CN113881810A - Novel detection method for pathogenic microorganisms of coronavirus - Google Patents

Abstract

The present invention provides a new coronavirus pathogenic microorganism detection method. Including nucleic acid extraction, amplification reaction, desalting, mass spectrometry detection and other steps. The detection method provided by the present invention combines a variety of specific primers and probe sequences, and can be used for rapid detection of the new coronavirus by using an electrospray mass spectrometer. This detection method has the advantages of high throughput, low cost, high specificity, and high sensitivity, which greatly reduces the probability of "false positives" during detection, and provides technical support for the accurate detection of pathogenic microorganisms of the new coronavirus.

Description

A novel coronavirus pathogenic microorganism detection method

technical field

The invention belongs to the technical field of novel coronavirus detection, and in particular relates to a novel coronavirus pathogenic microorganism detection method.

Background technique

New coronavirus (new coronavirus, referred to in this application as 2019 new coronavirus, also known as 2019-nCoV, SARS-CoV-2, its basic biological structure is shown in Figure 1). In order to control the current spread of the new coronavirus in a timely manner, high-throughput detection methods are extremely important for quickly diagnosing potential patients, quickly isolating them, and quickly taking measures.

At present, there are two conventional new coronavirus detection methods: 1. By detecting coronavirus antigens or antibody proteins. Timely detection of antigen or antibody protein in the patient's body can reflect that the patient has been infected, but it takes several days to several weeks for the body to produce specific antibodies to the virus, and the antibodies produced by patients with poor immune function are not enough to be recognized by the detection method. , prone to false negatives. The principle of antigen antibody protein detection is different from that of nucleic acid detection. It cannot amplify the signal by means of amplification, resulting in insufficient signal strength to identify patients with virus infection at an early stage. 2. The polymerase chain reaction (PCR) technology, which amplifies the signal through nucleic acid amplification reaction, is considered to be the most widely used pathogen detection technology. At present, pathogenic microorganisms mainly use RT-PCR method, but the throughput is low, requiring 2-3 hours/sample. It is very necessary to develop a rapid and high-throughput detection method for the new coronavirus.

On this basis, it has been found through clinical and experimental applications that mass spectrometry technology can perform high-sensitivity detection of 2019-nCoV nucleotides. Based on nucleic acid mass spectrometry methods, the existing methods, the iPLEX method of Agena Company in the United States and the RFMP method of GEneMatrix Company in South Korea: (1) iPLEX method, adding a single base extension step on the basis of PCR, resulting in short oligonucleotides The acid fragments were detected by MALDI-TOF mass spectrometry; (2) RFMP method, the short oligonucleotide fragments produced were detected by MALDI-TOF mass spectrometry by restriction enzyme digestion of multiple PCR products containing single nuclear polymorphism sites.

The technical defects of the iPLEX method and the RFMP method: (1) In the experimental process: the steps of single-base extension or restriction enzyme cleavage are added, which is cumbersome and time-consuming; (2) In the experimental principle: both are based on laser-assisted mechanism analysis - Time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry platform, when measuring macromolecular nucleic acid, the degree of sample ionization is weak and the sensitivity is poor, and it is only suitable for low molecular weight (less than 25kDa) nucleic acid determination.

Therefore, it is very necessary to develop a new rapid and high-throughput nucleic acid detection method for the new coronavirus.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a primer set for the long fragment of nucleocapsid protein, which is the first primer set for detecting the nucleocapsid protein of novel coronavirus;

The first primer set includes: a first forward primer and a first reverse primer;

Wherein, the nucleotide sequence of the first forward primer includes the following tag sequence:

TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGA.

Preferably, the nucleotide sequence of the first forward primer is:

TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGATAATCAGACAAGGAACTGATTA;

Described first reverse primer, its nucleotide sequence is:

CGAAGGTGTGACTTCCATG.

In addition, in order to solve the above problems, the present application also provides a primer set for short fragments of nucleocapsid protein, which is used to detect the nucleocapsid protein of novel coronavirus, which is the second primer set;

In the second primer set, the second forward primer and the second reverse primer are included; wherein,

Described second forward primer, its nucleotide sequence is:

GGGGAACTTCTCCTGCTAGAAT;

Described second reverse primer, its nucleotide sequence is:

AGCAAGAGCAGCATCACC.

In addition, in order to solve the above problems, the present application also provides a primer set for the envelope protein gene fragment, which is used to detect the novel coronavirus envelope protein, which is the third primer set;

In the third primer set, the third E protein forward primer and the third E protein reverse primer are included; wherein,

The third E protein forward primer, its nucleotide sequence is:

CGTGGTATTCTTGCTAGTTAC;

The third E protein reverse primer, its nucleotide sequence is:

CGCACACAATCGAAGCG.

In addition, in order to solve the above problems, the present application also provides a new coronavirus pathogenic microorganism detection method, which is to use the combination of specific primers and probe sequences of the new coronavirus, combined with polymerase chain reaction amplification-electrospray mass spectrometry detection methods, including:

Nucleic acid extraction to obtain a reaction solution to be tested containing DNA or cDNA;

Take a primer mixture containing a combination of specific primers and probe sequences of the novel coronavirus, and carry out an amplification reaction with the reaction solution to be tested to obtain a first PCR product;

Desalting the first PCR product to obtain a second PCR product;

Purify the second PCR product, and use the molecular weight after amplification of the gene detected by electrospray mass spectrometry to determine whether the DNA or cDNA in the reaction solution to be tested contains the corresponding target new type according to the molecular weight after amplification of the gene. coronavirus nucleic acid sequence;

Wherein, the primer mixture containing the combination of specific primers and probe sequences of the novel coronavirus is a primer mixture containing the following specific primer sets:

A single primer set of one of the first primer set as described above, the second primer set as described above, or the third primer set as described above; or,

Multiple mixed primer sets of the first primer set and the second primer set; or,

A plurality of mixed mixed primer sets of the first primer set and the third primer set.

Preferably, the "determining whether the DNA or RNA in the reaction solution to be tested contains the corresponding target novel coronavirus nucleic acid sequence according to the molecular weight after the amplification of the gene" includes:

When the specific primers and probe sequences of the novel coronavirus are combined into a primer mixture of the following components, it is determined that the DNA or cDNA in the reaction solution to be tested contains the corresponding target novel coronavirus sequence:

When the primer mixture is a single primer set and is the first primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or the molecular weight within the range of plus or minus 5Da deviation: the first positive The molecular weight of the positive chain corresponding to the primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da;

When the primer mixture is a single primer set and is the second primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or a molecular weight within the range of plus or minus 5Da deviation: the second positive The molecular weight of the positive chain corresponding to the primer is 16540 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 16720 Da;

When the primer mixture is a single primer set and is the third primer set, the molecular weight after gene amplification is shown as the following molecular weight, or the molecular weight within the range of plus or minus 5Da deviation: the third E protein The molecular weight of the positive chain corresponding to the forward primer is 16860 Da;

When the primer mixture is a mixed primer set and is a mixed primer set of the first primer set and the second primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or within its plus or minus 5Da deviation Molecular weight within the range: the molecular weight of the positive chain corresponding to the first forward primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; and the molecular weight of the positive chain corresponding to the second forward primer is 16720Da, the reverse chain molecular weight corresponding to the first reverse primer is 16540Da;

When the primer mixture is a mixed primer set and is a mixed primer set of the first primer set and the third primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or within its plus or minus 5Da deviation Molecular weight within the range: the molecular weight of the positive chain corresponding to the first forward primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; and the positive chain corresponding to the third E protein forward primer is The molecular weight is 16860Da.

Preferably, the "taking the primer mixture containing the combination of specific primers and probe sequences of the novel coronavirus, and carrying out an amplification reaction with the reaction solution to be tested to obtain the first PCR product" includes:

Configure the reaction system: take the primer mixture, the reaction solution to be tested, the DNA polymerase, and the template DNA, supplement with sterilized deionized water, add sealing solution, and place in a thermal cycler;

The reaction system configured in the thermal cycler is subjected to amplification reaction to obtain the first PCR product.

Preferably, the amplification conditions of the amplification reaction are: the first stage 42°C/15min, the second stage 92°C/3min, the third stage 92°C/15s, 53°C/10s, 60°C/35s, a total of 40 cycles ;

Each of the specific primer sets contains at least two complementary paired specific primers, wherein the concentration of each specific primer is 10 μmol/L.

Preferably, the "desalting the first PCR product to obtain the second PCR product" comprises:

Water and resin are added to the first PCR product, mixed and centrifuged to obtain the second PCR product.

In addition, in order to solve the above problems, the present application also provides a novel coronavirus pathogenic microorganism detection kit, including a primer mixture containing a combination of specific primers and probe sequences for the novel coronavirus;

The primer mixture includes the following specific primer sets: a single primer set of one of the above-mentioned first primer set, the above-mentioned second primer set and the above-mentioned third primer set; or,

A mixed primer set of the first primer set and the second primer set; or,

A mixed primer set of the first primer set and the third primer set.

The present invention provides 3 kinds of specific primers, a novel coronavirus pathogenic microorganism detection method, and a novel coronavirus pathogenic microorganism detection kit. Among them, the specific primers include: a primer set for detecting the long fragment of nucleocapsid protein of the new coronavirus nucleocapsid protein and a primer set for a short fragment of the nucleocapsid protein, and a primer set for detecting the envelope protein of the new coronavirus Primer sets for membrane protein gene fragments. The novel coronavirus pathogenic microorganism detection method includes the steps of nucleic acid extraction, amplification reaction, desalting, mass spectrometry detection and the like. The purpose is to provide a method for rapid identification of pathogenic microorganisms such as novel coronavirus by combining specific primer and probe sequences with polymerase chain reaction amplification-electrospray mass spectrometry. The method is simple to operate. After the sample is amplified by specific primers, probes and gene PCR, it can be directly detected on the machine. No single-base extension or restriction enzyme digestion steps are required, no additional steps are required, and the operation is simple, fast, and high throughput. .

Compared with the MALDI-TOF detection method, this method can be widely used in the detection of long fragments (above 25kDa) or short fragments (below 25kDa) nucleic acid fragments, and is not limited by the nucleic acid molecular weight detection region.

To sum up, the detection method provided by the present invention combines a variety of specific primers and probe sequences, and can be used for rapid detection of new coronavirus by using electrospray mass spectrometer. This detection method has the advantages of high throughput, low cost, high specificity, and high sensitivity, which greatly reduces the probability of "false positives" during detection, and provides technical support for the accurate detection of pathogenic microorganisms of the new coronavirus.

Description of drawings

Figure 1 is a schematic diagram of the biological structure of the new coronavirus;

Fig. 2 is the embodiment 1 in the application, adopts the single primer set of the first primer set, as the mass spectrometry detection result that the primer mixture liquid containing the specific primer of novel coronavirus and the probe sequence combination is detected by the reaction solution to be tested;

Fig. 3 is the embodiment 2 in the application, adopts the single primer set of the second primer set, as the mass spectrometry detection result of the reaction solution to be tested detected by the primer mixture containing the specific primer of the novel coronavirus and the probe sequence combination;

Fig. 4 is the embodiment 3 in the application, adopts the E protein primer set of the third primer set, as the mass spectrometry detection result of the reaction solution to be tested detected by the primer mixture containing the specific primer of the novel coronavirus and the combination of the probe sequences;

Fig. 5 is the embodiment 4 in the application, adopts the mixed primer set of the first primer set and the second primer set, as the primer mixture containing the specific primer of the novel coronavirus and the combination of the probe sequences, the mass spectrum of the reaction solution to be tested detected Test results;

Fig. 6 is the embodiment 5 in the application, adopts the mixed primer set of the first primer set and the third E protein primer set, as the primer mixture containing the specific primer of the novel coronavirus and the combination of the probe sequences to detect the reaction solution to be tested mass spectrometry results.

The realization, functional characteristics and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments.

Detailed ways

The technical solution of the present invention will be described in further detail below in conjunction with specific embodiments, but it does not constitute any limitation to the present invention. The limited revisions made by anyone within the scope of the claims of the present invention are still within within the scope of the claims.

This embodiment provides a primer set for a long fragment of nucleocapsid protein, which is used to detect the nucleocapsid protein of the novel coronavirus, which is the first primer set;

The first primer set includes: a first forward primer and a first reverse primer;

Wherein, the nucleotide sequence of the first forward primer includes the following tag sequence:

TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGA.

As mentioned above, the first primer set includes two primers, the first forward primer and the first reverse primer, wherein at least the nucleotide sequence of the first forward primer includes the above tag sequence.

The above, the primer set of the long fragment of nucleocapsid protein is the primer set of the long fragment of N protein, which is used to detect the new coronavirus nucleocapsid protein, and can be applied to the detection of long fragment N protein nucleic acid fragments in a targeted manner, especially It can detect nucleic acid fragments of long fragments above 25kDa. The selection range expands the limitations of the existing detection methods (MALDI-TOF related methods can only be aimed at below 25kDa), improves the sensitivity and the scope of application, and reduces the probability of false positives during detection.

Preferably, the nucleotide sequence of the first forward primer is:

TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGATAATCAGACAAGGAACTGATTA;

Described first reverse primer, its nucleotide sequence is:

CGAAGGTGTGACTTCCATG.

As mentioned above, the nucleotide sequence of the first forward primer contains the tag sequence "TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGA".

The present embodiment also provides a primer set for detecting a short fragment of the nucleocapsid protein N protein of the novel coronavirus, which is the second primer set;

In the second primer set, the second forward primer and the second reverse primer are included; wherein,

Described second forward primer, its nucleotide sequence is:

GGGGAACTTCTCCTGCTAGAAT;

Described second reverse primer, its nucleotide sequence is:

AGCAAGAGCAGCATCACC.

The above-mentioned primer set for short fragments of nucleocapsid protein is used to detect the primer set of short fragments of nucleocapsid protein of novel coronavirus. Compared with the primer set of long fragments of nucleocapsid protein, it can be specifically applied to the short fragments. The detection of nucleic acid fragments can make up for the wide applicability of long fragment detection, and can perform more accurate detection of short fragments, especially nucleic acid fragments detection of short fragments within the range of 25kDa or less.

This embodiment also provides a primer set for the envelope protein gene fragment, which is used to detect the novel coronavirus envelope protein, which is the third primer set;

In the third primer set, the third E protein forward primer and the third E protein reverse primer are included; wherein,

The third E protein forward primer, its nucleotide sequence is:

CGTGGTATTCTTGCTAGTTAC;

The third E protein reverse primer, its nucleotide sequence is:

CGCACACAATCGAAGCG.

It should be noted that according to existing research, from the perspective of biological structure, the new coronavirus particle is composed of five components: an RNA gene chain and four proteins. Among them, the outermost layer of the particle is the spike glycoprotein (S, SpikeProtein), and the bottom of the spike is composed of small envelope glycoprotein (E, Envelope Protein, E protein, which is the envelope protein in this application) and membrane glycoprotein ( M, Membrane Protein) viral envelope, the core hidden in the envelope is a helix composed of RNA gene chain and nucleocapsid protein (N, Nucleocapsid Protein, N protein, which is the nucleocapsid protein in this application). folded structure.

The above, N protein, namely the nucleocapsid protein of the new coronavirus (N protein, N, Nucleocapsid Protein), is the nucleocapsid protein described in the application, the helical folded structure formed by the combination of viral RNA gene chains, and is used to express the N protein. amino acid sequence. The N protein plays an important role in the viral replication process.

The above-mentioned, E protein, that is, the small envelope glycoprotein of the new coronavirus, is the envelope protein (E protein, E, Envelope Protein) described in this application, which is an integral part of the viral particle envelope. Amino acid sequence for expression of E protein. The main function of the E protein is to protect the RNA gene chain inside the virus. Envelope proteins mediate viral entry into host cells by binding to receptors, and can disrupt microvascular integrity by binding to endothelial cells, causing vascular leakage. In addition, envelope glycoproteins have important receptor binding sites and antigenic epitopes, which play a very important role in the process of virus infection and host immunity, and are also a hot spot in vaccine research. Therefore, using the genetic characteristics of the envelope protein, the new coronavirus can be detected more accurately, especially for mass spectrometry detection after the combination of E protein and other proteins and specific primer amplification, which can greatly reduce the existence of false positives in the detection. probability.

The present embodiment also provides a detection method for novel coronavirus pathogenic microorganisms, which is a detection method using the combination of specific primers and probe sequences of the novel coronavirus combined with polymerase chain reaction amplification-electrospray mass spectrometry, including:

Nucleic acid extraction to obtain a reaction solution to be tested containing DNA or RNA;

Take a primer mixture containing a combination of specific primers and probe sequences of the novel coronavirus, and carry out an amplification reaction with the reaction solution to be tested to obtain a first PCR product;

Desalting the first PCR product to obtain a second PCR product;

The second PCR product is purified, and the molecular weight after gene amplification detected by electrospray mass spectrometry is used to determine whether the DNA or RNA in the reaction solution to be tested contains the corresponding target new type according to the molecular weight after the gene amplification. coronavirus nucleic acid sequence;

Wherein, the primer mixture containing the combination of specific primers and probe sequences of the novel coronavirus is a primer mixture containing the following specific primer sets:

A single primer set of one of the first primer set as described above, the second primer set as described above, or the third primer set as described above; or,

Multiple mixed primer sets of the first primer set and the second primer set; or,

A plurality of mixed mixed primer sets of the first primer set and the third primer set.

As mentioned above, in the nucleic acid extraction step, the automatic nucleic acid extractor can be operated with reference to the instructions of the viral DNA/RNA extraction kit to extract the DNA or RNA of the sample to be tested, thereby extracting the reaction solution to be tested containing DNA or RNA. The specific extraction process is not repeated here.

It should be noted that the amplification reaction refers to PCR amplification of DNA, and PCR is polymerase chain reaction. The reaction is designed according to the principle of DNA denaturation and renaturation. The general reaction system includes a micro sample containing the target gene, a heat-resistant DNA polymerase, four dNTPs (deoxynucleotides) and two excess primers. The primers are generally 20-30bp in length (the primers provided in this example are synthetic).

Above, in the primer mixture containing the combination of specific primers and probe sequences of the novel coronavirus, it can be a primer mixture containing different specific primer groups, and the primer mixture can be different combinations, specifically as follows 5 options (see Table 1):

Table 1. Overview of five schemes

Among them, it should be noted that the molecular weights of the primers in the second primer set and the third primer set are relatively close during mass spectrometry detection. In order to achieve accurate detection results and exclude false positive detection results, the second primer set is avoided in this embodiment. The primer set and the third primer set are mixed for detection to eliminate the analysis obstacle when the molecular weight is close. The first primer set and the third primer set or the first primer set and the second primer set, in the two combination schemes, have a large difference in molecular weight, which can be used as mixed primers for joint detection, and multi-results and multi-molecular weights are used for verification analysis. The data are compared with each other and cross-reference, which helps to further improve the accuracy and reduce the occurrence of false positives.

This embodiment provides a new coronavirus pathogenic microorganism detection method. Including nucleic acid extraction, amplification reaction, desalting, mass spectrometry detection and other steps. The purpose of the invention is to provide a method for rapid identification of pathogenic microorganisms such as novel coronavirus by using polymerase chain reaction amplification-electrospray mass spectrometry (MultiTag-PCR-ESI-MS) combined with specific primer and probe sequences.

The method is simple to operate. After the sample is amplified by specific primers, probes and gene PCR, it can be directly detected on the machine. No single-base extension or restriction enzyme digestion steps are required, no additional steps are required, and the operation is simple, fast, and high throughput. .

Compared with the MALDI-TOF detection method, this method can be widely used in the detection of long fragments (above 25kDa) or short fragments (below 25kDa) nucleic acid fragments, and is not limited by the nucleic acid molecular weight detection region. During the detection, the nucleic acid fragments can be selected according to the length and detection area, or mixed by using the mixed primer set scheme, so as to achieve the effect of detecting nucleic acids with different fragment lengths, improve the detection efficiency, and improve the convenience. and adaptability.

For the mixed primer set scheme, including the mixed primer set of the first primer set and the second primer set, and the mixed primer set of the first primer set and the third primer set, the molecular weights of different primer sets are quite different during detection, which can be obviously Differentiate the molecular weight detection results in different primer sets, so it is possible to obtain multiple results for different gene fragment regions of the new coronavirus protein at the same time in one inspection process. The accuracy of the data greatly reduces the probability of false positive test results through a one-time inspection step.

In conclusion, the detection method provided in this embodiment combines a variety of specific primers and probe sequences, and can be used for rapid detection of new coronavirus by using electrospray mass spectrometer. This detection method has the advantages of high throughput, low cost, high specificity, and high sensitivity, which greatly reduces the probability of "false positives" during detection, and provides technical support for the accurate detection of pathogenic microorganisms of the new coronavirus.

Preferably, the "determining whether the DNA or RNA in the reaction solution to be tested contains the corresponding target novel coronavirus nucleic acid sequence according to the molecular weight after the amplification of the gene" includes:

When the specific primers and probe sequences of the novel coronavirus are combined into a primer mixture of the following components, it is determined that the DNA or RNA in the reaction solution to be tested contains the corresponding target novel coronavirus sequence:

When the primer mixture is a single primer set and is the first primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or the molecular weight within the range of plus or minus 5Da deviation: the first positive The molecular weight of the positive chain corresponding to the primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da;

When the primer mixture is a single primer set and is the second primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or a molecular weight within the range of plus or minus 5Da deviation: the second positive The molecular weight of the positive chain corresponding to the primer is 16540 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 16720 Da;

When the primer mixture is a single primer set and is the third primer set, the molecular weight after gene amplification is shown as the following molecular weight, or the molecular weight within the range of plus or minus 5Da deviation: the third E protein The molecular weight of the positive chain corresponding to the forward primer is 16860 Da;

When the primer mixture is a mixed primer set and is a mixed primer set of the first primer set and the second primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or within its plus or minus 5Da deviation Molecular weight within the range: the molecular weight of the positive chain corresponding to the first forward primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; and the molecular weight of the positive chain corresponding to the second forward primer is 16720Da, the reverse chain molecular weight corresponding to the first reverse primer is 16540Da;

When the primer mixture is a mixed primer set and is a mixed primer set of the first primer set and the third primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or within its plus or minus 5Da deviation Molecular weight within the range: the molecular weight of the positive chain corresponding to the first forward primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; and the positive chain corresponding to the third E protein forward primer is The molecular weight is 16860Da.

Above, according to the molecular weight after gene amplification to determine whether the DNA or RNA in the reaction solution to be tested contains the corresponding target novel coronavirus nucleic acid sequence, specifically refer to the following table:

Table 2. Different schemes contain the theoretical detection data of the molecular weight of the corresponding target new coronavirus nucleic acid sequence

The tolerance range of the molecular weight mass spectrometry detection results after the amplification of the corresponding primers in each primer set in Table 2 is ±5Da.

Preferably, the "taking the primer mixture containing the combination of specific primers and probe sequences of the novel coronavirus, and carrying out an amplification reaction with the reaction solution to be tested to obtain the first PCR product" includes:

Configure the reaction system: take 1 μL of the primer mixture, 12.5 μL of the reaction solution to be tested, 0.5-1 μL of DNA polymerase, and 2-5 μL of template DNA, supplement with sterile deionized water to 25 μL, add 20 μL of sealing solution, placed in a thermal cycler;

The reaction system configured in the thermal cycler is subjected to amplification reaction to obtain the first PCR product.

Preferably, the amplification conditions of the amplification reaction are: the first stage 42°C/15min, the second stage 92°C/3min, the third stage 92°C/15s, 53°C/10s, 60°C/35s, a total of 40 cycles ;

Each of the specific primer sets contains at least two complementary paired specific primers, wherein the concentration of each specific primer is 10 μmol/L.

Preferably, the "desalting the first PCR product to obtain the second PCR product" comprises:

16 μL of water and 6 mg of resin were added to the first PCR product, mixed and centrifuged to obtain the second PCR product.

In addition, this embodiment also provides a novel coronavirus pathogenic microorganism detection kit, which adopts the aforementioned novel coronavirus pathogenic microorganism detection method. Including nucleic acid extraction, amplification reaction, desalting, mass spectrometry detection and other steps. The purpose of the invention is to provide a method for rapid identification of pathogenic microorganisms such as novel coronavirus by combining specific primer and probe sequences with polymerase chain reaction amplification-electrospray mass spectrometry (MultiTag-PCR-ESI-MS).

The detection kit provided in this embodiment utilizes the combination of specific primers and probe sequences of the novel coronavirus, combined with the detection method of polymerase chain reaction amplification-electrospray mass spectrometry; Primer mixture of specific primer and probe sequence combination;

The primer mixture includes the following specific primer sets: a single primer set of one of the above-mentioned first primer set, the above-mentioned second primer set and the above-mentioned third primer set; or,

A mixed primer set of the first primer set and the second primer set; or,

a mixed primer set of the first primer set and the third primer set;

Each of the specific primer sets contains at least two complementary paired specific primers, wherein the concentration of each specific primer is 10 μmol/L.

The novel coronavirus pathogenic microorganism detection kit provided in this example. The purpose of the invention is to provide a kit for rapid identification of pathogenic microorganisms such as new coronaviruses by polymerase chain reaction amplification-electrospray mass spectrometry (MultiTag-PCR-ESI-MS) combined with specific primer and probe sequences, which can be used for Rapid detection of the new coronavirus. The novel coronavirus pathogenic microorganism detection kit has the advantages of high throughput, low cost, high specificity, and high sensitivity, which greatly reduces the probability of "false positives" during detection, and provides technical support for the accurate detection of novel coronavirus pathogenic microorganisms .

In order to better illustrate the detection method provided in this application and the scheme without specific primer sets, the following specific examples are provided.

experimental method:

Step 1, nucleic acid extraction: operate the automatic nucleic acid extractor according to the instructions of the viral DNA/RNA extraction kit to extract the DNA or RNA of the sample to be tested; obtain the reaction solution to be tested containing DNA or RNA;

Step 2, Amplification reaction:

(1) Reaction system: prepare a reaction system in a 200 μL PCR tube: take 1 μL of the primer mixture containing the combination of specific primers and probe sequences for novel coronavirus, 12.5 μL of the reaction solution to be tested, 0.5-1 μL of DNA polymerase, and template DNA 2-5 μL, make up to 25 μL with sterilized deionized water, then add 20 μL of sealing solution, and place the PCR reaction tube in a thermal cycler;

(2) Amplification conditions: the first stage of pre-denaturation, 42°C/15min; the second stage, 92°C/3min; the third stage, 92°C/15s, 53°C/10s, 60°C/35s; a total of 40 cycles;

Under this amplification condition, the PCR reaction tube is subjected to amplification reaction to obtain the first PCR product;

Step 3, desalting treatment: in the PCR tube containing the first PCR product, add 16 μL of pure water and 6 mg of resin, rotate vertically at low speed for 30 min, make the resin fully contact with the first PCR product, and centrifuge to make the resin sink to the bottom of the well to obtain the first PCR product. Two PCR products;

Step 4, mass spectrometry detection: after the second PCR product is purified, it is detected by electrospray mass spectrometry to analyze the molecular weight of the reporter group, thereby identifying pathogenic microorganisms such as the new coronavirus.

Experimental biochemical samples:

For the same new coronavirus-positive patient (confirmed case), on-site nasal swabs were collected and sampled, and then the experimental biochemical samples were obtained by sub-packaging, and the reaction solution to be tested was prepared.

Experimental equipment: Thermo Tsq quantiva (electrospray mass spectrometry, the specific clinical application is not limited to the brand model in this example)

Example 1:

The experimental methods, experimental equipment and experimental biochemical specimens as described above are used, and the following primer mixtures containing the combination of specific primers and probe sequences for the novel coronavirus are used, specifically:

Example 2:

The experimental methods, experimental equipment and experimental biochemical specimens as described above are used, and the following primer mixtures containing the combination of specific primers and probe sequences for the novel coronavirus are used, specifically:

Example 3:

The experimental methods, experimental equipment and experimental biochemical specimens as described above are used, and the following primer mixtures containing the combination of specific primers and probe sequences for the novel coronavirus are used, specifically:

Example 4:

The experimental methods, experimental equipment and experimental biochemical specimens as described above are used, and the following primer mixtures containing the combination of specific primers and probe sequences for the novel coronavirus are used, specifically:

Example 5:

The experimental methods, experimental equipment and experimental biochemical specimens as described above are used, and the following primer mixtures containing the combination of specific primers and probe sequences for the novel coronavirus are used, specifically:

Experimental results:

Table 3, embodiment 1-5 MultiTag-PCR-ESI-MS detection data

In this example, by using electrospray mass spectrometry to detect the molecular weight of the gene of the reaction solution to be tested in Examples 1-5, pathogenic microorganisms such as the new coronavirus are identified. For specific data, see Table 3, and refer to Figures 2-6. in,

In Example 1, referring to Fig. 2, it can be seen that the molecular weight of the gene after amplification by the first forward primer is 44910Da, and the molecular weight after amplification by the first reverse primer is 45180Da, according to which it is determined that the sample to be tested contains the new coronavirus. N protein nucleic acid sequence.

In Example 2, referring to Fig. 3, it can be seen that the molecular weight of the gene after amplification by the second forward primer is 16720Da, and the molecular weight after amplification by the second reverse primer is 16540Da, according to which it is determined that the sample to be tested contains the new coronavirus. N protein nucleic acid sequence.

In Example 3, referring to FIG. 4 , it can be seen that the molecular weight of the E protein gene fragment after amplification by the forward primer is 16860 Da, and accordingly it is determined that the sample to be tested contains the nucleic acid sequence of the new coronavirus N protein.

In Example 4, referring to FIG. 5, it can be seen that the molecular weight of the gene after amplification by the first N protein forward primer is 44910 Da, the molecular weight after amplification by the first N protein reverse primer is 45180 Da, and the second N protein forward primer has a molecular weight of 45180 Da. The molecular weight is 16720Da, and the molecular weight of the second N protein forward primer is 16540Da. Based on this, it is determined that the sample to be tested contains the nucleic acid sequence of the new coronavirus N protein.

In Example 5, referring to Figure 6, it can be seen that the molecular weight of the gene after amplification by the first forward primer is 44910Da, the molecular weight after amplification by the first reverse primer is 45180Da, and the molecular weight of the third E protein forward primer is 16860Da, Based on this, it is determined that the sample to be tested contains the nucleic acid sequence of the N protein of the new coronavirus.

To sum up, the present invention provides three specific primers, a novel coronavirus pathogenic microorganism detection method, and a novel coronavirus pathogenic microorganism detection kit. Among them, the specific primers include: a primer set for detecting the long fragment of nucleocapsid protein of the new coronavirus nucleocapsid protein and a primer set for a short fragment of the nucleocapsid protein, and a primer set for detecting the envelope protein of the new coronavirus Primer sets for membrane protein gene fragments. The novel coronavirus pathogenic microorganism detection method combines a variety of specific primers and probe sequences, and can be used for rapid detection of the novel coronavirus by using an electrospray mass spectrometer. Combining with this example, it can be seen that this detection method has the advantages of high throughput, low cost, high specificity, high sensitivity, etc., which greatly reduces the probability of "false positive" during detection, and provides technical support for the accurate detection of pathogenic microorganisms of the new coronavirus. .

The above are the preferred embodiments and corresponding embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but It is not limited to the adjustment of ratio, flow process and dosage, which all fall within the protection scope of the present invention. The above are the preferred embodiments and corresponding embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but It is not limited to the adjustment of ratio, flow process and dosage, which all fall within the protection scope of the present invention.

Claims (10)

1. a primer set of a long fragment of nucleocapsid protein, for the detection of novel coronavirus nucleocapsid protein, is characterized in that, is the first primer set; The first primer set includes: a first forward primer and a first reverse primer; Wherein, the nucleotide sequence of the first forward primer includes the following tag sequence: TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGA. 2. the primer set of nucleocapsid protein long fragment as claimed in claim 1, is characterized in that, described first forward primer, its nucleotide sequence is: TCATAGCACTTCGTTGTAGCTAGCCTATCGGTCAGATAATCAGACAAGGAACTGATTA; Described first reverse primer, its nucleotide sequence is: CGAAGGTGTGACTTCCATG. 3. a primer set of a short fragment of nucleocapsid protein, for detecting novel coronavirus nucleocapsid protein, is characterized in that, is the second primer set; In the second primer set, the second forward primer and the second reverse primer are included; wherein, Described second forward primer, its nucleotide sequence is: GGGGAACTTCTCCTGCTAGAAT; Described second reverse primer, its nucleotide sequence is: AGCAAGAGCAGCATCACC. 4. a primer set of an envelope protein gene fragment for detecting novel coronavirus envelope protein, characterized in that it is the third primer set; In the third primer set, the third E protein forward primer and the third E protein reverse primer are included; wherein, The third E protein forward primer, its nucleotide sequence is: CGTGGTATTCTTGCTAGTTAC; The third E protein reverse primer, its nucleotide sequence is: CGCACACAATCGAAGCG. 5. a novel coronavirus pathogenic microorganism detection method, is characterized in that, for utilizing the specific primer of novel coronavirus and probe sequence combination, and in conjunction with the detection method of polymerase chain reaction amplification-electrospray mass spectrometry, comprising : Nucleic acid extraction to obtain a reaction solution to be tested containing DNA or cDNA; Take a primer mixture containing a combination of specific primers and probe sequences of the novel coronavirus, and carry out an amplification reaction with the reaction solution to be tested to obtain a first PCR product; Desalting the first PCR product to obtain a second PCR product; Purify the second PCR product, and use the molecular weight after amplification of the gene detected by electrospray mass spectrometry to determine whether the DNA or cDNA in the reaction solution to be tested contains the corresponding target new type according to the molecular weight after amplification of the gene. coronavirus nucleic acid sequence; Wherein, the primer mixture containing the combination of specific primers and probe sequences of the novel coronavirus is a primer mixture containing the following specific primer sets: A single primer set of one of the first primer set of any one of claims 1-2, the second primer set of claim 3, or the third primer set of claim 4; or, Multiple mixed primer sets of the first primer set and the second primer set; or, A plurality of mixed mixed primer sets of the first primer set and the third primer set. 6. novel coronavirus pathogenic microorganism detection method as claimed in claim 5, is characterized in that, Described "according to the molecular weight after the amplification of the gene to determine whether the DNA or cDNA in the reaction solution to be tested contains the corresponding target novel coronavirus nucleic acid sequence" includes: When the specific primers and probe sequences of the novel coronavirus are combined into a primer mixture of the following components, it is determined that the DNA or cDNA in the reaction solution to be tested contains the corresponding target novel coronavirus nucleic acid sequence: When the primer mixture is a single primer set and is the first primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or the molecular weight within the range of plus or minus 5Da deviation: the first positive The molecular weight of the positive chain corresponding to the primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; When the primer mixture is a single primer set and is the second primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or a molecular weight within the range of plus or minus 5Da deviation: the second positive The molecular weight of the positive chain corresponding to the primer is 16540 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 16720 Da; When the primer mixture is a single primer set and is the third primer set, the molecular weight after gene amplification is shown as the following molecular weight, or the molecular weight within the range of plus or minus 5Da deviation: the third E protein The molecular weight of the positive chain corresponding to the forward primer is 16860 Da; When the primer mixture is a mixed primer set and is a mixed primer set of the first primer set and the second primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or within its plus or minus 5Da deviation Molecular weight within the range: the molecular weight of the positive chain corresponding to the first forward primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; and the molecular weight of the positive chain corresponding to the second forward primer is 16720Da, the reverse chain molecular weight corresponding to the first reverse primer is 16540Da; When the primer mixture is a mixed primer set and is a mixed primer set of the first primer set and the third primer set, if the molecular weight after gene amplification is shown as the following molecular weight, or within its plus or minus 5Da deviation Molecular weight within the range: the molecular weight of the positive chain corresponding to the first forward primer is 44910 Da, and the molecular weight of the reverse chain corresponding to the first reverse primer is 45180 Da; and the positive chain corresponding to the third E protein forward primer is The molecular weight is 16860Da. 7. novel coronavirus pathogenic microorganism detection method as claimed in claim 5, is characterized in that, described " get the primer mixture that contains the specific primer of novel coronavirus and probe sequence combination, carry out with described reaction solution to be tested. The amplification reaction to obtain the first PCR product" includes: Configure the reaction system: take the primer mixture, the reaction solution to be tested, the DNA polymerase, and the template DNA, supplement with sterilized deionized water, add sealing solution, and place in a thermal cycler; The reaction system configured in the thermal cycler is subjected to amplification reaction to obtain the first PCR product. 8. The method for detecting novel coronavirus pathogenic microorganisms according to claim 7, wherein the amplification conditions of the amplification reaction are: 42°C/15min in the first stage, 92°C/3min in the second stage, and 92°C in the third stage /15s, 53℃/10s, 60℃/35s, a total of 40 cycles; Each of the specific primer sets contains at least two complementary paired specific primers, wherein the concentration of each specific primer is 10 μmol/L. 9. The novel coronavirus pathogenic microorganism detection method of claim 5, wherein the "desalting the first PCR product to obtain the second PCR product" comprises: Water and resin are added to the first PCR product, mixed and centrifuged to obtain the second PCR product. 10. A novel coronavirus pathogenic microorganism detection kit, characterized in that it comprises a primer mixture containing a combination of specific primers for novel coronavirus and probe sequences; The primer mixture includes the following specific primer sets: the first primer set according to any one of claims 1-2, the second primer set according to claim 3 and the third primer set according to claim 4 A single primer set for one of the sets; or, A mixed primer set of the first primer set and the second primer set; or, A mixed primer set of the first primer set and the third primer set.

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