CN112831605A - Multienzyme isothermal amplification detection kit and application thereof - Google Patents
Multienzyme isothermal amplification detection kit and application thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention provides a multienzyme isothermal amplification detection kit and application thereof, and relates to the technical field of biomedical detection. The kit comprises at least one of a primer probe set for detecting influenza A virus H1N1, a primer probe set for detecting influenza A virus H3N2, a primer probe set for detecting influenza A virus H5N1, a primer probe set for detecting influenza B virus IVB and a primer probe set for detecting novel coronavirus SARS-CoV-2. The multienzyme isothermal amplification detection kit can specifically detect influenza A virus, influenza B virus and novel coronavirus, and has the advantages of high accuracy and high detection speed compared with the existing fluorescent quantitative PCR detection method.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a multienzyme isothermal amplification detection kit and application thereof.
Background
Multiplex Enzyme Isothermal Amplification (MIA) is based on the principle that a recombinase binds to a primer to form a protein-DNA complex, and can search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, strand exchange reaction formation occurs and DNA synthesis is initiated, and the target region on the template is exponentially amplified. The replaced DNA strand binds to SSB, preventing further replacement. In this system, a single synthesis event is initiated by two opposing primers. The entire process is carried out very quickly and detectable levels of amplification product are typically obtained within ten minutes.
Influenza is an acute viral respiratory infection caused by influenza virus, and has strong infectivity and high morbidity, wherein the influenza A virus has stronger epidemics and more serious symptoms than the influenza B virus. The novel coronavirus (SARS-CoV-2) is a new strain of coronavirus found in man in 2019. The symptoms caused by the virus are fever, hypodynamia, dry cough and gradual dyspnea, and severe patients show acute respiratory distress syndrome, septic shock, metabolic acidosis and blood coagulation dysfunction which are difficult to correct. The virus has a phenomenon of human transmission, the virus latency is generally 1-14 days, and the latency is infectious.
Among adults and children, fever-associated respiratory syndrome is the most common acute infectious disease, and nucleic acid detection is still the first option for confirmed diagnosis for patients and public health departments, and in addition, virus isolation and identification and serological detection of cases and hemagglutination and inhibition assay (HAI) and trace neutralization assay are also available, but since antibodies produced by human bodies are in the middle and later stages of disease development, early stealth carriers cannot be diagnosed and screened through antibody detection, and virus isolation requires 3-5 days to obtain culture results, and influenza virus identification results are completed 3-5 days after virus isolation, the methods can only be generally used as auxiliary detection means besides nucleic acid detection. Clinically, the disease is worsened or its spread is controlled due to the limited time and delay, which can have serious consequences.
Fluorescent quantitative PCR is the currently preferred protocol for detecting various respiratory viruses, but it has the following limitations: (1) false positive caused by aerosol pollution is easy to occur, and a professional trained PCR on-duty certificate technician, a professional PCR certification laboratory and expensive analysis equipment are required to be equipped; (2) the traditional fluorescent quantitative PCR sample nucleic acid extraction and amplification detection are carried out in two steps, and at least 3 hours are needed from sampling to detection result.
Therefore, it becomes necessary and urgent to develop a constant temperature amplification-based MIA detection method, which has high accuracy, high detection speed, and can specifically detect influenza A virus, influenza B virus and novel coronavirus through a multi-enzyme constant temperature amplification detection kit.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an MIA detection method and a kit based on isothermal amplification, the method has the advantages of high accuracy and high detection speed, and can be used for specifically detecting influenza A viruses, influenza B viruses and novel coronaviruses.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the multienzyme isothermal amplification detection kit provided by the invention comprises a primer probe group for detecting influenza A virus H1N 1;
and/or, a primer probe set for detecting influenza a virus H3N 2;
and/or, a primer probe set for detecting influenza a virus H5N 1;
and/or, a primer probe set for detecting influenza b virus IVB;
and/or, a primer probe set for detecting a novel coronavirus SARS-CoV-2.
Further, the kit comprises a primer probe group for detecting the influenza A virus H1N1, a primer probe group for detecting the influenza A virus H3N2, a primer probe group for detecting the influenza A virus H5N1, a primer probe group for detecting the influenza B virus IVB and a primer probe group for detecting the novel coronavirus SARS-CoV-2.
Further, the primers in the primer probe set for detecting influenza a virus H1N1 comprise:
the nucleotide sequence of the upstream primer H1N1-M-F1 is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream primer H1N1-M-R1 is shown as SEQ ID No. 2;
preferably, the probe in the primer probe set for detecting influenza a virus H1N1 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H1N1 is shown as SEQ ID No. 3;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H1N1 is as follows:
H1N1-M-P1：5’-CCCTAAATGGGAATGGGGACCCGAACAACA[i6FAMdT]G[iBHQ1dT]A[idSp]AGAGCAGTTAAACTA[iSpC3]-3’。
further, the primers in the primer probe set for detecting influenza a virus H3N2 comprise:
the nucleotide sequence of the upstream primer H3N2-HA-F1 is shown as SEQ ID No. 4;
the nucleotide sequence of the downstream primer H3N2-HA-R1 is shown as SEQ ID No. 5;
preferably, the probe in the primer probe set for detecting influenza a virus H3N2 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H3N2 is shown as SEQ ID No. 6;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H3N2 is as follows:
H3N2-HA-P1：GCCCTGGAGAACCAACATACAATTGATCTAAC[i6FAMdT]G[idSp]C[iBHQ1dT]CAGAAATGAACA[iSpC3]。
further, the primers in the primer probe set for detecting influenza a virus H5N1 comprise:
the nucleotide sequence of the upstream primer H5N1-M-F1 is shown as SEQ ID No. 7;
the nucleotide sequence of the downstream primer H5N1-M-R1 is shown as SEQ ID No. 8;
preferably, the probe in the primer probe set for detecting influenza a virus H5N1 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H5N1 is shown as SEQ ID No. 9;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H5N1 is as follows:
H5N1-M-P1：CGAGGACTGCAGCGTAGACGCTTTGTCCAGAA[i6FAMdT]G[idSp]C[iBHQ1dT]TAAATGGAAATGGAG[iSpC3]。
further, the primers in the primer probe set for detecting influenza B virus IVB comprise:
the nucleotide sequence of the upstream primer IVB-PA-F1 is shown as SEQ ID No. 10;
the nucleotide sequence of the downstream primer IVB-PA-R1 is shown as SEQ ID No. 11;
preferably, the probe in the primer probe set for detecting influenza B virus IVB is a modified fluorescent probe;
the sequence of the probe in the primer probe group for detecting the influenza B virus IVB is shown as SEQ ID No. 12;
the modified fluorescent probe in the primer probe group for detecting the influenza B virus IVB is as follows:
IVB-PA-P1：CACGGATGTTGTAACAGTTGTGACTT[i6FAMdT]CG[idSp]G[iBHQ1dT]TTAGTAGTACAGATCCTAG[iSpC3]。
further, the primer probe set for detecting the novel coronavirus SARS-CoV-2 comprises a first primer probe set, a second primer probe set and a third primer probe set;
preferably, the primers in the first primer probe set comprise:
the nucleotide sequence of the upstream primer SARS-CoV-2-N-F1 is shown in SEQ ID No. 13;
the nucleotide sequence of the downstream primer SARS-CoV-2-N-R1 is shown in SEQ ID No. 14;
more preferably, the probes in the first primer probe group are modified fluorescent probes, and the sequences of the probes in the first primer probe group are shown as SEQ ID No. 15;
the modified fluorescent probe in the first primer probe group is as follows:
SARS-CoV-2-N-P1：GCTTCAGCGTTCTTCGGAATGTCGCGCAT[i6FAMdT]GG[idSp]A[iBHQ1dT]GGAAGTCACACCTTCGGG[iSpC3]；
preferably, the primers in the second primer probe set comprise:
the nucleotide sequence of the upstream primer SARS-CoV-2-ORF1a/b-F1 is shown as SEQ ID No. 16;
the nucleotide sequence of the downstream primer SARS-CoV-2-ORF1a/b-R1 is shown as SEQ ID No. 17;
more preferably, the probes in the second primer probe group are modified fluorescent probes, and the sequences of the probes in the second primer probe group are shown as SEQ ID No. 18;
the modified fluorescent probe in the second primer probe group is as follows:
SARS-CoV-2-ORF1a/b-P1：GCGGTATGTGGAAAGGTTATGGCTGTAGT[iROXdT]G[idSp]GA[iBHQ1dT]CAACTCCGCGAAC[iSpC3]；
preferably, the primers in the third primer probe set comprise:
the nucleotide sequence of the upstream primer SARS-CoV-2-E-F1 is shown in SEQ ID No. 19;
the nucleotide sequence of the downstream primer SARS-CoV-2-E-R1 is shown in SEQ ID No. 20;
more preferably, the probes in the third primer probe group are modified fluorescent probes, and the sequences of the probes in the third primer probe group are shown as SEQ ID No. 21;
the modified fluorescent probe in the third primer probe group is as follows:
SARS-CoV-2-E-P1：CACTAGCCATCCTTACTGCGCTTCGATTGTG[iJOEdT]G[idSp]G[iBHQ1dT]ACTGCTGCAATATTGTT[iSpC3]。
the invention provides an application of the multienzyme isothermal amplification detection kit in preparation of products for detecting influenza A viruses;
and/or, the application in the preparation of products for detecting influenza B virus;
and/or, the application in preparing products for detecting novel coronavirus;
and/or, the application in the preparation of products for synchronously detecting influenza A virus, influenza B virus and novel coronavirus.
Further, the method of application comprises the steps of:
providing RNA of a sample to be detected, and then carrying out multi-enzyme isothermal amplification by using the multi-enzyme isothermal amplification detection kit;
preferably, the test sample comprises at least one of a viral cell culture fluid, a nasopharyngeal swab suspension, sputum, alveolar lavage fluid, autopsy lung tissue from dead cases, and tracheal secretions.
Furthermore, the initial concentration of the primers in the reaction system of the multi-enzyme isothermal amplification is 2-20 mmol/L, and the initial concentration of magnesium acetate is 50-280 mmol/L.
Compared with the prior art, the invention has the beneficial effects that:
the kit comprises at least one of a primer probe group for detecting influenza A virus H1N1, a primer probe group for detecting influenza A virus H3N2, a primer probe group for detecting influenza A virus H5N1, a primer probe group for detecting influenza B virus IVB and a primer probe group for detecting novel coronavirus SARS-CoV-2. The multienzyme isothermal amplification detection kit can specifically detect influenza A virus, influenza B virus and novel coronavirus, and has the advantages of high accuracy and high detection speed compared with the existing fluorescent quantitative PCR detection method.
The multienzyme isothermal amplification detection kit provided by the invention can be widely applied to detection of influenza A virus, influenza B virus and novel coronavirus.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of the sensitivity of fluorescence signal detection provided in example 4 of the present invention;
FIG. 2 is a graph showing the results of the specificity test provided in example 5 of the present invention;
FIG. 3 is an analysis chart of the accuracy fitness result of the MIA method and the quantitative PCR amplification result provided in example 6 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the invention, a multi-enzyme isothermal amplification detection kit comprises a primer probe set for detecting influenza a virus H1N 1;
and/or, a primer probe set for detecting influenza a virus H3N 2;
and/or, a primer probe set for detecting influenza a virus H5N 1;
and/or, a primer probe set for detecting influenza b virus IVB;
and/or, a primer probe set for detecting a novel coronavirus SARS-CoV-2.
The kit comprises at least one of a primer probe group for detecting influenza A virus H1N1, a primer probe group for detecting influenza A virus H3N2, a primer probe group for detecting influenza A virus H5N1, a primer probe group for detecting influenza B virus IVB and a primer probe group for detecting novel coronavirus SARS-CoV-2. The multienzyme isothermal amplification detection kit can specifically detect influenza A virus, influenza B virus and novel coronavirus, and has the advantages of high accuracy and high detection speed compared with the existing fluorescent quantitative PCR detection method.
Specifically, the beneficial effects of the present application can be summarized as follows:
(1) the invention takes different genes of different subtypes of influenza A virus, influenza B virus gene and novel coronavirus SARS-CoV-2, including SARS-CoV-2, for amplifying different subtypes of influenza A virus as detection targets, designs MIA detection primers and probes, and further establishes an MIA detection method based on constant temperature amplification. The MIA detection primer probe for various respiratory viruses has good application prospect not only for novel coronavirus and alphavirus, but also for other respiratory syndrome coronavirus and respiratory syncytial virus.
(2) The MIA primer and the probe for detecting various respiratory viruses can be used for completing detection of screening various respiratory viruses for clinical patients or clinics. The kit has the characteristics of specificity, low price, sensitivity, portability, use and the like. The kit can effectively and specifically detect a plurality of respiratory viruses with 5copies or more in single reaction.
(3) The MIA primer and the probe are used for detection, one to more than one different fluorescent signals can be detected in one reaction, and a plurality of samples can be detected simultaneously, so that the system design is effectively simplified, and the problems of inaccuracy and the like caused by different optical detection time of the samples are solved.
(4) Compared with the gold standard real-time fluorescent quantitative PCR, the kit has higher detection accuracy and higher sensitivity, and can complete detection within 15 minutes.
(5) The MIA detection primers and the MIA detection probes of the N gene, the E gene and the ORF1ab are adopted for detection, the accuracy is high, and false negative and missed detection caused by single gene detection can be avoided.
(6) The invention adopts multi-enzyme optimized proportioning and freeze drying to form dry powder enzyme and an amplification tube, thereby subtracting the reverse transcription process, optimizing the detection time and the operation steps and simplifying the method.
(7) The MIA primer and the probe of the invention have been verified by the detection of 70 cases of COVID-19 clinical real patient nucleic acid samples, the positive coincidence rate is 100%, the accuracy and the specificity are very high, and the MIA primer and the probe have persuasion of clinical application test effect.
In a preferred embodiment of the invention, the kit comprises a primer probe set for detecting influenza A virus H1N1, a primer probe set for detecting influenza A virus H3N2, a primer probe set for detecting influenza A virus H5N1, a primer probe set for detecting influenza B virus IVB, and a primer probe set for detecting novel coronavirus SARS-CoV-2.
As a preferred embodiment, the kit comprises a primer probe set for detecting influenza A virus H1N1, a primer probe set for detecting influenza A virus H3N2, a primer probe set for detecting influenza A virus H5N1, a primer probe set for detecting influenza B virus IVB, and a primer probe set for detecting novel coronavirus SARS-CoV-2. The kit can be used for synchronously detecting the influenza A virus, the influenza B virus and the novel coronavirus, and has the advantages of high accuracy and high speed, and the result can be obtained in about 15 minutes.
In a preferred embodiment of the present invention, the primer probe set for detecting influenza a virus H1N1 is a primer and a probe for amplifying M gene, and comprises:
the nucleotide sequence of the upstream primer H1N1-M-F1 is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream primer H1N1-M-R1 is shown as SEQ ID No. 2;
preferably, the probe in the primer probe set for detecting influenza a virus H1N1 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H1N1 is shown as SEQ ID No. 3;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H1N1 is as follows:
H1N1-M-P1：5’-CCCTAAATGGGAATGGGGACCCGAACAACA[i6FAMdT]G[iBHQ1dT]A[idSp]AGAGCAGTTAAACTA[iSpC3]-3’。
it should be noted that, the modification point of the fluorescent probe of the present application, wherein:
FAM/ROX/JOE is a fluorescent group;
BHQ1 is a quencher group;
dSp is a THF tetrahydrofuran modification site;
SpC3 is a 3' terminal spacer modification.
In a preferred embodiment of the present invention, the primer probe set for detecting influenza a virus H3N2 is a primer and a probe for amplifying an HA gene, and comprises:
the nucleotide sequence of the upstream primer H3N2-HA-F1 is shown as SEQ ID No. 4;
the nucleotide sequence of the downstream primer H3N2-HA-R1 is shown as SEQ ID No. 5;
preferably, the probe in the primer probe set for detecting influenza a virus H3N2 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H3N2 is shown as SEQ ID No. 6;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H3N2 is as follows:
H3N2-HA-P1：GCCCTGGAGAACCAACATACAATTGATCTAAC[i6FAMdT]G[idSp]C[iBHQ1Dt]CAGAAATGAACA[iSpC3]。
in a preferred embodiment of the present invention, the primer probe set for detecting influenza a virus H5N1 is a primer and a probe for amplifying M gene, and comprises:
the nucleotide sequence of the upstream primer H5N1-M-F1 is shown as SEQ ID No. 7;
the nucleotide sequence of the downstream primer H5N1-M-R1 is shown as SEQ ID No. 8;
preferably, the probe in the primer probe set for detecting influenza a virus H5N1 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H5N1 is shown as SEQ ID No. 9;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H5N1 is as follows:
H5N1-M-P1：CGAGGACTGCAGCGTAGACGCTTTGTCCAGAA[i6FAMdT]G[idSp]C[iBHQ1dT]TAAATGGAAATGGAG[iSpC3]。
in a preferred embodiment of the present invention, the primer probe set for detecting influenza b virus IVB is a primer and a probe for amplifying a PA gene, and comprises:
the nucleotide sequence of the upstream primer IVB-PA-F1 is shown as SEQ ID No. 10;
the nucleotide sequence of the downstream primer IVB-PA-R1 is shown as SEQ ID No. 11;
preferably, the probe in the primer probe set for detecting influenza B virus IVB is a modified fluorescent probe;
the sequence of the probe in the primer probe group for detecting the influenza B virus IVB is shown as SEQ ID No. 12;
the modified fluorescent probe in the primer probe group for detecting the influenza B virus IVB is as follows:
IVB-PA-P1：CACGGATGTTGTAACAGTTGTGACTT[i6FAMdT]CG[idSp]G[iBHQ1dT]TTAGTAGTACAGATCCTAG[iSpC3]。
in a preferred embodiment of the present invention, the primer probe set for detecting the novel coronavirus SARS-CoV-2 comprises a first primer probe set, a second primer probe set and a third primer probe set;
preferably, the first primer probe set is a primer and a probe for amplifying the N gene, and comprises:
the nucleotide sequence of the upstream primer SARS-CoV-2-N-F1 is shown in SEQ ID No. 13;
the nucleotide sequence of the downstream primer SARS-CoV-2-N-R1 is shown in SEQ ID No. 14;
more preferably, the probes in the first primer probe group are modified fluorescent probes, and the sequences of the probes in the first primer probe group are shown as SEQ ID No. 15;
the modified fluorescent probe in the first primer probe group is as follows:
SARS-CoV-2-N-P1：GCTTCAGCGTTCTTCGGAATGTCGCGCAT[i6FAMdT]GG[idSp]A[iBHQ1dT]GGAAGTCACACCTTCGGG[iSpC3]；
preferably, the second primer probe set is a primer and a probe for amplifying ORF1a/b gene, comprising:
the nucleotide sequence of the upstream primer SARS-CoV-2-ORF1a/b-F1 is shown as SEQ ID No. 16;
the nucleotide sequence of the downstream primer SARS-CoV-2-ORF1a/b-R1 is shown as SEQ ID No. 17;
more preferably, the probes in the second primer probe group are modified fluorescent probes, and the sequences of the probes in the second primer probe group are shown as SEQ ID No. 18;
the modified fluorescent probe in the second primer probe group is as follows:
SARS-CoV-2-ORF1a/b-P1：GCGGTATGTGGAAAGGTTATGGCTGTAGT[iROXdT]G[idSp]GA[iBHQ1dT]CAACTCCGCGAAC[iSpC3]；
preferably, the third primer probe set is a primer and a probe for amplifying an E gene, and comprises:
the nucleotide sequence of the upstream primer SARS-CoV-2-E-F1 is shown in SEQ ID No. 19;
the nucleotide sequence of the downstream primer SARS-CoV-2-E-R1 is shown in SEQ ID No. 20;
more preferably, the probes in the third primer probe group are modified fluorescent probes, and the sequences of the probes in the third primer probe group are shown as SEQ ID No. 21;
the modified fluorescent probe in the third primer probe group is as follows:
SARS-CoV-2-E-P1：CACTAGCCATCCTTACTGCGCTTCGATTGTG[iJOEdT]G[idSp]G[iBHQ1dT]ACTGCTGCAATATTGTT[iSpC3]。
according to one aspect of the invention, the application of the multi-enzyme isothermal amplification detection kit in preparation of products for detecting influenza A viruses is provided;
and/or, the application in the preparation of products for detecting influenza B virus;
and/or, the application in preparing products for detecting novel coronavirus;
and/or, the application in the preparation of products for synchronously detecting influenza A virus, influenza B virus and novel coronavirus.
The application of the kit for the detection of the multienzyme isothermal amplification of the present application is not intended for therapeutic purposes.
In a preferred embodiment of the invention, the use is for simultaneous detection of influenza a virus, influenza b virus and novel coronaviruses.
In a preferred embodiment of the invention, the method of application comprises the steps of:
providing RNA of a sample to be detected, and then carrying out multi-enzyme isothermal amplification by using the multi-enzyme isothermal amplification detection kit;
in the above preferred embodiment, the sample to be tested comprises at least one of a viral cell culture fluid, a nasopharyngeal swab suspension, a sputum, an alveolar lavage fluid, autopsy lung tissue in cases of death, and tracheal secretions.
In the preferred embodiment, the initial concentration of the primers in the reaction system of the multi-enzyme isothermal amplification is 2-20 mmol/L, and the initial concentration of the magnesium acetate is 50-280 mmol/L.
Preferably, the kit is used in a method comprising the following steps:
(1) and RNA template extraction: extracting RNA in a sample to be detected;
(2) and MIA amplification reaction: taking the RNA extracted in the step (1) as a template, and carrying out MIA amplification reaction in an MIA reaction tube by adopting an MIA primer and a probe in the kit;
(3) and detecting and analyzing MIA amplification products by fluorescence.
The kit comprises 5 groups of amplification reaction systems which respectively comprise a primer probe group for detecting influenza A virus H1N1, a primer probe group for detecting influenza A virus H3N2, a primer probe group for detecting influenza A virus H5N1, a primer probe group for detecting influenza B virus IVB and a primer probe group for detecting novel coronavirus SARS-CoV-2. Specifically, the reaction system for each set of MIA amplification reactions was calculated as 50 μ L:
1.0-10 mu L of template RNA;
1.0-5.0 mu L of upstream primer;
1.0-5.0 mu L of downstream primer;
0.5-5 mu L of probe primer;
rehydration Buffer (Rehydration Buffer) 29.5. mu.L;
0.5-2.5 mu L of magnesium acetate;
sterilizing RNase-free water and supplementing deionized water to 50 mu L;
the initial concentration of the upstream primer and the initial concentration of the downstream primer are both 1-10 mmol/L, and the initial concentration of the magnesium acetate is 50-280 mmol/L.
Further, the temperature of the MIA amplification reaction is 25-42 ℃, the amplification time is 1-60 min, and the optimal amplification time is 39 ℃ for 3-15 min.
More preferably, the fluorescence detection assay may utilize any instrument capable of identifying and collecting fluorescence signals, and the luminescent genes include, but are not limited to, FAM channels, and include fluorescence detection groups of Cy3, Cy5, Texas Red, Alexa flow, ROX, JOE, and the like.
More preferably, the isothermal reaction can be performed by any device capable of controlling temperature, including but not limited to a water bath, an incubator, and the like.
The technical solution of the present invention will be further described with reference to the following examples.
Note: in the present invention, unless otherwise specified, the terms H1N 1M gene, H3N2 HA gene, H5N 1M gene, IVB PA gene, SARS-CoV-2N gene, ORF1a/b gene and E gene are to be understood as whole gene fragments, and H1N 1M gene, H3N2 HA gene, H5N 1M gene, IVB PA gene, SARS-CoV-2N gene, ORF1a/b gene and E gene are not limited to the fragments related to the primer probe, and may be other primer-designable regions of the gene.
In the present invention, the fluorescent probe is not limited to FAM, and may be other fluorescence excitation groups such as GFP, Texas Red, DAPI, Alexa Fluor @488, FITC, Cy3, Cy5, Cy7, ROX, JOE, and Alexa Fluor @ 750;
in the present invention, the test sample may be a liquid sample such as virus cell culture fluid, nasopharyngeal swab suspension, sputum, alveolar lavage fluid, autopsy lung tissue in blood and death cases, tracheal secretion, etc. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art or related testers according to specific situations.
In the present invention, the dry powder enzyme composition: BST DNA polymerase, single-stranded DNA binding protein SSB, recombinase Cre capable of binding single-stranded nucleic acids (oligonucleotide primers), and reverse transcriptase are all commercially available.
In the following examples, reagents, instruments and the like used were all commercially available.
Example 1 primer and probe design:
according to the base sequences of different subtypes of genomes of novel coronavirus and alphavirus disclosed by NCBI, the base sequences of an M gene of H1N1, an HA gene of H3N2, an M gene of H5N1, a PA gene of IVB, an N gene of SARS-CoV-2, an ORF1a/b gene and an E gene are searched and compared, primers are artificially and creatively designed according to the primer design principle, and the optimal range of the primers is ensured by verifying the GC content and other parameters of the designed primers respectively.
Meanwhile, corresponding probes are respectively designed according to the design principle of the MIA probe primers. The designed multiple groups of primers are sent to the company of Biotechnology engineering (Shanghai) for synthesis, and RNase-free water is added to dilute the primers to 10 mmol/. mu.L for later use.
Specifically, the primers and probes for H1N 1M gene, H3N2 HA gene, H5N 1M gene, IVB PA gene, SARS-CoV-2N gene, ORF1a/b gene and E gene are as follows:
it should be noted that, in order to increase the accuracy of the scheme for detecting the novel coronavirus and avoid the occurrence of false positive, the applicant respectively makes 3 pairs of primer probe sets for the N gene, ORF1a/b gene and E gene in SARS-CoV-2, so that the detection rate is more accurate;
wherein, the primer probe group aiming at the N gene in the SARS-CoV-2 is a first primer probe group;
the primer probe group aiming at ORF1a/b gene in SARS-CoV-2 is a second primer probe group;
the primer probe group aiming at the E gene in SARS-CoV-2 is a third primer probe group.
The primers and probes can be respectively used for detecting the M gene of H1N1, the HA gene of H3N2, the M gene of H5N1, the PA gene of IVB, the N gene of SARS-CoV-2, the ORF1a/b gene and the E gene.
Example 2
A multi-enzyme isothermal amplification detection kit comprising the primers and probes of example 1;
the KIT also comprises a rehydration buffer solution, magnesium acetate, RNase-free water, sterilized deionized water, an RNA extraction KIT (QIAamp Viral RNA mini KIT of QIAGEN), and dry powder enzyme; the dry powder enzyme is the freeze-dried powder of BST DNA polymerase, single-stranded DNA binding protein SSB, recombinase Cre capable of binding single-stranded nucleic acid (oligonucleotide primer) and reverse transcriptase according to a certain proportion.
The RNA extraction kit is used for extracting RNA in a sample to be detected as a template; the RNA extraction KIT can be a conventional commercially available RNA extraction KIT, and in this example, the RNA extraction KIT used is a Viral RNA extraction KIT QIAamp Viral RNA mini KIT of QIAGEN.
The kit of this example comprises the following 7 MIA amplification reaction systems:
1. an MIA amplification reaction system for detecting influenza a virus H1N1, the reaction system comprising the primer probe set for detecting influenza a virus H1N1 in example 1;
the MIA amplification system for detecting the influenza A virus H1N1 comprises the following components in 50 mu L:
1.0-10 mu L of template RNA;
1.0-5.0 mu L of upstream primer;
1.0-5.0 mu L of downstream primer;
0.5-5 mu L of probe primer;
rehydration Buffer (Rehydration Buffer) 29.5. mu.L;
0.5-2.5 mu L of magnesium acetate;
dry powder enzyme;
sterilizing RNase-free water and supplementing deionized water to 50 mu L;
2. an MIA amplification reaction system for detecting influenza a virus H3N2, the reaction system comprising the primer probe set for detecting influenza a virus H3N2 in example 1;
the MIA amplification system for detecting the influenza A virus H3N2 is the same as the MIA amplification system of H1N1 except that the primer probe group for detecting the influenza A virus H3N2 is used for replacing the primer probe group for detecting the influenza A virus H1N 1.
3. An MIA amplification reaction system for detecting influenza a virus H5N1, the reaction system comprising the primer probe set for detecting influenza a virus H5N1 in example 1;
the MIA amplification system for detecting the influenza A virus H5N1 is the same as the MIA amplification system of H1N1 except that the primer probe group for detecting the influenza A virus H5N1 is used for replacing the primer probe group for detecting the influenza A virus H1N 1.
4. An MIA amplification reaction system for detecting influenza b virus IVB, the reaction system comprising the primer probe set for detecting influenza b virus IVB in example 1;
the MIA amplification system for detecting the influenza B virus IVB is the same as the MIA amplification system of H1N1 except that the primer probe group for detecting the influenza B virus IVB is used for replacing the primer probe group for detecting the influenza A virus H1N 1.
5. MIA amplification reaction system for detecting N gene of novel coronavirus SARS-CoV-2, the reaction system comprising the first primer probe set for detecting novel coronavirus SARS-CoV-2 in example 1;
the MIA amplification system for detecting the N gene in the novel coronavirus SARS-CoV-2 is the same as the MIA amplification system of H1N1 except that the first primer probe group for detecting the novel coronavirus SARS-CoV-2 is used for replacing the primer probe group for detecting the influenza A virus H1N 1.
6. MIA amplification reaction system for detecting ORF1a/b gene of novel coronavirus SARS-CoV-2, which contains the second primer probe set for detecting novel coronavirus SARS-CoV-2 in example 1;
the MIA amplification system for detecting ORF1a/b gene in the novel coronavirus SARS-CoV-2 is the same as the MIA amplification system of H1N1 except that the second primer probe group for detecting the novel coronavirus SARS-CoV-2 is used to replace the primer probe group for detecting the influenza A virus H1N 1.
7. MIA amplification reaction system for detecting E gene of novel coronavirus SARS-CoV-2, which contains the third primer probe set for detecting novel coronavirus SARS-CoV-2 in example 1;
the MIA amplification system for detecting the E gene in the novel coronavirus SARS-CoV-2 is the same as the MIA amplification system of H1N1 except that a third primer probe group for detecting the novel coronavirus SARS-CoV-2 is used for replacing a primer probe group for detecting the influenza A virus H1N 1.
The use method of the kit comprises the following steps:
(1) and RNA template extraction: extracting RNA in a sample to be detected, and taking the deionized water without the RNase as negative control;
(2) and MIA amplification reaction: and (2) carrying out MIA amplification reaction in an MIA reaction tube by using the RNA extracted in the step (1) as a template and adopting an MIA primer and a probe in the kit.
Example 3
Selecting identified common bacteria isolated from oral sputum and respiratory tract, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus (see attached Table 1)Culturing, and measuring the bacteria concentration with bacteria turbidimeter to 105CFU/mL, extracting with bacterial genome extraction kit from Qiagen, extracting nucleic acid template, measuring with Nanodrop, and adjusting concentration of bacterial nucleic acid template to 105copies/mL, non-specific amplification assay using the kit and MIA amplification method described in example 2.
Table 1:
in the above table, "-" indicates that no amplification occurred.
Example 4 sensitivity detection of MIA primers:
(1) the kit of example 2 is provided, and reagents are added according to the following reaction system, wherein gene detection primers and probes are not mixed and added, and amplification is performed respectively, specifically comprising:
respectively adding 2.1 mu L of upstream primer, 2.1 mu L of downstream primer, 0.6 mu L of probe, 29.5 mu L of Rehydration Buffer (Rehydration Buffer) and 3.2 mu L of RNase-free water sterilized deionized water into an MIA amplification tube (containing dry powder enzyme), adding 10 mu L of template after the dry powder in the amplification tube is completely dissolved, carrying out instantaneous centrifugation at 2,000rpm, and adding 2.5 mu L of activator magnesium acetate.
(2) Immediately placing the reaction tube in a preset instrument capable of collecting fluorescence signals, wherein the MIA reaction condition is 39 ℃, reacting for 30 minutes, and collecting the fluorescence signals every 30 seconds.
The specific sensitivity detection fluorescence signal diagram is shown in FIG. 1, and the SARA-CoV-2 detection sensitivity is 200copies/mL as can be seen from FIG. 1; the detection sensitivity of H1N1 is 200 copies/mL; the detection sensitivity of H3N2 is 300 copies/mL; the detection sensitivity of H5N1 is 300 copies/mL; IVB detection sensitivity 500 copies/mL.
Example 5 specific detection of MIA primers:
selecting nucleic acids (10) of SARS-CoV-2, H1N1, H3N2, H5N1 virus, etc4copies/mL) and PBS buffer as a template for specificity verification of the MIA primer.
The nucleic acid can be directly purchased from commercial influenza A virus, influenza B virus, new coronavirus pseudovirus reagent or gene synthesis corresponding plasmid, usually comprises plasmid DNA of H1N 1M gene, H3N2 HA gene, H5N 1M gene, IVB PA gene, SARS-CoV-2N gene, ORF1a/b gene and E gene fragment to be detected, and is diluted by 10 times into a series of concentration gradients including 10 times6copies/mL，105copies/mL，104copies/mL，103copies/mL，500copies/mL，300copies/mL，200copies/mL，102copies/mL，101copies/mL and 1 copies/mL.
Providing the kit of example 2, adding each reagent of the sample according to the following reaction system, wherein each gene detection primer and each probe are not added in a mixed manner, and are amplified respectively, specifically comprising: respectively adding 2.1 mu L of upstream primer, 2.1 mu L of downstream primer, 0.6 mu L of probe primer, 29.5 mu L of Rehydration Buffer (Rehydration Buffer) and 3.2 mu L of RNase-free water sterilized deionized water into an MIA amplification tube, adding 10 mu L of template after the dry powder in the amplification tube is completely dissolved, carrying out instantaneous centrifugation at 2,000rpm, and adding 2.5 mu L of activator magnesium acetate.
Immediately, the reaction tube was placed in a preset fluorescence signal collecting instrument at an MIA reaction condition of 39 ℃ for 30 minutes, and a fluorescence signal was collected every 30 seconds.
The specific specificity detection result is shown in FIG. 2, and it can be seen from A in FIG. 2 that the SARS-CoV-2 primer amplification detection only detects that SARS-CoV-2 is negative, and no non-specific amplification occurs; as shown in B in FIG. 2, the H1N1 primer amplification detection only detected that H1N1 was negative, and no non-specific amplification occurred; as can be seen from C in FIG. 2, the amplification detection of the H3N2 primer only detects that H3N2 is negative, and non-specific amplification does not occur; as can be seen from D in FIG. 2, the amplification detection of the H5N1 primer only detected that H5N1 was negative, and no non-specific amplification occurred; as can be seen from E in FIG. 2, the IVB primer amplification assay only detected IVB that were otherwise negative and no non-specific amplification occurred.
Example 6 comparative validation of the MIA method and the fluorescent quantitative PCR method
Influenza H1N1 virus (10) is selected5The correlation verification is carried out by using a copies/mL), H3N2 virus, H5N1 virus (105copies/mL) and SARS-CoV-2 virus as templates for MIA amplification and a fluorescent quantitative PCR method.
Adding each reagent in the MIA reaction according to the following reaction system, wherein each gene detection primer and each probe are not added in a mixing way and are respectively amplified, and the method specifically comprises the following steps: respectively adding 2.1 mu L of upstream primer, 2.1 mu L of downstream primer, 0.6 mu L of probe primer, 29.5 mu L of Rehydration Buffer (Rehydration Buffer) and 3.2 mu L of RNase-free water sterilized deionized water into an MIA amplification tube, adding 10 mu L of template after the dry powder in the amplification tube is completely dissolved, carrying out instantaneous centrifugation at 2,000rpm, and adding 2.5 mu L of activator magnesium acetate.
Immediately placing the MIA reaction tube in a preset instrument capable of collecting the fluorescence signals, wherein the MIA reaction condition is 39 ℃, reacting for 30 minutes, and collecting the fluorescence signals every 30 seconds.
Performing fluorescent quantitative PCR reaction, namely performing amplification by using a commercial quantitative detection kit according to detection primers and probes recommended by the world health organization, wherein the reaction system is as follows:
h1 or N1 gene:
5x
RT-PCR buffer 10μL；
dNTP mix 2μL；
5x Q-solution 10μL；
forward primer (5. mu.M) 6. mu.L;
reverse primer (5. mu.M) 6. mu.L;
2 mu L of enzyme mixed solution;
0.5 μ L of RNase inhibitor (20U/. mu.L);
9 mu L of deionized water;
5 μ L of viral nucleic acid;
50 μ L was accumulated.
The reaction conditions are as follows: reverse transcription at 30min 50 deg.C, and PCR enzyme activation (pre-denaturation) at 15min 95 deg.C;
and (3) circulating amplification:
denaturation for 30sec 94 ℃;
annealing for 30sec and 55 ℃;
extension for 30sec at 72 ℃ to collect fluorescence signal
40 cycles;
the final reaction is 2min 72 ℃.
FIG. 3 is a graph showing the results of the MIA method and the accuracy of the quantitative PCR amplification. As can be seen from fig. 3, the detection result of the MIA method and the detection result of the fluorescent quantitative PCR in this embodiment have no significant difference, the fitting rate is above 95%, and the current nucleic acid detection standard is the fluorescent quantitative PCR, which means that the detection efficiency of the established method is no inferior to that of the fluorescent quantitative PCR.
Application example 1 MIA method clinical sample test analysis
Selecting 300 cases of clinical samples to extract nucleic acid (70 cases of new coronary pneumonia infectors, 30 cases of negative controls, 100 positive samples of influenza A and influenza B infectors and 100 negative samples);
the test analysis of clinical samples was performed using the kits of example 2, and the specific results are shown in table 2:
table 2:
as can be seen from Table 2, the detection rate of 70 samples of new corolla pneumovirus confirmed by RT-PCR identification is 100%, and no amplification signal is found in the negative samples; the detection rate of 100 cases of nucleic acid samples of the influenza A is 100 percent, the detection rate of nucleic acid of the B samples is 96 percent (48 cases are detected, 2 cases are missed), and no detection signal is found in negative samples, so that the MIA method established by the invention is applied to the specific detection of nucleic acid detection of the influenza A and the B and the new coronavirus to reach 100 percent, the positive detection rate of clinical samples exceeds 95 percent, the method can be effectively applied to the primary detection of actual samples, and the application prospect is wide.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Hangzhou Baojin Biotechnology Co., Ltd
<120> multienzyme isothermal amplification detection kit and application thereof
<160> 21
<170> PatentIn version 3.5
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Claims (10)
1. The kit is characterized by comprising a primer probe group for detecting the influenza A virus H1N 1;
and/or, a primer probe set for detecting influenza a virus H3N 2;
and/or, a primer probe set for detecting influenza a virus H5N 1;
and/or, a primer probe set for detecting influenza b virus IVB;
and/or, a primer probe set for detecting a novel coronavirus SARS-CoV-2.
2. The kit of claim 1, wherein the kit comprises a primer probe set for detecting influenza a virus H1N1, a primer probe set for detecting influenza a virus H3N2, a primer probe set for detecting influenza a virus H5N1, a primer probe set for detecting influenza b virus IVB, and a primer probe set for detecting novel coronavirus SARS-CoV-2.
3. The kit according to claim 1 or 2, wherein the primers in the primer probe set for detecting influenza a virus H1N1 comprise:
the nucleotide sequence of the upstream primer H1N1-M-F1 is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream primer H1N1-M-R1 is shown as SEQ ID No. 2;
preferably, the probe in the primer probe set for detecting influenza a virus H1N1 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H1N1 is shown as SEQ ID No. 3;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H1N1 is as follows:
H1N1-M-P1：5’-CCCTAAATGGGAATGGGGACCCGAACAACA[i6FAMdT]G[iBHQ1dT]A[idSp]AGAGCAGTTAAACTA[iSpC3]-3’。
4. the kit according to claim 1 or 2, wherein the primers in the primer probe set for detecting influenza a virus H3N2 comprise:
the nucleotide sequence of the upstream primer H3N2-HA-F1 is shown as SEQ ID No. 4;
the nucleotide sequence of the downstream primer H3N2-HA-R1 is shown as SEQ ID No. 5;
preferably, the probe in the primer probe set for detecting influenza a virus H3N2 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H3N2 is shown as SEQ ID No. 6;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H3N2 is as follows:
H3N2-HA-P1：5’-GCCCTGGAGAACCAACATACAATTGATCTAAC[i6FAMdT]G[idSp]C[iBHQ1dT]CAGAAATGAACA[iSpC3]-3’。
5. the kit according to claim 1 or 2, wherein the primers in the primer probe set for detecting influenza a virus H5N1 comprise:
the nucleotide sequence of the upstream primer H5N1-M-F1 is shown as SEQ ID No. 7;
the nucleotide sequence of the downstream primer H5N1-M-R1 is shown as SEQ ID No. 8;
preferably, the probe in the primer probe set for detecting influenza a virus H5N1 is a modified fluorescent probe;
the sequence of the probe in the primer probe set for detecting the influenza A virus H5N1 is shown as SEQ ID No. 9;
the modified fluorescent probe in the primer probe set for detecting the influenza A virus H5N1 is as follows:
H5N1-M-P1：5’-CGAGGACTGCAGCGTAGACGCTTTGTCCAGAA[i6FAMdT]G[idSp]C[iBHQ1dT]TAAATGGAAATGGAG[iSpC3]-3’。
6. the kit according to claim 1 or 2, wherein the primers in the primer probe set for detecting influenza B virus IVB comprise:
the nucleotide sequence of the upstream primer IVB-PA-F1 is shown as SEQ ID No. 10;
the nucleotide sequence of the downstream primer IVB-PA-R1 is shown as SEQ ID No. 11;
preferably, the probe in the primer probe set for detecting influenza B virus IVB is a modified fluorescent probe;
the sequence of the probe in the primer probe group for detecting the influenza B virus IVB is shown as SEQ ID No. 12;
the modified fluorescent probe in the primer probe group for detecting the influenza B virus IVB is as follows:
IVB-PA-P1：5’-CACGGATGTTGTAACAGTTGTGACTT[i6FAMdT]CG[idSp]G[iBHQ1dT]TTAGTAGTACAGATCCTAG[iSpC3]-3’。
7. the kit according to claim 1 or 2, wherein the primer probe set for detecting the novel coronavirus SARS-CoV-2 comprises a first primer probe set, a second primer probe set and a third primer probe set;
preferably, the primers in the first primer probe set comprise:
the nucleotide sequence of the upstream primer SARS-CoV-2-N-F1 is shown in SEQ ID No. 13;
the nucleotide sequence of the downstream primer SARS-CoV-2-N-R1 is shown in SEQ ID No. 14;
more preferably, the probes in the first primer probe group are modified fluorescent probes, and the sequences of the probes in the first primer probe group are shown as SEQ ID No. 15;
the modified fluorescent probe in the first primer probe group is as follows:
SARS-CoV-2-N-P1：5’-GCTTCAGCGTTCTTCGGAATGTCGCGCAT[i6FAMdT]GG[idSp]A[iBHQ1dT]GGAAGTCACACCTTCGGG[iSpC3]-3’；
preferably, the primers in the second primer probe set comprise:
the nucleotide sequence of the upstream primer SARS-CoV-2-ORF1a/b-F1 is shown as SEQ ID No. 16;
the nucleotide sequence of the downstream primer SARS-CoV-2-ORF1a/b-R1 is shown as SEQ ID No. 17;
more preferably, the probes in the second primer probe group are modified fluorescent probes, and the sequences of the probes in the second primer probe group are shown as SEQ ID No. 18;
the modified fluorescent probe in the second primer probe group is as follows:
SARS-CoV-2-ORF1a/b-P1：5’-GCGGTATGTGGAAAGGTTATGGCTGTAGT[iROXdT]G[idSp]GA[iBHQ1dT]CAACTCCGCGAAC[iSpC3]-3’；
preferably, the primers in the third primer probe set comprise:
the nucleotide sequence of the upstream primer SARS-CoV-2-E-F1 is shown in SEQ ID No. 19;
the nucleotide sequence of the downstream primer SARS-CoV-2-E-R1 is shown in SEQ ID No. 20;
more preferably, the probes in the third primer probe group are modified fluorescent probes, and the sequences of the probes in the third primer probe group are shown as SEQ ID No. 21;
the modified fluorescent probe in the third primer probe group is as follows:
SARS-CoV-2-E-P1：5’-CACTAGCCATCCTTACTGCGCTTCGATTGTG[iJOEdT]G[idSp]G[iBHQ1dT]ACTGCTGCAATATTGTT[iSpC3]-3’。
8. the application of the multi-enzyme isothermal amplification detection kit according to any one of claims 1-7 in preparation of products for detecting influenza A viruses;
and/or, the application in the preparation of products for detecting influenza B virus;
and/or, the application in preparing products for detecting novel coronavirus;
and/or, the application in the preparation of products for synchronously detecting influenza A virus, influenza B virus and novel coronavirus.
9. The application according to claim 8, characterized in that the method of application comprises the steps of:
providing RNA of a sample to be detected, and then carrying out multi-enzyme isothermal amplification by using a multi-enzyme isothermal amplification detection kit;
preferably, the test sample comprises at least one of a viral cell culture fluid, a nasopharyngeal swab suspension, sputum, alveolar lavage fluid, autopsy lung tissue from dead cases, and tracheal secretions.
10. The application of the kit as claimed in claim 9, wherein the initial concentration of the primers in the reaction system of the multi-enzyme isothermal amplification is 2-20 mmol/L, and the initial concentration of the magnesium acetate is 50-280 mmol/L.
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