CN114182031A - Nucleic acid probe and application thereof - Google Patents
Nucleic acid probe and application thereof Download PDFInfo
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- CN114182031A CN114182031A CN202111578321.6A CN202111578321A CN114182031A CN 114182031 A CN114182031 A CN 114182031A CN 202111578321 A CN202111578321 A CN 202111578321A CN 114182031 A CN114182031 A CN 114182031A
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- nucleic acid
- acid probe
- plasmid
- pucm
- probe
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention provides a nucleic acid probe and application thereof. The 5 'end of the nucleic acid probe is marked with a fluorescent label, and the 3' end of the nucleic acid probe is marked with ddNTP.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a nucleic acid probe with two ends marked.
Background
Most of the existing colloidal gold detection is an immunodetection and diagnosis technology, and the detection technology based on nucleic acid amplification is relatively few. The nucleic acid colloidal gold detection technology can generate non-specific amplification and signals, so that most of nucleic acid colloidal gold detection cards can only realize single amplification, the design requirements for realizing double or even multiple amplification are greatly improved, and the application of the nucleic acid colloidal gold detection cards in the multi-index detection direction is limited.
Disclosure of Invention
One aspect of the present invention provides a nucleic acid probe, wherein an antigen label is labeled at the 5 'end and ddNTP is labeled at the 3' end.
In a specific embodiment, the antigen is one of FAM, Dig, FITC and HEX.
In a specific embodiment, the ddNTP is one of ddATP, ddGTP, ddCTP, and ddTTP.
In one embodiment, the nucleic acid probe has a sequence as shown in SEQ ID No.2 and/or SEQ ID No. 6.
The second aspect of the invention provides the use of a nucleic acid probe according to the first aspect of the invention in an immunoassay.
In a specific embodiment, the immunoassay is performed on a prawn acute hepatopancreatic necrosis pathogen and/or prawn enterocytozoon hepatica.
The invention has the beneficial effects that:
the invention simultaneously marks two ends of the probe, one end of the probe is marked with antigen, and the other end of the probe is sealed by ddNTP, thereby realizing the reduction of non-specific amplification, improving the detection specificity, reducing the design difficulty of the primer and the probe, and achieving the aim of realizing the multiple detection of the nucleic acid colloidal gold detection card.
Drawings
FIG. 1 shows a schematic of the insertion of amplification products into a biological anti-contamination test card.
FIG. 2 shows the results of detection in application example 1, wherein C1Representing the membrane scratching position of the goat anti-rabbit antibody for quality control; c2Represents the membrane-scratching position of digoxin antibody; t represents FAM antibody scoring position.
FIG. 3 shows the results of the detection of application example 2, in which C1Representing the membrane scratching position of the goat anti-rabbit antibody for quality control; c2Represents the membrane-scratching position of digoxin antibody; t represents FAM antibody scoring position.
FIG. 4 shows the result of detection of comparative application example 1, in which C1Representing the membrane scratching position of the goat anti-rabbit antibody for quality control; c2Represents the membrane-scratching position of digoxin antibody; t represents FAM antibody scoring position.
FIG. 5 shows the result of detection of comparative application example 2, in which C1Representing sheep resistanceThe membrane scratching position of the rabbit antibody is used for quality control; c2Represents the membrane-scratching position of digoxin antibody; t represents FAM antibody scoring position.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not to be construed as limiting the invention in any way.
The biological anti-pollution detection card is a closed anti-pollution colloidal gold detection test strip produced by Jiangsu Hunting matrix biotechnology limited, an anti-biotin antibody is spotted on a gold label pad in the test strip, and an FAM antibody, a DIG antibody and a goat anti-rabbit antibody are sequentially marked from the near end to the far end away from a sample hole.
Example 1
The nucleic acid sequence of the probe for detecting the gene (shown as SEQ ID No. 1) of Vibrio parahaemolyticus (Vibrio parahaemolyticus) pirB which is a pathogen of prawn acute hepatopancreatic necrosis (EMS) is shown as SEQ ID No. 2.
EMS pathogen nucleic acid probes were synthesized in Shanghai Biotechnology Ltd, and the synthesized probes were designated as JAP, labeled as digoxin (Dig) at the 5 'end and ddCTP at the 3' end.
The primer pair for amplifying the EMS pirB gene is AF-b (SEQ ID No.3) and AR (SEQ ID No.4), and is synthesized by Shanghai Biotechnology Co., Ltd, wherein the 5' end of the upstream primer AF-b is marked with biotin.
Preparation of positive test plasmid EMS plasmid: amplifying an EMS pirB virulence gene, inserting the amplified fragment into a pUCm-T vector (2710bp), transforming a ligation product into an Escherichia coli DH5 alpha competent cell, and carrying out positive colony screening through PCR and sequencing to obtain a recombinant plasmid pUCm-EMSPIR, wherein the corresponding positive recombinant strain is DH5 alpha/pUCm-EMSPIR.
The plasmid pUCm-EMSPIR was extracted from DH 5. alpha./pUCm-EMSPIR as a recombinant strain, the concentration and purity of the plasmid DNA were measured by an ultramicro spectrophotometer, and the number of copies of DNA contained in a unit volume of the plasmid was calculated according to Moore's law. Plasmid copy number ═ plasmid concentration × plasmid volume × 6.02 × 1023.00 ]/[ (vector length + target pirB gene fragment length) × 660.00 ]. The pUCm-EMSPIR plasmid was diluted to 10 pg/microliter with purified water.
Example 2
The nucleic acid sequence of the probe for detecting 18s rDNA (shown as SEQ ID No. 5) of prawn Enterobacter Hepaticae (EHP) is shown as SEQ ID No. 6.
An EHP pathogenic nucleic acid probe was synthesized by Shanghai Biotechnology Ltd, and the synthesized probe was named JEP, and 5-carboxyfluorescein (5-FAM) was labeled at the 5 'end and ddCTP was labeled at the 3' end.
The primer pair for amplifying the EHP18s rDNA is GCF-b (SEQ ID No.7) and GCR (SEQ ID No.8) which are synthesized by Shanghai Biotechnology Co., Ltd, wherein the upstream primer GCF-b 5' end is labeled with biotin.
Preparation of positive test plasmid EHP plasmid: the test plasmid contains the amplified fragments of the primers and probes. The recombinant plasmid pUCm-EHP18s is obtained from Jiangsu Hunt-matrix Biotechnology Co., Ltd at the early stage, EHP18s rDNA is taken for amplification, the amplified fragment is inserted into a pUCm-T vector, a ligation product is transformed into a competent cell, positive colony screening is carried out through PCR and sequencing, and the recombinant plasmid pUCm-EHP18s is obtained, wherein the corresponding positive recombinant strain is DH5 alpha/pUCm-EHP 18 s.
The pUCm-EHP18s plasmid was extracted from DH 5. alpha./pUCm-EHP 18s, the concentration and purity of the plasmid DNA was determined by ultraspectrophotometer, and the number of copies of DNA contained in the plasmid per unit volume was calculated according to Moore's law. Plasmid copy number ═ plasmid concentration × plasmid volume × 6.02 × 1023.00 ]/[ (vector length + target 18s rDNA fragment length) × 660.00 ]. The pUCm-EHP18s plasmid was diluted to 10 pg/microliter with purified water.
Comparative example 1
The nucleic acid sequence of the probe for detecting the gene of Vibrio parahaemolyticus (Vibrio parahaemolyticus) pirB of acute hepatopancreatic necrosis (EMS) of prawns is shown in SEQ ID No. 1.
An EMS pathogen nucleic acid probe is synthesized by Shanghai Biotechnology Limited, and is named as AP, only the 5 'end is marked with Dig, and the 3' end is not marked.
The rest is the same as example 1.
Comparative example 2
The nucleic acid sequence of the probe for detecting 18s rDNA gene of prawn Enterobacter Hepaticai (EHP) is shown as SEQ ID No. 4.
An EHP pathogenic nucleic acid probe, designated EP, was synthesized at Shanghai Biotech, Inc., and was labeled only with FAM at the 5 'end and not labeled at the 3' end.
The rest is the same as example 2.
Application example 1
Preparation of 20. mu.l amplification reaction: 20 μ M JAP 0.1 μ l, 20 μ M JEP 0.1 μ l, 20 μ M AF-b 0.2 μ l, 20 μ M AR 0.1 μ l, 20 μ M GCF-b 0.2 μ l, 20 μ M GCR 0.1 μ l, 2 XPCR premix Mix 10 μ l, ddH2O7.2. mu.l, 10 pg/. mu.l of pUCM-EMSPIR plasmid 2. mu.l.
PCR amplification procedure: 2min at 95 ℃; 5s at 95 ℃, 10s at 59 ℃ and 40 cycles; 5s at 95 ℃, 10s at 72 ℃ and 3 cycles; 1min at 95 ℃; storing at 60 deg.C for 2min and 4 deg.C.
As shown in FIG. 1, 20. mu.l of the amplification product was put into a well 1 of a biological anti-contamination test card (Jiangsu Hunter matrix), 100. mu.l of purified water was put into a well 2, and the well was pressed for 5 seconds by a card presser and left to stand for 3 minutes. The results are shown in FIG. 2, with the results interpreted through the interpretation window.
Application example 2
Example 1 was applied in the same manner as in the case of using 10 pg/. mu.l of pUCM-EHP18s plasmid instead of 10 pg/. mu.l of pUCM-EMSPIR plasmid.
The results are shown in FIG. 3.
Comparative application example 1
Preparation of 20. mu.l amplification reaction: mu.l of 20. mu.M AP 0.1. mu.l, 20. mu.M EP 0.1. mu.l, 20. mu.M AF-b 0.2. mu.l, 20. mu.M AR 0.1. mu.l, 20. mu.M GCF-b 0.2. mu.l, 20. mu.M GCR 0.1. mu.l, 2 XPCR premix Mix 10. mu.l, ddH2O7.2. mu.l, 10 pg/. mu.l of pUCM-EMSPIR plasmid 2. mu.l.
Other examples are the same as in example 1.
The results are shown in FIG. 4.
Comparative application example 2
Example 1 was applied in the same manner as in the above example, except that 10 pg/. mu.l of pUCM-EHP18s plasmid was used instead of 10 pg/. mu.l of pUCM-EMSPIR plasmid.
The results are shown in FIG. 5.
As can be seen from FIGS. 2 to 5, the dual-labeled probe has good detection specificity and no non-specific signal appears. And the single-labeled probe has nonspecific signals in detection, which causes interference to experimental results. The double-labeled probe can effectively improve the specificity of the colloidal gold nucleic acid detection test strip, reduce non-specific amplification and reduce the design difficulty of primers and probes in the multi-index detection of the nucleic acid colloidal gold test strip.
While the invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various changes can be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, and method to the essential scope and spirit of the present invention. All such modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
<110> Jiangsu Hunting battle Biotech Co., Ltd
<120> nucleic acid probe and use thereof
<130> LHA2160722
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 302
<212> DNA
<213> Vibrio parahaemolyticus (Vibrio parahaemolyticus)
<400> 1
caaaccaaga taacgtgtat gatgaagtga tgggtgctcg tagttggtgt acggttcacg 60
gctttgaaca tatgcttatt tggcaaaaaa tcaaagagtt gaaaaaagtt gatgtgtttg 120
ttcacagtaa tttaatttca tattcacctg ctgttggttt tcctagtggt aatttcaact 180
atattgctac aggtacggaa gatgaaatac ctcaaccatt aaaaccaaat atgtttgggg 240
aacgtcgaaa tcgtattgta aaaattgaat catggaacag tattgaaata cattattaca 300
at 302
<210> 2
<211> 45
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
tttttttttt tttttttttt ttttaggaaa accaacagca ggtga 45
<210> 3
<211> 38
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
tttttttttt tttttttttt gggtgctcgt agttggtg 38
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
acgatttcga cgttccccaa 20
<210> 5
<211> 432
<212> DNA
<213> Penaeus vannamei enterohepatis (Enterocytozoon hepatopenaei)
<400> 5
agcggaacgg atagggagca tggtataggt gggcaaagaa tgaaatctca agaccccacc 60
tggaccaacg gaggcgaaag cgatgctctt agacgtatct ggggatcaag gacgaaggct 120
agagtatcga aagtgattag acaccgctgt agttctagca gtaaactatg ccgacaatgc 180
tgggtgttgc gagagcgatg cttggtgtgg gagaaatctt agttttcggg ctctggggat 240
agtacgctcg caagggtgaa acttaaagcg aaattgacgg aaggacacta ccaggagtgg 300
attgtgctgc ttaatttaac tcaacgcggg aaaacttacc agggtcaagt ctatcgtaga 360
ttggagacat gaggtagaca agagtggtgc atggccgttg gaaattgatg gggcgacttt 420
tagcttaagt gc 432
<210> 6
<211> 44
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
tttttttttt tttttttttt ttttcatcgc tctcgcaaca ccca 44
<210> 7
<211> 44
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
tttttttttt tttttttttt tttgtgggca aagaatgaaa tctc 44
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
gcaccactct tgtctacctc a 21
Claims (6)
1. A nucleic acid probe comprising an antigen label labeled at the 5 'end and ddNTP labeled at the 3' end.
2. The nucleic acid probe of claim 1, wherein the antigenic marker is one of FAM, Dig, FITC and HEX.
3. The nucleic acid probe of claim 1, wherein the ddNTP is one of ddATP, ddGTP, ddCTP, and ddTTP.
4. The nucleic acid probe of claim 1, wherein the sequence of the nucleic acid probe is shown in SEQ ID No.2 and/or SEQ ID No. 6.
5. Use of a nucleic acid probe according to any one of claims 1 to 4 in an immunoassay.
6. The use of claim 5, wherein the subject of said immunoassay is prawn acute hepatopancreatic necrosis pathogen and/or prawn enterocytozoon hepatica.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111578321.6A CN114182031A (en) | 2021-12-22 | 2021-12-22 | Nucleic acid probe and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111578321.6A CN114182031A (en) | 2021-12-22 | 2021-12-22 | Nucleic acid probe and application thereof |
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| CN114182031A true CN114182031A (en) | 2022-03-15 |
Family
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| CN202111578321.6A Pending CN114182031A (en) | 2021-12-22 | 2021-12-22 | Nucleic acid probe and application thereof |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100248980A1 (en) * | 2007-12-04 | 2010-09-30 | Panagene Inc. | Method for Selective Labeling and Detection of Target Nucleic Acids Using Immobilized Peptide Nucleic Acid Probes |
| US20130190208A1 (en) * | 2010-09-20 | 2013-07-25 | Seegene, Inc. | Detection of target nucleic acid sequences by exonucleolytic activity using single-labeled immobilized probes on solid phase |
| US20160273029A1 (en) * | 2013-10-30 | 2016-09-22 | Mast Group Limited | Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification |
| US20200056227A1 (en) * | 2018-08-16 | 2020-02-20 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Shrimp disease detection assays and uses thereof |
| CN111020709A (en) * | 2019-12-31 | 2020-04-17 | 江苏猎阵生物科技有限公司 | Gene chip and kit |
| CN111073984A (en) * | 2019-12-30 | 2020-04-28 | 江苏猎阵生物科技有限公司 | Kit and method for detecting target nucleic acid |
-
2021
- 2021-12-22 CN CN202111578321.6A patent/CN114182031A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100248980A1 (en) * | 2007-12-04 | 2010-09-30 | Panagene Inc. | Method for Selective Labeling and Detection of Target Nucleic Acids Using Immobilized Peptide Nucleic Acid Probes |
| US20130190208A1 (en) * | 2010-09-20 | 2013-07-25 | Seegene, Inc. | Detection of target nucleic acid sequences by exonucleolytic activity using single-labeled immobilized probes on solid phase |
| US20160273029A1 (en) * | 2013-10-30 | 2016-09-22 | Mast Group Limited | Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification |
| US20200056227A1 (en) * | 2018-08-16 | 2020-02-20 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Shrimp disease detection assays and uses thereof |
| CN111073984A (en) * | 2019-12-30 | 2020-04-28 | 江苏猎阵生物科技有限公司 | Kit and method for detecting target nucleic acid |
| CN111020709A (en) * | 2019-12-31 | 2020-04-17 | 江苏猎阵生物科技有限公司 | Gene chip and kit |
Non-Patent Citations (1)
| Title |
|---|
| 黄纪徽;吴山楠;王丹云;张亮亮;陈平亚;程奇;张建勋;: "虾肝肠胞虫实时荧光RAA快速检测方法的建立", 养殖与饲料, no. 11, pages 17 - 20 * |
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