CN109971885A - Novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer, kit and application - Google Patents
Novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer, kit and application Download PDFInfo
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Abstract
The present invention relates to technical field of virus detection, in particular to the application of novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer pair and specific probe.The multiple fluorescence quantitative PCR system that the present invention establishes can accurately detect the single or mixed infection of novel goose astrovirus, goose's paramyxovirus, goose parvovirus, and the multiple fluorescence quantitative PCR system is optimized, optimal primer concentration and optimal concentration and probe concentration has been determined, keep detection accuracy higher, there is considerable advantage in viral identification field.
Description
Technical field
The present invention relates to a kind of virus detection techniques, and in particular to one kind is for identifying that novel goose astrovirus, goose pair are glutinous
Virus, fluorescence quantitative PCR detection primer, probe compositions and the detection method of goose parvovirus, belong to Protocols in Molecular Biology
Field.
Background technique
Astrovirus belongs to Astroviridae (astroviridae) Astrovirus (astrovirus), not according to host
It is same to be divided into two categories: mammal Astrovirus (Mamastrovirus) and birds Astrovirus (Avastrovirus).
Existing 3 categories (Avastrovirus 1-3) of fowl Astrovirus, including duck astrovirus (Duck astrovirus), bird kidney
Scorching virus (Avian nephritisa strovirus), turkey astrovirus (Turkey astrovirus) etc., turkey is starlike
Virus is its representative species.Astrovirus is single-stranded positive, the picornavirus without cyst membrane, genome length about 6.8kb, from
5 ' ends to 3 ' ends include 4 parts altogether: the 5 ' end noncoding regions, 3 open reading frame (Open reading of 1 85 nucleotide
Frames, ORFs) (ORF1a, ORF1b, ORF2), the 3 ' end noncoding regions of 1 80 nucleotide, 1 30 nucleotide it is more
Poly A tail.Since 2017, occur successively in the young gaggle on the ground such as China Shandong, Jiangsu, Henan, Guangdong and Anhui with
It is the communicable disease of cardinal symptom that uric acid mineralization and arthragra, which occur, in internal organ, which takes place mostly within 20 ages in days
Young goose, the death rate are up to 30%~50%.Huge economic loss is caused to the feeding goose industry in China.Through Identification of etiology, cause
The cause of disease that gout symptom occurs in young goose is novel astrovirus.
The cause of disease of goose paramyxovirus is that goose is glutinous viral (Goose paramyxovirus, GPMV), belongs to paramyxovirus section pair
Glutinous Tobamovirus.Virus is widely present in the internal organs of disease goose (spleen, liver, intestinal tube etc.).It observes under an electron microscope,
Virion is (average diameter is 120 nanometers) not of uniform size, and not just, there is intensive fine lug structure on surface to form, and viral internal is by capsule
Film is wrapped in helical symmetry nucleocapsid.Isolated strain can be bred rapidly after being inoculated with 10 age in days development of chick embryo, chicken embryo after inoculation 2
Death in~3 days.Goose sticks the red blood cell that virus can be aggregated chicken, and artificial infection chicken can cause death.According to epidemic characteristic, clinical symptoms
And pathological change, tentative diagnosis can be made.It makes a definite diagnosis, needs to carry out etiological diagnosis.
Goose parvovirus (Goose Parvovirus, GPV) is the pathogen of gosling plague.Since nineteen sixty-five, many states
Family once reported goose parvovirus (GPV) disease.The Clinical symptoms of gosling plague mainly with septic lesion performance based on, according to the course of disease
Length be divided into most acute, acute and subacute three types.The disease is fast with spread speed, morbidity and mortality are higher
Feature.Currently, the popular outburst of gosling blast is also difficult to control, once this infectious disease of happening and prevelence gosling plague, it can provisions goose
Industry causes huge economic loss.
With the fast development of molecular biotechnology, many detection methods are established for the nucleic acid of pathogenic microorganism, this packet
Include PCR detection technique, nucleic acid probe hybridization technology, loop-mediated isothermal amplification technique (LAMP) etc..Wherein round pcr is that molecule is raw
One of object important research means, this method is time saving, easy, economical and practical, substantially increases the detection efficiency of clinical sample.?
On the basis of substance PCR, the round pcr of various new is established, such as multiple PCR technique, Real-Time Fluorescent Quantitative PCR Technique, more
Weight fluorescent quantitative PCR technique, reverse transcription PCR technology etc..Wherein, real-time fluorescence quantitative PCR is acknowledged as the one of PCR diagnostic techniques
Secondary qualitative leap.Fluorescent quantitative PCR technique is the specific probe by fluorescent dye or fluorescent marker, is carried out to PCR product
Label tracking, monitors reaction process in real time.With the progress that PCR reacts, reaction product constantly accumulates, and fluorescence signal intensity is also etc.
Ratio increases.It is every to collect first order fluorescence strength signal by a circulation, it can thus be monitored and be produced by fluorescence intensity change
The variation of object amount is analyzed, available fluorescent amplification curve in conjunction with corresponding software, calculates the amount of sample to be tested original template.
Currently without use multiple fluorescence quantitative PCR method come and meanwhile detect novel goose astrovirus, goose's paramyxovirus, goose parvovirus
Record.
Summary of the invention
In order to solve above do not have to come using multiple fluorescence quantitative PCR method while to detect novel goose starlike in the prior art
The record of virus, goose's paramyxovirus, goose parvovirus, the present invention is with novel goose astrovirus, goose's paramyxovirus, goose parvovirus
Three kinds of viruses are research object, establish multiple fluorescence PCR detection method.
The present invention provides a kind of novel goose astrovirus, goose's paramyxovirus, the inspections of goose parvovirus multiple fluorescence quantitative PCR
Survey primer pair and specific probe.
The present invention also provides a kind of novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR
Detection kit.The kit has the specificity and specificity of height, and amplification efficiency is high, and high sensitivity, accuracy rate is high,
Favorable reproducibility, detection cycle is short, and energy real-time detection DNA amplification reaction, has very high feasibility and application prospect.
It is a further object to provide the applications of above-mentioned detection primer pair and specific probe or detection kit.
To achieve the above object, the present invention adopts the following technical solutions: one kind is for identifying novel goose astrovirus, goose pair
Glutinous viral, goose parvovirus fluorescence PCR detection reagent kit, it includes novel goose astrovirus primer pair and probe, goose
Paramyxovirus primer pair and probe, goose parvovirus primer pair and probe, in which:
The specific detection primer pair and specific probe of novel goose astrovirus are as follows:
GoAst-qF:5 '-ACGGACTGGACGCGTTATGA-3 '；
GoAst-qR:5 '-TGGCCACTTGGATTTCCCTT-3 '；
GoAst-probe:5 '-FAM-CCTCTCTTCTGGCGGATACG-MGB-3 '；
The specific detection primer pair and specific probe of goose's paramyxovirus are as follows:
GPMV-qF:5 '-TGGTATCATATCGCAAAATT-3 '；
GPMV-qR:5 '-CATTGCACGAATGTCTATCT-3 '；
GPMV-probe:5 '-FAM-TGGAGAAGCTGTATCCCTGA-MGB-3 '；
The specific detection primer pair and specific probe of goose parvovirus are as follows:
GPV-qF:5 '-GCAAGACTGTAACGAATGAA-3 '；
GPV-qR:5 '-CTCCAAGAACATCCAGATCT-3 '；
GPV-probe:5 '-FAM-AAACACTACTACAGCTCCTA-MGB-3 '.
Contain above-mentioned novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer pair
And specific probe be used for and meanwhile detect novel goose astrovirus, goose's paramyxovirus, goose parvovirus detection kit.
The novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer pair and
Detection kit described in specific probe is in the novel goose astrovirus of detection aquatic bird, the list of goose's paramyxovirus and goose parvovirus
Application in one infection or mixed infection.
The RNA reverse transcription is by the application, the preferably viral DNA and RNA in extraction sample to be tested tissue
CDNA, using the novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer pair and
Specific probe expands the viral DNA and cDNA.
Further, fluorescent quantificationally PCR detecting kit of the invention, wherein final concentration of 0.1~0.5 μM of each primer,
Final concentration of 0.05~0.25 μM of each probe.
Further, fluorescent quantificationally PCR detecting kit of the invention, it further include: 2 × TaqMan Master Mix,
DNA profiling and distilled water.
It is further preferred that above-mentioned fluorescent quantificationally PCR detecting kit, 16 μ L PCR amplification systems are as follows: 2 ×
TaqMan Master Mix, final concentration of 0.1~0.5 μM of each primer, final concentration of 0.05~0.25 μM of each probe, 0.5~
The 2 μ L of DNA profiling of 50ng/ μ L, distilled water supply 16 μ L, and preparation method is as shown in table 1.
1 PCR of table reacts amplification system
Further, above-mentioned fluorescent quantificationally PCR detecting kit further includes novel goose astrovirus positive reference substance, goose
Paramyxovirus positive reference substance, goose parvovirus positive reference substance, negative controls and blank control product.
The present invention also provides a kind of for identifying the detection of novel goose astrovirus, goose's paramyxovirus and goose parvovirus
Method, the specific steps are as follows:
1, the template ribonucleic acid or DNA of measuring samples are extracted；
2, PCR amplification
PCR amplification is carried out using above-mentioned fluorescent quantificationally PCR detecting kit, amplification program: 95 DEG C, 2min；94 DEG C,
10s；59 DEG C, 10s collects fluorescence signal herein；72 DEG C, 40s, 40 circulations, set up positive control, negative control and blank pair
According to.Real-time fluorescence quantitative PCR reaction system is made of the reaction system of 16 μ L, is reacted on 7500 fluorescence quantitative PCR instrument of ABI
It carries out, each sample does parallel laboratory test three times.The acquisition of Ct Value Data in reaction is selected absolute using the threshold value setting of correction
Quantitative experimental method carries out real-time fluorescence quantitative PCR reaction, and software automatically generates standard curve, according to the standard items of input
Copy number calculates the copy number of other samples automatically.
Beneficial effects of the present invention:
It is new that the multiple fluorescence quantitative PCR specific detection primer pair and specific probe that the present invention establishes can accurately detect aquatic bird
The single or mixed infection of type goose astrovirus, goose's paramyxovirus, goose parvovirus, specificity and specificity with height,
And amplification efficiency is high, high sensitivity, and accuracy rate is high, and favorable reproducibility, detection cycle is short, and detection, tool can be completed in 1.5 hours
There are very high feasibility and application prospect, provides scientific reliable side for Pathogenic Microorganisms On Tropical and reduction culturing economic loss
Method has considerable advantage in viral identification field.
Detailed description of the invention
Fig. 1 is the amplification curve diagram of novel goose astrovirus；
Fig. 2 be novel goose astrovirus be respectively 200ng, 20ng, 2ng, 0.2ng, 0.02ng, 0.002ng,
Sensitive amplification curve graph when 0.0002ng, 0.00002ng, it can be seen from the figure that its minimum detectability is
0.00002ng；
Fig. 3 is the amplification curve diagram of goose's paramyxovirus；
Fig. 4 be goose's paramyxovirus be respectively 200ng, 20ng, 2ng, 0.2ng, 0.02ng, 0.002ng, 0.0002ng,
Sensitive amplification curve graph when 0.00002ng, it can be seen from the figure that its minimum detectability is 0.00002ng；
Fig. 5 is the amplification curve diagram of goose parvovirus；
Fig. 6 be goose parvovirus be respectively 200ng, 20ng, 2ng, 0.2ng, 0.02ng, 0.002ng, 0.0002ng,
Sensitive amplification curve graph when 0.00002ng, it can be seen from the figure that its minimum detectability is 0.00002ng.
Specific embodiment
The present invention will be further elaborated combined with specific embodiments below, it should explanation, illustrate below be only
In order to explain the present invention, its content is not defined.
Experimental material used in the present invention, reagent and instrument:
1, experimental material: novel goose astrovirus (GoAst) detected, goose in goose's paramyxovirus (GPMV), the present invention
Parvovirus (GPV), goose influenza virus, goose tembusu virus, GsFJ2017 plants of goose astrovirus, goose astrovirus CastV-8
Strain.
2, main agents centrifugal column type viral DNA/RNA extracts kit is purchased from the limited public affairs of Beijing Quan Shijin biotechnology
Department；HiScript cDNA the first chain synthetic agent box is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；Primer and probe by
The Primer Express Software v2.0 of ABI company is designed, and is synthesized by Huada gene company.
3, instrument: 7500 fluorescence quantitative PCR instrument of ABI is ABI Products, and PCR instrument is Bio-Rad Products,
5424D type supercentrifuge is Eppendorf Products.
Embodiment 1
1.1 design of primers and synthesis
Sequence designs 3 groups of spies that can expand 3 kinds of goose cause of diseases referring to the sequence of each target gene in Gene Bank database
Specific primer and probe, primer and probe are designed by the Primer Express Software v2.0 of ABI company, by Hua Da base
Because company synthesizes, sets -20 DEG C and save backup.
The nucleotide sequence of 3 groups of primers and probe is as shown in table 2 below:
The nucleotide sequence of 2 primer of table and probe
1.2 viral nucleic acids extract and cDNA synthesis
It is extracted respectively using Easypure Viral DNA/RNA Kit (centrifugal column type viral DNA/RNA extracts kit)
Three kinds of viral DNA/RNA carry out reverse transcription to three kinds of viral RNAs with HiScript cDNA the first chain synthetic agent box: using 40
μ L system sequentially adds 16 μ L of RNA template, 2 μ L of random primer, 2 × RTMIX, 20 μ L, HiScript Enzyme in EP pipe
2 μ L of Mix, total volume totally 40 μ L are placed in progress cDNA synthesis in PCR instrument, with 25 DEG C of 5min, 50 DEG C of 45min, 85 DEG C of 5min, 4
The program of DEG C 5min carries out a circulation, saves backup for -20 DEG C after cDNA synthesis.
1.3 the optimization of quantitative fluorescent PCR reaction condition
Reaction system is shown in Table 3.
3 RT-PCR of table reacts amplification system (16 μ L)
1.4 fluorescent quantitative PCR conditions are as follows: 95 DEG C of 2min；94 DEG C of 10s, 59 DEG C, 10s, 72 DEG C 40s, 40 circulations.
The specific test of 1.5 kits
Specific test is carried out using the kit, respectively with novel goose astrovirus, goose's paramyxovirus, the tiny disease of goose
Poison, goose influenza virus (H5 hypotype, H7 hypotype, H9 hypotype), goose tembusu virus, GsFJ2017 plants of goose astrovirus, He Exing
CastV-8 plants of shape virus are template, using the kit, carry out quantitative fluorescent PCR using the PCR reaction condition of optimization, as a result
It was found that only novel goose astrovirus, goose's paramyxovirus and goose parvovirus can effectively expand, attached drawing 1,3,5, goose influenza are seen
Virus, goose tembusu virus, GsFJ2017 plants of goose astrovirus and CastV-8 plants of goose astrovirus cannot be expanded effectively, be said
Bright research designed probe and primer have stronger specificity.The result is shown in tables 4.
The test of 4 specificity verification of table
1.6 application kit progress sensitivity tests
The genome of standard items of novel goose astrovirus, goose's paramyxovirus, goose parvovirus is quantitatively arrived into 10ng/ respectively
μ L, by 10 × gradient dilution, each gradient take 2.0 μ L be template quantity, (that is: 20ng, 2ng, 0.2ng, 0.02ng,
0.002ng, 0.0002ng, 0.00002ng) real-time fluorescence quantitative PCR detection is carried out, assess detection limit of the invention.See Fig. 2,
Fig. 4 and Fig. 6, the results showed that this method also has fabulous linear relationship when sample concentration is diluted to 0.00002ng, shows the examination
Agent box has high sensitivity.
The detection of 1.7 clinical samples
Are collected by 100 parts of clinical samples and is detected for Shandong, Henan, Jiangsu Province gaggle, acquires the liver of dead goose sample
Dirty, renal tissue is centrifuged after being ground with physiological saline according to the ratio of 1:3, is extracted using centrifugal column type viral DNA/RNA
Kit extracts the DNA/RNA of virus, is reacted using optimal quantitative fluorescent PCR reaction condition, and positive rate is calculated.Simultaneously
It is detected using conventional PCR method.As a result as shown in table 5 below: to detect 13 parts of GPMV (positive rate 13%), GoAst 8
Part (positive rate 8%), GPV10 parts (positive rate 10%), wherein GPMV and GPV mixed infection case 2；Utilize conventional list
One PCR method detects same group of clinical sample, similarly, detects 13 parts of GPMV (positive rate 13%), GoAst
8 parts (positive rate 8%), GPV10 parts (positive rate 10%), wherein GPMV and GPV mixed infection case 2.The two result
Coincidence rate be 100%.
Table 5
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the limit of embodiment
System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be
Equivalence replacement mode, is included within the scope of the present invention.
Claims (8)
1. a kind of novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR detection primer pair and special
Property probe, it is characterised in that:
The specific detection primer pair and specific probe of novel goose astrovirus are as follows:
GoAst-qF:5 '-ACGGACTGGACGCGTTATGA-3 '；
GoAst-qR:5 '-TGGCCACTTGGATTTCCCTT-3 '；
GoAst-probe:5 '-FAM-CCTCTCTTCTGGCGGATACG-MGB-3 '；
The specific detection primer pair and specific probe of goose's paramyxovirus are as follows:
GPMV-qF:5 '-TGGTATCATATCGCAAAATT-3 '；
GPMV-qR:5 '-CATTGCACGAATGTCTATCT-3 '；
GPMV-probe:5 '-FAM-TGGAGAAGCTGTATCCCTGA-MGB-3 '；
The specific detection primer pair and specific probe of goose parvovirus are as follows:
GPV-qF:5 '-GCAAGACTGTAACGAATGAA-3 '；
GPV-qR:5 '-CTCCAAGAACATCCAGATCT-3 '；
GPV-probe:5 '-FAM-AAACACTACTACAGCTCCTA-MGB-3 '.
2. one kind contains novel goose astrovirus, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative described in claim 1
PCR detection primer pair and specific probe are used for while detecting novel goose astrovirus, goose's paramyxovirus, goose parvovirus
Detection kit.
3. a kind of novel goose astrovirus described in claim 1, goose's paramyxovirus, goose parvovirus multiple fluorescence quantitative PCR
Detection primer pair and specific probe or detection kit as claimed in claim 2 are in the novel goose astrovirus of detection aquatic bird, goose
The Simple infection or the application in mixed infection of paramyxovirus and goose parvovirus.
4. application according to claim 3, it is characterised in that the viral DNA and RNA in sample to be tested tissue are extracted, it will
The RNA reverse transcription is cDNA, more using novel goose astrovirus described in claim 1, goose's paramyxovirus, goose parvovirus
Weight fluorescence quantitative PCR detection primer pair and specific probe expand the viral DNA and cDNA.
5. application according to claim 4, it is characterised in that the quantitative fluorescent PCR reaction system used when being extended is total
Volume is 16 μ L, in which: 2 × PCR MIX, 8 μ L；Concentration is the 0.2 μ L of upstream primer of 50 pM/ μ L；Concentration is 50 pM/
The 0.2 μ L of downstream primer of μ L；Concentration is the 0.1 μ L of specific probe of 50 pM/ μ L；1 μ L of template；Add aqua sterilisa to totality
Product is 16 μ L.
6. application according to claim 4, it is characterised in that amplification reaction condition are as follows: amplification program be 95 DEG C of 2 min, 94
DEG C 10 s, 59 DEG C of 10 s, 72 DEG C of 40 s, 40 circulations.
7. application according to claim 4, which is characterized in that the extracting method is specially the internal organ for acquiring dead aquatic bird
Tissue is used as clinical sample to be detected, is centrifuged after viscera tissue and physiological saline are ground according to the ratio of mass ratio 1:3,
The viral DNA and RNA in tissue are extracted using centrifugal column type viral DNA/RNA extracts kit.
8. application according to claim 4, it is characterised in that reverse transcription reaction condition are as follows: use HiScript cDNA first
Chain synthetic agent box to the RNA carry out reverse transcription: use 40 μ L systems, sequentially added in EP pipe 16 μ L of RNA template, with
Machine primer 2 μ L, 2 × RTMIX, 20 μ L, 2 μ L of HiScript Enzyme Mix, total volume totally 40 μ L, are placed in PCR instrument
CDNA synthesis is carried out, a circulation is carried out with the program of 25 DEG C of 5min, 50 DEG C of 45 min, 85 DEG C of 5 min, 4 DEG C of 5min.
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