CN112981008A - Primer group, probe group and kit for multiple recombinase polymerase amplification technology for detecting novel coronavirus - Google Patents

Abstract

The invention specifically relates to a primer group, a probe group and a kit thereof for a multiple recombinase polymerase amplification technology for detecting novel coronavirus, and belongs to the field of in-vitro nucleic acid molecule detection. The invention designs primers and probes of the N gene, the ORF1ab gene and the E gene of the novel coronavirus and the human ACTB gene, can simultaneously detect the target gene and the human reference gene of the novel coronavirus, and realizes double, triple or even quadruple detection of the novel coronavirus. The invention can complete amplification within 20 minutes at 42 ℃, can effectively avoid the false negative problem in the experimental process, can realize the reaction result in a real-time fluorescence detection mode, and can also be combined with a lateral chromatography test strip to perform naked eye interpretation on the detection result. The method has the advantages of short detection time, strong specificity, high sensitivity and good repeatability, realizes quality monitoring in the processes of sample collection, extraction and amplification through the detection of the ginseng genes, is favorable for further control of epidemic situations, and has good practical value.

Description

Primer group, probe group and kit for multiple recombinase polymerase amplification technology for detecting novel coronavirus

Technical Field

The invention belongs to the field of in-vitro nucleic acid molecule detection, and particularly relates to a primer group, a probe group and a kit thereof for a multiple recombinase polymerase amplification technology for detecting novel coronavirus.

Background

The novel coronavirus (SARS-CoV-2) is a novel virus appearing in the end of 2019, has high infectivity, rapidly causes wide spread worldwide in a short term, and is still a public health emergency facing the world. Even worse, the current approach to the treatment of patients with Novel Coronavirus Pneumonia (NCP) is still symptomatic treatment, and no suitable drug is available to inhibit virus replication. And at present, no vaccine suitable for being inoculated by people of all ages exists, the existing vaccine is only suitable for healthy people of 18-59 years old, and a plurality of groups such as the old, infants, hypertension, diabetes, leukemia patients, pregnant women and the like are not in the scope suitable for inoculation, and the groups are just the key protection objects. Therefore, how to diagnose SARS-CoV-2 quickly and accurately is of great significance to the prevention and control of the current epidemic situation.

However, for the detection of SARS-CoV-2, real-time fluorescent quantitative PCR (RT-PCR) is currently used. However, this method has some limitations such as a long time required for amplification (often 1.5 to 2 hours, and even a rapid PCR requires about 1 hour for amplification, and usually 5 to 6 hours for completion of detection), and high requirements for template purity required for amplification in RT-PCR, and further, high requirements for operators. Importantly, RT-PCR techniques require sophisticated and expensive nucleic acid amplification equipment. In resource-poor areas, the number of nucleic acid amplification instruments is small or even lacking. Once epidemic situation occurs in local area, RT-PCR can hardly achieve the requirement of large-scale crowd screening and isolating infected persons in time. Therefore, when the global epidemic is not yet over, the RT-PCR technology is not favorable for large-scale detection when the epidemic is outbreaked again, and also does not accord with the principle of rapid and accurate report issuing required by clinical laboratories. In conclusion, there is still a need to develop a simpler and faster detection method for SARS-CoV-2. The recombinase polymerase amplification technology is a constant temperature amplification technology, can realize the amplification of target genes under the condition of 37-42 ℃, and the amplification result can be realized by a real-time fluorescent quantitative detection mode, and can also be combined with a lateral chromatography test strip to carry out naked eye interpretation on the detection result, thereby meeting the requirements of different occasions.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a primer set and a probe set for multiplex recombinase polymerase amplification technology for detecting a novel coronavirus, a second object of the present invention is to provide a real-time fluorescence detection kit for detecting a novel coronavirus, and a third object of the present invention is to provide a lateral chromatography test strip detection kit for detecting a novel coronavirus.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a primer set and a probe set for a multiple recombinase polymerase amplification technique for detecting novel coronavirus, wherein the primer set comprises one or more of a primer pair for amplifying human ACTB gene and a primer pair for amplifying N gene, ORF1ab gene or E gene of the novel coronavirus; the probe set comprises one or more of a probe for amplifying human ACTB gene and a probe for detecting N gene, ORF1ab gene and E gene of the novel coronavirus;

the primer pair for amplifying the novel coronavirus N gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 19; the primer pair for amplifying the novel coronavirus ORF1ab gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 7, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 12; the primer pair for amplifying the novel coronavirus E gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 36, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 40; the primer pair for amplifying the human ACTB gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 22, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 26;

the probe nucleotide sequence for detecting the novel coronavirus N gene is SEQ ID NO. 21; the probe nucleotide sequence for detecting the novel coronavirus ORF1ab gene is SEQ ID NO. 16; the nucleotide sequence of the probe for detecting the novel coronavirus E gene is SEQ ID NO. 45; the nucleotide sequence of the probe for detecting the human ACTB gene is SEQ ID NO. 30.

As one of the preferred technical schemes, the probe in the probe group has four modification sites: tetrahydrofuran and analogues thereof are used for replacing original bases at positions which are about 30 bases away from the 5 'end and about 15 bases away from the 3' end to serve as recognition sites of exonuclease; a T base at the upstream of tetrahydrofuran and analogues thereof is marked with a fluorescent group, a T base at the downstream is marked with a quenching group corresponding to the fluorescent group, and the distance between the two groups is 2-4 nt; the 3' end is labeled with a blocking group that blocks probe extension.

As one of the preferred technical schemes, the fluorescent group comprises FAM, VIC, CY5 or ROX.

As one of the preferable technical schemes, the quenching group comprises BHQ1, BHQ2 or BHQ 3.

As one of the preferred technical solutions, the blocking group comprises C3-Spacer, amine group, biotin-triethylene glycol or phosphate group.

In a preferred embodiment, the reverse primers of the N gene, ORF1ab gene, E gene, or human ACTB gene in the primer set are labeled with antigen tag 1, the forward primers of the N gene, ORF1ab gene, E gene, or human ACTB gene are labeled with antigen tags 2, 3, 4, and 5, respectively, and the antigen tag 1 is different from the antigen tags 2, 3, 4, and 5.

As one of the preferred technical schemes, the antigen tag 1 comprises biotin, FAM, CY5, TAMRA or digoxin.

As one of the preferred technical schemes, the antigen tag 2 comprises biotin, FAM, CY5, TAMRA or digoxin.

As one of the preferred technical schemes, the antigen tag 3 comprises biotin, FAM, CY5, TAMRA or digoxin.

As one of the preferred technical schemes, the antigen tag 4 comprises biotin, FAM, CY5, TAMRA or digoxin.

As one of the preferred technical schemes, the antigen tag 5 comprises biotin, FAM, CY5, TAMRA or digoxin.

2. A real-time fluorescence detection kit for detecting novel coronavirus comprises a dissolving buffer solution, freeze-dried enzyme powder, a magnesium acetate solution, a primer group and a probe group; the dissolving buffer comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 500 ng/mu L of 100-one recombinant enzyme, 400 ng/mu L of 100-one recombinant enzyme cofactor, 900 ng/mu L of 400-one-stranded DNA binding protein, 50-200 ng/mu L of DNA polymerase, 500 ng/mu L of 100-one exonuclease, 50-100 ng/mu L of reverse transcriptase, 1-3mM ATP, 30-100mM creatine phosphate, 300 ng/mu L of 200-one creatine kinase, 500 mu M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol.

As one of the preferable technical schemes, the using method of the kit comprises the following steps:

1) preparing a reaction system: 29.4 mu L of dissolving buffer solution, wherein the final concentration of the forward primer and the reverse primer of each gene in the primer group is 200 and 600 nM; the final concentration of each probe in the probe group is 60-180nM, and 50 muL in total;

2) adding the prepared reaction system into the freeze-dried enzyme powder, mixing uniformly, adding 2.5 mu L of 28mM magnesium acetate solution into a tube cover, centrifuging, and carrying out real-time fluorescence PCR amplification at 42 ℃ for 20 minutes.

3. A lateral chromatography test paper strip detection kit for detecting novel coronavirus comprises a dissolving buffer solution, freeze-dried enzyme powder, a magnesium acetate solution, an amplification product diluent, a lateral chromatography test paper strip, a goat anti-mouse monoclonal antibody, a monoclonal antibody of an anti-human ACTB gene antigen label, a monoclonal antibody of 1 or more SARS-CoV-2 target gene antigen labels, a primer group and a probe group; the dissolving buffer comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 100-500 ng/. mu.L recombinase, 100-400 ng/. mu.L recombinase cofactor, 400-900 ng/. mu.L single-stranded DNA binding protein, 50-200 ng/. mu.L DNA polymerase, 50-100 ng/. mu.L reverse transcriptase, 1-3mM ATP, 30-100mM creatine phosphate, 200-300 ng/. mu.L creatine kinase, 200-500. mu.M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol.

As one of the preferred technical schemes, the dilution of the amplification product does not contain enzyme water or Tris buffer.

As one of the preferable technical schemes, the using method of the kit comprises the following steps:

1) configuring a reaction system, 29.4 mu L of dissolving buffer solution, the primer group and the probe group, wherein the final concentration of the forward primer and the reverse primer of each gene in the primer group is 200-600 nM; the final concentration of each probe in the probe group is 60-180nM, and 50 muL in total;

2) adding the prepared reaction system into the freeze-dried enzyme powder, mixing uniformly, adding 2.5 mu L of 28mM magnesium acetate solution into a tube cover, centrifuging, and keeping the constant temperature at 42 ℃ for reaction for 20 minutes;

3) marking a goat anti-mouse monoclonal antibody, a monoclonal antibody of a human ACTB gene antigen label to be detected and monoclonal antibodies of 1 or more SARS-CoV-2 target gene antigen labels on a lateral chromatography test strip;

4) mu.L of amplification product was taken, 190. mu.L of amplification product diluent was added and mixed well, a lateral flow strip was inserted vertically therein, and the result was observed after 5 minutes.

The invention has the beneficial effects that:

the existing method for detecting SARS-CoV-2 is characterized by that except for RT-PCR, the report in the constant-temperature amplification is also increased, but the existent constant-temperature amplification detection technology is mostly only aimed at single-tube single-target detection of SARS-CoV-2, and lacks correspondent internal reference gene to indicate validity of sample collection process and sample extraction process, so that it is not favourable for explaining detection result, and is difficult to avoid false negative result brought by experimental operation. On the basis of following the principle of fast SARS-CoV-2 detection, the present invention establishes a multiple recombinase polymerase amplification technology for detecting SARS-CoV-2, and is used for simultaneously detecting SARS-CoV-2 target gene and human ACTB gene. According to the design principle of primers and probes of the recombinase polymerase amplification technology, corresponding primer and probe sequences are respectively designed and a multiple reaction system is optimized to obtain the optimal target gene amplification condition, so that the problem of monitoring the clinical sampling effectiveness is mainly solved, and the multiple real-time fluorescent recombinase polymerase amplification technology is established. The result shows that the established reaction system has good sensitivity and strong specificity, and except that the nucleic acid of the clinical simulation sample of the positive pseudovirus and the SARS-CoV-2 has obvious amplification, the fluorescence value of other respiratory viruses, bacteria, human coronavirus and negative quality control substances is not increased. Under the reaction condition of 42 ℃, the amplification reaction is completed within 20 minutes, the operation process is simple, the application range is wide, the kit can be widely applied to various fluorescence detection devices, and the detection result can be visually read by combining a lateral chromatography test strip mode. The invention has the advantages of short detection time, strong specificity and good repeatability, can monitor the quality of the sample extraction and amplification process through the detection result of the human reference gene, and has good practical value.

Drawings

For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 is a diagram showing the result of SARS-CoV-2 gene primer screening by real-time fluorescence recombinase polymerase amplification;

FIG. 2 is a diagram showing the results of the reaction specificity test in the real-time fluorescent recombinase polymerase amplification technique;

FIG. 3 is a diagram showing the detection result of the real-time fluorescent dual recombinase polymerase amplification technique reaction system;

FIG. 4 is a diagram showing the detection result of the real-time fluorescent triple recombinase polymerase amplification technique reaction system;

FIG. 5 is a diagram showing the detection result of the real-time fluorescence quadruple recombinase polymerase amplification technique reaction system;

FIG. 6 is a diagram showing the result of dual detection of SARS-CoV-2 by recombinase polymerase amplification technology combined with lateral chromatography test paper;

FIG. 7 is a diagram showing the result of the triple detection of SARS-CoV-2 by the recombinase polymerase amplification technique in combination with a lateral chromatography test strip;

FIG. 8 is a diagram showing the results of the quadruple detection of SARS-CoV-2 by the recombinase polymerase amplification technique combined with the lateral chromatography test strip.

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. Through the embodiments, the present invention can be more clearly understood by scientific researchers, and certain changes and modifications can be made on the basis of the embodiments to obtain different research effects of the experimental methods in the following embodiments, which are conventional methods unless otherwise specified. The reagents involved in the experimental process are all conventional reagents, and the use of the reagents is all referred to the product use instruction.

Example 1

SARS-CoV-2 specific primer and probe design and screening for real-time fluorescent recombinase polymerase amplification technology

(1) Design of SARS-CoV-2 specific primer and probe

According to SARS-CoV-2 nucleic acid sequence published by CDC and WHO, the N gene, ORF1ab gene, E gene and human ACTB gene of SARS-CoV-2 are sequence-compared, and several correspondent specific primers and probes are designed in the conserved region, and the sequences of candidate primers and probes are shown in Table 1.

The design requirements of recombinase polymerase amplification technology primers are as follows: the overall principle is similar to that of PCR, for example, GC content cannot exceed 70% or be less than 30%, primer dimer formation between primers is avoided, etc. The exo probe design used requires: does not overlap with the recognition site of the specific primer, has the length of 46-52nt, and avoids palindromic sequence, internal secondary structure and continuous repeated base in the sequence; the exo probe has four modification sites: 1. tetrahydrofuran is used for replacing the original base at the position 30-35nt away from the 5' end to be used as the recognition site of the exonuclease; 2. a fluorescent group (such as FAM, VIC and ROX used in the embodiment of the invention) is marked on the T base at the upstream of the tetrahydrofuran site, a quenching group (such as BHQ1 used in the embodiment of the invention) is marked on the T base at the downstream, and the distance between the two groups is 2-4 nt; 3. the 3' end is labeled with a blocking group (e.g., C3-Spacer used in the examples of the present invention) that blocks probe extension.

TABLE 1 candidate primer and probe sequences for N gene, ORF1ab gene, E gene and human ACTB gene

(2) Screening of SARS-CoV-2 specific primer and probe

Reaction system: 400nM of forward and reverse primers for N gene/ORF 1ab gene/E gene/human ACTB gene, 29.4. mu.L of lysis buffer (40mM Tris buffer, 100mM potassium acetate), 120nM of probe for N gene/ORF 1ab gene/E gene/human ACTB gene, 2. mu.L of LSARS-CoV-2 pseudovirus extract, and a complement of 50. mu.L of enzyme-free water. The prepared reaction system was added to lyophilized enzyme powder (500 ng/. mu.L recombinase, 300 ng/. mu.L recombinase, 400 ng/. mu.L single-stranded DNA binding protein, 100 ng/. mu.L DNA polymerase, 150 ng/. mu.L exonuclease, 100 ng/. mu.L reverse transcriptase, 3mM ATP, 50mM phosphocreatine, 300 ng/. mu.L creatine kinase, 500. mu.M dNTPs, 5.5% w/v polyethylene glycol 20000, 2mM dithiothreitol), well dissolved and mixed, 2.5. mu.L 280mM magnesium acetate was added to a tube cap, centrifuged, transferred to a real-time quantitative PCR apparatus (Bio-rad, USA, CFX96) for amplification, and run at 42 ℃ for 20 minutes.

As shown in FIG. 1, different primer combinations were effective in amplification under the same reaction conditions, but the amplification effects were different, and by combining the two indexes of reaction threshold time and relative fluorescence intensity, the primer set with an earlier positive threshold time, a higher relative fluorescence unit, and a typical S-shaped curve was used as the optimal primer set, so that the optimal primer set for the N gene was R1F1, the optimal primer set for the ORF1ab gene was R4F7, the optimal primer set for the E gene was R4F6, and the optimal primer set for the human ACTB gene was R1F1, and the optimal primer sets and probes for each gene are shown in Table 2.

TABLE 2 optimal primer set and Probe for each Gene

(3) Real-time fluorescent recombinase polymerase amplification technology reaction specificity test

The N gene, ORF1ab gene, E gene optimal primer set and each genome probe screened above are used for detecting other pathogens, including human coronavirus (OC43, HKU1, 229E, NL63, SARS), middle east respiratory syndrome coronavirus (MERS-CoV), influenza A virus (Flu A), influenza B virus (Flu B), human parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV), human cytomegalovirus (HMPV), pseudomonas aeruginosa (Pae), Klebsiella pneumoniae (Kpn), staphylococcus aureus (Sau), Acinetobacter baumannii (Aba).

The result of real-time fluorescence PCR according to the reaction system and the reaction conditions is shown in figure 2, except that the nucleic acid of the clinical simulation sample of the positive pseudovirus and the SARS-CoV-2 is obviously amplified, the fluorescence value of other respiratory viruses, bacteria, human coronavirus and negative quality control products is not increased, the method of the invention can specifically detect the SARS-CoV-2, and the cross reaction is not generated between the SARS-CoV-2 and other pathogens.

Example 2

Real-time fluorescence multiplex recombinase polymerase amplification technology for detecting SARS-CoV-2

(1) Real-time fluorescent double recombinase polymerase amplification technology

The N gene, ORF1ab gene and E gene were combined with human ACTB gene, respectively, according to the optimal primer set for N gene, ORF1ab gene, E gene, human ACTB gene and each genome probe selected in example 1, and real-time fluorescence detection was performed, wherein the probe for N gene was labeled with FAM fluorophore, the probe for ACTB gene was labeled with ROX fluorophore, and the synthesized pseudovirus was used as a positive sample in the reaction system.

The reaction system amounted to 50. mu.L: mu.L of lysis buffer (40mM Tris buffer, 100mM potassium acetate), 600nM forward and reverse N gene/ORF 1ab gene/E gene primer, 180nM N gene/ORF 1ab gene/E gene probe, 200nM ACTB gene forward and reverse primer, 60nM ACTB probe, 2. mu.L of SARS-CoV-2 pseudovirus-extracted sample, 10. mu.L of SARS-CoV-2 pseudovirus-extracted sample⁴-10⁵Copy ACTB sample, the remaining volume was made up with enzyme free water. The prepared premix is added into freeze-dried enzyme powder (500 ng/. mu.L recombinase, 300 ng/. mu.L recombinase, 400 ng/. mu.L single-stranded DNA binding protein, 100 ng/. mu.L DNA polymerase, 150 ng/. mu.L exonuclease, 100 ng/. mu.L reverse transcriptase, 3mM ATP, 50mM phosphocreatine, 300 ng/. mu.L creatine kinase, 500. mu.M dNTPs, 5.5% w/v polyethylene glycol 20000 and 2mM dithiothreitol), fully dissolved and mixed, 2.5. mu.L 28mM magnesium acetate is added into a tube cover, the reaction is triggered by centrifugation, and the mixture is transferred to a real-time fluorescence quantitative PCR instrument for real-time fluorescence amplification, and the operation is carried out for 20 minutes at 42 ℃.

As a result, as shown in FIG. 3, the fluorescence value of the N gene increased about 7 minutes after the start of the reaction, the fluorescence value of the ACTB gene increased about 8 minutes, and a typical amplification curve was obtained within 10 minutes after the amplification reaction, which confirmed that the reaction system was successfully constructed.

(2) Real-time fluorescent triple recombinase polymerase amplification technology

Three sets of triple reaction systems were constructed according to the optimal primer sets for the N gene, ORF1ab gene, E gene, human ACTB gene and each genome probe selected in example 1: n gene, ORF1ab gene, and ACTB gene; n gene, E gene and ACTB gene; the E gene, ORF1ab gene and ACTB gene. Wherein target probes of the N gene and the ORF1ab gene are respectively marked by FAM and VIC fluorophores, ACTB gene is marked by ROX fluorophore, and the synthesized pseudovirus is used as a positive sample of the reaction system.

The reaction system amounted to 50. mu.L: 29.4. mu.L of lysis buffer (40mM Tris buffer, 100mM potassium acetate), 500nM NylThe forward primer, the reverse primer, the 180nM N gene probe, the 200nM ACTB gene forward primer, the reverse primer, the 180nM ACTB probe, the 300nM ORF1ab gene forward primer, the reverse primer, the 180nM ORF1ab gene probe, 2 uL SARS-CoV-2 pseudovirus sample, 10⁴-10⁵Copy ACTB sample, the remaining volume was made up with enzyme free water. The prepared premix is added into freeze-dried enzyme powder (500 ng/. mu.L recombinase, 300 ng/. mu.L recombinase, 400 ng/. mu.L single-stranded DNA binding protein, 100 ng/. mu.L DNA polymerase, 150 ng/. mu.L exonuclease, 100 ng/. mu.L reverse transcriptase, 3mM ATP, 50mM creatine phosphate, 300 ng/. mu.L creatine kinase, 500. mu.M dNTPs, 5.5% w/v polyethylene glycol 20000 and 2mM dithiothreitol), fully dissolved and mixed, 2.5. mu.L 28mM magnesium acetate is added into a tube cover, the mixture is transferred to a real-time fluorescence quantitative PCR instrument for amplification through centrifugal trigger reaction, and the mixture is operated at 42 ℃ for 20 minutes.

As a result, as shown in FIG. 4, the fluorescence values of both the N gene and ORF1ab gene increased at about 6 minutes after the start of the reaction, and the fluorescence value of the ACTB gene increased at about 10 minutes, confirming that the reaction system was successfully constructed.

(3) Real-time fluorescent quadruple recombinase polymerase amplification technology

A quadruple reaction system was constructed according to the optimum primer set for the N gene, ORF1ab gene, E gene, human ACTB gene and each genome probe selected in example 1: n gene, ORF1ab gene, E gene, and human ACTB gene. Wherein the target probes of the N gene, the ORF1ab gene and the E gene are respectively marked by FAM, VIC and CY5 fluorescent reporter groups, the ACTB gene is marked by ROX fluorescent reporter groups, and the synthesized pseudovirus is used as a positive sample of a reaction system.

The reaction system amounted to 50. mu.L: mu.L of lysis buffer (40mM Tris buffer, 100mM potassium acetate), 400nM forward and reverse primers for the N gene, 200nM forward and reverse ACTB genes, 120nM ACTB probe, 400nM forward and reverse ORF1ab gene, 120nM ORF1ab gene probe, 400nM forward and reverse E gene, 120nM E gene probe, 2. mu.L of SARS-CoV-2 pseudovirus-extracted sample, 10. mu.L of SARS-CoV-2 pseudovirus-extracted sample⁴-10⁵Copy ACTB sample, the remaining volume was made up with enzyme free water. Adding the prepared premix intoFreeze-dried enzyme powder (500 ng/. mu.L recombinase, 300 ng/. mu.L recombinase, 400 ng/. mu.L single-stranded DNA binding protein, 100 ng/. mu.L DNA polymerase, 150 ng/. mu.L exonuclease, 100 ng/. mu.L reverse transcriptase, 3mM ATP, 50mM creatine phosphate, 300 ng/. mu.L creatine kinase, 500. mu.M dNTPs, 5.5% w/v polyethylene glycol 20000, 2mM dithiothreitol), well dissolved and mixed, 2.5. mu.L 28mM magnesium acetate is added into a tube cover, the mixture is transferred to a real-time fluorescence quantitative PCR instrument for amplification through centrifugal trigger reaction, and the mixture is operated at 42 ℃ for 20 minutes.

As a result, as shown in FIG. 5, the fluorescence values of the N gene, ORF1ab gene, E gene and ACTB gene were increased about 10 minutes after the start of the reaction, and the success of the reaction system construction was confirmed.

Example 3

Multiple detection of SARS-CoV-2 by combining recombinase polymerase amplification technology with lateral chromatography test paper strip

The primers and probes for the optimal SARS-CoV-2 target gene determined in example 1 were modified, and the 5 '-ends of the reverse primers for ACTB gene, N gene, ORF1ab gene and E gene were labeled with digoxin antigen tags, while the 5' -ends of the forward primers were labeled with TAMRA, biotin, CY5 and FAM antigen tags, respectively. A goat anti-mouse monoclonal antibody is marked on a lateral chromatography test strip as a quality control line, and a monoclonal antibody (a gold-marked anti-TAMRA antibody) of an anti-human ACTB gene antigen label to be detected and 1 or more monoclonal antibodies (an N gene, an ORF1ab gene and an E gene) of an anti-SARS-CoV-2 target gene antigen label are marked, wherein the monoclonal antibodies are a gold-marked anti-biotin antibody, a gold-marked anti-CY 5 antibody and a gold-marked anti-FAM antibody respectively. The sequences of the primer pairs involved are shown in Table 3.

TABLE 3 optimal primer set for each gene

Primer and method for producing the same	Oligonucleotide sequence (5 '-3')
		ACTB-F1	TAMRA-CTCCATCCTGGCCTCGCTGTCCACCTTCCAG(SEQ ID NO:22)
ACTB-R1	digoxin-AATCTCATCTTGTTTTCTGCGCAAGTTAGG (SEQ ID NO:26)
		ORF21-F7	CY5-CCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTT(SEQ ID NO:7)
ORF21-R4	digoxin-CACCGCAAACCCGTTTAAAAACGATTGTGC (SEQ ID NO:12)
		N21-F1	Biotin-CAGCAGTAGGGGAACTTCTCCTGCTAGAAT (SEQ ID NO:17)
N21-R1	digoxin-GGCCTTTACCAGACATTTTGCTCTCAAGCTG (SEQ ID NO:19)
		F6.nCov.E	FAM-CCATCCTTACTGCGCTTCGATTGTGTGCGTACTG(SEQ ID NO:36)
R4.nCov.E	digoxin-GACCAGAAGATCAGGAACTCTAGAAGAATTCAGATTT (SEQ ID NO:40)

(1) Dual detection

The reaction system amounted to 50. mu.L: mu.L lysis buffer (40mM Tris buffer, 100mM potassium acetate), 600nM forward and reverse primers for N gene, 200nM forward and reverse primers for ACTB gene, 2. mu.L SARS-CoV-2 pseudovirus, 10⁴-10⁵Copy ACTB sample, the remaining volume was made up with enzyme free water. The prepared premix was added to lyophilized enzyme powder (500 ng/. mu.L recombinase, 300 ng/. mu.L recombinase cofactor, 400 ng/. mu.L single-stranded DNA-binding protein, 100 ng/. mu.L DNA polymerase, 100 ng/. mu.L reverse transcriptase, 3mM ATP, 50mM phosphocreatine, 300 ng/. mu.L creatine kinase, 500. mu.M dNTPs, 5.5% w/v polyethylene glycol 20000, 2mM dithiothreitol) to dissolve it sufficiently and mix well, 2.5. mu.L 28mM magnesium acetate was added to the vial cap, and the reaction was triggered by centrifugation at 42 ℃ for 20 minutes. And taking 10 mu L of amplification product, adding 190 mu L of dilution of the amplification product, uniformly mixing, vertically inserting a lateral chromatography test strip into the dilution, and observing the experiment result after 5 minutes.

The result is shown in fig. 6, clear bands are presented at the quality control line of the test strip, so that the accuracy of the detection result is ensured, clear bands are presented at the detection lines of the ACTB gene and the N gene, and the combination of the amplification product containing digoxin and TAMRA double-antigen markers and the amplification product containing digoxin and biotin double-antigen markers with the gold-labeled anti-TAMRA antibody and the gold-labeled anti-biotin antibody on the lateral chromatography test strip is proved.

The method is adopted to establish a dual reaction system of the E gene and the ACTB gene, the ORF1ab gene and the ACTB gene, and the result shows that clear strips are formed at the test strip quality control line, the internal reference gene test line and the SARS-CoV-2 target gene test line.

(2) Triple detection

Establishing a triple reaction system, wherein the total reaction system is 50 mu L, and the system comprises: mu.L of lysis buffer (40mM Tris buffer, 100mM potassium acetate), 500nM of forward and reverse N-gene primers, 200nM of forward and reverse ACTB gene primers, 300nM of forward and reverse ORF1ab gene primers, 2. mu.L of SARS-CoV-2 pseudovirus-extracted sample, 10. mu.L of the same, and the like⁴-10⁵Copy ACTB sample, the remaining volume was made up with enzyme free water. The prepared premix was added to lyophilized enzyme powder (500 ng/. mu.L recombinase, 300 ng/. mu.L recombinase cofactor, 400 ng/. mu.L single-stranded DNA binding protein, 100 ng/. mu.L DNA polymerase, 100 ng/. mu.L reverse transcriptase, 3mM ATP, 50mM phosphocreatine, 300 ng/. mu.L creatine kinase, 500. mu.M dNTPs, 5.5% w/v polyethylene glycol 20000, 2mM dithiothreitol) The mixture is fully dissolved and uniformly mixed, 2.5 mu L of 28mM magnesium acetate is added into a tube cover, the reaction is triggered by centrifugation, the mixture is transferred to a real-time fluorescent quantitative PCR instrument for amplification, and the reaction is carried out for 20 minutes at 42 ℃. And taking 10 mu L of amplification product, adding 190 mu L of dilution of the amplification product, uniformly mixing, vertically inserting a lateral chromatography test strip into the dilution, and observing the experiment result after 5 minutes.

The results are shown in figure 7, clear bands are presented at the test strip quality control line, which ensures the accuracy of the results, and clear bands are presented at the detection lines of ACTB gene, SARS-CoV-2N gene and ORF1ab gene, which proves that the amplification product containing digoxin and TAMRA double-antigen markers, the amplification product containing digoxin and biotin double-antigen markers and the amplification product containing digoxin and CY5 double-antigen markers are respectively combined with the anti-TAMRA antibody, the anti-biotin antibody and the anti-CY 5 antibody marked by gold on the lateral chromatography test strip.

The method is adopted to establish a triple reaction system of the N gene, the E gene and the ACTB gene, the ORF1ab gene, the E gene and the ACTB gene, and the result shows that clear bands are formed at the test strip quality control line, the reference gene detection line and the SARS-CoV-2 target gene detection line.

(3) Quadruple detection

Establishing a quadruple reaction system, wherein the total amount of the reaction system is 50 mu L: comprises 29.4 mu L of dissolving buffer solution (40mM Tris buffer solution, 100mM potassium acetate), 400nM of N gene forward and reverse primers, 200nM ACTB gene forward and reverse primers, 400nM ORF1ab gene forward and reverse primers, 400nM E gene forward and reverse primers, 2 mu L of SARS-CoV-2 pseudovirus extraction sample, 10 mM of SARS-CoV-2 pseudovirus extraction sample⁴-10⁵Copy ACTB sample, the remaining volume was made up with enzyme free water. The prepared premix is added into freeze-dried enzyme powder (500 ng/muL recombinase, 300 ng/muL recombinase, 400 ng/muL single-strand DNA binding protein, 100 ng/muL DNA polymerase, 100 ng/muL reverse transcriptase, 3mM ATP, 50mM phosphocreatine, 300 ng/muL creatine kinase, 500 muM dNTPs, 5.5% w/v polyethylene glycol 20000 and 2mM dithiothreitol) to be fully dissolved and mixed, 2.5 muL 28mM magnesium acetate is added into a tube cover, the mixture is transferred to a real-time fluorescence quantitative PCR instrument for amplification through centrifugal trigger reaction, and the reaction is carried out for 20 minutes at 42 ℃. Taking 10 μ LAnd adding 190 mu L of amplification product diluent into the amplification product, uniformly mixing, vertically inserting a lateral chromatography test strip into the amplification product, and observing the experiment result after 5 minutes.

The results are shown in FIG. 8, clear bands are presented at the quality control line of the test strip, which ensures the accuracy of the results, and the detection lines of the ACTB gene, the SARS-CoV-2N gene, the ORF1ab gene and the E gene all present clear bands, which proves that the amplification product containing digoxin and TAMRA double-antigen markers, the amplification product containing digoxin and biotin double-antigen markers, the amplification product containing digoxin and CY5 double-antigen markers and the amplification product containing digoxin and FAM double-antigen markers are respectively combined with the anti-TAMRA antibody, the anti-biotin antibody, the anti-CY 5 antibody and the anti-FAM antibody marked with gold on the lateral chromatography test strip.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Sequence listing

<110> China people liberation army special medical center

<120> a primer set, probe set and kit for multiple recombinase polymerase amplification technology for detecting novel coronavirus

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<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<212> DNA

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

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<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

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aacaaataaa gccatgccaa tctcatcttg 30

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<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

<400> 32

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<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

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<210> 34

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<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

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<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

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<210> 36

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<213> Artificial sequence (Artificial sequence)

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<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

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Claims (10)

1. A primer group and a probe group of a multiple recombinase polymerase amplification technology for detecting novel coronavirus are characterized in that: the primer group comprises one or more of a primer pair for amplifying the human ACTB gene and a primer pair for amplifying the novel coronavirus N gene, ORF1ab gene or E gene; the probe set comprises one or more of a probe for amplifying human ACTB gene and a probe for detecting N gene, ORF1ab gene and E gene of the novel coronavirus;

the primer pair for amplifying the novel coronavirus N gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 19; the primer pair for amplifying the novel coronavirus ORF1ab gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 7, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 12; the primer pair for amplifying the novel coronavirus E gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 36, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 40; the primer pair for amplifying the human ACTB gene comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 22, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 26;

the probe nucleotide sequence for detecting the novel coronavirus N gene is SEQ ID NO. 21; the probe nucleotide sequence for detecting the novel coronavirus ORF1ab gene is SEQ ID NO. 16; the nucleotide sequence of the probe for detecting the novel coronavirus E gene is SEQ ID NO. 45; the nucleotide sequence of the probe for detecting the human ACTB gene is SEQ ID NO. 30.

2. The primer set and probe set of claim 1, wherein the probes in the probe set have four modification sites: tetrahydrofuran and analogues thereof are used for replacing original bases at positions which are about 30 bases away from the 5 'end and about 15 bases away from the 3' end to serve as recognition sites of exonuclease; a T base at the upstream of tetrahydrofuran and analogues thereof is marked with a fluorescent group, a T base at the downstream is marked with a quenching group corresponding to the fluorescent group, and the distance between the two groups is 2-4 nt; the 3' end is labeled with a blocking group that blocks probe extension.

3. The primer set, probe set of claim 2, wherein the quencher group comprises BHQ1, BHQ2 or BHQ 3.

4. The primer set and probe set of claim 2, wherein the blocking group comprises C3-Spacer, amine group, biotin-triethylene glycol, or phosphate group.

5. The primer set and the probe set as claimed in claim 1, wherein the reverse primer of the N gene, the ORF1ab gene, the E gene or the human ACTB gene in the primer set is labeled with antigen tag 1, the forward primer of the N gene, the ORF1ab gene, the E gene or the human ACTB gene is labeled with antigen tags 2, 3, 4 and 5 respectively, and the antigen tag 1 is different from the antigen tags 2, 3, 4 and 5.

6. The primer set and probe set of claim 5, wherein the antigen tag 1 comprises biotin, FAM, CY5, TAMRA or digoxin.

7. A real-time fluorescence detection kit for detecting a novel coronavirus, which is characterized by comprising a dissolution buffer solution, freeze-dried enzyme powder, a magnesium acetate solution, a primer set and a probe set according to any one of claims 2 to 4; the dissolving buffer comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 500 ng/mu L of 100-one recombinant enzyme, 400 ng/mu L of 100-one recombinant enzyme cofactor, 900 ng/mu L of 400-one-stranded DNA binding protein, 50-200 ng/mu L of DNA polymerase, 500 ng/mu L of 100-one exonuclease, 50-100 ng/mu L of reverse transcriptase, 1-3mM ATP, 30-100mM creatine phosphate, 300 ng/mu L of 200-one creatine kinase, 500 mu M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol.

8. The kit according to claim 7, wherein the kit is used in a method comprising:

1) preparing a reaction system: 29.4 μ L of a lysis buffer, the primer set and the probe set as claimed in any one of claims 2 to 4, wherein the final concentration of the forward primer and the reverse primer of each gene in the primer set is 200 and 600 nM; the final concentration of each probe in the probe group is 60-180nM, and 50 muL in total;

2) adding the prepared reaction system into the freeze-dried enzyme powder, mixing uniformly, adding 2.5 mu L of 28mM magnesium acetate solution into a tube cover, centrifuging, and carrying out real-time fluorescence PCR amplification at 42 ℃ for 20 minutes.

9. A lateral chromatography test paper strip detection kit for detecting novel coronavirus, which is characterized by comprising a dissolution buffer solution, freeze-dried enzyme powder, a magnesium acetate solution, an amplification product diluent, a lateral chromatography test paper strip, a goat anti-mouse monoclonal antibody, a monoclonal antibody of an anti-human ACTB gene antigen label, a monoclonal antibody of 1 or more SARS-CoV-2 target gene antigen labels, a primer group and a probe group as described in claim 5 or 6; the dissolving buffer comprises 30-50mM Tris buffer and 50-150mM potassium acetate; the freeze-dried enzyme powder comprises 100-500 ng/. mu.L recombinase, 100-400 ng/. mu.L recombinase cofactor, 400-900 ng/. mu.L single-stranded DNA binding protein, 50-200 ng/. mu.L DNA polymerase, 50-100 ng/. mu.L reverse transcriptase, 1-3mM ATP, 30-100mM creatine phosphate, 200-300 ng/. mu.L creatine kinase, 200-500. mu.M dNTPs, 5-10% w/v polyethylene glycol 20000 and 1-5mM dithiothreitol.

10. The kit according to claim 9, wherein the kit is used in a method comprising:

1) configuring a reaction system, 29.4 μ L of a dissolution buffer solution, the primer group and the probe group described in claim 5 or 6, wherein the final concentration of the forward primer and the reverse primer of each gene in the primer group is 200-600 nM; the final concentration of each probe in the probe group is 60-180nM, and 50 muL in total;

2) adding the prepared reaction system into the freeze-dried enzyme powder, mixing uniformly, adding 2.5 mu L of 28mM magnesium acetate solution into a tube cover, centrifuging, and keeping the constant temperature at 42 ℃ for reaction for 20 minutes;

3) marking a goat anti-mouse monoclonal antibody, a monoclonal antibody of a human ACTB gene antigen label to be detected and monoclonal antibodies of 1 or more SARS-CoV-2 target gene antigen labels on a lateral chromatography test strip;

4) mu.L of amplification product was taken, 190. mu.L of amplification product diluent was added and mixed well, a lateral flow strip was inserted vertically therein, and the result was observed after 5 minutes.

Images