CN111321252B

CN111321252B - Novel coronavirus nucleic acid detection primer pair with mutation resistance, kit and application thereof

Info

Publication number: CN111321252B
Application number: CN202010306852.9A
Authority: CN
Inventors: 谷立川; 胡玮; 谢晓鸿; 谢时灵; 董宏杰; 张俊梅; 王宏伟; 张坤迪; 张冯瑜; 何青; 王峰
Original assignee: Qingdao Exploration Microorganism Technology Co ltd; Shandong Shidasi Medical Technology Co ltd; Shandong Stars Bioindustry Co ltd
Current assignee: Qingdao Exploration Microorganism Technology Co ltd; Shandong Shidasi Medical Technology Co ltd; Shandong Stars Bioindustry Co ltd
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2021-06-15
Anticipated expiration: 2040-04-17
Also published as: CN111321252A

Abstract

The invention provides a novel coronavirus nucleic acid detection primer pair with mutation resistance, a kit and application thereof, belonging to the technical field of biological engineering and molecular detection. The invention ensures that the primer pair has excellent mutation resistance by optimal design, thereby effectively avoiding the reduction of detection sensitivity and even the generation of false negative phenomenon caused by the gene mutation of the new coronavirus, and realizing the rapid, high-sensitivity and high-accuracy real-time fluorescence quantitative PCR detection of the novel coronavirus SARS-CoV-2; is convenient for basic level operation and application, thus having great application value for the diagnosis of the novel coronavirus SARS-CoV-2 epidemic disease and epidemiological investigation.

Description

Novel coronavirus nucleic acid detection primer pair with mutation resistance, kit and application thereof

Technical Field

The invention belongs to the technical field of biological engineering and molecular detection, and particularly relates to a novel coronavirus nucleic acid detection primer pair with mutation resistance, a kit and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The new respiratory diseases outbreak since 12 months in 2019, more than two million cases have been diagnosed globally in 4 middle months in 2020, and the main clinical symptoms are fever, hypodynamia and dry cough. The etiology of the coronavirus is confirmed to be a novel coronavirus through separation and identification, and the international committee on virus taxonomy designates the coronavirus as SARS-CoV-2. This is the seventh coronavirus currently found in humans, of which there are mainly 6, of which 229E, OC43, NL63, HKU1 are four of the less pathogenic, which cause mild respiratory disease, and the two remaining SARS and MERS coronaviruses, which cause severe respiratory disease.

The novel coronavirus genome is 27-31kb in size and has important structural characteristics specific to positive strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, the 3' end is provided with a PolyA tail structure, and the RNA is a single-segment positive-strand RNA virus which belongs to the order of nested viruses, the family of coronaviridae and the subfamily of orthocoronavirus.

According to the rules of the current new coronavirus pneumonia diagnosis and treatment scheme (trial seventh edition), suspected cases have one of the following etiological or serological evidences: the real-time fluorescence RT-PCR detects the positive nucleic acid and virus gene sequencing of the novel coronavirus, and the novel coronavirus is highly homologous with the known novel coronavirus, the serum novel coronavirus specific IgM and IgG antibodies are positive, and the serum novel coronavirus specific IgG antibodies are converted from negative to positive or are increased by 4 times or more than the acute stage in the recovery stage, so that the novel coronavirus pneumonia patient can be diagnosed. However, the virus genome sequencing has high requirements on technology, equipment and operators, a long window period is needed for specific antibody detection, and the nucleic acid amplification method has the advantages of short window period and high detection sensitivity, so that the detection of novel coronavirus nucleic acid by real-time fluorescence PCR becomes the first choice in various hospitals at present.

However, when using the nucleic acid diagnostic products of various companies, there are problems that the medium and experts continuously reflect that the detection positive rate of the novel coronavirus nucleic acid is low, positive results appear after repeated detection and multiple negative results appear in some cases, negative results appear in repeated detection of throat swab specimens but positive results are finally detected in respiratory lavage fluid specimens, and nucleic acid detection is negative when novel coronavirus pneumonia characteristics appear in CT image screening. The false negative of the fluorescent quantitative PCR result of the new coronavirus is caused by a plurality of reasons, such as: infected patients are in the early stage of disease attack, the virus content is less, the virus content of upper respiratory tract samples is less than that of lower respiratory tract samples, the samples are damaged in the storage and transportation process, the quality of nucleic acid extraction and amplification systems of various manufacturers is uneven, and experimental operators are not professional enough. However, the inventors have found that it is important to ignore the fact that the genome of the virus is mutated.

RNA viruses, compared to DNA viruses, have genetic material that is single-stranded RNA, and are structurally less stable than double-stranded DNA, and are more susceptible to mutations during replication. In one infection, one virus will multiply millions of times, and thus the probability of viral mutation will be greatly enhanced. There are two major effects of viral variability, one being that it may escape surveillance by the human immune system and not be killed by immune cells. Secondly, the two results are not easy to be detected by the existing detection technology, and both results can cause the spread of virus carriers everywhere, thereby causing large-scale infection.

All current detection sites of nucleic acid diagnostic kits include: nucleocapsid protein gene N, spike protein gene (S), small envelope protein gene (E) and ORF1ab gene. In clinical use, the specificity of the N gene is relatively poor, and a plurality of samples only have positive N genes, while the other gene is negative. However, since the N gene is easy to detect due to its small molecular weight and high copy number, many diagnostic kits select the N gene as one of the target genes in order to avoid missed detection.

According to data published by the biological information center of China, by 4 months and 16 days in 2020, 3783 strains of viruses have mutation in sequenced novel coronaviruses, wherein the single nucleotide mutation frequency accounts for 96.95%, and the specific mutation sites are distributed as shown in figure 1, so that a detection primer with mutation resistance is designed, and the detection of the novel coronaviruses is very slow.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel coronavirus nucleic acid detection primer pair with mutation resistance, a kit and application thereof. The invention takes the novel coronavirus nucleocapsid protein N gene as a detection target gene, further analyzes the amino acid sequence and the protein three-dimensional structure of the coding protein, selects an amino acid site which has important influence on the protein structure, namely once the amino acid changes, the virus is dead or the activity is greatly reduced, and simultaneously, the nucleotide mutation at the 3 'tail end of a primer can generate fatal influence on the PCR process, so the 3' tail end of the primer is designed to be arranged at the nucleotide site which is not easy to mutate, and in conclusion, the novel coronavirus nucleic acid detection primer pair with the mutation resistance is obtained, thereby completing the invention.

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

in a first aspect of the present invention, there is provided a primer pair, wherein the primer pair is any one of the following (1) to (3):

(1) a specific primer pair A;

(2) a specific primer pair B;

(3) a specific primer pair A and a specific primer pair B;

the specific primer pair A consists of a primer F1 and a primer R1;

the specific primer pair B consists of a primer F2 and a primer R2;

the primer F1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;

the primer R1 is a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.

The primer F2 is b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

b2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and have the same functions as the sequence 1;

the primer R2 is the following b3) or b 4):

b3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;

b4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 2.

The application of the primer pair is c1) or c2) as follows:

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

c2) and identifying or assisting in identifying whether the sample to be detected contains the novel coronavirus SARS-CoV-2.

In a second aspect of the invention, the application of the primer pair in the preparation of a kit is provided; the use of the kit is c1) or c2) as follows:

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

In a third aspect of the present invention, a kit is provided, which comprises the above primer pair; the use of the kit is c1) or c2) as follows:

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

The kit also includes a fluorescent probe.

Specifically, the kit comprises any one of the following (1) to (3):

(1) a specific primer pair A and a fluorescent probe A;

(2) a specific primer pair B and a fluorescent probe B;

(3) specific primer pair A and fluorescent probe A, and

a specific primer pair B and a fluorescent probe B.

The specific primer pair A consists of a primer F1 and a primer R1;

the fluorescent Probe A is a Probe Probe 1.

The specific primer pair B consists of a primer F2 and a primer R2;

the fluorescent Probe B is a Probe Probe 2.

The primer F1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

the primer R1 is a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

The Probe1 is a5) or a6) as follows:

a5) a single-stranded DNA molecule shown in sequence 3 of the sequence table;

a6) and (b) the DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 2.

The primer F2 is b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

the primer R2 is the following b3) or b 4):

b3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;

The Probe2 is b5) or b6) as follows:

b5) a single-stranded DNA molecule shown in sequence 6 of the sequence table;

b6) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 6 and has the same function as the sequence 2.

The specific primer pair A and the fluorescent probe A, and the specific primer pair B and the fluorescent probe B are used for detecting the new coronavirus N gene; in practical application, the specific primer pair A and the fluorescent probe A can be used in a combined manner, the specific primer pair B and the fluorescent probe B can be used for detecting the new coronavirus, and the specific primer pair A and the fluorescent probe A or the specific primer pair B and the fluorescent probe B can be used independently for detecting the new coronavirus; of course, the detection can also be carried out by combining other existing new coronavirus detection primers and probes.

Further, the 5' ends of the Probe robe1 and the Probe Probe2 are both labeled with fluorescent groups; the 3' ends of the Probe Probe1 and the Probe Probe2 are both labeled with a quencher group. In the present invention, the fluorescence cluster is specifically HEX, and the quenching cluster is specifically BHQ 2.

Further, the molar ratio of the primer F1, the primer R1 and the Probe Probe1 is 1:1: 1;

further, the molar ratio of the primer F2, the primer R2 and the Probe Probe2 is 1:1: 1.

The preparation method of the kit also belongs to the protection scope of the invention.

Therefore, in the fourth aspect of the present invention, the above-mentioned kit is prepared as follows (I) or (II):

(I) packaging the primers and the probes of the primer pairs separately;

(II) mixing the primers and the probes of the primer pairs together in proportion.

In a fifth aspect of the present invention, there is provided a method for identifying or assisting in identifying a novel coronavirus SARS-CoV-2, comprising the steps of: performing fluorescent quantitative PCR by using cDNA of a virus to be detected as a template and adopting the primer pair and/or the kit;

if the positive amplification curve is displayed, the virus to be tested is the candidate new coronavirus SARS-CoV-2, and if the above condition is not satisfied, the virus to be tested is the candidate non-new coronavirus SARS-CoV-2.

In the sixth aspect of the present invention, there is provided a method for identifying or assisting in identifying whether a sample to be tested contains the novel coronavirus SARS-CoV-2, comprising the following steps: carrying out fluorescence quantitative PCR by using cDNA of a sample to be detected as a template and adopting the primer pair and/or the kit;

if the positive amplification curve is displayed, the sample to be tested is suspected to contain the novel coronavirus SARS-CoV-2, and if the positive amplification curve is not displayed, the sample to be tested is suspected to not contain the novel coronavirus SARS-CoV-2.

In any of the above methods, the reaction procedure of the fluorescence quantitative PCR at least comprises: pre-denaturation: 2min at 95 ℃; and (3) PCR reaction: 95 ℃ for 15s, 60 ℃ for 30s, 45 cycles.

In any of the above methods, the reaction system of the fluorescence quantitative PCR comprises: buffer mix 5. mu.L, primer F1 or F20.5. mu.L, primer R1 or R20.5. mu.L, fluorescent probe A or B0.5. mu. L, cDNA template 9. mu.L, add water to 20. mu.L.

In any of the above methods, the sample includes, but is not limited to, throat fluid, alveolar lavage, saliva, sputum, blood, urine, and stool.

The sample collection subject may be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, cats, monkeys, chimpanzees, etc.; humans are preferred.

The invention has the beneficial technical effects that: the primer pair and the kit provided by the invention are adopted for detection, the detection method is simple, convenient and quick, the repeatability is good, the sensitivity is high, the specificity is strong, and particularly, due to the mutation resistance of the primer pair, the reduction of the detection sensitivity and the generation of false negative phenomena caused by the gene mutation of the new coronavirus can be effectively avoided, so that the real-time fluorescent quantitative PCR detection of the novel coronavirus SARS-CoV-2 with quickness, high sensitivity and high accuracy is realized; is convenient for basic level operation and application, thus having great application value for the diagnosis of the novel coronavirus SARS-CoV-2 epidemic disease and epidemiological investigation.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a diagram showing the mutation of the new coronavirus gene.

FIG. 2 is a structural diagram of tryptophan at the end of primer N5-PF 3' in example 1 of the present invention;

FIG. 3 is a structural diagram of tryptophan at the end of primer N5-PR 3' in example 1 of the present invention;

FIG. 4 is a structural diagram of tryptophan at the end of primer N6-PF 3' in example 1 of the present invention;

FIG. 5 is a diagram showing the structure of the tryptophan at the end of the primer N6-PR 3' in example 1 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention 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 exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

As mentioned above, the detection sites of all the present novel coronavirus nucleic acid diagnostic kits include: nucleocapsid protein gene N, spike protein gene (S), small envelope protein gene (E) and ORF1ab gene. In clinical use, the specificity of the N gene is relatively poor, and a plurality of samples only have positive N genes, while the other gene is negative. However, since the N gene is easy to detect due to its small molecular weight and high copy number, many diagnostic kits select the N gene as one of the target genes in order to avoid missed detection. Although most of mutations occur in the ORF1ab region, the ORF1ab gene is very large and is not favorable for designing a detection primer with mutation resistance, so the N gene is selected as a target point for primer design.

PCR primer design is based on genome sequence, and if the primer is designed in the conservation area of genome, it has the advantage of relatively small influence of virus variation, and the disadvantage of difficulty in distinguishing virus with high homology with SARS-CoV-2, such as SARS. If the primers are designed in the non-conserved region of the genome, the advantages are that SARS-CoV-2 and other coronaviruses can be distinguished, the defect is that the possibility of nucleotide mutation at the position is high, and false negative can occur in the detection of the kit. Nucleotide mutations at the 3' end of the primers will have a fatal effect on the PCR process, and the detection primers of the N gene are verified in the invention. As a result, it was found that the detection efficiency of the existing primer is actually drastically decreased once the nucleotide on the N gene that is paired with the 3 '-end of the primer is mutated, and thus the present invention utilizes this rule to place the 3' -end of the primer at a nucleotide site that is not easily mutated.

The design idea of the invention is to search for suitable primer sites based on protein structure and genetic code rule. The N gene is used as a target detection gene, the amino acid sequence and the three-dimensional structure of the protein are further analyzed, and a detection primer with the mutation resistance is designed. First, it is known that the mutation at the 3 'end of the primer has the greatest effect, and therefore the 3' end of the primer is selected to encode tryptophan W or methionine M. Since these two amino acids have only one codon, amino acid changes must be caused by nucleotide mutations. On the basis, the three-dimensional structure of the protein is comprehensively considered, amino acid sites which have important influence on the protein structure are selected, and once the amino acid changes, the virus is killed or the activity is greatly reduced.

The N gene has two homologous structures which cover the N-terminal (1SSK) and C-terminal domains (2 CJR). According to structural analysis and sequence comparison, the methionine at position 101, the tryptophan at position 108, the tryptophan at position 132, the tryptophan at position 301 and the proline at position 302 of the N gene of the novel coronavirus are found to have important influence on the protein structure. Thus the first primer pair forward 3 'ends with W108 and the reverse primer 3' ends with W132. The second primer pair, forward 3', ends with W301, but P is also non-mutable since the following P302 is important for the structure. P has four codons, which are: CCT, CCA, CCC, CCG, where the two bases are variable and the first base is not. Thus, the forward 3 'end of the second primer pair extends to the first nucleotide C encoding P, and the reverse primer 3' ends with M322, thereby completing the present invention.

In view of this, in an exemplary embodiment of the present invention, there is provided a primer pair, which is any one of the following (1) to (3):

(1) a specific primer pair A;

(2) a specific primer pair B;

(3) a specific primer pair A and a specific primer pair B;

the specific primer pair A consists of a primer F1 and a primer R1;

the specific primer pair B consists of a primer F2 and a primer R2;

the primer F1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

the primer R1 is a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

The primer F2 is b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

the primer R2 is the following b3) or b 4):

b3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;

The application of the primer pair is c1) or c2) as follows:

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

In another embodiment of the invention, the application of the primer pair in the preparation of a kit is provided; the use of the kit is c1) or c2) as follows:

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

In another embodiment of the present invention, a kit is provided, which comprises the above primer pair; the use of the kit is c1) or c2) as follows:

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

The kit also includes a fluorescent probe.

In still another embodiment of the present invention, the kit comprises any one of the following (1) to (3):

(1) a specific primer pair A and a fluorescent probe A;

(2) a specific primer pair B and a fluorescent probe B;

(3) specific primer pair A and fluorescent probe A, and

a specific primer pair B and a fluorescent probe B.

The specific primer pair A consists of a primer F1 and a primer R1;

the fluorescent Probe A is a Probe Probe 1.

The specific primer pair B consists of a primer F2 and a primer R2;

the fluorescent Probe B is a Probe Probe 2.

The primer F1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

the primer R1 is a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

The Probe1 is a5) or a6) as follows:

a5) a single-stranded DNA molecule shown in sequence 3 of the sequence table;

The primer F2 is b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

the primer R2 is the following b3) or b 4):

b3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;

The Probe2 is b5) or b6) as follows:

b5) a single-stranded DNA molecule shown in sequence 6 of the sequence table;

In another embodiment of the present invention, the 5' ends of the Probe1 and the Probe2 are labeled with fluorescent groups; the 3' ends of the Probe Probe1 and the Probe Probe2 are both labeled with a quencher group. In the present invention, the fluorescence cluster is specifically HEX, and the quenching cluster is specifically BHQ 2.

In still another embodiment of the present invention, the molar ratio of the primer F1, the primer R1 and the Probe Probe1 is 1:1: 1;

in still another embodiment of the present invention, the molar ratio of the primer F2, the primer R2 and the Probe Probe2 is 1:1: 1.

Therefore, in another embodiment of the present invention, the method for preparing the above kit is (I) or (II):

(I) packaging the primers and the probes of the primer pairs separately;

In another embodiment of the present invention, there is provided a method for identifying or aiding in identifying a novel coronavirus SARS-CoV-2, comprising the steps of: performing fluorescent quantitative PCR by using cDNA of a virus to be detected as a template and adopting the primer pair and/or the kit;

In another embodiment of the present invention, a method for identifying or assisting in identifying whether a sample to be tested contains the novel coronavirus SARS-CoV-2 is provided, comprising the following steps: carrying out fluorescence quantitative PCR by using cDNA of a sample to be detected as a template and adopting the primer pair and/or the kit;

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

1. Experimental Material

The RT-PCR template used for testing is N gene pseudovirus and is provided by Xiamen Shangshan corporation;

the kit for RNA extraction is purchased from Baoriji medical technology (Beijing) Co., Ltd, and has a product number of 9766;

primers and probes were purchased from Huada Qinglan Biotech (tin-free) Ltd;

RT-PCR in-silico Mix was purchased from Thermo Scientific;

the RT-PCR instrument used for the test was ABI 7500.

2. Primer design

To verify the influence of the 3' mutation of the N gene primer on the PCR process, the following primers were designed:

N-PF1(T-A)	5’-GGGGAACTTCTCCTGCTAGAAA-3’(SEQ ID NO.7)
		N-PF2(T-C)	5’-GGGGAACTTCTCCTGCTAGAAC-3’(SEQ ID NO.8)
N-PF3(T-G)	5’-GGGGAACTTCTCCTGCTAGAAG-3’(SEQ ID NO.9)
		N-PR1(G-A)	5’-CAGACATTTTGCTCTCAAGCTA-3’(SEQ ID NO.10)
N-PR2(G-T)	5’-CAGACATTTTGCTCTCAAGCTT-3’(SEQ ID NO.11)
		N-PR3(G-C)	5’-CAGACATTTTGCTCTCAAGCTC-3’(SEQ ID NO.12)

based on the nucleotide sequence of the N gene, the following primers are designed by integrating the genetic code rule and the three-dimensional structure of the protein:

3. reaction system

The total RT-PCR reaction was 20. mu.L, 9. mu.L of template was added during the reaction, and DEPC water was used as a negative control.

Composition (I)	Addition amount (μ L)	Final concentration (μ M)
			Buffer mix	5	1×
PF	0.5	0.25
			PR	0.5	0.25
Probe	0.5	0.25
			Form panel	9	-
Water (W)	4.5	-
			In total	20	-

4. Step of amplification

The first step is as follows: UNG incubation is carried out, DNA in a PCR system is eliminated, and nonspecific amplification can be prevented;

the second step is that: reverse transcriptase reaction, which is to carry out reverse transcription on template RNA of a sample, pseudovirus and the like to generate DNA;

the third step: pre-denaturation, namely denaturing the template DNA and simultaneously inactivating UNG enzyme and reverse transcriptase;

the fourth step: PCR amplification reaction, annealing and extension at 60 ℃ for fluorescence detection.

Content of research

1. Function of primer 3' end amino acid in protein three-dimensional structure

Two pairs of detection primers N5 and N6 are designed to be respectively positioned at the N end and the C end of the amino acid sequence of the N gene, and the N gene is found to have two homologous structures respectively covering the N end (1SSK) and the C end domain (2CJR) by searching in a protein structure database. Therefore, the two homologous structures are used for analogically analyzing the effect of the 3' end amino acid of the two pairs of detection primers in the three-dimensional structure of the protein.

2. Effect of primer 3' end mutation

Taking 1 μ LN gene pseudovirus (concentration: 1X 10)⁸copies/mL), diluted to 500. mu.L with DEPC water, and mixed well with shaking. Extracting RNA from pseudovirus by using 200 mu L of preservation solution, and performing RT-PCR detection by respectively using primers of non-mutated PF and PR, non-mutated PF3 ', non-mutated PR and non-mutated PR 3'.

3. Determination of minimum detection limit

Pseudovirions containing the RNA fragment of the SARS-CoV-2N gene were diluted with a DEPC water gradient and used for minimal detection limit studies. The concentration gradient is: 100000copies/mL, 20000copies/mL, 10000copies/mL, 9000copies/mL, 1000copies/mL, 800copies/mL, 600copies/mL each concentration gradient is repeated a minimum of three times, the lowest concentration level which can be detected by 100% is taken as an estimation detection limit, and a plurality of gradient concentration samples are prepared around the concentration: 700copies/mL, 500copies/mL, 400copies/mL, at least 20 times of detection per concentration, and the lowest concentration level with 90% -95% positive detection rate is used as the determined lowest detection limit.

Results of the study

1. Primer 3' end amino acid structure

1) 3' terminal tryptophan structure of N5 primer

W86 and W110 are both located at the core part of the three-dimensional structure of the protein, L34, L42, Y65 and V109 around W86, and Y64, Y89, Y101, L91, L99 and A103 around W110 are all nonpolar amino acids which are gathered together to form a hydrophobic core which is very important for maintaining the stability of the structure of the protein, and the side chains of W110 and A103 form hydrogen bonds to further stabilize the conformation of the protein.

2) N6-primer 3' end tryptophan structure

F287, L292, Y299, P303 and I305 around W302, and F308, L332, L340, I358 and V351 nonpolar amino acids around M323 form a hydrophobic region to stabilize the protein structure, and a hydrogen bond is formed between W302 and Y299 to stabilize the protein conformation.

2. Effect of primer 3' end mutation

As can be seen from the above table, when the 3 'end of the detection primer is mutated, the Ct value amplified by RT-PCR is obviously increased, which indicates that when the N gene is mutated so as not to match with the 3' end of the existing detection primer, the detection efficiency of the existing detection primer is sharply reduced.

3. Determination of the minimum detection Limit

According to determination, when the primers and the probes are detected by using the N gene recommended by the Chinese CDC, the lowest detection limit is 500 copies/mL; when the primer N5 with the anti-mutation performance is used, the lowest detection limit is 10000copies/mL, and the sensitivity is reduced to some extent; when the primer N6 with the mutation resistance is used, the lowest detection limit is 500copies/mL, and the sensitivity is consistent with that of the existing detection primer.

Therefore, the primers N5 and N6 with the mutation resistance can be matched with other detection primers of each kit, and the detection rate of the N gene mutation virus can be improved.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shandong Shi Dasi Bio Inc. Shandong Shi Dasi medical science and technology Co., Ltd

Qingdao exploration Microorganism Technology Co.,Ltd.

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<211> 22

<212> DNA

<213> Artificial sequence

<400> 6

cagcgcttca gcgttcttcg ga 22

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<400> 7

ggggaacttc tcctgctaga aa 22

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<400> 8

ggggaacttc tcctgctaga ac 22

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<400> 9

ggggaacttc tcctgctaga ag 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> 10

cagacatttt gctctcaagc ta 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<400> 11

cagacatttt gctctcaagc tt 22

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<400> 12

cagacatttt gctctcaagc tc 22

Claims

1. A primer set, characterized in that the primer set is any one of the following (1) to (3):

(1) a specific primer pair A;

(2) a specific primer pair B;

(3) a specific primer pair A and a specific primer pair B;

the specific primer pair A consists of a primer F1 and a primer R1;

the specific primer pair B consists of a primer F2 and a primer R2;

the primer F1 is a single-stranded DNA molecule shown in sequence 1 of the sequence table;

the primer R1 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

the primer F2 is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

the primer R2 is a single-stranded DNA molecule shown in a sequence 5 of a sequence table.

2. Use of the primer pair according to claim 1 for the preparation of a kit for the following c1) or c 2):

c1) identifying or assisting in identifying novel coronavirus SARS-CoV-2;

3. A kit comprising the primer set according to claim 1.

4. The kit of claim 3, wherein the kit further comprises a fluorescent probe.

5. The kit according to claim 3, comprising any one of the following (1) to (3):

(1) the specific primer pair A and the fluorescent probe A in the claim 1;

(2) the specific primer pair B and the fluorescent probe B in the claim 1;

(3) the specific primer pair A and the fluorescent probe A as defined in claim 1, and

the specific primer pair B and the fluorescent probe B in claim 1.

6. The kit of claim 5, wherein the fluorescent Probe A is Probe Probe 1;

the Probe Probe1 is a single-stranded DNA molecule shown in sequence 3 of the sequence table;

or the fluorescent Probe B is a Probe Probe2, and the Probe Probe2 is a single-stranded DNA molecule shown in a sequence 6 of the sequence table.

7. The kit of claim 6, wherein the 5' ends of the Probe1 and the Probe2 are labeled with a fluorescent group; the 3' ends of the Probe Probe1 and the Probe Probe2 are both labeled with a quencher group.

8. The method for producing a kit according to any one of claims 4 to 7, which is characterized by comprising (I) or (II):

(I) packaging the primers and the probes of the primer pairs separately;