CN108315479A - Ankara real-time fluorescence quantitative PCR primer pair and kit - Google Patents

Ankara real-time fluorescence quantitative PCR primer pair and kit Download PDF

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Publication number
CN108315479A
CN108315479A CN201810228347.XA CN201810228347A CN108315479A CN 108315479 A CN108315479 A CN 108315479A CN 201810228347 A CN201810228347 A CN 201810228347A CN 108315479 A CN108315479 A CN 108315479A
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ankara
fadv
real
quantitative pcr
time fluorescence
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黄雯晶
黄淑坚
李智丽
黄兴国
刘佑明
梅敏敏
李晓文
李文锋
傅禹铭
黄浩琪
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Foshan University
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Foshan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—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
    • 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

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Abstract

The present invention provides a kind of Ankara real-time fluorescence quantitative PCR primer pairs, including 4 R of FAdV 4 F and FAdV.The primer pair can be used for preparing Ankara real-time fluorescence quantitative PCR detection kit, to carry out the detection of viral level to Ankara.The kit includes positive control standard items, PCR reaction solution and negative control；The positive control standard items are the recombinant plasmid pMD18 FAdV 4 containing 4 sequences of FAdV；The PCR reaction solution includes the template DNA of extraction, primer pair, I fluorescent dye solutions of SYBR Green, Taq enzyme, ddH2O, I fluorescent dye solutions of SYBR Green include SYBR Green I fluorescent dyes, dNTP, reaction buffer；Distilled water is as negative control.The Ankara real-time fluorescence quantitative PCR detection kit prepared with the primer pair of the present invention, detection method is simple and efficient, it is reproducible, susceptibility is high, high specificity, the detection of big throughput sample may be implemented, quick, high sensitive real-time fluorescence quantitative PCR can be carried out to Ankara and is detected.

Description

Ankara real-time fluorescence quantitative PCR primer pair and kit
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of Ankara real-time fluorescence quantitative PCR primer Pair and kit.
Background technology
Ankara disease is that one kind caused by 4 type (FAdV-4) of aviadenovirus C kinds serotype is gone out with hydropericardium, liver The infectious disease that blood, enlargement are characterized.It was most found in place of the Pakistan Karachi close to Ankara early in 1987, therefore claims " Ankara disease ".According to its pathogeneticing characteristic, also referred to as " chicken hydropericardium-hepatitis syndrome ".Ankara disease was in 2015 Summer causes broiler chicken group largely to fall ill in the Shenxian County of Liaocheng Prefecture first, then propagates to meat elsewhere rapidly Chicken, Sanhuang chicken, numb chicken etc..The death rate is up to 10%~80%.And the diseased region of the disease constantly expands, and is made to poultry husbandry At certain economic loss.Currently, established
The existing detection method of FAdV-4 is also more single, main still to utilize conventional polymeric enzyme chain reaction method (PCR) It is detected, quick, sensitivity etc. still has some deficits.
Invention content
The present invention is intended to provide a kind of real-time fluorescence quantitative PCR detects the primer pair of Ankara disease.
The technical solution used in the present invention is：
A kind of Ankara real-time fluorescence quantitative PCR primer pair, including FAdV-4-F：5′- CTTCGCAAGTCTCAGTA-3 ' (SEQ ID № 1) and FAdV-4-R：5′-GTGCCCGTGTTATCCA-3′(SEQ ID №2). The primer pair is the Hexon whole genome sequence [accession number according to the FAdV-4 delivered on Gen Bank： EU931692.1] conserved sequence and design.
The primer pair of the present invention can be used for preparing Ankara real-time fluorescence quantitative PCR detection kit, to peace OK a karaoke club virus (FAdV-4) carries out the detection of viral level.The kit includes positive control standard items, PCR reaction solution and feminine gender Control；The positive control standard items are the recombinant plasmid pMD18-FAdV-4 containing FAdV-4 sequences；The PCR reaction solution packet Include the template DNA of extraction, foregoing primer pair, I fluorescent dye solutions of SYBR Green, Taq enzyme, ddH2O, the SYBR I fluorescent dye solutions of Green include SYBR Green I fluorescent dyes, dNTP, reaction buffer；Distilled water is as negative right According to.
Ankara (FAdV-4) real-time fluorescence quantitative PCR detection kit prepared with the primer pair of the present invention, inspection Survey method is simple and efficient, reproducible, and susceptibility is high, and the detection of big throughput sample may be implemented in high specificity, can be to Ankara Virus carries out quick, high sensitive real-time fluorescence quantitative PCR detection.
Description of the drawings
Fig. 1 is quantitative fluorescent PCR standard curve；
Fig. 2 is the solubility curve of fluorescent quantitative PCR product；
Fig. 3 is quantitative fluorescent PCR specific test figure, and wherein label 1 is negative control, and 2 be FAdV-4 positives DNA, and 3 are Egg Drop syndrome virus (EDSV), 4 be newcastle disease virus (NDV), and 5 be H5 subtype avian influenza virus, and 6 be H9 subtype avian influenza diseases The nucleic acid of poison, 7 be the c DNA of H9 subtype avian influenza virus；
Fig. 4 is that the sensitivity of 10 dilution standard samples tests amplification curve diagram.
Specific implementation mode
Below by specific implementation mode combination attached drawing, invention is further described in detail.But those skilled in the art It will be understood that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment Particular technique or condition person, according to technology described in document in the art or condition (such as with reference to J. Pehanorm Brookers etc. It writes, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Institute Production firm person is not specified with reagent or instrument, being can be with conventional products that are commercially available.
Embodiment 1, the foundation of fluorescence quantifying PCR method
FAdV-4 genome target fragment PCR amplifications：
The chick embryo allantoic liquid for taking collection collects supernatant and extracts viral nucleic acid after 3000r/min, 4 DEG C of centrifugation 10min. PCR reaction systems are：Primix Ex TaqTM enzymes 12.5 μ L, each 1 μ L of upstream and downstream primer (10 μm of ol/L), 2 μ L of DNA profiling, Add sterilizing tri-distilled water to 25 μ L of total volume.After above-mentioned each component mixing brief centrifugation, following journey is executed in temperature gradient PCR instrument Sequence：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 30s, 47 DEG C of annealing 54.5s, 72 DEG C of extension 60s；It carries out 34 to recycle, last 72 DEG C Extend 10min.5 μ L reaction products are taken to carry out 1% agarose gel electrophoresis.
The preparation of FAdV-4 positive control standard items：
After purpose PCR product electrophoresis, with San Prep pillar DNA plastic recovery kits recycle, according to target fragment with Ratio of the pMD-18T carrier molecules than 5: 1 is attached, converts, bacterium colony PCR is identified, sequence analysis, and to positive control standard Product carry out a small amount of extraction with after purification, are saved backup in -20 DEG C.
Real-time quantitative PCR reacts and the foundation of standard curve and regression equation：
After a small amount of extraction positive control standard items are purified, its light absorption value at 260nm and 280nm is measured, is calculated According to molecular weight calculating target gene copy number is calculated, it is dilute to carry out 10 times of gradients to positive recombinant plasmid for plasmid concentration and purity It releases, the standard items as real-time quantitative PCR.Reaction condition and PCR system are as shown in table 1：
1 reaction condition of table and PCR system
Embodiment 2, the identification of FAdV-4 target fragments PCR amplification and recombinant plasmid
The target fragment that length in the Hexon conserved sequences of FAdV-4 is 114bp is purified and is connect with pMD-18T carriers Afterwards, connection product is transformed into DH5 α competent cells, while purifying culture is carried out to converting successful bacterium colony, and carry out PCR And sequencing identification, it is consistent with expected results.Sequencing results show that the homology with the corresponding region of Du CV genomes is 100%, structure is successfully named as pMD18-FAdV-4 containing target gene recombinant plasmid.
Embodiment 3, real-time quantitative PCR reaction and the drafting of standard curve
It is respectively 0.165 He that pMD18-FAdV-4 plasmids by a small amount of extraction and after purification, which measure OD260nm and OD280nm, 0.089, the recombinant plasmid copy number of respective concentration is 2.40 × 108Recombinant plasmid is carried out 10 times of gradient dilutions by copy/μ L, Take 100~106The recombinant plasmid of copy number establishes standard curve, and abscissa represents the logarithm (X) of plasmid copy number, and ordinate is Ct values (Y)；Derive that corresponding regression equation is y=-3.1245x+34.197, regression equation and standard curve on this basis Related coefficient (R2) it is 0.997, amplification efficiency 1.090, the results are shown in Figure 1.The dissolving of fluorescent quantitative PCR product Curve is shown in Fig. 2.The solution temperature Tm values of each dilution of standard items are between 86.0~86.5 DEG C, primer free dimer and Fei Te Specific amplification product occurs, and the primer specificity of established PCR method is good.
Embodiment 4, specific test
Specificity, sensitivity and repetitive test：
Using the fluorescence quantifying PCR method of foundation to FAdV-4 positives DNA, Egg Drop syndrome virus (EDSV), newcastle disease Viral (NDV), H5 subtype avian influenza virus, the nucleic acid of H9 subtype avian influenza virus or c DNA are expanded, while setting up feminine gender Control, the results are shown in Figure 3, and solubility curve is single, shows that this method has good specificity.
Embodiment 5, sensitivity experiment
It will have calculated that 10 times of gradient dilutions of recombinant plasmid of copy number, make 10 dilutions, the recombination of each gradient concentration Plasmid carries out fluorescent quantitative PCR under optimum reaction condition, as a result sees Fig. 4 respectively as template.As shown in Figure 4, it can examine It is 6.72 × 10 to measure the minimum copy number come-1Copy/μ L.
Embodiment 6, repetitive test
With 6.72 × 103、6.72×104、6.72×105The FAdV-4 recombinant plasmid samples of copy/dilutions of μ L 3 are made For template, with optimum reaction condition carry out quantitative fluorescent PCR batch in and batch between repetitive test, the results are shown in Table 2.It can by table 2 Know, the coefficient of variation of repetitive test is respectively less than 2% in 3 times batches and between criticizing.Show that the method for the quantitative fluorescent PCR established has Preferable repeatability.
2 quantitative fluorescent PCR of table batch in criticize between repetitive test result
Quantitative fluorescent PCR is special come the real time measure by continuously monitoring the power of fluorescence signal in PCR reaction process Property product amount, and accordingly infer detection pathological material of disease initial toxic amount, not only have the efficient spy of the amplification of Standard PCR technology Point also has the characteristics that the specificity of height, the hypersensitivity of spectral technique and accuracy, while avoiding the steps such as follow-up electrophoresis Suddenly, reduce the error that pollution and manual operation are brought.Using this method detection Egg Drop syndrome virus (EDSV), Newcastle Disease Malicious (NDV), H5 subtype avian influenza virus, the nucleic acid of H9 subtype avian influenza virus or c DNA results are feminine gender, illustrate this method With good specificity；In repetitive test, the coefficient of variation of repetitive test result is respectively less than 2% in batch, between criticizing, table Bright repeatability is good.This experiment minimum concentrations are 6.72 × 10-1Copy/μ L, improves on sensitivity.The fluorescence Quantifying PCR method can utilize its standard curve accurate quantitative analysis, that is, detect the original viral amount contained in sample.
SEQUENCE LISTING
<110>Foshan Science ＆. Technology College
<120>Ankara real-time fluorescence quantitative PCR primer pair and kit
<130> 2018
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<400> 1
cttcgcaagt ctcagta 17
<210> 2
<211> 16
<212> DNA
<213>Artificial sequence
<400> 2
gtgcccgtgt tatcca 16

Claims (2)

1. a kind of Ankara real-time fluorescence quantitative PCR primer pair, which is characterized in that shown in SEQ ID № 1 FAdV-4-R shown in FAdV-4-F and SEQ ID № 2.
2. a kind of Ankara real-time fluorescence quantitative PCR kit, which is characterized in that including positive control standard items and PCR Reaction solution；The positive control standard items are the recombinant plasmid pMD18-FAdV-4 containing FAdV-4 sequences；The PCR reaction solution Template DNA including extraction, primer pair as described in claim 1, I fluorescent dye solutions of SYBR Green, Taq enzyme, ddH2O。
CN201810228347.XA 2018-05-10 2018-05-10 Ankara real-time fluorescence quantitative PCR primer pair and kit Pending CN108315479A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207637A (en) * 2018-09-30 2019-01-15 河南牧业经济学院 A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method
CN110616278A (en) * 2019-08-09 2019-12-27 山东省农业科学院家禽研究所 Specific primer and kit for detecting FAV-8 and FAV-11

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Publication number Priority date Publication date Assignee Title
CN105886502A (en) * 2016-05-19 2016-08-24 浙江大学 Primer pair for preparing kit for detecting type-4 avian adenovirus and application thereof
CN105907893A (en) * 2016-06-20 2016-08-31 河南省动物疫病预防控制中心 Fluorogenic quantitative PCR detection reagent and preparation method and application thereof
CN106811551A (en) * 2017-03-22 2017-06-09 中国农业科学院上海兽医研究所 The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method
CN107354238A (en) * 2017-09-05 2017-11-17 天津市畜牧兽医研究所 A kind of specific primer and application for being used to detect aviadenovirus IV types

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Publication number Priority date Publication date Assignee Title
CN105886502A (en) * 2016-05-19 2016-08-24 浙江大学 Primer pair for preparing kit for detecting type-4 avian adenovirus and application thereof
CN105907893A (en) * 2016-06-20 2016-08-31 河南省动物疫病预防控制中心 Fluorogenic quantitative PCR detection reagent and preparation method and application thereof
CN106811551A (en) * 2017-03-22 2017-06-09 中国农业科学院上海兽医研究所 The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method
CN107354238A (en) * 2017-09-05 2017-11-17 天津市畜牧兽医研究所 A kind of specific primer and application for being used to detect aviadenovirus IV types

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卢受昇等: "禽腺病毒I群C种4型病毒的荧光PCR诊断方法的建立", 《中国兽医杂志》 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207637A (en) * 2018-09-30 2019-01-15 河南牧业经济学院 A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method
CN110616278A (en) * 2019-08-09 2019-12-27 山东省农业科学院家禽研究所 Specific primer and kit for detecting FAV-8 and FAV-11
CN110616278B (en) * 2019-08-09 2022-08-12 山东省农业科学院家禽研究所 Specific primer and kit for detecting FAV-8 and FAV-11

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Application publication date: 20180724