CN108384899B - Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof - Google Patents
Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof. The fluorescent quantitative PCR kit contains primers shown by SEQ ID NO.1-SEQ ID NO.2, probes shown by SEQ ID NO.3, a standard substance and a fluorescent quantitative PCR reaction reagent. The fluorescent quantitative PCR kit can detect the novel goose astrovirus, has strong specificity, only specifically binds with the novel goose astrovirus, and has no cross reaction with other goose-origin viruses; and the detection sensitivity is high, and the detection sensitivity is 4 multiplied by 10 in a standard product0‑4×109The copy range has excellent linear relation, the detection range can reach 10 orders of magnitude, novel goose astrovirus disease goose tissues and virus carrying geese without clinical symptoms can be efficiently detected, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of avian virus detection, in particular to a fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof.
Background
Astrovirus (AStV) is a single-stranded positive-strand RNA virus which has no envelope, is spherical and has a diameter of 28-30nm, the genome of the virus has infectivity and a length of 6.4-7.9kb, and the infection of the virus has certain prevalence and can cause enteritis and diarrhea of human beings or animals, and some diseases are accompanied by vomit and abdominal pain. The astrovirus family can be divided into mammalian astrovirus and avian astrovirus according to different infection hosts, and the avian astrovirus can cause poultry to generate various diseases, and the clinical manifestations of the avian astrovirus are different due to different virus strains, virulence or infection hosts. Goose astrovirus (GAstV), which mainly attacks young goslings, has already occurred in a plurality of provinces, so that the death rate of the goslings is increased, and the healthy development of the Goose industry in China is greatly damaged.
In 2017, in 2-12 months, the gosling group in Shandong, Anhui, Jiangsu, Liaoning, Henan, Guangdong and the like in China outbreaks an infectious disease which is mainly characterized by gout. The disease mainly occurs to goslings of 5-20 days old, and the death rate can reach 50% at most. The body cavities and joints of the sick goslings are seriously deposited with urate, the goslings are tired in lying and difficult to take, the growth of the goslings is slow, the feed-meat ratio is increased, the slaughtering qualification rate of the meat geese is obviously reduced, and the serious economic loss is caused to the meat goose breeding industry.
Research shows that the outbreak of the disease is caused by a novel goose astrovirus infection, and no vaccine can be used for preventing the disease at present; conventional antiviral and antibacterial treatment methods are ineffective. Under the current situation, the effective measure for controlling the disease is to monitor the infection of the novel goose astrovirus, so as to discover infected goose groups as soon as possible and take isolation or killing measures on the infected goose groups, thereby reducing the loss caused by the infection of the novel goose astrovirus.
At present, the detection method of astrovirus mainly comprises three major categories of cytological diagnosis, immunological diagnosis and molecular biological diagnosis, wherein the cytological diagnosis technology mainly utilizes cell isolation culture and electron microscope observation. The method is relatively complex in operation, long in detection period and high in detection condition requirement. The immunological diagnosis technology mainly comprises enzyme-linked immunosorbent assay (ELISA) and immunofluorescence methods, and the methods have high sensitivity but high requirements on antibodies. The molecular biological diagnosis technology mainly comprises a traditional Polymerase Chain Reaction (PCR) method and a fluorescence PCR method, and the traditional PCR method can only qualitatively detect the virus infection, can not quantitatively detect the virus infection and has lower sensitivity; the fluorescent quantitative PCR technology has the advantages of high sensitivity, high speed, strong specificity and the like, and is widely applied to the aspects of gene expression level analysis, qualitative and quantitative detection of pathogens and the like. However, the novel goose astrovirus is reported for the first time, and the fluorescent quantitative PCR detection of the novel goose astrovirus still belongs to the technical blank.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in the first aspect of the invention, a group of primers and probes are provided, wherein the nucleotide sequences of the primers are shown as SEQ ID NO.1-SEQ ID NO. 2; the nucleotide sequence of the probe is shown as SEQ ID NO. 3. The method comprises the following specific steps:
a forward primer: ORF 2-F: 5'-CCAGGTCAAGATACAATG-3', respectively; (SEQ ID NO.1)
Reverse primer: ORF 2-R; 5'-GTGTGTTCCAAGTGTAAA-3', respectively; (SEQ ID NO.2)
A fluorescent probe: ORF 2-tag: 5 '-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ 1-3'; (SEQ ID NO.3)
In a second aspect of the invention, the application of the primer and the probe in preparing a reagent or a kit for detecting the novel goose astrovirus is provided.
The novel goose astrovirus is preserved in China center for type culture Collection in 2018, 4 and 26 months, and the preservation number is CCTCC NO: v201808, address: wuhan, Wuhan university, China.
In a third aspect of the invention, a fluorescent quantitative PCR kit for detecting the novel goose astrovirus is provided, and the fluorescent quantitative PCR kit contains the primer and the probe. The preservation number of the goose astrovirus is CCTCC NO: and V201808.
Further, the fluorescent quantitative PCR kit further comprises: standard substance and fluorescent quantitative PCR reaction reagent.
Preferably, the standard is prepared by the following method:
the primers shown in SEQ ID NO.1-SEQ ID NO.2 are adopted to amplify the primer with the preservation number of CCTCC NO: v201808, obtaining an amplification product, connecting the amplification product to a pMD18-T vector, screening positive clones, extracting plasmid DNA, and preparing the standard product.
The fluorescent quantitative PCR reaction reagent comprises: one Step RT-PCR Buffer, TaKaRa Ex Taq HS, PrimeScript RT Enzyme Mix II and ROX reference dye.
In a fourth aspect of the invention, a detection reagent for detecting a novel goose astrovirus is provided, wherein the detection reagent contains the primer and the probe.
In a fifth aspect of the present invention, there is provided a use of the above-mentioned fluorescent quantitative PCR kit or the above-mentioned detection reagent in any one of the following (1) to (3):
(1) carrying out epidemiological investigation on the goose infected with the novel goose astrovirus;
(2) monitoring the new goose astrovirus contamination in blood or serum products;
(3) accurately detecting the copy number of the novel goose astrovirus and determining the infection process of the goose astrovirus.
The sixth aspect of the present invention provides a method for detecting goose astrovirus by using the fluorescent quantitative PCR kit, comprising the following steps:
(1) performing gradient dilution on the standard substance, performing TaqMan fluorescence PCR reaction, and drawing a fluorescence quantitative standard curve according to the concentration and Ct value of the standard substance;
(2) extracting total RNA of a sample to be detected, carrying out TaqMan fluorescence PCR reaction, collecting a fluorescence signal of the sample to be detected, carrying out data processing on the fluorescence signal to obtain a Ct value and an amplification curve, and carrying out qualitative and quantitative detection on the sample to be detected.
Preferably, in the step (1) and the step (2), the procedure of the TaqMan fluorescence PCR reaction is as follows:
5min at 42 ℃; 10s at 95 ℃; thereafter, 40 cycles of 95 ℃ for 5 seconds, 53 ℃ for 30 seconds and 72 ℃ for 30 seconds were carried out.
The invention has the beneficial effects that:
(1) aiming at the newly discovered novel goose astrovirus capable of causing gosling gout, the invention designs the fluorescent quantitative PCR kit capable of detecting the novel goose astrovirus, the kit has strong specificity, only specifically combines with the novel goose astrovirus, and has no cross reaction with other goose-origin viruses such as gosling plague virus, goose tembusu virus, goose influenza virus and the like.
(2) The detection sensitivity is high, and the standard product is 4X 100-4X 109The copy range has excellent linear relation, and the detection range can reach 10 orders of magnitudeThe detection method has the advantages that 4 copies can be detected at least, the detection sensitivity is high, novel goose astrovirus disease goose tissues and virus carrying geese without clinical symptoms can be efficiently detected, and the detection efficiency is improved.
(3) The kit can monitor the novel goose astrovirus infection so as to discover infected geese as soon as possible and take isolation or killing measures on the infected geese to reduce the loss caused by the novel goose astrovirus infection; the novel goose astrovirus can also be subjected to epidemiological investigation on goose infection, and the goose astrovirus pollution in blood or serum products can be monitored.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, the gosling group in Shandong, Anhui, Jiangsu, Liaoning, Henan, Guangdong and the like in China outbreaks an infectious disease which is mainly characterized by gout in 2017. The symptoms of the disease are different from the prior astrovirus infection, and the symptoms of gout appear in goose flocks for the first time, so that the infectious disease is probably caused by the novel astrovirus. There is currently no drug or method available to effectively control this new astrovirus infection. Based on the fluorescent quantitative PCR kit, the fluorescent quantitative PCR kit capable of detecting the novel goose astrovirus is provided, so that infected goose groups can be found as soon as possible, isolation or killing measures can be taken on the infected goose groups, and loss caused by infection of the novel goose astrovirus can be reduced.
As the astrovirus is an RNA virus and has a plurality of segments, different strains have certain differences in antigen structure, pathogenicity, cell culture characteristics and host specificity, and are easy to mutate during genetic evolution. The astrovirus isolated from different hosts therefore has a great variability, differing in pathogenicity, genetic sequence and coding for major proteins.
The inventor of the application separates a novel virus N-AStV-SDPY from tissues such as the liver, the spleen, the kidney and the like of a gosling died from diseases, and identifies the novel virus N-AStV-SDPY as a goose astrovirus. The invention carries out whole genome sequencing on the strain N-AStV-SDPY, and carries out homology analysis and genetic evolution analysis with the astrovirus reported at present, and the result shows that the homology of each gene fragment of the strain N-AStV-SDPY is less than 70 percent; genetic evolution analysis shows that the strain N-AStV-SDPY is in a variation branch; the above results illustrate that: the strain N-AStV-SDPY is different from other astrovirus and is a new species of avian astrovirus. The novel goose astrovirus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: and V201808. Therefore, the novel goose astrovirus to be detected by the invention and the existing reported astrovirus have variability, and the detection of the novel goose astrovirus is more difficult.
For the fluorescent quantitative PCR detection, how to design primer and probe sequences is the key to ensure the detection effectiveness. Although there are many primer design software and primer design principles in the prior art, the primer and probe combination with strong specificity and high sensitivity is not simply obtained by primer design software, which requires technicians to continuously select and optimize a target region by using professional knowledge, then perform primer design according to the optimized target region, and repeatedly screen, optimize and redesign the designed primer. Specifically, the difficulty of designing primers and probes is that the novel goose astrovirus to be detected is different from the existing reported avian astrovirus, and how to specifically detect the novel goose astrovirus of the application and avoid cross reaction with other avian astrovirus (particularly the reported goose astrovirus) is the difficulty of designing the primers and probes of the invention. For the detection of avian astrovirus, some primers and probes are designed according to the gene sequence characteristics of polymerase 1b protein of goose astrovirus, some primers and probes are designed according to the target sequence of open reading frame 2 coding caspid protein of the astrovirus, and the like, so that how to select a target region for designing the primers and the probes for the detection of the astrovirus is not known uniformly at present. The invention optimizes the target region for primer and probe design in the test process, and the result shows that the specificity detection of the novel goose astrovirus can be realized by using the sequence conserved region of ORF2 gene of the novel goose astrovirus for primer and probe design.
The primer and the probe designed by the invention are matched for use and complement each other, the reaction condition of the fluorescent quantitative PCR is further fully considered in the design process, the mutual interference between the primer and the probe is avoided, and the primer and the probe are finally designed. Wherein:
a forward primer: ORF 2-F: 5'-CCAGGTCAAGATACAATG-3', respectively; (SEQ ID NO.1)
Reverse primer: ORF 2-R; 5'-GTGTGTTCCAAGTGTAAA-3', respectively; (SEQ ID NO.2)
A fluorescent probe: ORF 2-tag: 5 '-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ 1-3'; (SEQ ID NO.3)
The 5 'end of the fluorescent probe is marked with a fluorescent reporter group FAM, and the 3' end of the fluorescent probe is marked with a fluorescent quenching group BHQ 1; the length of the amplified fragment is 83 bp.
In the test process, a plurality of groups of different primers and probes are designed according to other conserved region sequences of the goose astrovirus gene and other primer design principles, and are respectively combined, and the specificity and the amplification efficiency of the primers and the probes are respectively detected after reaction so as to screen the optimal primer and probe combination for clinical detection. For example:
a first group:
a forward primer: 5'-GGCCAATATTCAACAACA-3' is used as a reference material;
reverse primer: r; 5'-CCTTCCTTATTGACACAAG-3', respectively;
a fluorescent probe: 5 '-FAM-TGTGTAATGTCTGGCTCACCCA-Eclipse-3'.
Second group:
a forward primer: 5'-GACGCGTGCCACAAGGA-3' is used as a reference material;
reverse primer: r; 5'-CCACGCTTGATCTTATCTGTAAGC-3', respectively;
a fluorescent probe: 5 '-FAM-AGCAAGGCGCGCATTCCCC-BHQ-3'.
As a result, it was found that the specificity and sensitivity of the detection of the novel goose astrovirus by using the primer and probe combination (SEQ ID NO.1-SEQ ID NO.3) of the present invention are optimal. And other primer and probe combinations can not effectively distinguish the novel goose astrovirus from other goose-derived viruses, and false positive or false negative easily occurs.
The invention also optimizes the fluorescent quantitative PCR condition in the test process, reduces the operation to the maximum extent in the clinical application of the kit, ensures the sensitivity, reduces various pollutions to the maximum extent, and ensures the science and the accuracy of the result.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.
Example 1: design of fluorescent quantitative PCR primer and probe
The whole genome sequence of the novel goose astrovirus (with the preservation number of CCTCC NO: V201808) is obtained according to the second-generation sequencing technology, and is shown as SEQ ID NO. 4. Specific primer sequences and fluorescent probe sequences are designed aiming at the sequence conserved region of ORF2 gene, and the sequences are designed as follows:
a forward primer: ORF 2-F: 5'-CCAGGTCAAGATACAATG-3', respectively; (SEQ ID NO.1)
Reverse primer: ORF 2-R; 5'-GTGTGTTCCAAGTGTAAA-3', respectively; (SEQ ID NO.2)
A fluorescent probe: ORF 2-tag: 5 '-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ 1-3'; (SEQ ID NO.3)
The 5 'end of the fluorescent probe is marked with a fluorescent reporter group FAM, and the 3' end of the fluorescent probe is marked with a fluorescent quenching group BHQ 1; the length of the amplified fragment is 83bp (shown in SEQ ID NO. 5).
Example 2: establishment of fluorescent quantitative PCR detection method
(1) Extraction of viral RNA:
30mg of suspected diseased gosling kidney and liver mixed homogenate tissue is taken, and the classical Trizol method or the commercialized kit is adopted to extract the total RNA.
(2) Establishing a fluorescent PCR reaction system:
the One-Step fluorescence quantitative kit used was One Step primer PrimerScript having a product number of RR064A from TakaraTM2 XOne Step RT-PCR Buffer III-10 muL, 1 XTaKaRa Ex Taq HS (5U/. mu.L) -0.4 muL, PrimeScript RT Enzyme Mix II-0.4 muL, PCR Forward Primer (10 muM) -0.4 muL, PCR Reverse Primer (10 muM) -0.4 muL, Probe-0.8 muL, ROX Reference Dye or Dye II (50 x) -0.4 muL, Total RNA-2 muL of the sample to be detected extracted in the Step (1) and RNase Free dH are sequentially added into a 200 muL PCR reaction tube2O was supplemented to 20. mu.L system. Placing in an ABI 7300Fast fluorescent quantitative PCR instrument for reaction at 42 ℃ for 5 min; fluorescence was collected after 40 cycles of 95 ℃ for 10s, 95 ℃ for 5s, 53 ℃ for 30s, and 72 ℃ for 30 s.
(3) Preparation of a standard curve:
in order to accurately quantify the copy number of the virus in the sample, a plasmid containing the amplified fragment of interest was prepared as a standard to draw a standard curve. Firstly, designing forward and reverse primers (shown in SEQ ID NO.1 and SEQ ID NO.2) of ORF2 gene, and amplifying a novel goose astrovirus genome to obtain an amplification product (shown in SEQ ID NO. 5) of 83 bp.
Connecting the plasmid to a pMD18-T vector according to a classical molecular cloning operation method, named as pMD-ORF2, screening positive clones, extracting plasmids, and measuring the plasmid concentration by using a spectrophotometer to obtain a standard substance. Performing 10-time gradient dilution on the standard substance, performing TaqMan fluorescence PCR reaction, and automatically drawing a fluorescence quantitative standard curve by instrument software according to the concentration and Ct value of the standard substance; the method specifically comprises the following steps: y-3.173 x +40.059, correlation coefficient R20.992, a good linear relationship is presented; wherein x represents the copy number logarithm of the sample and y represents the Ct value.
(4) And (3) judging a detection result:
and (3) collecting a fluorescence signal according to the fluorescent quantitative PCR reaction, and processing data by using instrument software to obtain an amplification curve and a Ct value. And taking the Ct value corresponding to the lowest concentration of the standard and the amplification curve as a judgment basis.
And a result judgment method comprises the following steps: if the fluorescence signal of the sample to be detected exceeds the threshold value and Ct is less than or equal to 30.0, and the amplification curve is smooth S type, the novel goose-star virus in the sample to be detected can be judged.
According to the fluorescent quantitative standard curve, the novel goose astrovirus in the sample can be quantitatively determined.
Example 3: optimization of composition and experimental parameters of kit and investigation of specificity, sensitivity and repeatability
1. The kit comprises the following components:
the kit of the embodiment is a fluorescent quantitative PCR kit and is used for detecting the novel goose astrovirus (preservation number is CCTCC NO: V201808). The kit comprises: a forward primer (10. mu.M) shown as SEQ ID NO.1 designed in example 1, a reverse primer (10. mu.M) shown as SEQ ID NO.2 designed in example 1, a probe shown as SEQ ID NO.3 designed in example 1, a standard and a fluorescent quantitative PCR reaction reagent.
The standard substance is prepared by the following method:
the primers shown in SEQ ID NO.1-SEQ ID NO.2 are adopted to amplify the primer with the preservation number of CCTCC NO: v201808, obtaining an amplification product (shown in SEQ ID NO. 5) of 83bp, connecting the amplification product to a pMD18-T vector, screening positive clones, extracting plasmid DNA, and preparing the standard product.
The fluorescent quantitative PCR reaction reagent comprises: 2 Xone Step RT-PCR Buffer III, 1 XTaKaRa Ex Taq HS (5U/. mu.l), PrimeScript RT Enzyme Mix II, ROX Reference Dye or Dye II (50X), RNase Free dH2O。
2. Optimization of experimental parameters:
optimized exploration is carried out on the fluorescent quantitative PCR reaction condition of the primer (SEQ ID NO.1-2) in the kit, and the result shows that the primer is 10 mu M and the reaction condition is 5min at 42 ℃; 10s at 95 ℃; and then, 40 cycles of 95 ℃ for 5s, 53 ℃ for 30s and 72 ℃ for 30s are carried out, so that the kit has the best sensitivity and detection effect.
3. Specificity, sensitivity and reproducibility investigation of the kit
(1) Specificity test
The specific test of the kit is carried out by applying the kit, and the specific test is carried out by using gosling plague virus, goose tembusu virus, goose influenza virus, goose astrovirus GsFJ2017 strain (GenBank accession number is MF576430), goose astrovirus CastV-8 strain and a preservation number detected by the invention as CCTCC NO: v201808, using the kit to perform fluorescence quantitative PCR, finding that neither gosling plague virus, goose tembusu virus, goose influenza virus, goose astrovirus GsFJ2017 strain (GenBank accession number is MF576430) nor goose astrovirus CastV-8 strain can be effectively amplified; only the preservation number is CCTCC NO: the novel goose astrovirus of V201808 can be effectively amplified.
The test results show that the specificity of the kit is 100%, the kit has strong specificity, and primers and probes in the kit are specifically combined with the novel goose astrovirus and do not have cross reaction with other goose-derived viruses.
(2) Repeatability test
The kit is applied to carry out a repeatability test of the kit. Clinically collected kidney tissue samples of goslings taking gout as a main symptom are taken, the kit is used for carrying out repeated detection, and the Coefficient of Variation (CV) corresponding to three repeated samples in one experiment is less than 0.5%, which shows that the goslings have extremely high repeatability.
(3) Sensitivity test
The standard substance is diluted into different concentrations, the kit is adopted for detection, and common PCR detection is used as comparison. As a result, the fluorescent quantitative PCR result shows that the kit can detect 4 copies of standard substance and is 100-109The copy range has excellent linear relation, and the detection range can reach 10 orders of magnitude; the kit is shown to have extremely high sensitivity.
Example 4: clinical application experiment 1 of the kit of the present invention
(1) Extraction of viral RNA:
30mg of suspected diseased gosling kidney and liver mixed homogenate tissue is taken, and the classical Trizol method or the commercialized kit is adopted to extract the total RNA.
(2) Establishing a fluorescent PCR reaction system:
2 Xone Step RT-PCR Buffer III-10 μ L, 1 XTaKaRa Ex Taq HS (5U/. mu.L) -0.4 μ L, PrimeScript RT Enzyme Mix II-0.4 μ L, PCR Forw ard Primer (10 μ M) -0.4 μ L, PCR Reverse Primer (10 μ M) -0.4 μ L, Probe-0.8 μ L, ROX Reference Dye or Dye II (50X) -0.4 μ L, Total RNA-2 μ L of the sample to be detected extracted in the Step (1) is added into a 200 μ L PCR reaction tube in sequence, and the sample is supplemented into a 20 μ L system by RNase Free H2O. Placing in an ABI 7300Fast fluorescent quantitative PCR instrument for reaction at 42 ℃ for 5 min; 10s at 95 ℃; thereafter, fluorescence was collected at 95 ℃ for 5 seconds, 53 ℃ for 30 seconds, and 72 ℃ for 30 seconds for 40 cycles.
And a result judgment method comprises the following steps: if the fluorescence signal of the sample to be detected exceeds the threshold value and Ct is less than or equal to 30.0, and the amplification curve is smooth S type, the novel goose-star virus in the sample to be detected can be judged. The results are shown in Table 1.
Table 1: fluorescence quantitative PCR detection result of disease material for detection in diseased goose farm
The result shows that the novel goose astrovirus can be detected in the disease material of the goose farm in 7 different areas by adopting the kit.
Moreover, 40 identified samples are detected by adopting the kit, 36 samples contain the novel goose astrovirus, and the detection accuracy is 100% consistent with the identification result.
Example 5: clinical application experiment 2 of the kit of the present invention
Clinically collected gosling infected with the novel goose astrovirus and suffering from gout is taken, kidney and liver mixed tissues are taken, 5 times of normal saline is added, then tissue homogenate is carried out, after repeated freeze thawing is carried out for 3 times, supernatant is taken and inoculated to GKC cells, and toxicity is collected after stable cell lesion occurs.
Extraction of viral RNA as described above:
400 mu L of cell samples infected by the virus to be detected are taken, and the total RNA is extracted by a classical Trizol method or a commercial kit.
Establishing a fluorescent PCR reaction system:
2 Xone Step RT-PCR Buffer III-10 μ L, 1 XTaKaRa Ex Taq HS (5U/. mu.L) -0.4 μ L, PrimeScript RT Enzyme Mix II-0.4 μ L, PCR Forw ard Primer (10 μ M) -0.4 μ L, PCR Reverse Primer (10 μ M) -0.4 μ L, Probe-0.8 μ L, ROX Reference Dye or Dye II (50X) -0.4 μ L, Total RNA-2 μ L of the sample to be detected extracted in the Step (1) is added into a 200 μ L PCR reaction tube in sequence, and the sample is supplemented into a 20 μ L system by RNase Free H2O. Placing in an ABI 7300Fast fluorescent quantitative PCR instrument for reaction at 42 ℃ for 5 min; fluorescence was collected after 40 cycles of 95 ℃ for 10s, 95 ℃ for 5s, 53 ℃ for 30s, and 72 ℃ for 30 s.
And a result judgment method comprises the following steps: if the fluorescence signal of the sample to be detected exceeds the threshold value and Ct is less than or equal to 30.0, and the amplification curve is smooth S type, the novel goose-star virus in the sample to be detected can be judged. The results are shown in Table 2.
Table 2: fluorescence quantitative PCR detection result of novel goose astrovirus infected cell sample
Through detection, the sample contains the novel goose astrovirus, and the detection method accords with expectation, and shows that the detection method is accurate and reliable.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof
<130> 2018
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
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ccaggtcaag atacaatg 18
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<213> Artificial sequence
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gtgtgttcca agtgtaaa 18
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
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agtccttcca cgatagccaa cat 23
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<211> 7252
<212> DNA
<213> Goose astrovirus (Goose astrovirus)
<400> 4
tgtagcctct gtgttttcgc ttcctgcgca tcaaggcacg ccttctcacc caaattcata 60
aggtttgcac caatatcggg tgaccatgtt ggtttggtga catcatcggc gaaaaagaga 120
ccatacctcc cattgtcaac cccttttcct cacttgacac tcgtgaggaa aggaagactg 180
atgagattca gtcaggcctt gagaaggtct tccacttcaa tggtgttaat gaattataca 240
ccaagatgaa aggcttttat ggtcgcactc cagcctggag tgctctcatg aagtgtaatg 300
ccatctatct taaagattgg aaaacagcta taggggttga caatgggagg tatggtctct 360
ttttcgccga tgatgtcacc aaaccaacat ggtcacccga tattggtgca aaccttatga 420
atttgggtga gaaggcgtgc cttgatgcgc aggaagcgaa aacacagagg ctacaatgct 480
cgctgaaacg atgtggagct cttgtccaac aagttcttga gaagagcaaa caagcaaagg 540
aacaaatgca aaaagccgag gaattacaga agaagattga tcaaattaat gaaagcaaca 600
aggttctaca tcgcaaaatg caggagagaa atgttgagta ttctcagaag gtcataactc 660
gtattcatga gctgcaaaaa gacagagacc tgtggatgtc gcgtgctgct cattacgagc 720
aggagaacgt gcggctacgt attcaactgg ggacatatga tccgaacagg tctaagctga 780
tgactttcct agcatggatg tgtgttgcga tcattgcttt cagcttgatt caattggcgg 840
atgctagtga gattaaggat aacagaacag aaaattgttt cccaacatct ggctgtgttc 900
tctcagacgt tccttggact aagtcgtaca cctttatgga gctaatggac aagtgtgtga 960
ccggtactgc catcatacct aaaaacacat ttaatggtga agggcttgag ctggagtgta 1020
ttaagaattt tggcaagaaa tggtgcaaag gaagggttga gaagctcata ccgcattatt 1080
gtgatagaga taatactttt gatgccctct atcagtatta tgttgaaaca gtagcaattg 1140
ctgttgactt ctttaaagtt gcggtgcagt atcggattga caattggatt gtggctctct 1200
ttagtctagt attgactggg aagatggaga agtttcttaa gacgctaccc tttgtaattt 1260
tggcatggtg gtttaagttg cccatctttg ttatgagcat tgccacgaca atctttcctg 1320
tgcatgctct gccattcttg ttcttccagg tgtgctttcc aaactttgtc atcatcactg 1380
gttttctctt gtggatgacc tctatattag tggcttttta ttggtttgat gggactgcaa 1440
ttttggttga ggtttcttat gcggtattct atactattat cttctttgtg tggtctacag 1500
cattgacaat tatttcaact ttgtcattga cagtccctgt acagatttta ttattctgtg 1560
tttcattaac catttattgt ggcacaaagt ttgcgtgttc cactattaca gtcactaatc 1620
ctgacggtac tgtgtcgaaa tactcaagag ttgccaaaat gaaggatact gttgccatac 1680
agtgcaaaaa gatggtgagc tttgggcagg cccgtggtgt gataccagca actccagtga 1740
aggtgaattc cattgtcgtt gttgaaggaa aaaaaggttc aggggttggc ttccggttca 1800
tgaattatat aattacagca ggacacgtca cattaggctc tgactgggta acagtaaaac 1860
atgctggcac agtagccaaa acaaaagtcc tgaagcagat tgaggtgttt gagtgcgtcg 1920
atacaatagc ggttataaag ctgccccccg aattacagaa tttgaaaccc ataaaactcg 1980
caaaacgtgt tgagtccaca tacttaacat tgacagcttt tgatcctaat tttcaaaatg 2040
ttgtgtcata cactggctgg tgtattattg atggaaattg gatcagtaat gcatttaata 2100
ctcaatttgg aaacagtggt ggcccatacg ttgatgcaga gggtaaattg gttgggattc 2160
atcttggaac tcagggagtt atgtcccaag gtattgttgt ggtggatgcc cttaaaacgc 2220
atttcacgct cgcagaccag tgcaaggatt ttgattatga tggcttcctg gagaaagtta 2280
ttgcaggaac aaagacatca catgaagcaa ttctcaaagg tattgaggaa ttgcgagagg 2340
acgtcgacac gctcaagaac aagtgcaaga attatgatga gtactggctt gttcaaacta 2400
tttttggcca gaagaaaaag ggcaaaacaa agaaaacagc ccgggggaat aaacatcttg 2460
taacaaagag attcctgagt aaaggccact tcatgaaact caaaatgctc accgatgagg 2520
aatataatca tatgattgag caaggtttca ccgctgatga gatacgtgag gcagtcaatg 2580
agctgagaga acaagcatgg ctcaactact gcattgataa tgatattgat gaagaaggtg 2640
cggaagagtg gtatgaagat atgattgaag atgaccaaat taatgaaaag attgatgaag 2700
ccattgagaa agcaatggaa gaaaggggcg agttttatca agtcaaaaaa aggaaaacat 2760
ttgcacaaca agcattatta cacctcatcc gtgtcagtaa agccaggagc caaactgcaa 2820
agcaagaggt tcagaaagaa aacgcagatc agctttacga gatgttcaaa tctgccgtaa 2880
aggatgaaca agttgatgaa ggcacaacct cttacgcact cttcgccaat ggtgatcaga 2940
ttgagatggt cgaaggaaag gaacttgatt ttgaaaaggt aaagcttatc cacgttgatg 3000
gtgaacaaaa aaatgagacc attccgggga cgaaagtcac tgagatttct actggccctg 3060
agaataagaa gaacattttg cgcaagaagg acactcaaat taatccagaa caagctcaac 3120
ctcttgagca gcgcaagagg atctgcaagt ggtgtgggtc gagccaaaag catgactttc 3180
gtgattgcaa gttccagcgt gagaaaaggt tttgtgtctt ctgtgcaaag atgcactcat 3240
tgtttgatgg tcatcaacgg gacgtcttgt gcgacgtttg tgggaagaag ttcaagagtg 3300
tagaggagct tgaacagcac gtaactcgtg aaacgtgtga aaaaaactag ataaggggga 3360
cgatgccatc tgggtccccc gcataccacc tgatgattct cacatttttt cttggcagga 3420
tgatattatt gagtgtgata agaaaatcac tttgcccgac tatatagata taattggttt 3480
tatacctgta gataaattag ttgtcagagc aaagaccatt catgacccct tattaaactt 3540
ggttgagcat tggcaacaag atgaatatgc tcctactaaa tgggactata aagcctatac 3600
caagatgttt gaaaaatttt tctatcatga acctcaggat tttgtaaaca attttccaga 3660
gctggtcatc ttaagtgata gtgttgtgtt agaagaacat tcttatatgg ccaattccaa 3720
tttaacaccc ataatggcaa ctaagaagaa tgtcgatagt acccctggtt atccaaaatt 3780
taagtacttt gacacagaga aggactatct tgaacaatgt ggctggggtg agtacttgaa 3840
ggctgtatca gatattgatg agactgtgca aaataggccc ctctggtggt gttttctcaa 3900
aaatgaggtc ctcaaaaaga agaaaatcat ggataatgat attcgtatga tcttgtgtgc 3960
tgatccagta tatacaagga ttggagccat gtttgagcag gaccagaatg agaaaatgaa 4020
gcaacagaca gaacggcggg ctgcacaagt tggttggaca cccttttttg gtggaataca 4080
tcagcgagta tctaggctca ttggtggtgg tgacaggttt tttgtagaga cggactggac 4140
gcgttatgat ggtacgttgc ccaagcctct cttctggcgg atacgacaga tgcgttactt 4200
tttcctgtcc aaccatcata agacaccaca gcttaagaaa ctctatgatt ggtatgtcaa 4260
aaacttagtt gaaaaaatta tattattacc aactggggag gtttgtacag ttaagaaggg 4320
aaatccaagt ggccaatatt caacaacagt ggacaacaat atgtgtaatg tctggctcac 4380
ccattttgag atagcttatc tttattggaa acagcatggg tcattgccga cgctcagatt 4440
actcagggat aatgtcacca tgatttgcta tggcgatgat aggcttgtgt caataaggaa 4500
gggttttatt aattacgaca cgaatgtcgt catagatatg tataaaaata tttttggtat 4560
gtgggttaag ccagaaaatg tcaaggtctc tgatgatatt gagggtatga ctttctgtgg 4620
tcttacgttg gttaaaagga gtgatggtag atttgttgga gttcccaatg ttgataagat 4680
attgtcaaca cttgaaaatc ctgttagaaa attacccaac ttagaatcac tctggggtaa 4740
attggtttct ttaaggatct tgtgcgaaaa tgcacccaat agtgttagag agtttcttga 4800
taaacaaatc aacacggttg aggagcacgc tgccagtgaa aatattaact tacctgaggt 4860
cgggcccgac ttcttttctc aaatctggtg agtggcggac cgaaataaaa tggcagacag 4920
ggcggtggcc ccgcgcgaga aggtgaccaa gaaggttaca aaagtagtca ccgttaagaa 4980
aaaacaccca aaaaagaaac caaagcagaa agtatataag ccccaaaaat tacccatgaa 5040
ggccgagagg aagcttgaga aagaagtgaa aggtttgaag aaaagagtag ctggaccacc 5100
cgttaatgac aaaatgacta ccacgataac acttggtcag atcactggga attcaacaga 5160
cacactcgac cggaagcata aatacttcac aaatccactc atgatgaaaa accaggaaaa 5220
tgggcaaaca gcaactcctc taagtataag ggcctcacaa tataacttgt ggaggatcag 5280
aaagctgcat atccgccttg ttccacttgc tggtagggcc aatattcttg gctcggttgt 5340
cttcctagat atagaacagg aagctaacac agctgggcca gagtccatag ataccataaa 5400
agctcggccg catcttgaac tcccgattgg ctcaaaacat ctttggaggg ttcaacccag 5460
gctgatgcag ggaccccggc aaggatggtg gaatgtagac cccggggatt cacctactga 5520
ctcacttggt ccagcaatca atatgtggac atatttgaaa acagtaaatg cactttcaac 5580
acgggcgcag gcacaacaag ttccctatac ttctgccctc ttcctggttg aagcaacggt 5640
cacttatgag ttctcgaact atggtccaaa gccagggctc tcactcatga ccagtgaaac 5700
actttcagca tcagggaaaa cggcaaccct tgtaaatacc caggatggtg cacttgcttt 5760
aacagttagt ggcgcattgc agagattcct cgatgagaag gagcaacaca ggcgcgtttc 5820
aaatgcgcag acctctggtg tcggtgaggt gttttgggct gtttccacag aggtggtcga 5880
gaccgtagcg agtgcacttg gaggctgggg ttggctcttg aaaggaggat ggttcgtgat 5940
cagaaaattg tttggtgctg cttcaaattc tggttccact tatctgatct attcctcagt 6000
ttcagacgcc cagatagaca gcaggattta tcagacagtt ccaccgaaca cgccactaca 6060
gctggctgca aatacagtta agcttgtaca gctaacacag ccaaatgtga atacaactgg 6120
acaaggtacc actgtccttt cacgggatgc cgattacctg cctctgccag tggcaccaat 6180
gcaggttact ccctcgcttg tgtacaactt ccagggtgaa aggcagagca ctacagaatc 6240
gtgctcattc ctggtgtttg gaataccaca ggcagaatcc aggtcaagat acaatgcaaa 6300
tataaccttc aatgttggct atcgtggaag gacttcaaca tcatttacac ttggaacaca 6360
caattggtgg gctgttatga cactttcaca aacaggagta atttttgcac caccggctgt 6420
gggcacaggg gtctgtaata cattggctac agccatacaa cacttaaacc ctgagcttga 6480
aacagcagtc ctgcgtgtga ataccagtac aacatctact ggtgggctaa taacggaact 6540
caggaatcgg ctcaacatcg ctgatgggga ctatgtgatt tcaatgggtg atccgcaagg 6600
aaataggtca gcactgtact ttaggaattc agaccagaaa tgggtgtggc tctgggctgg 6660
cgactctaac cctggtgaaa ctttccaaag ttttaagatg ccagtcctaa ttaattggtc 6720
agtgtcagat tcccaggaac aatataatgc tagagtcagg atggtacagt atgctaatgc 6780
acaacagcag actttgacag accctgagga agatgatgat cccctctctg atgtcacttc 6840
gctttttgat ccaacagcgg aagatgagac tgacttccac ctagcagtct cactcaagac 6900
ctctgactac ttaaaagaag aggctgagta ctggaaagcg aaggcgcagg ccttgcttat 6960
ggagaaggca ctaagtgcac cacaagcagg gacagtccgc tttgagaagg gcggacatga 7020
gtgatctctt cactagcagc cgcggccacg ccgagtagga tcgagggtac agctgcacct 7080
tctcattagg cttgagggag aatcggccct ctctgcctta aattaacccg gtgacagtca 7140
ggtggtcacc cacacaaatc gcgtgcgggt acccctgacc gggtttttgt ttaaagaatc 7200
aaatgtgatt tttaagcatt taaaatcttt aaaaaaaaaa aaaaaaaaaa aa 7252
<210> 5
<211> 83
<212> DNA
<213> Artificial sequence
<400> 5
ccaggtcaag atacaatgca aatataacct tcaatgttgg ctatcgtgga aggacttcaa 60
catcatttac acttggaaca cac 83
Claims (4)
1. The application of the primer and the probe in preparing a reagent for detecting the novel goose astrovirus; the novel goose astrovirus is characterized in that the preservation number of the novel goose astrovirus is CCTCC NO: v201808;
the nucleotide sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO. 2; the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. A fluorescent quantitative PCR kit for detecting novel goose astrovirus is characterized in that the fluorescent quantitative PCR kit contains primers and probes;
the nucleotide sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO. 2; the nucleotide sequence of the probe is shown as SEQ ID NO. 3;
the fluorescent quantitative PCR kit also comprises: a standard substance and a fluorescent quantitative PCR reaction reagent; the standard substance is prepared by the following method:
the primers shown in SEQ ID NO.1-SEQ ID NO.2 are adopted to amplify the primer with the preservation number of CCTCC NO: v201808, obtaining an amplification product, connecting the amplification product to a pMD18-T vector, screening positive clones, extracting plasmid DNA, and preparing the standard product.
3. The fluorescent quantitative PCR kit of claim 2, wherein the fluorescent quantitative PCR reaction reagents comprise: one Step RT-PCR Buffer, TaKaRa Ex Taq HS, PrimeScript RT Enzyme Mix II and ROX reference dye.
4. The use of the fluorescent quantitative PCR kit of claim 2 or 3 in monitoring the contamination of a new goose astrovirus in a serum product, wherein the new goose astrovirus has the preservation number CCTCC NO: and V201808.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109762062B (en) * | 2018-09-19 | 2022-02-25 | 天津瑞普生物技术股份有限公司 | Preparation method of goose gout egg yolk antibody |
| CN109457055A (en) * | 2019-01-03 | 2019-03-12 | 山东省农业科学院家禽研究所 | Detect the primer sets and kit of goose source astrovirus |
| CN111088402A (en) * | 2020-01-13 | 2020-05-01 | 华南农业大学 | Novel goose astrovirus detection primer group and kit |
| CN111826473B (en) * | 2020-09-03 | 2023-11-07 | 佛山科学技术学院 | Primer pair for fluorescent quantitative PCR detection of goose type 2 astrovirus and application thereof |
| CN112662814B (en) * | 2021-01-22 | 2023-12-22 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Goose astrovirus nucleic acid CRISPR-Cas13a detection system, RPA primer pair and crRNA |
| CN114214466B (en) * | 2022-02-09 | 2022-07-22 | 江西省农业科学院畜牧兽医研究所 | Novel goose astrovirus and duck tembusu virus multiplex fluorescence quantitative PCR detection primer probe set, kit and application |
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| CN107299155A (en) * | 2017-08-18 | 2017-10-27 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection |
| CN107385111A (en) * | 2017-08-18 | 2017-11-24 | 福建省农业科学院畜牧兽医研究所 | The real-time fluorescence quantitative PCR detection primer and its kit of a kind of goose astrovirus |
| CN107475458A (en) * | 2017-09-29 | 2017-12-15 | 福建省农业科学院畜牧兽医研究所 | Goose astrovirus ring mediated isothermal amplification detection primer group and kit |
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| CN107385111A (en) * | 2017-08-18 | 2017-11-24 | 福建省农业科学院畜牧兽医研究所 | The real-time fluorescence quantitative PCR detection primer and its kit of a kind of goose astrovirus |
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