CN114182046B - Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof - Google Patents
Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof Download PDFInfo
- Publication number
- CN114182046B CN114182046B CN202110949243.XA CN202110949243A CN114182046B CN 114182046 B CN114182046 B CN 114182046B CN 202110949243 A CN202110949243 A CN 202110949243A CN 114182046 B CN114182046 B CN 114182046B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- primer
- nucleotide sequence
- probe
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of detection of microorganisms and nucleic acid genomes, in particular to a primer combination and a probe for detecting nucleic acid of human herpesvirus (EB virus) which is common clinically by utilizing an isothermal nucleic acid amplification technology, and particularly relates to an isothermal nucleic acid detection primer probe combination of the EB virus, a kit and application thereof. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
Description
Technical Field
The invention relates to the field of detection of microorganisms and nucleic acid genomes, in particular to a pathogen nucleic acid detection primer probe combination of human herpesvirus, a kit and application thereof.
Background
EB virus (Epstein-Barr virus, EBV) is a double stranded DNA virus in the human herpesvirus family and can cause a variety of common clinical diseases, EBV infection being the primary cause of infectious mononucleosis, common in children and closely associated with B-lymphoma related diseases and nasopharyngeal carcinoma. Like other human herpesviruses, EB virus infection can also cause nervous system infections, respiratory tract diseases, and the like. EB virus also has the characteristic of latency, and after infection of human body, it will often latency to human B lymphocyte, and when the human body has low immunity, it is easy to reactivate and cause related diseases, especially in neonates with incomplete development, in patients with HIV infection and in patients receiving organ transplantation, and seriously endanger the life and health of human beings.
Currently, the etiology identification of EB virus infection in clinic mainly includes immunological detection and molecular biological detection. Immunological detection is suitable for epidemiological investigation, but has the problems of long time consumption, unstable window period and the like. Although the amphotropic agglutination test may suggest an EBV infection, it is often negative in children, affecting the clinical doctor's judgment of the disease. In recent years, quantitative polymerase chain reaction (Quantitative Polymerase chain reaction, qPCR) is gradually becoming a "gold standard" for pathogen nucleic acid detection, and quantitative detection of pathogen nucleic acid provides early diagnosis and a basis for prevention and treatment in clinic. However, expensive and sophisticated equipment is difficult in basic applications, and there is a need to develop a simple, rapid, convenient, and applicable method of nucleic acid detection.
Disclosure of Invention
In view of this, the present invention provides a pathogen nucleic acid detection primer probe combination of human herpesvirus (EB virus), a kit and applications thereof. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe combination, which comprises a first primer probe combination and/or a second primer probe combination;
the first primer probe combination is an isothermal nucleic acid amplification system combination containing exogenous internal references, and comprises:
(I) The upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 4; or (b)
A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), (II) and/or (III), and having the same or similar function as the nucleotide sequence shown in (I), (II) and/or (III); or (b)
(V) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I), (II), (III) and/or (IV); and/or
The second primer probe combination is an isothermal nucleic acid amplification system combination containing endogenous reference, and comprises the following components:
(I) The upstream primer has a nucleotide sequence shown as SEQ ID No. 5; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 6; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 7; or (b)
A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), (II) and/or (III), and having the same or similar function as the nucleotide sequence shown in (I), (II) and/or (III); or (b)
(V) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I), (II), (III) and/or (IV).
In some embodiments of the invention, the primer probe combinations further comprise a target gene probe having:
(I) A nucleotide sequence shown as SEQ ID No. 3; or (b)
(II) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I); or (b)
(III) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I) and/or (II).
In some embodiments of the invention, the second primer probe combination is an isothermal nucleic acid amplification system combination containing endogenous internal controls, further comprising:
(I) The upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 3; or (b)
A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), (II) and/or (III), and having the same or similar function as the nucleotide sequence shown in (I), (II) and/or (III); or (b)
(V) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I), (II), (III) and/or (IV).
Based on the research, the invention also provides application of the primer combination in preparing a reagent or a kit for detecting the EB virus. In some embodiments of the invention, the human herpesvirus is epstein barr virus. In some embodiments of the invention, the detected amplification temperature is 39℃and the amplification time is 30min.
In addition, the invention also provides a detection reagent for human herpesvirus, which comprises the primer probe combination and acceptable auxiliary agents. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
More importantly, the invention also provides a detection kit for human herpesvirus, which comprises the primer probe combination and acceptable auxiliary agents or carriers. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
The Recombinase-mediated isothermal nucleic acid amplification technology (RAA) is an isothermal nucleic acid amplification technology with independent intellectual property rights in China which is newly appeared in recent years, and is widely applied to the pathogen nucleic acid detection field and single nucleotide polymorphism detection, wherein the RAA technology is tightly combined with a primer under the constant temperature condition to form an enzyme and primer polymer, when the primer searches a sequence which is completely matched with the primer on a template DNA, the template double strand is opened under the action of a single-strand DNA binding protein, template synthesis is started under the action of the DNA polymerase to form the DNA double strand, the exponential amplification is realized by repeating the cycle for 30min, the amplification can be rapidly completed, and the result can be judged by the detection of a specific fluorescent probe in the fastest minutes. The internal quality control (IC) includes exogenous internal reference and endogenous internal reference, and is an important system monitoring of nucleic acid detection reaction, in which IC is introduced into the nucleic acid detection reaction system, and the whole reaction system is monitored in real time to avoid false negative results caused by misoperation, incorrect temperature, incorrect mixture of the reaction system, poor enzyme activity or the existence of inhibitory substances in the sample matrix. Exogenous internal reference is to add a section of internal reference template into the reaction system or specimen. The endogenous internal reference is derived from human genome in the sample, different primer pairs are used for respectively amplifying and detecting target genes and human operation genes, more importantly, the whole process from nucleic acid extraction to result interpretation can be monitored in real time, the amplification reaction can be monitored, and the sample acquisition quality and the nucleic acid extraction quality can be interpreted.
The invention adopts two different forms of IC to be introduced into the RAA reaction system, thereby realizing the real-time nucleic acid detection and monitoring of the EB virus. In particular to a primer combination and a probe for detecting human herpesvirus (EB virus) nucleic acid which are common in clinic by utilizing an isothermal nucleic acid amplification technology. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
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.
FIGS. 1 (A) to 1 (B) are graphs showing analysis of exogenous internal reference concentration by the EBV technique using isothermal nucleic acid amplification detection of exogenous internal reference, and the target gene is 10 0 Copying/reacting. Wherein, 1:1, 2:2,3:4;4:10;
FIGS. 2 (A) to 2 (B) are graphs showing analysis of exogenous internal reference concentration by an EBV technique for isothermal nucleic acid amplification detection containing exogenous internal reference, the target gene being 5 copies/reaction; wherein, 1:1, 2:2,3:4;4:10;
FIG. 3 (A) to FIG. 3 (B) are graphs showing analysis of exogenous internal reference concentration by the EBV technique using isothermal nucleic acid amplification detection of exogenous internal reference, and the target gene is 10 1 Copying/reacting; wherein, 1:1, 2:2,3:4;4:10;
FIGS. 4 (A) to 4 (B) show the containing outer partSensitivity analysis of the EBV technique for isothermal nucleic acid amplification detection of the source internal control. Wherein, 1: negative, 2:5,3:10 1 ，4：10 2 ，5:10 3 ,6:10 4 ;
FIGS. 5 (A) to 5 (B) are specific analysis charts showing an EBV technique for isothermal nucleic acid amplification detection containing exogenous internal references;
FIG. 6 (A) to FIG. 1 (B) are sensitivity analysis diagrams showing an EBV technique for isothermal nucleic acid amplification detection containing an endogenous reference having a concentration of 2 ✕ 10 4 Copy/. Mu.L. Wherein, 1:10 4 ，2:10 3 ，3：10 2 4：10 1 ,5:2,6: negative;
FIGS. 7 (A) to 7 (B) are sensitivity analysis diagrams showing an EBV technique for isothermal nucleic acid amplification detection containing an endogenous reference having a concentration of 2 ✕ 10 2 Copy/. Mu.L; wherein, 1:10 4 ，2:10 3 ，3：10 2 4：10 1 ,5:2,6: negative;
FIGS. 8 (A) to 8 (B) are diagrams showing sensitivity analysis of an EBV technique for isothermal nucleic acid amplification detection containing an endogenous reference at a concentration of 2 copies/. Mu.L; wherein, 1:10 4 ，2:10 3 ，3：10 2 4：10 1 ,5:2,6: negative;
FIGS. 9 (A) to 9 (B) are specific analysis charts showing an EBV technique for isothermal nucleic acid amplification detection containing endogenous reference;
note that: all panels a represent EBV target gene amplification panels (FAM fluorescence) of the invention and all panels B represent reference DNA amplification panels (HEX fluorescence) corresponding to panels a; the X-axis represents time (min) and the Y-axis represents fluorescence value (mv).
Detailed Description
The invention discloses a pathogen nucleic acid detection primer probe combination of human herpesvirus, a kit and application thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
In order to achieve the above object, the inventors have found a conserved sequence having high homology based on the EB virus genome sequence and the human genome (RNasP) sequence, and designed specific primer pairs and target probes suitable for isothermal nucleic acid amplification techniques. The specific DNA sequences of the two target genes are provided by comparison, and the fragment positions of the two target genes are 177631-177771bp (EBV) of a sequence disclosed by GenBank accession number M80571.1 and 554-708bp (RNaseP) of a sequence disclosed by GenBank accession number U77664.1.
The invention provides a primer probe combination, which comprises a first primer probe combination and/or a second primer probe combination;
the first primer probe combination is an isothermal nucleic acid amplification system combination containing exogenous internal references, and comprises:
(I) The upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 4; or (b)
A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), (II) and/or (III), and having the same or similar function as the nucleotide sequence shown in (I), (II) and/or (III); or (b)
(V) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I), (II), (III) and/or (IV); and/or
The second primer probe combination is an isothermal nucleic acid amplification system combination containing endogenous reference, and comprises the following components:
(I) The upstream primer has a nucleotide sequence shown as SEQ ID No. 5; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 6; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 7; or (b)
A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), (II) and/or (III), and having the same or similar function as the nucleotide sequence shown in (I), (II) and/or (III); or (b)
(V) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I), (II), (III) and/or (IV).
In some embodiments of the invention, the primer probe combinations further comprise a target gene probe having:
(I) A nucleotide sequence shown as SEQ ID No. 3; or (b)
(II) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I); or (b)
(III) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I) and/or (II).
In some embodiments of the invention, the second primer probe combination in the primer probe combination is an isothermal nucleic acid amplification system combination containing endogenous internal controls further comprising:
(I) The upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 3; or (b)
A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), (II) and/or (III), and having the same or similar function as the nucleotide sequence shown in (I), (II) and/or (III); or (b)
(V) a nucleotide sequence having at least 80% identity to the nucleotide sequences shown in (I), (II), (III) and/or (IV).
Based on the research, the invention also provides application of the primer combination in preparing a reagent or a kit for detecting human herpesvirus. In some embodiments of the invention, the primer probe combination, the human herpesvirus is epstein barr virus.
In the embodiment of the invention, the isothermal nucleic acid amplification system adopted by the invention is specifically configured as follows: buffer solution, freeze-dried powder, target gene forward primer, target gene reverse primer, target gene probe, internal reference forward primer, internal reference reverse primer, internal reference probe, internal reference and target gene template, non-ribozyme water and magnesium acetate.
More importantly, the invention also provides a detection reagent for human herpesvirus, which comprises the primer probe combination and acceptable auxiliary agents. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
The invention also provides a detection kit for human herpesvirus (EB virus), which comprises the primer probe combination and acceptable auxiliary agents or carriers. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
The invention adopts two forms of internal references. One is exogenous internal reference (recombinant DNA) consisting of plant virus fragment and target gene fragment (EBV fragment in the present invention), wherein the complementary region of EBV target gene probe (SEQ ID NO. 3) is replaced by plant virus fragment, and other sequence positions are reserved. In this case, the exogenous internal DNA is added in advance to the isothermal nucleic acid amplification reaction system, and the EBV target gene and the exogenous internal DNA are detected by fluorescence using the same primer sequences and the probes. The other is an endogenous reference, which is a fragment of human genomic DNA, in which case the EBV target gene and the exogenous reference are amplified using respective primers and probes, respectively.
In some embodiments of the invention, the amplification detection temperature is 39-42 ℃, preferably 39 ℃, and the amplification detection time is 15-30 min, preferably 30min.
Interpretation of the results: the system was mixed and placed in a fluorescence detector with FAM (target gene) and HEX (reference) channels. And setting the curve amplification slope of the fluorescence detector to be more than or equal to 20 in the amplification time range, and judging positive if the curve amplification slope has an obvious amplification curve.
Wherein, FAM and HEX are positive, and the positive result is judged.
FAM is positive, HEX is negative, and positive result is judged.
FAM is negative, HEX is positive, and negative result is judged.
Both FAM and HEX were negative and judged as invalid.
The invention provides primer combinations and probes for the detection of pathogen nucleic acids of two clinically common human herpesviruses using isothermal nucleic acid amplification techniques. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
The pathogen nucleic acid detection primer probe combination, the kit and the application thereof of the human herpesvirus provided by the invention can be purchased from the market. RAA amplification detection kit (fluorescence method) was purchased from Jiangsu Qihai Inc.
The invention is further illustrated by the following examples:
and downloading and comparing all EBV whole genome sequences with the genome sequences of RNaseP, and selecting the sequences with high conservation. According to the RAA primer and probe design principle, primers and probes are manually designed for the EBV BARF1 gene and the RPP38 gene. The length of the primer and the probe ranges from 30 bp to 35bp and from 46 bp to 52bp respectively. The probe consisted of upstream oligonucleotides carrying 5'-FAM (target gene probe) and 5' -HEX (IC probe), respectively linked to adjacent downstream oligonucleotides via THF spacer. The 3' end of the downstream oligonucleotide carries a C3' spacer (polymerase extension blocking group), and the specificity of the primers and probes was analyzed and evaluated using oligo7.0 software and NCBI's Primer BLAST software, the sequences of which are shown in SEQ ID No.1-SEQ ID No. 7. All primers and probes were synthesized by professional company, the primers were purified by PAGE and the probes were purified by HPLC. The three probes are double-labeled probes, the target gene probe uses FAM, the IC probe uses HEX, the quenching groups are BHQ-1, and tetrahydrofuran is arranged between the two fluorescent groups.
The exogenous internal reference sequence is a selected EBV target gene sequence, and the target gene probe position is replaced by a plant virus sequence fragment. The target probe positions are replaced with rose-loop sequences in the selected EBV genome sequence. The professional company is entrusted to synthesize, construct and return target genes and two reference target sheets for system establishment and optimization in the subsequent invention.
EBV target gene sequence 5'-3': (as shown in SEQ ID No. 8)
ATGGCCAGGTTCATCGCTCAGCTCCTCCTGTTGGCCTCCTGTGTGGCCGCCGGCCAGGCTGTCACCGCTTTCTTGGGTGAGCGAGTCACCCTGACCTCCTACTGGAGGAGGGTGAGCCTCGGTCCAGAGATTGAGGTCAGCTGGTTTAAACTGGGCCCAGGAGAGGAGCAGGTGCTTATTGGGCGCATGCACCACGATGTCATCTTTATAGAGTGGCCTTTCAGGGGCTTCTTTGATATCCACAGAAGTGCCAACACCTTCTTTTTAGTAGTCACCGCTGCCAACATCTCCCATGACGGCAACTACCTGTGCCGCATGAAACTGGGCGAGACCGAGGTCACCAAGCAGGAACACCTGAGCGTGGTGAAGCCTCTAACGCTGTCTGTCCACTCCGAAAGGTCTCAGTTCCCAGACTTCTCTGTCCTTACTGTGACATGCACCGTGAATGCATTTCCCCATCCCCACGTCCAGTGGCTCATGCCCGAGGGCGTGGAGCCCGCACCAACTGCGGCAAATGGCGGTGTTATGAAGGAAAAGGATGGGAGCCTCTCTGTTGCTGTTGACCTGTCACTTCCCAAGCCCTGGCACCTGCCAGTGACCTGCGTTGGGAAAAATGACAAGGAGGAAGCCCACGGGGTTTATGTTTCTGGATACTTGTCGCAATAA
5'-3' of exogenous internal reference sequence (shown as SEQ ID No. 9)
ATGGCCAGGTTCATCGCTCAGCTCCTCCTGTTGGCCTCCTGTGTGGCCGCCGGCCAGGCTGTCACCGCTTTCTTGGGTGAGCGAGTCACCCTGACCTCCTACTGGAGGAGGGTGAGCCTCGGTCCAGAGATTGAGGTCAGCTGGTTTAAACTGGGCCCAGGAGAGGAGCAGGTGCTTATTGGGCGCATGCACCACGATGTCATCTTTATAGAGTGGCCTTTCAGGGGCTTCTTTGATATCCACAGAAGTGCCAACACCTTCTTTTTAGTAGTCACCGCTGCCAACATCTCCCATGACGGCAACTACCTGTGCCGCATGAAACTGGGCGAGACCGAGGTCACCAAGCAGGTAAGGTGCTAGACTAAAATTGTTGGGACTTTGAATCTCTGAAGTAAAAGGAGGTCTCAGTTCCCAGACTTCTCTGTCCTTACTGTGACATGCACCGTGAATGCATTTCCCCATCCCCACGTCCAGTGGCTCATGCCCGAGGGCGTGGAGCCCGCACCAACTGCGGCAAATGGCGGTGTTATGAAGGAAAAGGATGGGAGCCTCTCTGTTGCTGTTGACCTGTCACTTCCCAAGCCCTGGCACCTGCCAGTGACCTGCGTTGGGAAAAATGACAAGGAGGAAGCCCACGGGGTTTATGTTTCTGGATACTTGTCGCAATAA
Endogenous reference sequence 5'-3': as shown in SEQ ID No.10
TGGACTTCAGAAGATTGAAGATAAGAAGAAAAAGAACAAAACACCTTTTCTGAAAAAAGAAAGCAGAGAGAAATGCAGCATTGCTGTTGATATTAGTGATAATCTGAAGGAGAAGAAAACAGATGCTAAGCAGCAAGTGTCAGGGTGGACGCCTGCACACGTCAGGAAGCAGCTTGTCATTGGCGTTAACGAAGTTACCAGAGCCCTGGAAAGGAGGGAACTGCTGTTAGTTCTGGTGTGTAAATCAGTCAAGCCTGCCATGATCACCTCACACTTGATTCAGTTAAGCCTAAGCAGAAGTGTCCCTGCCTGTCAGGTCCCCCGGCTCAGTGAGAGAATCGCCCCCGTCATTGGCTTAAAATGTGTTCTAGCCTTGGCGTTCAAAAAGAACACCACTGACTTTGTGGACGAAGTAAGAGCCATCATCCCCAGAGTCCCCAGTTTAAGTGTACCATGGCTTCAAGACAGAATTGAAGATTCTGGGGAAAATTTAGAGACTGAACCTCTGGAAAGCCAAGACAGAGAGCTTTTGGACACTTCATTTGAAGATCTGTCAAAACCTAAGAGAAAGCTTGCTGACGGTCGGCAGGCTTCTGTAACATTACAACC
The primers and probes were synthesized using the primer probes (SEQ ID No.1-SEQ ID No. 4) designed in example 1. The template was constructed using the synthesized EBV target gene DNA and exogenous internal reference DNA of example 1.
The reagent adopts a RAA fluorescent detection kit for Jiangsu Qiyan, and the whole isothermal nucleic acid amplification system (50 ul) is configured as follows: 25 mu L of buffer solution, a forward primer (shown as SEQ ID No.1 and 420 nM) of a target gene, a reverse primer (shown as SEQ ID No.2 and 420 nM), a target gene probe (shown as SEQ ID No.3 and 120 nM), an exogenous reference probe (shown as SEQ ID No.4 and 120 nM), exogenous reference DNA, 1 mu L of a template and no nuclease water are adjustable in volume. The mixed reaction system was added to a reaction tube (single-strand binding protein 500 ng/. Mu. L, DNA polymerase 90 ng/. Mu.L, recombinase 400 ng/. Mu.L, exonuclease 85 ng/. Mu.L and UvsY protein 70 ng/. Mu.L) previously filled with lyophilized powder, and finally 14mM magnesium acetate was added.
Interpretation of the results: after the system was mixed, the reaction tube was placed in a fluorescence detector with FAM (target gene) and HEX (reference) channels. And setting the curve amplification slope of the fluorescence detector to be more than or equal to 20 in the amplification time range, and judging positive if the curve amplification slope has an obvious amplification curve. The amplification effect is mainly judged by the fluorescence signal intensity and the peak time.
Wherein, FAM and HEX are positive, and the positive result is judged.
FAM is positive, HEX is negative, and positive result is judged.
FAM is negative, HEX is positive, and negative result is judged.
Both FAM and HEX were negative and judged as invalid, suggesting a retest.
Confirmation of exogenous reference concentration
According to experience, when the exogenous internal reference DNA concentration is respectively 10 copies/. Mu.L, 4 copies/. Mu.L, 2 copies/. Mu.L and 1 copy/. Mu.L, the EBV target gene standard plasmid (1, 5, 10 copies/reaction) with low copy is detected, so that the amplification of the exogenous internal reference can not influence the normal amplification of the target gene and the proper exogenous internal reference DNA concentration is confirmed.
The results show that when the copy number of the EBV target gene is 10 copies, exogenous internal references with 4 different concentrations are judged to be positive, the normal amplification of the target gene is not affected, and the amplification effect of the target gene is not obviously different. As shown in fig. 3A-3B. When the copy number of the EBV target gene was 5 copies, the exogenous internal references at 4 different concentrations were judged positive and did not affect the normal amplification of the target gene. Wherein, the amplification effect (fluorescence value and peak time) of the target gene (1 copy) in the presence of 1 copy/. Mu.L of the exogenous reference DNA concentration is superior to that of the target gene in the presence of the other 3 exogenous reference DNA concentrations. As shown in fig. 3A-3B. When the copy number of the EBV target gene was 1 copy, 4 exogenous internal references of different concentrations were judged positive, while the target gene was negative. As shown in fig. 3A-3B. The above examples were repeated to obtain the same amplification results, and the reproducibility was good. Thus, the amplification of the internal reference according to the present invention does not affect the amplification of the target gene, and the amplification effect of the target gene is optimized. The final selection was made at a suitable exogenous reference DNA concentration of 1 copy per microliter.
The isothermal nucleic acid amplification technique containing exogenous internal reference for detecting EBV according to the present invention was analyzed for sensitivity and specificity according to the system and exogenous internal reference DNA concentrations determined in this example.
An EBV target gene template of example 1 was used (10 4 、10 3 、10 2 、10 1 、5、10 0 Copy/reaction) for sensitivity analysis. When the concentration of the exogenous reference DNA is 1 copy/mu L, the amplification of the reference DNA does not affect the normal amplification of the target gene, and the sensitivity of the target gene is 5 copies/reaction. The above results were repeated, and the reproducibility was good. The results are shown in FIGS. 4A-4B.
Clinical sample positive nucleic acids using other viruses collected in the present invention include EBV, human herpesvirus type 1, type 2, type 5, type 6, human hepatitis B virus, human hepatitis C virus and West Nile virus, and human genome. The results show that the method has no cross reaction with other pathogens, and the method has good specificity. As shown in fig. 5A-5B.
Example 3 establishment of an isothermal nucleic acid amplification detection EBV technical System with endogenous reference, sensitivity analysis, specificity analysis
In contrast to example 2, the primers and probes were synthesized using the primers probes designed in example 1 (SEQ ID No.1-SEQ ID No.3 and SEQ ID No.5-SEQ ID No. 7). The template was constructed using the synthesized EBV target gene DNA and endogenous reference DNA of example 1.
The reagent adopts a RAA fluorescent detection kit for Jiangsu Qiyan, and the whole isothermal nucleic acid amplification system (50 ul) is configured as follows: the volumes of the buffer 25. Mu.L, the target gene forward primer (shown as SEQ ID No.1, 420 nM), the reverse primer (shown as SEQ ID No.2, 420 nM), the target gene probe (shown as SEQ ID No.3, 120 nM), the endogenous reference forward primer (shown as SEQ ID No.4, 120 nM), the endogenous reference reverse primer (shown as SEQ ID No.4, 120 nM), the endogenous reference probe (shown as SEQ ID No.4, 50 nM), the nuclease-free water and the template are adjustable. The mixed reaction system was added to a reaction tube (single-strand binding protein 500 ng/. Mu. L, DNA polymerase 90 ng/. Mu.L, recombinase 400 ng/. Mu.L, exonuclease 85 ng/. Mu.L and UvsY protein 70 ng/. Mu.L) previously filled with lyophilized powder, and finally 14mM magnesium acetate was added.
Interpretation of the results: after the system was mixed, the reaction tube was placed in a fluorescence detector with FAM (target gene) and HEX (reference) channels. And setting the curve amplification slope of the fluorescence detector to be more than or equal to 20 in the amplification time range, and judging positive if the curve amplification slope has an obvious amplification curve. The amplification effect is mainly judged by the fluorescence signal intensity and the peak time.
Wherein, FAM and HEX are positive, and the positive result is judged.
FAM is positive, HEX is negative, and positive result is judged.
FAM is negative, HEX is positive, and negative result is judged.
Both FAM and HEX were negative and judged as invalid, suggesting a retest.
According to the system determined in this example, the isothermal nucleic acid amplification technique containing exogenous internal reference for detecting EBV in the present invention was analyzed for sensitivity and specificity.
Empirically, the EBV target gene template of example 1 in real time was used (10 4 、10 3 、10 2 、10 1 2 copies/reaction), sensitivity analysis was performed in the presence of 3 different endogenous references. The results showed that the endogenous reference concentration was 2 ✕ 10 4 The sensitivity of the detection results can reach 2 copies per reaction by copying/mu L and 8 times of repeated detection results. The results are shown in FIGS. 6A-6B; at an endogenous reference concentration of 2 ✕ 10 2 The sensitivity of the detection results can reach 2 copies per reaction by copying/mu L and 8 times of repeated detection results. The results are shown in FIGS. 7A-7B; the detection result is repeated for 8 times at the endogenous reference concentration of 2 copies/mu L, and the sensitivity can reach 2 copies per reaction. The results are shown in FIGS. 8A-8B.
Clinical sample positive nucleic acids using other viruses collected in the present invention include EBV, human herpesvirus type 1, type 2, type 5, type 6, human hepatitis B virus, human hepatitis C virus, and West Nile virus. The results show that the method has no cross reaction with other pathogens, and the method has good specificity. As shown in fig. 9A-9B.
Example 4 evaluation of clinical samples
125 clinical samples of patients suspected of EBV infection were collected, DNA was extracted using Qiagen mini DNA Kit kit, and evaluation of clinical samples was performed on both EBV IC-RAA methods.
1. Samples were DNA detected using the commercial polymerase chain reaction quantification kit of EBV.
2. The reaction procedures are as follows: 93 ℃ for 2min;93 ℃,45s,55 ℃,1min,10 cycles; 93 ℃,30s,55 ℃,45s,30 cycles.
3. The reaction components are as follows: 42 mu LPCR reaction solution, 3 mu LTaq enzyme, 5 mu LDNA.
4. Interpretation of the results: when CT is less than or equal to 30, positive; CT > 30, negative.
5. The collected specimens were examined according to the isothermal nucleic acid amplification detection EBV technique system containing the exogenous internal reference established in example 2 and the isothermal nucleic acid amplification detection EBV technique system containing the endogenous internal reference established in example 3.
6. The results are interpreted with reference to examples 2 and 3.
The test results are shown in the following table:
the collected clinical samples were evaluated using the established isothermal nucleic acid amplification detection EBV technical system containing exogenous internal reference in example 2 and the established isothermal nucleic acid amplification detection EBV technical system containing endogenous reference in example 3, respectively, and showed good consistency and good reproducibility compared with the commercial quantitative PCR kit.
Clinical sample evaluation of the two methods shows that the internal references in two different forms are detected as positive results, the target gene EBV amplification is not affected, and the method is suitable for monitoring the amplification of a RAA reaction system. In RAA technology containing endogenous internal reference, the sample nucleic acid DAN extraction effect is indirectly proved to be good through the evaluation of clinical samples. The internal references in two different forms can be successfully applied to the RAA technology, and the invention has good effect and good repeatability.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Feng Zhishan
<120> pathogen nucleic acid detection primer probe combinations of human herpesvirus, kits and uses thereof
<130> MP21019590
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ctacctgtgc cgcatgaaac tgggcgagac cga 33
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
catgtcacag taaggacaga gaagtctggg 30
<210> 3
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n(32)=i6famdt
<220>
<221> misc_feature
<222> (33)..(33)
<223> n(33)=idsp
<220>
<221> misc_feature
<222> (34)..(34)
<223> n(34)=ibhq1dt
<220>
<221> misc_feature
<222> (49)..(49)
<223> n(49)=c3-spacer
<400> 3
gaacacctga gcgtggtgaa gcctctaacg cnnnctgtcc actccgaan 49
<210> 4
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n(32)=hexdt
<220>
<221> misc_feature
<222> (34)..(34)
<223> n(34)=idsp
<220>
<221> misc_feature
<222> (36)..(36)
<223> n(36)=bhq-dt
<220>
<221> misc_feature
<222> (52)..(52)
<223> n(52)=c3-spacer
<400> 4
gtaaggtgct agactaaaat tgttgggact tngnanctct gaagtaaaag gn 52
<210> 5
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gcttaaaatg tgttctagcc ttggcgttca 30
<210> 6
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cagaggttca gtctctaaat tttccccaga 30
<210> 7
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n(32)=i6famdt
<220>
<221> misc_feature
<222> (34)..(34)
<223> n(34)=idsp
<220>
<221> misc_feature
<222> (36)..(36)
<223> n(36)=ibhq1dt
<220>
<221> misc_feature
<222> (52)..(52)
<223> n(52)=c3-spacer
<400> 7
gtaaggtgct agactaaaat tgttgggact tngnanctct gaagtaaaag gn 52
<210> 8
<211> 666
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atggccaggt tcatcgctca gctcctcctg ttggcctcct gtgtggccgc cggccaggct 60
gtcaccgctt tcttgggtga gcgagtcacc ctgacctcct actggaggag ggtgagcctc 120
ggtccagaga ttgaggtcag ctggtttaaa ctgggcccag gagaggagca ggtgcttatt 180
gggcgcatgc accacgatgt catctttata gagtggcctt tcaggggctt ctttgatatc 240
cacagaagtg ccaacacctt ctttttagta gtcaccgctg ccaacatctc ccatgacggc 300
aactacctgt gccgcatgaa actgggcgag accgaggtca ccaagcagga acacctgagc 360
gtggtgaagc ctctaacgct gtctgtccac tccgaaaggt ctcagttccc agacttctct 420
gtccttactg tgacatgcac cgtgaatgca tttccccatc cccacgtcca gtggctcatg 480
cccgagggcg tggagcccgc accaactgcg gcaaatggcg gtgttatgaa ggaaaaggat 540
gggagcctct ctgttgctgt tgacctgtca cttcccaagc cctggcacct gccagtgacc 600
tgcgttggga aaaatgacaa ggaggaagcc cacggggttt atgtttctgg atacttgtcg 660
caataa 666
<210> 9
<211> 669
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atggccaggt tcatcgctca gctcctcctg ttggcctcct gtgtggccgc cggccaggct 60
gtcaccgctt tcttgggtga gcgagtcacc ctgacctcct actggaggag ggtgagcctc 120
ggtccagaga ttgaggtcag ctggtttaaa ctgggcccag gagaggagca ggtgcttatt 180
gggcgcatgc accacgatgt catctttata gagtggcctt tcaggggctt ctttgatatc 240
cacagaagtg ccaacacctt ctttttagta gtcaccgctg ccaacatctc ccatgacggc 300
aactacctgt gccgcatgaa actgggcgag accgaggtca ccaagcaggt aaggtgctag 360
actaaaattg ttgggacttt gaatctctga agtaaaagga ggtctcagtt cccagacttc 420
tctgtcctta ctgtgacatg caccgtgaat gcatttcccc atccccacgt ccagtggctc 480
atgcccgagg gcgtggagcc cgcaccaact gcggcaaatg gcggtgttat gaaggaaaag 540
gatgggagcc tctctgttgc tgttgacctg tcacttccca agccctggca cctgccagtg 600
acctgcgttg ggaaaaatga caaggaggaa gcccacgggg tttatgtttc tggatacttg 660
tcgcaataa 669
<210> 10
<211> 609
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tggacttcag aagattgaag ataagaagaa aaagaacaaa acaccttttc tgaaaaaaga 60
aagcagagag aaatgcagca ttgctgttga tattagtgat aatctgaagg agaagaaaac 120
agatgctaag cagcaagtgt cagggtggac gcctgcacac gtcaggaagc agcttgtcat 180
tggcgttaac gaagttacca gagccctgga aaggagggaa ctgctgttag ttctggtgtg 240
taaatcagtc aagcctgcca tgatcacctc acacttgatt cagttaagcc taagcagaag 300
tgtccctgcc tgtcaggtcc cccggctcag tgagagaatc gcccccgtca ttggcttaaa 360
atgtgttcta gccttggcgt tcaaaaagaa caccactgac tttgtggacg aagtaagagc 420
catcatcccc agagtcccca gtttaagtgt accatggctt caagacagaa ttgaagattc 480
tggggaaaat ttagagactg aacctctgga aagccaagac agagagcttt tggacacttc 540
atttgaagat ctgtcaaaac ctaagagaaa gcttgctgac ggtcggcagg cttctgtaac 600
attacaacc 609
Claims (5)
1. The primer probe combination is characterized by comprising a first primer probe combination;
the first primer probe combination is an isothermal nucleic acid amplification system combination containing exogenous internal references:
(I) The nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; and
(II) the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; and
(III) the nucleotide sequence of the probe is shown as SEQ ID No. 4;
the first primer probe combination also comprises a target gene probe, and the nucleotide sequence of the target gene probe is shown as SEQ ID No. 3.
2. The use of the primer probe combination according to claim 1 for preparing a reagent or a kit for detecting EB virus.
3. The use according to claim 2, wherein the detected amplification temperature is 39 ℃ and the amplification time is 30min.
4. An EB virus detecting reagent comprising the primer probe assembly according to claim 1.
5. The kit for detecting EB virus, which comprises the primer probe combination according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110949243.XA CN114182046B (en) | 2021-08-18 | 2021-08-18 | Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110949243.XA CN114182046B (en) | 2021-08-18 | 2021-08-18 | Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114182046A CN114182046A (en) | 2022-03-15 |
| CN114182046B true CN114182046B (en) | 2024-01-30 |
Family
ID=80601275
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110949243.XA Active CN114182046B (en) | 2021-08-18 | 2021-08-18 | Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114182046B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116516069B (en) * | 2023-05-10 | 2024-03-22 | 武汉大学 | RPA kit for rapidly detecting Epstein-Barr virus in cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016011280A1 (en) * | 2014-07-16 | 2016-01-21 | Tangen Biosciences, Inc. | Isothermal methods for amplifying nucleic acid samples |
| CN110106285A (en) * | 2019-03-19 | 2019-08-09 | 中国疾病预防控制中心病毒病预防控制所 | A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection |
| CN111154913A (en) * | 2018-11-08 | 2020-05-15 | 中山大学 | Primer and crRNA for Epstein-Barr virus DNA detection and its application |
| CN113122660A (en) * | 2021-05-19 | 2021-07-16 | 冯志山 | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit |
-
2021
- 2021-08-18 CN CN202110949243.XA patent/CN114182046B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016011280A1 (en) * | 2014-07-16 | 2016-01-21 | Tangen Biosciences, Inc. | Isothermal methods for amplifying nucleic acid samples |
| CN111154913A (en) * | 2018-11-08 | 2020-05-15 | 中山大学 | Primer and crRNA for Epstein-Barr virus DNA detection and its application |
| CN110106285A (en) * | 2019-03-19 | 2019-08-09 | 中国疾病预防控制中心病毒病预防控制所 | A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection |
| CN113122660A (en) * | 2021-05-19 | 2021-07-16 | 冯志山 | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit |
Non-Patent Citations (3)
| Title |
|---|
| Detection of low-load Epstein-Barr virus;Jing‑yi Li;《AMB Express》;20220611;第1-8页 * |
| Rapid Internal Control Reference Recombinase-Aided;GAO Yuan;《Biomed Environ Sci》;20210820;第34卷(第8期);第650-655页 * |
| 人乳头瘤病毒6型和11型通...等温扩增技术检测方法的建立;何安娜;《新发传染病电子杂志》;20220515;第7卷(第2期);第25-29页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114182046A (en) | 2022-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| CN110982944A (en) | Novel coronavirus visual constant temperature rapid detection kit | |
| CN113755565B (en) | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever | |
| CN108060263B (en) | A primer-probe combination and quantitative PCR kit for simultaneous detection of three Cryptococcus species | |
| CN112981011B (en) | Primer composition for detecting SARS-CoV-2 and its application | |
| CN110387439B (en) | Primers, probes, kit and method for adenovirus detection and typing | |
| Reddy et al. | Quantitative detection and differentiation of human herpesvirus 6 subtypes in bone marrow transplant patients by using a single real-time polymerase chain reaction assay | |
| KR20240157771A (en) | Methods for Enriching and Detecting Low Abundance Mutant DNA | |
| CN111286559B (en) | Primers, probes and kits for detection of African swine fever virus | |
| CN112695134A (en) | Novel coronavirus COVID-19 nucleic acid detection primer group, probe group, detection kit and detection method | |
| CN105648114B (en) | Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and kit for detecting new variant type highly pathogenic porcine reproductive and respiratory syndrome virus and detection method | |
| CN117265185B (en) | Adenovirus rapid detection method based on One-Tube RPA-CRISPR/Cas12a | |
| CN114182046B (en) | Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof | |
| CN112662808A (en) | Novel coronavirus COVID-19 nucleic acid detection kit and detection method thereof | |
| CN114634996B (en) | Primer probe combination, kit and application thereof for detecting bovine respiratory diseases | |
| CN115058543A (en) | Primer group and kit for identifying Delta variant strain and Omicron variant strain | |
| CN114250322A (en) | Dual Real-Time PCR Kit for Detection of Mink Circovirus and Mink Aleutian Virus | |
| CN113122660B (en) | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit | |
| CN112646927A (en) | Fluorescent RAA kit for detecting bovine sarcoidosis virus, detection method and application thereof | |
| CN109735645B (en) | Real-time fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting Sporothrix globosum | |
| CN114317831B (en) | Kit for detecting novel coronavirus Omicron mutant strain | |
| CN111004868A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting goat intranasal tumor virus | |
| CN106929601B (en) | High-sensitivity human papilloma virus 6,11 type nucleic acid detection kit | |
| CN105296668B (en) | Primer, probe and kit for specifically detecting type 3 ungulate bocavirus parvovirus | |
| CN116814857A (en) | Cat parvovirus and kit thereof and fluorescent recombinase polymerase amplification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |