CN106868221A - Porcine pseudorabies pyrosequencing detection method and primer special - Google Patents
Porcine pseudorabies pyrosequencing detection method and primer special Download PDFInfo
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Abstract
The invention discloses porcine pseudorabies pyrosequencing detection method and primer special.The present invention is with PRV gD GFP conserved sequences as research object, design a pair of universal amplification primers and a sequencing primer with biotin labeling, pyrosequencing reaction is carried out after obtaining purity PCR primer higher through PCR amplifications, obtain one section of short sequence of high specificity, this section of sequence can be as the target sequence of PRV identifications, directly it is determined that PRV from this section of gene order.The method can be diagnosed as the routine screening of porcine pseudorabies, improve detection efficiency, it is to avoid the appearance of false positive results, and PRV is identified from gene order intuitively, and the infected pigs to PRV carry out examination identification faster.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of porcine pseudorabies pyrosequencing detection method and special
Primer.
Background technology
Porcine pseudorabies (Pseudorabies, PR) are caused by Pseudorabies virus (Pseudorabies virus, PRV)
Various domestic animals and the acute infectious disease that can infect of wild animal, with heating, miscarriage, stillbirth, very itch (except pig) and brain ridge
It is cardinal symptom that marrow is scorching, wherein the harm to pig is maximum, and pig is unique natural reservoir (of bird flu viruses).The disease betides U.S. in 1813 first
State, then involves the countries and regions such as America and Europe, and now the disease has been in worldwide prevalence.Since the second half year in 2012, China PR morbidities
Rate substantially rises, and great economic loss is caused to pig industry.
Porcine pseudorabies virus belong to herpetoviridae, also known as the type of herpesvirus suis I.So far, in porcine pseudorabies
11 kinds of glycoprotein are had discovered that in poison, wherein, outside secretion egg is returned in addition to gG is, other 10 kinds of structural proteins are positioned at capsule
On film.Wherein gD is the major structural protein of PRV, it not only induction body fluid be immunized, and inducing cellular immune, while being virus
The main molecules of the virus infected cell of particle surface, play a significant role in viruses adsorption and during invading host cell.
The detection of the method in epidemic disease such as including virus purification and identification, molecular biology of traditional epidemic disease etiological diagnosis method
Method has played huge effect, but also possesses some simultaneously and cannot meet the defect for fast and accurately detecting requirement, i.e., cannot
Golden standard --- the gene order of molecule diagnosis is obtained, consequently, it is possible to causing the result of false positive.If desired for carrying out positive knot
Fruit confirms, then expand purpose fragment must be connected on specific sequencing vector, could be obtained after conversion, picking positive colony
To complete sequencing confirmation, time-consuming effort.
Pyrosequencing techniques are the short chain sequencing technologies that Quantitative Sequence relatively advanced in recent years is determined, and can realize height
Throughput sample intuitively sequence analysis.Golden standard --- the gene sequence that molecule is diagnosed can be obtained by pyrosequencing techniques
Row, it is to avoid the appearance of false positive results；Especially so that in the laboratory for not possessing veterinary biological level of security, can use should
Method is to viral Rapid identification parting.
The content of the invention
It is an object of the present invention to provide a kind of primer set for detecting PRV.
The primer set that the present invention is provided, including following primer pair：
It is single-stranded shown in sequence 2 in single strand dna and sequence table of the primer pair as shown in sequence in sequence table 1
DNA molecular is constituted.
In above-mentioned primer set, a prime end biotin labeling in the primer pair.
In above-mentioned primer set, the primer set is also classified as the sequencing primer of sequence 3 in sequence table including nucleotides sequence.
Complete PCR reagent containing above-mentioned primer set or the examination containing the primer set or the complete PCR reagent
Agent box is also the scope of protection of the invention；
Or the DNA molecular shown in sequence 4 81-135 is also the scope of protection of the invention；.
In above-mentioned complete PCR reagent,
The complete PCR reagent is made up of PCR reagent 1 and PCR reagent 2；
The PCR reagent 1 is the reagent containing primer pair described in above-mentioned primer set；
Or, concentration of the every primer in the PCR reagent 1 is 20 μM in the primer pair；
The PCR reagent 2 is the reagent containing sequencing primer described in above-mentioned primer set；
Or, concentration of the sequencing primer in the PCR reagent 2 is 0.3 μM.
DNA molecular shown in above-mentioned primer set or above-mentioned complete PCR reagent or kit or sequence 4 81-135
Application in detecting or aiding in detection PRV is also the scope of protection of the invention；
Or, above-mentioned primer set or above-mentioned complete PCR reagent or kit or the DNA shown in sequence 4 81-135
Application of the molecule in detection or auxiliary detection PRV product is prepared is also the scope of protection of the invention；
Or, above-mentioned primer set or above-mentioned complete PCR reagent or kit or the DNA shown in sequence 4 81-135
Whether molecule is also the model protected of the invention containing the application in PRV in detecting or aiding in detection sample to be tested
Enclose；
Or, above-mentioned primer set or above-mentioned complete PCR reagent or kit or the DNA shown in sequence 4 81-135
Whether molecule is also the present invention containing the application in PRV product in detection or auxiliary detection sample to be tested is prepared
The scope of protection.
Whether another object of the present invention contains porcine pseudorabies in being to provide a kind of detection or auxiliary detection sample to be tested
The method of poison.
The method that the present invention is provided, comprises the following steps：
1) with the single strand dna group in the single strand dna shown in sequence 1 in sequence table and sequence table shown in sequence 2
Into primer pair sample to be tested is entered performing PCR amplification, obtain pcr amplification product；
2) sequencing primer that sequence 3 in sequence table is classified as with nucleotides sequence carries out pyrophosphoric acid survey to the pcr amplification product
Sequence, obtains pcr amplification product sequencing result；
If the homology of the DNA molecular shown in the pcr amplification product sequencing result and sequence 4 81-135 is more than etc.
In 80%, then contain in the sample to be tested or candidate contains porcine pseudorabies virus, if the pcr amplification product sequencing result
Homology with the DNA molecular shown in sequence 4 81-135 is less than 80%, then do not contained in the sample to be tested or not candidate
Contain porcine pseudorabies virus.
Whether the 3rd purpose of the present invention contains PRV in being to provide a kind of detection or auxiliary detection sample to be tested
Method.
The method that the present invention is provided, comprises the following steps：
Detect in the nucleic acid of sample to be tested whether containing the DNA molecular shown in sequence 4 81-135 or with its homology
DNA molecular more than or equal to 80%, if it does, then the sample to be tested contains or candidate contains PRV, if being free of
Have, then the sample to be tested is not contained or candidate does not contain PRV.
In the above method, whether divide containing the DNA shown in sequence 4 81-135 in the nucleic acid of the detection sample to be tested
The method of son or the DNA molecular with its homology more than or equal to 80% comprises the following steps：The step of claim 7 1) and step
2) pcr amplification product sequencing result, is obtained, analyzes whether the pcr amplification product sequencing result contains sequence 4 81-135
Shown DNA molecular.
In the above method, the annealing temperature of the PCR amplifications is 55 DEG C, and annealing time is 30S；
Or, the template of the PCR amplifications is the nucleic acid of the sample to be tested.
The present invention designs a pair of universal amplification primers and a band with PRV gD GFP conserved sequences as research object
There is the sequencing primer of biotin labeling, pyrosequencing reaction is carried out after obtaining purity PCR primer higher through PCR amplifications, obtain
One section of short sequence of high specificity, this section of sequence can be as the target sequence of PRV identifications, directly from this section of gene order just
Can be determined that PRV.The method can be diagnosed as the routine screening of porcine pseudorabies, improve detection efficiency, it is to avoid false positive knot
The appearance of fruit, intuitively identifies PRV from gene order, and the infected pigs to PRV carry out examination identification faster.
Brief description of the drawings
Fig. 1 is the PCR amplifications of PRV gD genes.
Fig. 2 is PRV gD GFP pyrosequencing results.
Fig. 3 is the PCR/RT-PCR specific amplification results of PRV gD genes.
Fig. 4 is the sensitivity results of pyrosequencing.
Fig. 5 is pyrosequencing replica test result.
Fig. 6 is common detection methods result of the test.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Porcine pseudorabies virus (PRV), pig parvoviral (PPV), Porcine epidemic diarrhea virus in following embodiments
(PEDV), the Virus Sample such as porcine reproductive and respiratory syndrome virus (PRRSV) and sample of nucleic acid are by academy of agricultural sciences of Shandong Province herding beast
Doctor research institute, Shandong Animal Disease Prevention and Control Center and Shandong Linyi Jin Luoxincheng animal husbandry Co., Ltd provide.
PrimeScriptTM One Step RT-PCR Kit、TaKaRa ExHot Start Version、100bp DNA
Ladder Marker and corresponding reagent etc. are purchased from Dalian TaKaRa companies；Pyrosequencing instrument PyroMarkTM ID System
Biotage companies of Sweden are purchased from Vacuum Prep Tool；(strepto- is affine for Streptavidin Sepharose beads
Element coating magnetic bead, product article No. 65305) purchased from Invitrogen companies, PyroGold SQA Reagents 1 × 96DNA sequences
Row assay kit (including Enzyme mixture, Substrate mixture, dATP α S, dGTP, dCTP, dTTP),
Annealing Buffer (annealing buffer, product article No. 979009), Binding buffer (combination buffer, product goods
Number 979006), Denatureation buffer (denaturation buffer, product article No. 979007), Washing buffer (washings
Buffer solution, product article No. 979008) etc. be QIAGEN Products.Paramagnetic particle method RNA extracts kits and instrument for extracting nucleic acid by
Xi'an Tianlong Science ＆ Technology Co., Ltd. provides.Primer synthesizes and is sequenced by the completion of Shanghai Sheng Gong bioengineering Co., Ltd.
Embodiment 1, the design and the foundation of method of the pyrosequencing detection primer special of identification PRV
First, the pyrosequencing of identification PRV detects the design of primer special
The Partial Fragment (sequence 4) of the conserved sequence according to the PRV gD GFPs announced in GenBank, designs a pair
(F is located at 58-75 of sequence 4 for universal amplification primers F and R；R is located at 128-145 of sequence 4) and sequencing primer S (sequences
66-80 of row 4) (as shown in table 1), wherein primer R its 5 ' end carry out biotin labeling, it is contemplated that amplified fragments (sequence 4
81-135) size be 88bp.Primer synthesizes by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 1 is the amplimer and sequencing primer of PRV
2nd, the foundation of the pyrosequencing detection method of identification PRV
1st, the extraction of viral nucleic acid DNA
The PRV viruses that will be collected be (Chen Chao's, Liu Cun, Li Haie etc.《The immune pig farm porcine pseudorabies virus in Shandong Province are separated
Identification and the sequence analysis of gE virulence genes》, Chinese animal doctor's journal, 2016,36 (6)：902-907) sample utilizes magnetic bead nucleic acid
Extraction method extracts nucleic acid, and the DNA of extraction is immediately available for PCR amplifications or puts -80 DEG C saving backup.
2nd, PCR reactions
With viral DNA as template, with the sense primer F and anti-sense primer R of above-mentioned one design, enter performing PCR amplification and to anti-
Answer condition to optimize, obtain the PCR primer of 88bp sizes.
Above-mentioned PCR reaction systems (50 μ L) are：TaKaRa Ex Taq HS (5units/ μ L) 0.25 μ L, 10 × Ex Taq
Buffer 5 μ L, dNTP Mixture (2.5mM each) 4 μ L, primers F (20 μM, final concentration of 0.4 μM in reaction system) 1
μ L, primer R (20 μM, final concentration of 0.4 μM in reaction system) 1 μ L, the 34.75 μ L of μ L, ddH2O of template DNA 4.
The reaction condition of above-mentioned PCR amplification is：98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 1min, 45 circulations；72 DEG C of extensions
5min。
Take 5 μ L PCR primers and enter row agarose gel electrophoresis detection amplification, it is biological that PCR primer is sent into Shanghai life work
Engineering Co., Ltd is sequenced.
Result is as shown in figure 1, M is 100bp DNA Ladder Marker；1 is pcr amplified fragment；As can be seen that amplification
Clip size is 88bp, consistent with expected purpose clip size, and the sequencing result analysis shows purpose band and PRV gD eggs
White genetic fragment homology is up to 100%.
3rd, the preparation of single-stranded template is sequenced
Pyrosequencing operation explanation according to Gene Company Limited prepares sequencing single-stranded template,
Combination buffer (the Binding that magnetic bead is coated with containing 200 μ g Streptavidins is added in the PCR primer that 50 μ L above-mentioned 1 are obtained
Buffer), normal temperature concussion mixes 10min, vacuum prep tool (vavuum pump) are cleaned in the ultra-pure water after 30s crawl with
The magnetic bead that PCR primer is combined, then cleans 5s with 70% ethanol；Denatureation buffer (denaturation buffer) wash 5s；
Finally to cleaning 10s in Washing buffer (lavation buffer solution), vacuum prep tool (vavuum pump) are put into pre-
First containing in 0.3 μM of orifice plate of the PSQ of the annealing buffer of sequencing primer 96, shake discharges magnetic bead.This orifice plate of PSQ 96 is put
In 80 DEG C of 2min on thermal cycler, taking-up naturally cools to room temperature, obtains that single-stranded template is sequenced.
4th, pyrosequencing reaction
Come setting program and the given dosage of program according to the explanation of pyrosequencing instrument, in corresponding hole in reagent cabin
Enzymatic mixture (archaeal dna polymerase, the ATP for adding PyroGold SQA 1 × 96DNA of Reagents Sequencing Kits to provide
Sulfurylase, luciferase and apyrase), substrate A PS and four kinds of dNTP (dATP α S, dCTP, dGTP,
DTTP), 96 orifice plates for being placed with sequencing single-stranded template that reagent cabin and above-mentioned 3 obtain are put into pyrosequencing instrument carries out burnt phosphorus
Sour sequencing reaction, obtains the sequencing result (sequence 4 81-135) of PCR primer：ACGGCGTGAACGTCCTCACC
GACTTCATGGTGGCGCTCCCCGAGGGGCAAGAGTG。
Through online BLAST sequence homology analysis, the sequence results confirm to belong to PRV gD GFPs, are sequenced with expected
As a result (sequence 4 81-135) is consistent, and coincidence rate is up to 100%.
If the similitude of the DNA molecular sequence shown in sample to be tested PCR primer sequencing result and sequence 4 81-135 is big
In equal to 80%, showing that both belong to homologous gene, illustrate to contain in sample to be tested or candidate contains porcine pseudorabies virus, if
Sample to be tested PCR sequencing results are less than 80% with the similitude of the DNA molecular sequence shown in sequence 4 81-135, then show
Both are not belonging to homologous gene, illustrate not containing in sample to be tested or candidate does not contain porcine pseudorabies virus.
The sequence can be used as the target sequence of PRV Rapid identifications, and its pyrosequencing result is sequenced with common Sanger and ties
Really completely the same, coincidence rate is 100%, and test method has preferable accuracy and specificity (as shown in Figure 2).
The specific test of embodiment 2, pyrosequencing
The nucleic acid samples with PRV, PEDV, TGEV, PRRSV carry out Jiao as template according to the two of embodiment 1 method respectively
Phosphoric acid sequencing reaction, obtains pyrosequencing product.
Pyrosequencing product electrophoresis result is as shown in figure 3, M:100bp DNA Ladder Marker；1:PRRSV；2:
PEDV；3:PPV；4:PRV, it can be seen that only PRV obtains the result of aim sequence.
Pyrosequencing product sends to sequencing, as a result the pyrosequencing product sequencing result and aim sequence of only PRV
Fragment (sequence 4 81-135) coincidence rate is 100%；Other viral nucleic acid samples do not obtain any sequence results, specifically
Property show embodiment 1 primer set have preferably specificity.
The sensitivity test of embodiment 3, pyrosequencing
PRV sample of nucleic acid (200ng/ μ L) is carried out into 10 times of gradient dilutions respectively, as template, according to embodiment 1
Two method carries out pyrosequencing reaction, obtains pyrosequencing product.
Pyrosequencing product electrophoresis, as a result as shown in figure 4, M:100bp DNA Ladder Marker；1-7:It is respectively
Template dilution factor is 10-1、10-2、10-3、10-4、10-5、10-6、10-7；The minimum detection of nucleic acids of PCR is limited to 0.2pg/ μ L (dilutions
Spend is 10-6When), the PCR primer for now being expanded can be used for pyrosequencing, obtain preferable sequence results.
Advanced performing PCR amplification is needed due to carrying out pyrosequencing, therefore the sensitivity of pyrosequencing that is to say that PCR is expanded
Sensitivity, judge the sensitivity of pyrosequencing according to this.
Embodiment 3, replica test
The PCR primer (amplified production before pyrophosphoric acid reaction) that the two of embodiment 12 amplifications are obtained is carried out 3 times respectively
Pyrosequencing, comparative sequences result determines the repeatability and stability of the method.
Machine testing, testing result in 3 repeatability are carried out to PCR primer as shown in figure 5, a, b, c are respectively 3 repeatability
Upper machine testing is results, it can be seen that 3 sequencing results are consistent, it was demonstrated that the method has repeatability and stability well.
The comparison application of embodiment 4, pyrosequencing detection method in clinical sample
1st, pyrosequencing detection method of the invention
20 samples (pathological material of disease, body fluid, tissue fluid, blood equal samples) extraction DNA of PEDV doubtful to clinical acquisitions, and with
This carries out pyrosequencing reaction for template according to the one of embodiment 2 method, obtains pyrosequencing product.
Pyrosequencing product is sent into Shanghai Sheng Gong bioengineering Co., Ltd carries out sequencing, with target sequence
Row sequence 4 is compared, and 16 homologys are 100% in 20 samples, and remaining 4 sample homology is not 100%, therefore, make a definite diagnosis
16 PRV positives, 4 PRV feminine genders.
2nd, common detection methods
20 samples (pathological material of disease, body fluid, tissue fluid, blood equal samples) extraction DNA of PEDV doubtful to clinical acquisitions, and with
This is template, and (sequence 4 is covered with the primer pair in the PCR mentioned in GB GB/T 18641-2002
Whole DNA sequence dna) amplification, obtain pcr amplification product.
Primer pair F1：CAGGAGGACGAGCTGGGGCT；
R1：GTCCACGCCCCGCTTGAAGCT.
Pcr amplification product is through agarose gel electrophoresis as shown in fig. 6, M:DNA Marker 2,000, band is from top to bottom
It is followed successively by 2000,1000,750,500,250,100bp；- and+it is respectively negative and positive control；1-20 is respectively sample PCR
Amplified production electrophoretogram, it can be seen that 6,14,20 is feminine gender, and remaining is positive findings, has 17 positive bands, 3 feminine genders
Band, then positive products are sent to professional sequencing company carry out sequencing identification, prove that 19 is false positive after sequence verification, it is cloudy
Property result (there may be sample contamination or non-specific amplification occur and cause), do not measure aim sequence, it is final to determine there is 16
The individual positive, 4 feminine genders.
Compared by two methods, both results are finally consistent, but the knot that pyrosequencing method of the invention is obtained
Fruit quick and precisely, saves detection time, as a result very clear, and sequence results are intuitively seen from the result after upper machine, without
Further checking can be judged, it is to avoid the appearance of false positive results.
Sequence table
<110>Jinan Entry-Exit Inspection and Quarantine Bureau, People's Republic of China
<120>Porcine pseudorabies pyrosequencing detection method and primer special
<160> 4
<170> PatentIn version 3.5
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<211> 18
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gtaccggcgc ctggtgtc 18
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<212> DNA
<213>Artificial sequence
<220>
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<400> 2
ggcgaacggg cactcttg 18
<210> 3
<211> 15
<212> DNA
<213>Artificial sequence
<220>
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<400> 3
gcctggtgtc cgtcg 15
<210> 4
<211> 217
<212> DNA
<213>Artificial sequence
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cacggaggac gagctggggc tgctcatggt ggccccgggg cggttcaacg agggccagta 60
ccggcgcctg gtgtccgtcg acggcgtgaa cgtcctcacc gacttcatgg tggcgctccc 120
cgaggggcaa gagtgcccgt tcgcccgcgt ggaccaacac cgcacgtaca agttcggcgc 180
gtgctggagc gacgacagct tcaagcgggg cgtggac 217
Claims (10)
1. a kind of primer set for detecting PRV, including following primer pair：
Single stranded DNA point in single strand dna and sequence table of the primer pair as shown in sequence in sequence table 1 shown in sequence 2
Son composition.
2. primer set according to claim 1, it is characterised in that：A prime end biotin in the primer pair
Mark.
3. primer set according to claim 1 and 2, it is characterised in that：The primer set also includes nucleotide sequence
It is the sequencing primer of sequence 3 in sequence table.
4. complete PCR reagent containing any primer sets of claim 1-3 or containing the primer set or it is described into
Cover the kit of PCR reagent；
Or the DNA molecular shown in sequence 4 81-135.
5. complete PCR reagent according to claim 4, it is characterised in that：
The complete PCR reagent is made up of PCR reagent 1 and PCR reagent 2；
The PCR reagent 1 is the reagent containing primer pair described in any primer sets of claim 1-3；
Or, concentration of the every primer in the PCR reagent 1 is 20 μM in the primer pair；
The PCR reagent 2 is the reagent containing sequencing primer described in any primer sets of claim 1-3；
Or, concentration of the sequencing primer in the PCR reagent 2 is 0.3 μM.
6. any primer sets of claim 1-3 or complete PCR reagent or kit or sequence 4 described in claim 4
The application of DNA molecular shown in 81-135 in detecting or aiding in detection PRV；
Or, any primer sets of claim 1-3 or complete PCR reagent or kit or sequence 4 described in claim 4
DNA molecular shown in 81-135 is preparing the application in detecting or aiding in detection PRV product；
Or, any primer sets of claim 1-3 or complete PCR reagent or kit or sequence 4 described in claim 4
DNA molecular answering in detecting or aiding in detection sample to be tested whether containing PRV shown in 81-135
With；
Or, any primer sets of claim 1-3 or complete PCR reagent or kit or sequence 4 described in claim 4
Whether the DNA molecular shown in 81-135 contains PRV product in detection or auxiliary detection sample to be tested is prepared
In application.
7. it is a kind of detection or auxiliary detection sample to be tested in whether the method containing PRV, comprise the following steps：
1) with the single strand dna composition in the single strand dna shown in sequence 1 in sequence table and sequence table shown in sequence 2
Primer pair enters performing PCR amplification to sample to be tested, obtains pcr amplification product；
2) sequencing primer that sequence 3 in sequence table is classified as with nucleotides sequence carries out pyrosequencing to the pcr amplification product, obtains
To pcr amplification product sequencing result；
If the homology of the DNA molecular shown in the pcr amplification product sequencing result and sequence 4 81-135 is more than or equal to
80%, then in the sample to be tested contain or candidate contain porcine pseudorabies virus, if the pcr amplification product sequencing result with
The homology of the DNA molecular shown in sequence 4 81-135 is less than 80%, then do not contained in the sample to be tested or candidate does not contain
There are porcine pseudorabies virus.
8. it is a kind of detection or auxiliary detection sample to be tested in whether the method containing PRV, comprise the following steps：
Detect and whether be more than containing the DNA molecular shown in sequence 4 81-135 or with its homology in the nucleic acid of sample to be tested
DNA molecular equal to 80%, if it does, then the sample to be tested contains or candidate contains PRV, if not containing,
The sample to be tested is not contained or candidate does not contain PRV.
9. method according to claim 8, it is characterised in that：Whether contain sequence in the nucleic acid of the detection sample to be tested
The method of 4 DNA moleculars shown in 81-135 or the DNA molecular with its homology more than or equal to 80% comprises the following steps：
The step of claim 7 1) and step 2), pcr amplification product sequencing result is obtained, analyze the pcr amplification product sequencing result
Whether the DNA molecular shown in sequence 4 81-135 is contained.
10. according to any described method in claim 7-9, it is characterised in that：
The annealing temperature of the PCR amplifications is 55 DEG C, and annealing time is 30S；
Or, the template of the PCR amplifications is the nucleic acid of the sample to be tested.
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| 孙涛等: "焦磷酸测序技术在确证猪甲型H1N1流感病毒中的应用", 《中国动物检疫》 * |
| 蒋增海: "猪繁殖与呼吸综合征病毒和猪伪狂犬病病毒的分离、鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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