CN111996294A - Primer pair and kit for quantitatively detecting eel herpesvirus - Google Patents

Primer pair and kit for quantitatively detecting eel herpesvirus Download PDF

Info

Publication number
CN111996294A
CN111996294A CN202011061436.3A CN202011061436A CN111996294A CN 111996294 A CN111996294 A CN 111996294A CN 202011061436 A CN202011061436 A CN 202011061436A CN 111996294 A CN111996294 A CN 111996294A
Authority
CN
China
Prior art keywords
eel
kit
qpcr
herpesvirus
primer pair
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
Application number
CN202011061436.3A
Other languages
Chinese (zh)
Inventor
李英英
葛均青
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Biotechnology of Fujian Academy of Agricultural Science
Original Assignee
Institute of Biotechnology of Fujian Academy of Agricultural Science
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institute of Biotechnology of Fujian Academy of Agricultural Science filed Critical Institute of Biotechnology of Fujian Academy of Agricultural Science
Priority to CN202011061436.3A priority Critical patent/CN111996294A/en
Publication of CN111996294A publication Critical patent/CN111996294A/en
Pending legal-status Critical Current

Links

Images

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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a primer pair and a kit for quantitatively detecting eel herpes virus, belonging to the technical field of molecular biology. The primer pair provided by the invention is based on eel herpes virus envelope protein ORF49 gene; by using the quantitative detection kit for the eel herpes virus provided by the invention, the rapid and accurate quantitative detection of a suspected eel herpes virus disease sample can be realized according to the latent infection characteristic of the eel herpes virus, the kit has the characteristics of high sensitivity, strong specificity, good repeatability and the like, the detection rate of the eel herpes virus in asymptomatic eels is improved, and the kit has important significance for the prevention and control of eel 'detachment septicemia syndrome' in production.

Description

Primer pair and kit for quantitatively detecting eel herpesvirus
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer pair and a kit for quantitatively detecting eel herpes virus.
Background
Anguilla anguilla herpesvirus (AngHV) is an important viral pathogen for the culture of Anguilla anguilla, and is produced by Sano et al Japanese Anguilla anguilla for the first time from Japan (Anguilla japonica) And (4) separating in vivo. Thereafter, from all over the worldThe eel herpes virus is separated from the bodies of different varieties of eels, and the serology and gene sequence analysis show that the eel herpes virus separated from Asia and Europe is the same virus strain. In the previous studies, the subject group used PCR method to detect Eel herpes virus from Eel cultivated in Fujian province, and used Eel ovarian cell line (EO) to separate Eel herpes virus from Eel "detachment septicemia syndrome" disease material, which is the first isolation of the virus from continental of China.
Eel 'detasseling septicemia syndrome' is a main epidemic disease in the young eel breeding stage, diseased eels often show 'detasseling', 'red head', 'septicemia' and other symptoms, and the eel disease-removing septicemia syndrome has the characteristics of high morbidity, strong infectivity, large death amount and the like; if adult eels get ill, the disease is acute, the disease symptoms are serious, the symptoms of septicemia are often presented, a large amount of death is caused, and serious economic loss is caused to breeding owners. The challenge experiment and epidemiological analysis show that the eel herpes virus is the pathogenic pathogen of eel 'descemet septicemia syndrome'. Epidemiological investigation shows that eel herpesvirus can be latently infected, the pathogenicity of the eel herpesvirus is closely related to environment, water temperature and the like, the eel herpesvirus does not attack when the water temperature is low, and the incidence rate also rises when the water temperature rises.
Eel herpesvirus belongs to the order Herpesvirales (Herpesvirales), subfamily of the fish herpesvirus (Allophesviridae), genus Cyprinus (Herpesviridae)Cyprinivirus) The genome is linear double-stranded DNA, the total length of the genome is 248 kb, the genome contains 136 Open Reading Frames (ORF), and the genome consists of a unique long sequence and 11 kb terminal repetitive sequences on both sides. Gene sequence analysis shows that the homology of eel herpesvirus with other virus sequences and gene sequences of fish herpesvirus subfamily is lower and is only 19-58%.
At present, the established eel herpesvirus detection method has complex operation of an in-situ hybridization method, needs to use isotope labeling and may have potential safety hazard to the health of operators; both the immunoperoxidase monolayer test and the immune indirect fluorescent antibody test need the antibody of the eel herpesvirus and are not easy to obtain; the common PCR method cannot accurately quantify the virus, and agarose electrophoresis is needed to observe the amplification result after amplification, so that the operation steps are multiple and long-time. With the development of molecular biology technology, the qPCR method is widely used for pathogen detection due to its characteristics of rapidness, sensitivity, accuracy and the like. Aiming at the characteristic of latent infection of the herpes viruses of the eels, the invention provides a primer and a kit for quantitatively detecting the herpes viruses of the eels by utilizing an SYBR Green qPCR method, which are different from a TaqMan probe method, have the advantages of strong specificity, high sensitivity, low cost, simplicity in operation and the like, provide a new detection means for quickly and accurately quantitatively detecting the herpes viruses of the eels, improve the positive detection rate of the herpes viruses of the eels in asymptomatic eels, and are beneficial to the early prevention of the 'detachment septicemia syndrome' of the eels.
Disclosure of Invention
The invention aims to provide a primer pair and a kit for quantitatively detecting eel herpesvirus, and the kit can be used for quickly and quantitatively detecting the eel herpesvirus in eel tissue and cell samples and has the characteristics of strong specificity, high sensitivity, good repeatability and the like.
The technical scheme adopted by the invention for solving the technical problems is as follows: a specific primer pair for quantitatively detecting eel herpes virus is provided, the primer pair is preferably obtained from 10 designed primers, the target gene for the primer pair is eel herpes virus ORF49, and the primer sequence is as follows:
AngHV_qF: 5´-CCGTGGTGGTGCCTCTTATCTACA-3´
AngHV_qR: 5´-TCCTCCGCGGCGTCCACAG-3´
the invention also provides a kit for quantitatively detecting eel herpes virus, which comprises the primer pair, a positive plasmid standard product containing eel herpes virus ORF49 gene, a negative control product, and SYBRPremix Ex Taq TM II (Tli RNaseH Plus) (2X) and ROX Reference Dye II (50X) (available from TaKaRa).
The concentration of positive quality granule standard substance in the kit is 1 × 109 Copy/. mu.L, negative control sterile ddH2O。
The method for quantitatively detecting eel herpes virus adopted by the kit is a qPCR method, and comprises the following steps:
(1) establishing a qPCR reaction standard curve: diluting the positive plasmid standard substance to 10 times according to a 10-fold ratio8~101Copying/mu L of 8 concentration gradients, using the concentration gradients as a template, carrying out qPCR amplification by using a specific primer pair detected by the qPCR of the herpes anguilla viruses, and making a standard curve by using the logarithm of the copy number of the template as an abscissa and the corresponding Ct value as an ordinate;
(2) diluting the sample DNA to a standard curve range, carrying out qPCR reaction by taking a specific primer pair detected by eel herpesvirus qPCR as a primer to obtain a sample Ct value, and calculating the virus copy number in the sample according to the standard curve;
(3) and (4) judging a result: the Ct value is less than 35, and if a typical amplification curve appears, the result is judged to be positive; the negative control has no typical amplification curve, and if the negative control has amplification, the experimental result is invalid.
The reaction system and conditions of the qPCR are as follows: reaction system: 2 XMix 10. mu.L, upstream and downstream primers (10. mu.M/. mu.L) each 0.8. mu.L, Dye II 0.4. mu.L, DNA template 1. mu.L, ddH2O7. mu.L. Amplification conditions: pre-denaturation at 95 ℃ for 30 s; 95 ℃ for 5 s, 60 ℃ for 34 s, 40 cycles.
The fluorescent dye used in the qPCR detection method is SYBR Green.
And carrying out sensitivity, specificity and repeatability detection on the established qPCR method, respectively detecting eel tissue samples suspected to suffer from the detasseling septicemia syndrome collected in the early stage by using a PCR method and a qPCR method, and comparing the positive detection rates of the two detection methods.
In the qPCR detection method for herpes viruses of eels, the linear relation between the plasmid copy number and the Ct value in the established standard curve is y = -3.2859x + 37.427, and the correlation coefficient R20.999, and the amplification efficiency was 100.855%.
According to the qPCR detection method for the eel herpes virus, the minimum detection copy number of the eel herpes virus is 10.
According to the qPCR detection method for the eel herpes virus, the coefficient of variation in Ct values and between groups of the qPCR detection method for the eel herpes virus is less than 2%.
The kit is applied to the rapid and quantitative detection of eel herpes viruses in eel tissue and cell samples.
The invention has the beneficial effects that:
(1) the primer pair provided by the invention is obtained through optimization, and the targeted gene is eel herpes virus ORF 49.
(2) Based on the primer pair, the kit for quantitatively detecting the eel herpes virus is provided, and the kit has high sensitivity which is 100 times that of common PCR; strong specificity, no amplification to several common eel DNA viruses and negative control; the repeatability is good, the Ct value variation coefficient (C.V.) in the group is 0.05% -0.42%, and the Ct value C.V. between groups is 1.07% -1.13%, and both are less than 2%.
(3) The kit for quantitatively detecting the eel herpes virus provided by the invention can be applied to detecting suspected eel herpes virus infected samples, has a positive detection rate obviously higher than that of common PCR (polymerase chain reaction), provides a new detection method for quickly and accurately quantitatively detecting the number of the eel herpes virus in the samples, and can be applied to diagnosis and epidemiological research of eel herpes virus diseases.
(4) The qPCR detection kit for quantitatively detecting the eel herpesvirus provided by the invention uses an SYBR Green dye method, is different from a TaqMan probe method, and has the characteristics of low cost, wide application and the like.
Drawings
FIG. 1 is electrophoresis diagram of amplified fragment of eel herpesvirus ORF49 gene; wherein: lane M is DL1000 DNA marker; lane 1 is eel herpesvirus, target fragment size is 693 bp, lane 2 is negative control.
FIG. 2 is a standard curve for the qPCR detection method of the present invention.
FIG. 3 shows the melting curve of qPCR assay of the present invention, 108~101The melting curves of the copy plasmids have only one melting peak, and the Tm value is 88.34-88.95 ℃.
FIG. 4 shows the results of sensitivity detection of qPCR of the present invention, wherein 1 to 9 are sequentially 1X 108~1×100 10 copies/. mu.L of recombinant plasmid, negativeAnd (6) comparison.
FIG. 5 shows the sensitivity detection result of the conventional PCR of the present invention, wherein a lane M is DL500 DNA marker; lanes 1-9 are sequentially 1X 108~1×100 Copies/. mu.L of recombinant plasmid, lane 10 is a negative control.
FIG. 6 shows the specific detection result of qPCR of the present invention, in which 1 is AngHV, 2 is recombinant plasmid pET-32a-ORF49, 3-5 are CyHV-3, EIV and RGV in turn, and 6 is negative control.
Detailed Description
The present invention will be described in detail with reference to examples.
The virus strain, cell line and material to be tested
Eel herpesvirus (AngHV), Eel Iridovirus (EIV), frog iridovirus (RGV) were isolated and stored by the institute of biotechnology, the farm institute of Fujian province. 25 eel tissue samples suspected to be infected with eel herpesvirus were collected and stored in eel farms in Fujian province between 2008 and 2019 by the institute of biotechnology of the farm institute of Fujian province.
Example 1: primer design and screening of kit for quantitatively detecting eel herpesvirus
By referring to the sequences (NC _ 013668.3) of genes ORF8, ORF49, ORF78 and ORF95 of eel herpesvirus in Genbank, DNASTARGAGEN V7.1 was used to design 10 specific primers for eel herpesvirus, which were synthesized by Shanghai Biotechnology Limited as candidate primers for the detection method of the present invention.
Inoculating AngHV into an Eel ovarian cell line (EO), collecting cells after the cells generate typical lesions, extracting DNA as a template, carrying out qPCR amplification by using a synthesized primer, and screening out a primer pair with good amplification efficiency and strong specificity according to an amplification curve and a Ct value for establishing a subsequent detection method.
Table 1 screened primers for quantitatively detecting eel herpesvirus
Figure DEST_PATH_IMAGE002
Example 2: kit for quantitatively detecting eel herpesvirus
1. Preparation of Positive plasmid Standard
Specific primer pairs 49F (5 '-GGATCCATGATTTGTTGGGCGG-3') and 49R (5 '-GAATTCTCATACGCAGCTCCCAAG-3') were designed and synthesized based on the sequence (NC-013668.3) of the eel herpesvirus ORF49 gene in GenBank. Taking eel herpesvirus DNA as a template to carry out PCR amplification, wherein the reaction system is as follows:Premix Taq12.5. mu.L of each of the upstream and downstream primers, 1. mu.L of the template, ddH2O9.5. mu.L, equal amount of ddH in negative control2O replaces the template; the reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 45 s; 72 ℃ for 10 min.
And detecting the amplification product by using 1.5% agarose gel electrophoresis, cutting the gel, recovering and purifying a target fragment, and performing sequencing verification. Cloning the target fragment to a pET-32a vector, extracting plasmid DNA by using a plasmid extraction kit, and then carrying out enzyme digestion and sequencing identification to obtain a plasmid pET-32a-ORF 49; the concentration and purity were determined by ultraminimetric UV spectrophotometer, and the Copy Number of the plasmid per μ L of the extracted DNA sample was calculated using DNA/RNA Copy Number Calculator (http:// www.endmemo.com/bio/dnacopy. php). The plasmid was stored at-80 ℃ until use. The concentration of the plasmid was determined to be 122.46 ng/. mu.L and the calculated copy number of the plasmid was 1.8X 1010 copies/μL。
2. Establishing a standard curve
The DNA of plasmid pET-32a-ORF49 was diluted 10-fold to 108~1018 concentration gradients per μ L as template, using SYBR with primer pair AngHV _ qF/AngHV _ qRPremix Ex Taq TM II (Tli RNaseH Plus) kit for qPCR amplification, each concentration setting 3 replicates, negative control with sterile ddH2O replace the template to make a standard curve. The reaction system and the reaction conditions are as follows, and the reaction system is 20 mu L: 2 XMix 10. mu.L, upstream and downstream primers (10. mu.M/. mu.L) each 0.8. mu.L, Dye II 0.4. mu.L, DNA template 1. mu.L, ddH2O7. mu.L. Amplification conditions: pre-denaturation at 95 ℃ for 30 s; 5 s at 95 ℃, 34 s at 60 ℃ and 40 cycles。
Drawing a standard curve (figure 2) by using the copy number and the Ct value of the plasmid, wherein the linear relation of the copy number and the Ct value of the plasmid is y = -3.2859x + 37.427, and the correlation coefficient R20.999 and 100.855% amplification efficiency, indicating that Ct value of the qPCR is 108~101The copy ORF49 gene has a good linear relationship. Melting curve analysis showed (FIG. 3), 108~101The melting curves of the copy plasmids have only one melting peak, the Tm value is 88.34-88.95 ℃, and the reaction products are specific amplification of ORF49 and do not generate non-specific amplification or primer dimer.
3. Sensitivity test
And (3) respectively carrying out qPCR (quantitative polymerase chain reaction) and common PCR amplification by using the plasmid pET-32a-ORF49 diluted in a gradient manner as a template and utilizing a designed primer pair AngHV _ qF/AngHV _ qR, and analyzing the lowest eel herpes virus copy number detected by the two methods.
By 108~100Copies of 10 fold dilutions of plasmid pET-32a-ORF49 were subjected to qPCR and conventional PCR amplifications, respectively. The results showed that the lowest detected copy number of ordinary PCR was 103(FIG. 4), and the minimum detectable copy number of qPCR can be up to 101(FIG. 5); the sensitivity of the qPCR method is 100 times higher than that of the ordinary PCR.
4. Specificity detection
Respectively inoculating AngHV, CyHV-3, RGV and EIV into cells, collecting the cells after most cells have typical cytopathic effect, extracting DNA by using a blood/cell/tissue genome DNA extraction kit, using the extracted DNA as a template, amplifying by using an established qPCR method, and evaluating the specificity of the detection method. The results showed that amplification curves were generated for both AngHV and plasmid pET-32a-ORF49, whereas there was no amplification signal for CyHV-3, EIV, RGV and the negative control (FIG. 6).
5. Repeatability detection
Get 104、105、106Copying/mu L of plasmid pET-32a-ORF49 with 3 concentrations, performing qPCR amplification as a template, setting 3 repeats for each concentration, and analyzing the repeatability in the group according to the coefficient of variation of the Ct value; the experiment was repeated 3 times and the reproducibility between groups was analyzed.
TABLE 2 detection repeatability of eel herpesvirus qPCR
Figure DEST_PATH_IMAGE004
As can be seen from Table 2, the intra-group variation coefficients are less than 1%, respectively 0.42%, 0.05% and 0.19%; the variation coefficients among the groups are less than 2 percent and are respectively 1.13 percent, 1.07 percent and 1.13 percent, which shows that the method has good repeatability and can ensure the stability and reliability of the detection result.
Example 3: application of eel herpes virus quantitative detection kit
Taking 25 eel tissue samples suspected to be infected with eel herpes virus collected and stored in a laboratory between 2008 and 2019, extracting DNA (deoxyribonucleic acid) as a template, and detecting by using the quantitative detection kit for eel herpes virus provided by the invention; at the same time, the samples were subjected to normal PCR amplification with the primer pair AngHV _ qF/AngHV _ qR (25. mu.L of amplification system:Premix Taq12.5. mu.L of each of the upstream and downstream primers (10. mu.M/. mu.L), 1. mu.L of DNA template, ddH2O9.5. mu.L. Amplification conditions: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 30 s; extending for 5min at 72 ℃) and comparing the detection rates of the eel herpes viruses.
TABLE 3 quantitative detection kit for herpes virus of eel and common PCR method for detecting suspected eel 'deschemilla vulgaris' sample
Figure DEST_PATH_IMAGE006
As can be seen from table 3, 25 suspected eel "detasseling septicemia syndrome" pathogens collected from 2008 to 2019 in the laboratory are detected by using the quantitative detection kit for eel herpesvirus and the ordinary PCR method provided by the invention, 19 positive samples are detected by the ordinary PCR method, and the positive detection rate is 76%; 24 positive samples are detected by the eel herpes virus quantitative detection kit, the positive detection rate is 96%, and the positive detection rate of the eel herpes virus quantitative detection kit is obviously higher than that of a common PCR method.
The primer pair and the kit for quantitatively detecting the eel herpes virus, which are provided by the invention, have the advantages of high sensitivity, strong specificity, good repeatability and good application effect, can be used for quickly and quantitatively detecting the eel herpes virus, and have important significance for preventing and controlling eel 'deshesia septicemia syndrome'.
SEQUENCE LISTING
<110> institute of biotechnology of academy of agricultural sciences of Fujian province
<120> primer pair and kit for quantitatively detecting eel herpes virus
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence
<400> 1
ccgtggtggt gcctcttatc taca 24
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
tcctccgcgg cgtccacag 19
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
ggatccatga tttgttgggc gg 22
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence
<400> 4
gaattctcat acgcagctcc caag 24

Claims (5)

1. A specific primer pair for quantitatively detecting eel herpesvirus is characterized in that the primer sequence is as follows:
AngHV qF: 5´-CCGTGGTGGTGCCTCTTATCTACA-3´；
AngHV qR: 5´-TCCTCCGCGGCGTCCACAG-3´。
2. a kit for quantitatively detecting eel herpesvirus is characterized by comprising a primer pair as claimed in claim 1, a positive plasmid standard substance containing an eel herpesvirus ORF49 gene sequence and a negative control substance, wherein the used fluorescent dye is SYBR Green.
3. The kit for quantitatively detecting eel herpesvirus according to claim 2, wherein the concentration of the positive plasmid standard substance is 1 x 109 Copy/. mu.L, negative control sterile ddH2O。
4. The kit for quantitatively detecting eel herpesvirus according to claim 2, wherein the kit for quantitatively detecting eel herpesvirus is a kit for detecting by qPCR method, comprising the following steps:
(1) establishing a qPCR reaction standard curve: diluting the positive plasmid standard substance to 10 times according to a 10-fold ratio8~101Copying/. mu.L 8 concentration gradients, using the concentration gradients as a template, carrying out qPCR amplification on AngHV qF/qR by using a specific primer pair detected by qPCR of the eel herpesvirus, taking the logarithm of the template copy number as an abscissa and the corresponding Ct value as an ordinate, and making a standard curve, wherein the linear regression equation of the obtained standard curve is as follows: y = -3.2859x + 37.427;
(2) diluting the sample DNA to a standard curve range, carrying out qPCR reaction by taking a specific primer pair detected by eel herpesvirus qPCR as a primer to obtain a sample Ct value, and calculating the virus copy number in the sample according to the standard curve;
(3) and (4) judging a result: the Ct value is less than 35, and if a typical amplification curve appears, the result is judged to be positive; the negative control has no typical amplification curve, and if the negative control has amplification, the experimental result is invalid.
5. The method of claim 4, wherein the reaction system and conditions of qPCR are as follows: 2 XMix 10. mu.L, 10. mu.M/. mu.L upstream and downstream primers 0.8. mu.L each, Dye II 0.4. mu.L, DNA template 1. mu.L, ddH2O7 mu L; amplification conditions: pre-denaturation at 95 ℃ for 30 s; 95 ℃ for 5 s, 60 ℃ for 34 s, 40 cycles.
CN202011061436.3A 2020-09-30 2020-09-30 Primer pair and kit for quantitatively detecting eel herpesvirus Pending CN111996294A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011061436.3A CN111996294A (en) 2020-09-30 2020-09-30 Primer pair and kit for quantitatively detecting eel herpesvirus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011061436.3A CN111996294A (en) 2020-09-30 2020-09-30 Primer pair and kit for quantitatively detecting eel herpesvirus

Publications (1)

Publication Number Publication Date
CN111996294A true CN111996294A (en) 2020-11-27

Family

ID=73475068

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011061436.3A Pending CN111996294A (en) 2020-09-30 2020-09-30 Primer pair and kit for quantitatively detecting eel herpesvirus

Country Status (1)

Country Link
CN (1) CN111996294A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112852987A (en) * 2021-03-16 2021-05-28 青岛农业大学 PCR (polymerase chain reaction) specific primer and detection method for detecting eel dermatophytosis

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007119557A (en) * 2005-10-26 2007-05-17 Daisan Kogyo Kk Lubricant composition for resin conveyer and method for using the same
CN108315487A (en) * 2018-04-16 2018-07-24 福建省农业科学院生物技术研究所 A kind of primer sets, kit and its application of detection common eel herpesviral
CN110863067A (en) * 2019-11-29 2020-03-06 厦门海关技术中心 Primer pair and kit for detecting eel herpes virus
CN110894550A (en) * 2018-09-13 2020-03-20 杭州众测生物科技有限公司 RAA constant temperature fluorescence detection method and reagent for eel Herpes Virus (HVA)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007119557A (en) * 2005-10-26 2007-05-17 Daisan Kogyo Kk Lubricant composition for resin conveyer and method for using the same
CN108315487A (en) * 2018-04-16 2018-07-24 福建省农业科学院生物技术研究所 A kind of primer sets, kit and its application of detection common eel herpesviral
CN110894550A (en) * 2018-09-13 2020-03-20 杭州众测生物科技有限公司 RAA constant temperature fluorescence detection method and reagent for eel Herpes Virus (HVA)
CN110863067A (en) * 2019-11-29 2020-03-06 厦门海关技术中心 Primer pair and kit for detecting eel herpes virus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FRANS RIJSEWIJK等: "Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene", 《JOURNAL OF VIROLOGICAL METHODS》, vol. 124, no. 1, 31 March 2005 (2005-03-31), pages 87 - 94 *
S J VAN BEURDEN等: "Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1", 《JOURNAL OF FISH DISEASES》, vol. 39, no. 1, 31 January 2016 (2016-01-31), pages 95 - 104 *
杨金先等: "鳗鲡疱疹病毒的生物学及理化特性", 《水产学报》, vol. 44, no. 9, 27 September 2020 (2020-09-27), pages 1435 - 1440 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112852987A (en) * 2021-03-16 2021-05-28 青岛农业大学 PCR (polymerase chain reaction) specific primer and detection method for detecting eel dermatophytosis

Similar Documents

Publication Publication Date Title
CN110760620A (en) Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method
CN110760617B (en) Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild virus
CN111020062A (en) Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain
CN111996191A (en) Primer group and kit for simultaneously identifying African swine fever wild strain and gene deletion strain based on multiple qPCR technology
CN108315483B (en) Combination of primers and probes for distinguishing wild strains and vaccine strains of duck tembusu virus
CN113046489B (en) Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof
CN113755565A (en) Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild viruses and vaccine strains
CN113564280A (en) RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof
CN114634996B (en) Primer probe combination and kit for detecting bovine respiratory disease and application of primer probe combination and kit
CN106947834B (en) Multiplex PCR method for detecting six duck susceptibility viruses
CN113122655A (en) TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for African swine fever virus EP402R gene
CN114350848A (en) Dual fluorescent probe primer combination and kit for identifying African swine fever type I strain and type II strain and application of dual fluorescent probe primer combination and kit
CN107190103B (en) Multiplex PCR primer group, kit and method for simultaneously detecting three fish viruses
CN111996294A (en) Primer pair and kit for quantitatively detecting eel herpesvirus
CN116656845A (en) Triple fluorescent quantitative PCR detection kit for diagnosing brucella vaccine immunity and natural infection and detection method thereof
NL2031088B1 (en) Locked Nucleic Acid-modified One-step Nested PCR Primers Set and Kit for African Swine Fever Virus
CN110643740A (en) Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for Palimam serogroup virus
CN114058740B (en) Fluorescent PCR amplification primer probe set capable of identifying gene I type and gene II type African swine fever virus and detection kit
CN112626278B (en) Primer and probe for identifying canine distemper virus wild strain and vaccine strain and application
CN114182046B (en) Pathogen nucleic acid detection primer probe combination of human herpesvirus, kit and application thereof
CN112941240B (en) Primer pair, kit and method for detecting goose astrovirus and goose goblet virus
CN111826473B (en) Primer pair for fluorescent quantitative PCR detection of goose type 2 astrovirus and application thereof
CN112266979A (en) RPA detection primer based on watermelon mosaic virus conserved region, detection method and application thereof
CN107164511B (en) Method for rapidly detecting haemophilus parasuis serotype 4
CN110735005A (en) SIV and PRRSV multiple RT-PCR rapid detection kit and primer

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

Application publication date: 20201127

RJ01 Rejection of invention patent application after publication