal amplification enzyme 12.5 mu L;

2 mu L of template;

0.5 mu L of fluorescent dye;

0.01. mu.L of each of SEQ ID NO 1 and SEQ ID NO 2 at a concentration of 0.1. mu.M or 0.01. mu.L of each of SEQ ID NO 9 and SEQ ID NO 10 at a concentration of 0.1. mu.M; 0.12. mu.L of each of SEQ ID NO 3 and SEQ ID NO 4 at a concentration of 1.2. mu.M or 0.01. mu.L of each of SEQ ID NO 11 and SEQ ID NO 12 at a concentration of 0.12. mu.M;

make up to 20 μ L with deionized water.

Images