CN110117678A - EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit - Google Patents
EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit Download PDFInfo
- Publication number
- CN110117678A CN110117678A CN201910514352.1A CN201910514352A CN110117678A CN 110117678 A CN110117678 A CN 110117678A CN 201910514352 A CN201910514352 A CN 201910514352A CN 110117678 A CN110117678 A CN 110117678A
- Authority
- CN
- China
- Prior art keywords
- ehp
- shiv
- probe
- detection
- time fluorescence
- 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.)
- Pending
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
- C12Q1/68—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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kits, belong to aquatic animal technical field of detection of pathogeny.The primer combination of probe uses dual real-time fluorescence quantitative PCR detecting method, can disposably carry out the amplification of two kinds of cause of disease DNA using the special primer for checking and probe of EHP, SHIV simultaneously.Detection kit provided by the invention be it is a kind of easily and fast, detection limit it is low, and high specificity high sensitivity accuracy rate is high and can detect the dual real-time fluorescence quantitative PCR detection of prawn shrimp liver sausage born of the same parents worm, haemocyte irido virus, the early monitoring suitable for prawn subclinical infection simultaneously.
Description
Technical field
The invention belongs to aquatic animal technical field of detection of pathogeny, and in particular to EHP and SHIV dual real-time fluorescence is quantitative
PCR detection primer probe combinations and kit.
Background technique
Prawn haemocyte irido virus (Shrimp Hemocyte Iridovirus, SHIV) earliest by Lightner and
Redman is in 1993 in rough prawn (the Protrachypene precipua of original in Ecuadorian prawn culturing field
Burkenroad it being found in), sick shrimp symptom is that vigor is poor, and the obvious atrophy of hepatopancrease, muscle whitens, and enteron aisle is rubescent, is broken,
Jejunum sky stomach, the gill, step and swimmeret black.2017, Chinese Academy of Sciences Huanghai Sea research institute scientific research personnel was in Zhejiang Serious Mortality
A kind of new virus is found on Penaeus Vannmei, it is not belonging to established five categories under Iridoviridae, infects the disease according to shrimp
Clinical symptoms and pathological symptom after poison are named as shrimp haemocyte irido virus (SHIV).And shrimp liver sausage born of the same parents worm (Enterocy
Tozoon hepatopenaei, EHP) be cultured prawn especially Penaeus Vannmei at this stage biggest threat, SHIV and EHP
It is two kinds of new hair epidemic diseases during China's prawn culturing, the former causes shrimp largely dead, and the latter leads to prawn slow growth, ginseng
, only just there is mortality just or under stressed condition with white once in a while in poor uneven or even growth retardation, so cultivation is thrown
Enter high result to get little, causes huge economic loss to aquaculture.
Currently, shrimp disease type is more, diagnostic means mainly include pathology and biological method, immunologic diagnosis method point
Son hybridization, Electronic Speculum observation and round pcr.Established Standard PCR technology is mostly a kind of cause of disease of one-time detection, detection it is time-consuming compared with
Long, detection sensitivity and accuracy need to be further increased.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection primers
Probe combinations and kit can not only detect two kinds of cause of diseases of EHP and SHIV simultaneously, but also detection sensitivity is high, accuracy is good.
A kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe provided by the invention, including detection
The primer combination of probe of EHP and the primer combination of probe of detection SHIV；
The primer combination of probe of the detection EHP includes forward primer EHP-F, such as SEQ as shown in SEQ ID No.1
Reverse primer EHP-R shown in the ID No.2 and probe EHP-Pro as shown in SEQ ID No.3；
The primer combination of probe of the detection SHIV includes forward primer SHIV-F, such as SEQ as shown in SEQ ID No.4
Reverse primer SHIV-R shown in the ID No.5 and probe SHIV-Pro as shown in SEQ ID No.6.
The present invention provides the primer combination of probe in preparation EHP and SHIV dual real-time fluorescence quantitative PCR detection
Application in reagent.
The present invention provides a kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection kits, including the primer to visit
Needle combination.
It preferably, further include qPCR Probe MasterMix.
Preferably, in same detection architecture, the final concentration of SHIV-F or SHIV-R stand alone as 0.4pmol/ μ L, SHIV-
The final concentration of final concentration of 0.2pmol/ the μ L, EHP-F or EHP-R of Pro stand alone as the final concentration of 0.35pmol/ μ L, EHP-Pro
For 0.25pmol/ μ L.
Preferably, as follows for detecting the amplification program of EHP and SHIV dual real-time fluorescence quantitative PCR: 95 DEG C of initial denaturations
5min；95 DEG C of denaturation 10s, 60 DEG C of 30s of annealing temperature, totally 40 recycle, and carry out fluorescence signal acquisition at the end of each circulation.
EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe provided by the invention, based on dual real-time
Quantitative fluorescent PCR detects two kinds of cause of diseases of EHP and SHIV simultaneously, has the characteristics that time-consuming is less, while substantially increasing detection knot
The sensibility and accuracy of fruit.Embodiment with shrimp haemocyte irido virus (Shrimp Hemocyte Iridovirus, SHIV),
Shrimp liver sausage born of the same parents worm (Enterocy tozoon hepatopenaei, EHP), infectious subcutaneous and haematopoietic necrosis virus
(Infectious hypodermal and hematopoietic necrosis virus, IHHNV) and guttate morphea syndrome
The genome of viral (White spot syndrome virus, WSSV) is template, carries out real-time fluorescence quantitative PCR amplification, knot
Fruit shows that only two kinds of corresponding fluorescence signals of virus of SHIV, EHP are the positive, and the detection of other viruses is feminine gender, illustrates institute
Primer combination of probe is stated with stronger specificity.Early diagnosis to cause of disease, convenient for taking effectively preventing measure in time.Through
Cross sensibility the experimental results showed that, detection SHIV and EHP regular-PCR and SYBR Green Real-time PCR method with building
It compares, double fluorescent quantitative PCR primer combination of probe provided by the invention is 100 times of regular-PCR method sensibility.
Detailed description of the invention
Fig. 1 is SHIV Real-time PCR standard curve；
Fig. 2 is EHP Real-time PCR standard curve；
Fig. 3 is the specific detection result of dual real-time fluorescence PCR system；
Fig. 4 is the sensitivity Detection of fluorescence quantitative RT-RCR system；Wherein SHIV (a to g) and EHP (1 to 7) template copy
Number is followed successively by 1 × 107、1×106、1×104、1×103、1×102With 1 × 10.
Fig. 5 is EHP regular-PCR sensitivity Detection result；Wherein EHP (2 to 8) template copy numbers 1 × 10,1 × 102、1×
103、1×104、1×105、1×106With 1 × 107；1: negative control；M is MarkerDL1000；
Fig. 6 is SHIV regular-PCR sensitivity Detection, note: SHIV (1 to 7) template copy numbers 1 × 107、1×106、1×
105、1×104、1×103、1×102With 1 × 10；1 is negative control；M is Marker DL1000.
Specific embodiment
A kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe provided by the invention, including detection
The primer combination of probe of EHP and the primer combination of probe of detection SHIV；The primer combination of probe of the detection EHP includes such as SEQ
Forward primer EHP-F shown in ID No.1, the reverse primer EHP-R as shown in SEQ ID No.2 and such as SEQ ID No.3 institute
The probe EHP-Pro shown；The primer combination of probe of the detection SHIV includes the forward primer as shown in SEQ ID No.4
SHIV-F, the reverse primer SHIV-R as shown in SEQ ID No.5 and the probe SHIV-Pro as shown in SEQ ID No.6.
The EHP-F and EHP-R is using the genome of EHP as the segment of template amplification 92bp long；The SHIV-F and SHIV-
R is using the genome of SHIV as the segment of template amplification 114bp long.The present invention does not have special limit to the source of the primed probe
System, using primed probe source known in the art.Different fluorescent bases is respectively adopted in the EHP-Pro and SHIV-Pro
Group's label.The present invention is not particularly limited the type of the fluorescence probe, using fluorophor known in the art.
In embodiments of the present invention, primer combination of probe commission Shanghai Hui Rui Biotechnology Co., Ltd synthesis.
The present invention provides the primer combination of probe in preparation EHP and SHIV dual real-time fluorescence quantitative PCR detection
Application in reagent.
The present invention provides a kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection kits, including the primer to visit
Needle combination.In the present invention, every kind of primer and probe preferably individually dispenses in the kit.The concentration of every kind of primer or probe
Preferably 100 times of working concentration.
In the present invention, it is also preferable to include qPCR Probe Master Mix for the kit.The present invention is to the qPCR
The ingredient of Probe Master Mix is not particularly limited, and is detected using known in the art for real-time fluorescence quantitative PCR
With component.In embodiments of the present invention, the qPCR Probe Master Mix only praises biotechnology purchased from Nanjing promise and has
Limit company.
In the present invention, the application method of the kit, comprising the following steps:
A. the genomic DNA of sample to be detected is extracted；
B. using the genomic DNA as template, real-time fluorescence quantitative PCR detection is carried out with the primer combination of probe, is obtained
To amplification curve；
C. it is calculated according to SHIV Real-time PCR standard curve and EHP Real-time PCR standard curve to be detected
The content of SHIV or EHP in sample.
The present invention is not particularly limited the extracting method of the genomic DNA of sample to be detected, and use is known in the art
Extracting method.In embodiments of the present invention, the sample to be detected is Mabio viral DNA/RNA extracts kit.
In the present invention, the reaction system of the real-time fluorescence quantitative PCR detection is preferably as follows: AceQ qPCR Probe
Totally 4.9 μ L, template DNA are 2 μ by MasterMix10 μ L, SHIV-F, SHIV-R, SHIV-Pro, EHP-F, EHP-R and EHP-Pro
L, it is 20 μ L that the water of no RNase, which supplies reaction system, and wherein the final concentration of SHIVP-F or SHIV-R is independently preferably 0.4pmol/
The final concentration of μ L, SHIV-Pro are preferably that the final concentration of 0.2pmol/ μ L, EHP-F or EHP-R are independently preferably 0.35pmol/ μ
The final concentration of L, EHP-Pro are preferably 0.25pmol/ μ L.For detecting the amplification of EHP and SHIV dual real-time fluorescence quantitative PCR
Program is preferably as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s of annealing temperature, totally 40 circulations, each circulation are tied
Fluorescence signal acquisition is carried out when beam.
In the present invention, the system of SHIV Real-time PCR standard curve and EHP Real-time PCR standard curve
Preparation Method is not particularly limited, using the preparation method of Real-time PCR standard curve known in the art.
Below with reference to embodiment to a kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection primer provided by the invention
Probe combinations and kit are described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1, viral nucleic acid extracts
Morbidity shrimp tissue (hepatopancrease, the cheek, enteron aisle, muscle) is taken, homogenized is carried out, obtains tissue homogenate, specific side
Method is as follows:
1) about 3 steel balls, big steel ball 1 (diameter 4.5mm), 2 (diameters of small steel ball are first added in 2mL homogenate tube
2.5mm)；
2) it takes morbidity shrimp tissue (hepatopancrease, the cheek, enteron aisle, muscle) about (100mg) pathological material of disease into pipe, 500 μ LPBS is added；
3) homogenate tube is placed in tissue homogenizer and is homogenized 1min；
4) tissue homogenate is frozen at least 30min in -40 DEG C, then puts back to normal-temperature dissolution, multigelation 2~3 times, frozen every time
Melt and is placed in tissue homogenizer homogenate 1 time；
5) it is centrifuged 5min under the conditions of 10,000 × g, about 200 μ L supernatants are shifted in 1.5mL centrifuge tube, are numbered spare.
2, real-time fluorescence PCR detection
1) nucleic acid extraction
It is operated according to Mabio viral DNA/RNA extracts kit specification, positive control is also required to extract simultaneously, protects
It is stored in -20 DEG C of standby inspections.
The design of double fluorescent quantitative PCR primer and probe
2) specific primer and fluorescence probe of EHP and SHIV are designed, primed probe is limited by the farsighted biotechnology of Shanghai brightness
Company's synthesis, specifying information are shown in Table 1.
1 primer probe sequence information of table
3) preparation of standard items
Take the total nucleic acid of said extracted.The amplification of SHIV and EHP target gene is carried out respectively.Amplification condition: 94 DEG C of initial denaturations
3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 40 recycle；72 DEG C of extension 8min.After reaction,
It takes 5 μ LPCR reaction products to carry out agarose gel electrophoresis, amplifies SHIV (114bp) and EHP (92bp) target gene.It will obtain
After the two PCR product agarose gel electrophoresis obtained, gel extraction purifying is cloned into pMD18-T carrier respectively, is transformed into
DH5 α competent cell, screening positive clone recombinant plasmid serve the sequencing identification of Hai Huirui Biotechnology Co., Ltd.For mirror
Fixed correct recombinant plasmid, is respectively designated as pMD-SHIV and pMD-EHP.Concentration mensuration, copy number meter are carried out to positive plasmid
It calculates, packing, -80 DEG C save backup.
The preparation result of standard items
The PCR testing result of recombinant plasmid shows, the size of the amplified production of SHIV and EHP is respectively 114bp and 92bp,
It is in the same size with expection.Sequencing result shows shrimp liver sausage born of the same parents worm objective gene sequence, the shrimp haemocyte irido virus purpose of clone
Gene order respectively with shrimp liver sausage born of the same parents worm reference sequences FJ496356.1, shrimp haemocyte irido virus reference sequences KY681040.1
Similitude up to 100%, and clone and show that target gene is successively inserted into carrier.
4) foundation of real-time fluorescence quantitative PCR standard curve
The DNA standard items (pMD-SHIV or pMD-EHP) of the SHIV and EHP of step 3) preparation are done 10 times to be serially diluted,
Use the DNA of different dilution gradients as template, the DNA of each gradient sets up 3 repetitions.It prepares reaction system and carries out real-time fluorescence
Quantitative pcr amplification, reaction system are 20 μ L:AceQ qPCR Probe Master Mix10 μ L, SHIV-F, SHIV-R, SHIV-
Totally 4.9 μ L, template DNA are 2 μ L by Pro, EHP-F, EHP-R and EHP-Pro, and it is 20 μ L that the water of no RNase, which supplies reaction system,
The final concentration of middle SHIVP-F or SHIV-R is independently preferably that the final concentration of 0.4pmol/ μ L, SHIV-Pro are preferably 0.2pmol/ μ
The final concentration of L, EHP-F or EHP-R are independently preferably that the final concentration of 0.35pmol/ μ L, EHP-Pro are preferably 0.25pmol/ μ L.
The reaction condition of quantitative fluorescent PCR is as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s of annealing temperature, totally 40 are followed
Ring carries out fluorescence signal acquisition at the end of each circulation.
Result figure 1 and Fig. 2.The quantitative fluorescent PCR reaction normal product of SHIV and EHP, the gradient concentration of DNA mixed liquor from
1×107L~1 × 10 copies/ μ1The examination criteria curve of 7 linear gradients of copies/ μ L is in isometry and collimation, tool
There is preferable gradient (Fig. 1,2).
Embodiment 2
Specific test
Respectively with SHIV, EHP of extraction, infectious subcutaneous and haematopoietic necrosis virus (Infectious
Hypodermal and hematopoietic necrosis virus, IHHNV), white spot syndrome virus (White spot
Syndrome virus, WSSV) nucleic acid be template, according to standard curve establish method carry out PCR amplification respectively, verify its spy
It is anisotropic.
Real-time fluorescence quantitative PCR amplification is carried out by template of the genome of SHIV, EHP, IHHNV and WSSV, is as a result shown
Show, the only corresponding fluorescence signal of both SHIV, EHP is the positive, and the detection of other viruses is feminine gender, illustrates what this was constructed
The specificity of dual real-time fluorescence quantifying PCR method is relatively strong (Fig. 3).
Embodiment 3
Sensitivity tests by the template for measuring DNA concentration progress 10 times be serially diluted, with it is established it is dual in real time it is glimmering
Fluorescent Quantitative PCR method measures its sensibility.
By the DNA standard items of SHIV and EHP, measure its concentration with nucleic acid-protein detector, calculate its copy number, then into
10 times of serial dilutions of row obtain 1 × 107L~1 × 10 copies/ μ1Copies/ μ L, using this research establish SHIV and
EHP dual real-time fluorescence is quantitatively expanded, and the kinetic enrichment for obtaining SHIV and EHP sonde method real-time fluorescence quantitative PCR is bent
Line (Fig. 4), the minimum amount of DNA that can be detected in 40 range of Ct < be 10copies/ μ L, therefore establish the method sensibility
It can achieve 10copies/ μ L.With detection SHIV and the EHP regular-PCR and SYBR Green of this laboratory early period of building
Real-time PCR method compares, and is 100 times (Fig. 5,6) of regular-PCR method sensibility.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>the general detection Science and Technology Ltd. of Zhuhai section skill
<120>EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aacgttaaag ggtctcacgg 20
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gcttgatcgg catccttg 18
<210> 3
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aacaaatctc gagcccaata cgaa 24
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tagcagtaaa ctatgccgac a 21
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tatccgttcc gctactct 18
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aactcaatgg cttttaacac cac 23
Claims (6)
1. a kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe, which is characterized in that including detecting EHP
Primer combination of probe and detection SHIV primer combination of probe；
The primer combination of probe of the detection EHP includes forward primer EHP-F, such as SEQ ID as shown in SEQ ID No.1
Reverse primer EHP-R shown in the No.2 and probe EHP-Pro as shown in SEQ ID No.3；
The primer combination of probe of the detection SHIV includes forward primer SHIV-F, such as SEQ ID as shown in SEQ ID No.4
Reverse primer SHIV-R shown in the No.5 and probe SHIV-Pro as shown in SEQ ID No.6.
2. primer combination of probe described in claim 1 is in preparation EHP and SHIV dual real-time fluorescence quantitative PCR detecting reagent
Application.
3. a kind of EHP and SHIV dual real-time fluorescence quantitative PCR detection kit, which is characterized in that including described in claim 1
Primer combination of probe.
4. kit according to claim 3, which is characterized in that further include qPCR Probe MasterMix.
5. kit according to claim 3 or 4, which is characterized in that in same detection architecture, SHIV-F or SHIV-R
Final concentration final concentration of 0.2pmol/ the μ L, EHP-F or EHP-R that stand alone as 0.4pmol/ μ L, SHIV-Pro final concentration it is only
Found the final concentration of 0.25pmol/ μ L for 0.35pmol/ μ L, EHP-Pro.
6. kit according to claim 3, which is characterized in that quantitative for detecting EHP and SHIV dual real-time fluorescence
The amplification program of PCR is as follows:
95 DEG C of initial denaturation 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s of annealing temperature, totally 40 recycle, and carry out at the end of each circulation
Fluorescence signal acquisition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910514352.1A CN110117678A (en) | 2019-06-14 | 2019-06-14 | EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910514352.1A CN110117678A (en) | 2019-06-14 | 2019-06-14 | EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110117678A true CN110117678A (en) | 2019-08-13 |
Family
ID=67524180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910514352.1A Pending CN110117678A (en) | 2019-06-14 | 2019-06-14 | EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110117678A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110616279A (en) * | 2019-09-29 | 2019-12-27 | 中国水产科学研究院东海水产研究所 | Kit for synchronously and quantitatively detecting 3 important shrimp pathogens |
| CN113897463A (en) * | 2021-11-29 | 2022-01-07 | 中国科学院合肥物质科学研究院 | Multiplex fluorescence PCR detection kit and method for simultaneously detecting WSSV, IHHNV, EHP and SHIV of prawns |
| CN117512221A (en) * | 2023-12-07 | 2024-02-06 | 拱北海关技术中心 | Dual real-time fluorescent PCR detection primers, probes, kit and method for white spot syndrome virus and octopoda iridovirus 1 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048022A (en) * | 2016-06-16 | 2016-10-26 | 徐锦余 | Kit for detecting enterocytozoonhepatopenaei |
| CN106244728A (en) * | 2016-08-23 | 2016-12-21 | 广东海大畜牧兽医研究院有限公司 | A kind of PEDV and TGEV dual RT PCR detection method and probe primer thereof combine and test kit |
| CN106636471A (en) * | 2017-01-16 | 2017-05-10 | 山东省海洋生物研究院 | Multiple-PCR detection method and kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns |
-
2019
- 2019-06-14 CN CN201910514352.1A patent/CN110117678A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048022A (en) * | 2016-06-16 | 2016-10-26 | 徐锦余 | Kit for detecting enterocytozoonhepatopenaei |
| CN106244728A (en) * | 2016-08-23 | 2016-12-21 | 广东海大畜牧兽医研究院有限公司 | A kind of PEDV and TGEV dual RT PCR detection method and probe primer thereof combine and test kit |
| CN106636471A (en) * | 2017-01-16 | 2017-05-10 | 山东省海洋生物研究院 | Multiple-PCR detection method and kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns |
Non-Patent Citations (3)
| Title |
|---|
| 刘宝彬等: "凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量 PCR 检测", 《渔业科学进展》 * |
| 谢芝勋等: "WSSV和IHHNV二重实时荧光PCR检测方法的建立", 《水生生物学报》 * |
| 陈蒙蒙: "实时荧光定量 PCR 在凡纳滨对虾病原 VPAHPND及 SHIV 检测中的应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110616279A (en) * | 2019-09-29 | 2019-12-27 | 中国水产科学研究院东海水产研究所 | Kit for synchronously and quantitatively detecting 3 important shrimp pathogens |
| CN113897463A (en) * | 2021-11-29 | 2022-01-07 | 中国科学院合肥物质科学研究院 | Multiplex fluorescence PCR detection kit and method for simultaneously detecting WSSV, IHHNV, EHP and SHIV of prawns |
| CN113897463B (en) * | 2021-11-29 | 2023-11-07 | 中国科学院合肥物质科学研究院 | Multiplex fluorescence PCR detection kit and method for simultaneously detecting penaeus monodon WSSV, IHHNV, EHP, SHIV |
| CN117512221A (en) * | 2023-12-07 | 2024-02-06 | 拱北海关技术中心 | Dual real-time fluorescent PCR detection primers, probes, kit and method for white spot syndrome virus and octopoda iridovirus 1 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106947838A (en) | African swine fever virus nonstructural gene real-time fluorescence LAMP detection primer group, kit and detection method | |
| CN111206120A (en) | Primer probe group for detecting novel coronavirus SARS-CoV-2 and detection method | |
| CN110117678A (en) | EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit | |
| CN103966358B (en) | A kind of mandarin fish infectious spleen and kidney necrosis virus fluorescent quantificationally PCR detecting kit and detection method | |
| CN106191316B (en) | Multiplex PCR detects seven kinds of diarrhea virus primer sets and kit and its detection method | |
| CN103773861B (en) | Clonorchis sinensis and angiostrongylus cantonensis dual real-time fluorescence PCR detection reagent, test kit and detection method thereof | |
| CN103866050B (en) | Porcine epidemic diarrhea virus fluorescent quantitative PCR detection method and primer thereof | |
| CN106048022A (en) | Kit for detecting enterocytozoonhepatopenaei | |
| CN107828914B (en) | RAA constant temperature fluorescence detection method and reagent for Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) | |
| CN108060269A (en) | DPO primer sets and its application for the detection of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and porcine rotavirus | |
| CN110106264A (en) | Detection causes the fluorescence quantifying PCR method of Acute Hepatic pancreatic necrosis vibrio parahaemolytious | |
| CN108866244A (en) | Detect RPA primer and probe, kit and its method of prawn irido virus | |
| CN105603123A (en) | Real-time fluorescence RPA reagent kit and test strip RPA reagent kit for rapidly detecting porcine parvovirus and application of reagent kits | |
| CN111876502A (en) | Method for identifying Brucella S2 vaccine strain by dual real-time fluorescent quantitative PCR and kit used by same | |
| CN103993089B (en) | A kind of multiple fluorescence quantitative PCR detection kit and detection method thereof of vancomycin-resistant enterococcus | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| CN110257561B (en) | Reagent for detecting deer epidemic hemorrhagic fever virus, detection method and application | |
| CN108486279A (en) | A method of pigeon with newcastle disease is detected based on fluorescent quantitative PCR technique | |
| CN105506177A (en) | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof | |
| CN105256071A (en) | Method for quickly detecting carp herpes virus type III | |
| CN104561373B (en) | Kit for detecting goldfish haematopoietic necrosis virus and application thereof | |
| CN109097454A (en) | A kind of detection method of HEK293 gDNA residual quantity | |
| CN102154523B (en) | Primer for detecting human BK viral nucleic acid, fluorescent probe and application thereof | |
| CN108588274A (en) | Prawn steals dead nodavirus（CMNV）RAA constant temperature fluorescence detection method and reagent | |
| CN108384892A (en) | A kind of LAMP primer group, kit and the method for detection huichun viremia virus |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190813 |