CN109554508A - For identifying the detection primer and probe of anomaly PEDV and TGEV - Google Patents
For identifying the detection primer and probe of anomaly PEDV and TGEV Download PDFInfo
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Abstract
The invention belongs to epizootiology detection field, the present invention provides one group for identifying the real-time fluorescence quantitative PCR detection primer and probe of anomaly PEDV and TGEV.According to variant porcine epidemic diarrhea virus N gene, a pair of of detection primer and probe are designed, the primer sequence is as shown in SEQ ID NO.1-3；According to pig infectious gastroenteritis virus S gene, a pair of of detection primer and probe are designed, the primer sequence is as shown in SEQ ID NO.4-6.A kind of real time fluorescence quantifying PCR method that can quickly identify anomaly PEDV and TGEV is established according to the primer and probe.With other common pig source pathogen nonspecific react does not occur for this method.Method of the invention can rapid differential diagnosis, time saving and energy saving cost-saving in a short time, while there is high specificity, the characteristics of stability is good, high sensitivity, particularly suitable for the antidiastole in clinically mixed infection sample.
Description
Technical field
The present invention provides one group for identifying the real-time fluorescence quantitative PCR detection primer and spy of anomaly PEDV and TGEV
Needle belongs to epizootiology detection field.
Background technique
Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and pig transmissible stomach and intestine
Scorching virus (Transmissible gastroenteritis virus, TGEV) is the main pathogen for causing pig virus diarrhoea.
Infected piglet main clinic symptoms show as diarrhea, vomiting and dehydration, and each age level pig can infect.Since the two is drawn
Symptom, pathological change and the epidemiology risen is very much like, and often mixed infection, only relies on clinical symptoms and histopathology
Be difficult to antidiastole.In recent years its because its infectiousness it is strong, the death rate is high, causes immeasurable economic damage to pig-breeding industry
It loses, the serious development for perplexing pig breeding industry.
PEDV is linear single strand plus RNA virus, belongs to the more virales of Buddhist nun, coronaviridae, coronavirus genus.Virion
Son is in pleomorphism, and size variation is different, a diameter of 95-190nm, there is that petal-shaped fibre is prominent on cyst membrane, fibre dash forward it is short and small and intensive, it is fine
Dash forward about long 18-23nm, radially distributes around from core, queueing discipline is like imperial crown.The disease is in 1971 first in Britain
Occur, Belgian scientist is separated to virus from pathological material of disease within 1978, is named as CV777.China's change new in discovery in 2010
Special-shaped PEDV strain, has broken out serious epidemic situation.TGEV is linear single strand plus RNA virus, belongs to coronaviridae, coronal disease
Poison belongs to.Virion diameter is 90-200nm, and oval, round or polygon has cyst membrane, and surface has rodlike fine prominent.The disease
Betided Illinois, America first in 1933, nineteen forty-six determines the pathogen of the disease and done detailed report.China
This disease occurred in 1958.Since this 2 kinds of pathogen clinically often show as mixed infection, at the same anomaly PEDV strain with
The genetic distance of classical CV777 strain farther out, thus to the identification of epidemic disease, diagnosis brings very big difficulty, therefore is badly in need of establishing
The differential diagnostic method of specificity.
Currently, PEDV and the conventional method of two kinds of TGEV viral antidiastoles mainly have virus purification, immunohistochemistry
The conventional diagnostic techniques with Electronic Speculum etc., and conventional method is since cumbersome, time-consuming and higher cost, application range receives greatly greatly
To limitation.With the development of molecular biology, PCR detection method, application is more and more extensive, and TaqMan fluorescence probe method is glimmering in real time
Fluorescent Quantitative PCR (Quantitative Real-time PCR) is that a specific fluorescence is added while primer is added to visit
Needle, when probe is complete, the fluorescence signal of reporter group transmitting is quenched group absorptions, to fluorescence signal occur, passes through collection
The accumulation of fluorescence signal forms fully synchronized with PCR product.Therefore Real-Time Fluorescent Quantitative PCR Technique can be determined when detecting cause of disease
Property detection cause of disease, and can also be realized by the collection of fluorescence signal quantitative analysis viral level number.Multi-fluorescence is fixed
Amount PCR is a kind of special quantitative fluorescent PCR form, and the feature protruded is that a real-time fluorescence quantitative PCR reaction can be simultaneously
A variety of cause of diseases are detected, is able to achieve to complicated cause of disease or there are the cause of diseases of Multi-genotype to carry out identification detection.
Clinically, pig virus diarrhoea epidemic disease is often in mixed infection state, main cause of disease have anomaly PEDV and
The clinical symptoms of TGEV, the two infection are similar, are difficult from antidiastole in clinical symptoms and pathological change.Therefore, foundation can
Simultaneously to the identification detection method of anomaly PEDV and TGEV, both simplified operation program, save the cost, can clinically be carried out big
The detection of sample, epidemiological survey and the relevant pathogenesis of development to pig virus diarrhoea virus have important
Meaning, especially identifying in the pathogen in the sample of mixed infection has unique advantage and use value in detection.
Summary of the invention
The present invention provides one group for identifying the real-time fluorescence quantitative PCR detection primer and spy of anomaly PEDV and TGEV
Needle.Identification detection method that can simultaneously to anomaly PEDV and TGEV is established, both simplified operation program, save the cost, faced
The detection that large sample can be carried out on bed, epidemiological survey and the relevant pathogenesis of development to pig virus diarrhoea virus are ground
Study carefully and have great importance, especially identifying in detection in the pathogen in the sample of mixed infection has unique advantage and make
With value.
The present invention is achieved through the following technical solutions:
For detecting the N gene of anomaly PEDV:
Upstream primer PEDV-F (5'- 3'): CTCCCGAGTGTAGTTGAGATTG
Downstream primer PEDV-R (5'- 3'): CTCCACGACCCTGGTTATTT
Fluorescence probe PEDV-P (5'-3'): FAM-CAACCCAACACACCTCCTACTTCACG-BHQ1；
For detecting the S gene of TGEV:
Upstream primer TGEV-F (5'- 3'): TGAGGGTGCTGGCTTTGAT
Downstream primer TGEV-R (5'- 3'): CAAGAGTGACACCACCCGTT
Fluorescence probe TGEV-P (5'- 3'): VIC-CACTGTGGCACCCTTACCTGATTGT-BHQ1.
Wherein, the 5 ' ends of the fluorescence probe PEDV-P of anomaly PEDV are marked with fluorescent reporter group FAM, the fluorescence of TGEV
5 ' the ends of probe TGEV-P are marked with fluorescent reporter group VIC；3 ' the ends of the fluorescence probe PEDV-P of anomaly PEDV and TGEV
Fluorescence probe TGEV-P 3 ' end mark quenching group BHQ1.
For identifying the real time fluorescence quantifying PCR method of anomaly PEDV and TGEV, including the primer and probe.
Dual Taqman real-time fluorescence quantitative PCR reaction system are as follows:
Dual TaqMan real time fluorescence quantifying PCR method reaction condition are as follows: 94 DEG C of 3 min；94 DEG C of 5s, 57 DEG C of 15s, 72 DEG C
30s, 40 circulations.
After real-time fluorescence quantitative PCR, test result is analyzed, selects the channel FAM to see there is positive amplification letter at 520nm
Number, show that there are anomaly PEDV infection in test sample；It selects the channel VIC to see there is positive amplification signal at 550nm, shows
There are TGEV infection in test sample；If the selection channel VIC at the channel FAM and 550 is selected to see there is positive amplification type at 520nm
Number, show to exist simultaneously anomaly PEDV and TGEV infection in test sample.
The present invention provides one group for identifying the real-time fluorescence quantitative PCR detection primer and spy of anomaly PEDV and TGEV
Needle, the real time fluorescence quantifying PCR method of foundation have the following advantages that and effect:
1, detect simultaneously: the method for foundation can detect anomaly PEDV and TGEV simultaneously, and operation sequence is simple, save the cost.
After real-time fluorescence quantitative PCR, test result is analyzed, selects the channel FAM to see there is positive amplification signal at 520nm, shows
There are anomaly PEDV infection in test sample；It selects the channel VIC to see there is positive amplification signal at 550nm, shows to detect sample
There are TGEV infection in product；If the selection channel VIC at the channel FAM and 550 is selected to see there is positive amplification model, table at 520nm
Anomaly PEDV and TGEV infection are existed simultaneously in bright test sample.
2, detection is quick, efficient: the detection method can pass through after reaction without carrying out traditional detected through gel electrophoresis
Real-time fluorescence quantitative PCR instrumental transmission is judged to the digital signal of computer.It is only needed from nucleic acid extraction to result judgement
100min, and 96 sample detections can be once carried out simultaneously.
3, quantitative accurate: by preparing standard items, drawing standard curve, according to anomaly PEDV in detection measuring samples and
The Ct value of TGEV directly infects it anomaly PEDV and TGEV and carries out accurate quantitative analysis.
4, high sensitivity: detectable 10 copies minimum to anomaly PEDV；Detectable 10 copies minimum to TGEV.
5, high specificity: justify with other common pig source pathogen, such as swine fever virus, reproductive and respiratory syndrome virus, Pseudorabies virus, pig
Nonspecific reaction does not occur for 2 type of circovirus virus, encephalitis B virus, parvovirus.Only anomaly PEDV and TGEV are detected and occurred
Specific positive result.
Anomaly PEDV and TGEV dual real-time fluorescence quantitative PCR detection are carried out to 65 parts of clinical inspection diarrhea pathological material of diseases, used
Nucleic acid extraction kit is commercialized and extracts corresponding nucleic acid, according to the dual real-time fluorescence quantitative PCR system and reaction item of foundation
Part is detected.As a result, it has been found that selecting the channel FAM to see there are 30 parts of positive amplification signals at 520nm, show 30 parts of test samples
In there are anomaly PEDV infection, positive rates 46.15%；The channel VIC is selected to see there are 5 parts of positive amplification signals at 550nm,
Show that there are TGEV infection, positive rates 7.69% in 5 parts of test samples；Wherein there are also 2 parts of samples to select FAM logical at 520nm
It selects the channel VIC to have amplified signal at road and 550nm, shows that there are anomaly PEDV and TGEV mixing to feel in 2 parts of samples
Dye, positive rate 3.08%.
Detailed description of the invention
The amplification kinetic curve of Fig. 1 anomaly PEDV real time fluorescence quantifying PCR method.
The standard curve of Fig. 2 anomaly PEDV real time fluorescence quantifying PCR method.
The specific test of Fig. 3 anomaly PEDV real time fluorescence quantifying PCR method, wherein 1 is PEDV, 2 be TGEV, 3-8
It is ddH for CSFV, PRV, PRRSV, PPV, JEV, PCV-2,92O。
The amplification kinetic curve of Fig. 4 TGEV real time fluorescence quantifying PCR method.
The standard curve of Fig. 5 TGEV real time fluorescence quantifying PCR method.
The specific test of Fig. 6 TGEV real time fluorescence quantifying PCR method, wherein 1 is PEDV, 2 be TGEV, and 3-8 is
CSFV, PRV, PRRSV, PPV, JEV, PCV-2,9 be ddH2O。
The specific test of Fig. 7 dual real-time fluorescence quantifying PCR method, wherein 1 is PEDV, 2 be TGEV, and 3-8 is
CSFV, PRV, PRRSV, PPV, JEV, PCV-2,9 be ddH2O。
Specific embodiment
Be described further below the present invention, the case study on implementation introduced in this description be only it is exemplary, not to the present invention
Range be construed as limiting.Those skilled in the art should be understood that without departing from the principle of the invention and method, right
The details and form of technical solution of the present invention carry out part modifications or substitutions, but are belonged to based on this modifications or substitutions of the invention
In protection scope.
Embodiment 1
One, experimental method and step
1, correlation test cause of disease
Test swine fever virus, reproductive and respiratory syndrome virus, Pseudorabies virus, porcine circovirus 2 type, encephalitis B virus, parvovirus, variation
The positive nucleic acid of type PEDV and TGEV are identified and are saved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.
2, primer and probe designs
According to PEDV(AJ1102 plants of the anomaly retrieved in GenBank) N gene and TGEV(WH-1) S gene order,
The primer and probe for anomaly PEDV and TGEV of specificity is designed, sequence is as follows:
For detecting the N gene of anomaly PEDV:
Upstream primer PEDV-F (5'- 3'): CTCCCGAGTGTAGTTGAGATTG
Downstream primer PEDV-R (5'- 3'): CTCCACGACCCTGGTTATTT
Fluorescence probe PEDV-P (5'- 3'): FAM-CAACCCAACACACCTCCTACTTCACG-BHQ1
For detecting the S gene of TGEV:
Upstream primer TGEV-F (5'- 3'): TGAGGGTGCTGGCTTTGAT
Downstream primer TGEV-R (5'- 3'): CAAGAGTGACACCACCCGTT
Fluorescence probe TGEV-P (5'- 3'): VIC-CACTGTGGCACCCTTACCTGATTGT-BHQ1
Wherein, the 5 ' ends of the fluorescence probe PEDV-P of anomaly PEDV are marked with fluorescent reporter group FAM, the fluorescence probe of TGEV
5 ' the ends of TGEV-P are marked with fluorescent reporter group VIC；3 ' the ends of the fluorescence probe PEDV-P of anomaly PEDV are glimmering with TGEV's
3 ' the ends of light probe TGEV-P mark quenching group BHQ1.
Above-mentioned primer is synthesized in Sangon Biotech (Shanghai) Co., Ltd..
1, the foundation of anomaly PEDV and TGEV double fluorescent quantitative PCR detection method
The building of 3.1 anomaly PEDV positive criteria products
According to PEDV(AJ1102 plants of anomaly) N gene order conservative feature, utilize primer-design software Oligo design special
Specific primer, primer sequence are as follows: PEDV-F (5'-3'): CTCCCGAGTGTAGTTGAGATTG and PEDV-R (5'-3'):
CTCCACGACCCTGGTTATTT, for expanding the N genetic fragment of about 175bp, primer is by giving birth to work bioengineering (Shanghai) share
Co., Ltd's synthesis.
PEDV nucleic acid RNA is extracted with commercialization Viral nucleic acid extraction reagent box, using RNA as template, according to EasyScript
One-Step RT-PCR SuperMix specification carries out reverse transcription and obtains cDNA product, reaction system are as follows: EasyScript
One-Step RT-PCR SuperMix 10 μ L, ddH26 μ L, RNA template of O, 4 μ L adds up to 20 μ L.Reaction condition are as follows: 25 DEG C
5min, 45 DEG C of 30min；According to 2 × TransTaq-T PCR SuperMix(+dye) specification progress PCR reaction, reaction system
It is prepared with reference to kit specification, reaction system is 50 μ L, wherein 2 × TransTaq-T PCR SuperMix reaction solution, 25 μ
L, each 1 μ L of upstream and downstream primer (PEDV-F, PEDV-R), 1 μ L of cDNA template supplement 22 μ L of deionized water to 50 μ L of final volume.Instead
Answer condition are as follows: 94 DEG C of 5 min；94 DEG C of 50s, 54 DEG C of 30s, 72 DEG C of 30s, 35 circulations；72 DEG C of extension 10min after circulation terminates.
PCR product is identified with 1.0% agarose gel electrophoresis, using Ago-Gel QIAquick Gel Extraction Kit to specific purpose
Segment carries out gel extraction.Connection kit specification is cloned by N gene according to pEASY-T1 Simple Cloning Kit
Segment is cloned on pEASY-T1 carrier, and 8 single bacteriums of random picking fall within ampicillin (content is 100 μ g/mL) resistance
After 15 hours of LB liquid medium culture, corresponding plasmid is extracted using the small extracts kit of rapid plasmid.Using PCR amplification
When primer and condition PCR identification is carried out to the plasmid of extraction, by the positive recombinant plasmid filtered out send raw work bioengineering (on
Sea) limited liability company carries out sequencing identification.Sequencing result is carried out BLAT analysis in NCBI row to verify, expected from Pass Test
Positive criteria product (T-PEDV) of the positive recombinant plasmid as real-time fluorescence quantitative PCR, is placed in -20 DEG C after packing and saves backup.
The building of 3.2 TGEV positive criteria products
According to TGEV(WH-1 plants of anomaly) S gene order conservative feature, designed using primer-design software Oligo special
Property primer, primer sequence be TGEV-F (5'- 3'): TGAGGGTGCTGGCTTTGAT and TGEV-R (5'- 3'):
CAAGAGTGACACCACCCGTT, for expanding the N genetic fragment of about 149bp, primer is by giving birth to work bioengineering (Shanghai) share
Co., Ltd's synthesis.
TGEV nucleic acid RNA is extracted with commercialization Viral nucleic acid extraction reagent box, using RNA as template, according to EasyScript
One-Step RT-PCR SuperMix specification carries out reverse transcription and obtains cDNA product, reaction system are as follows: EasyScript
10 6 μ L, RNA template of μ L, ddH2O of One-Step RT-PCR SuperMix, 4 μ L adds up to 20 μ L.Reaction condition are as follows: 25 DEG C
5min, 45 DEG C of 30min；According to 2 × TransTaq-T PCR SuperMix(+dye) specification progress PCR reaction, reaction system
It is prepared with reference to kit specification, reaction system is 50 μ L, wherein 2 × TransTaq-T PCR SuperMix reaction solution, 25 μ
L, each 1 μ L of upstream and downstream primer (TGEV-F, TGEV-R), 1 μ L of cDNA template supplement 22 μ L of deionized water to 50 μ L of final volume.Instead
Answer condition are as follows: 94 DEG C of 5 min；94 DEG C of 50s, 54 DEG C of 30s, 72 DEG C of 30s, 35 circulations；72 DEG C of extension 10min after circulation terminates.
PCR product is identified with 1.0% agarose gel electrophoresis, using Ago-Gel QIAquick Gel Extraction Kit to specific purpose
Segment carries out gel extraction.Connection kit specification is cloned by N gene according to pEASY-T1 Simple Cloning Kit
Segment is cloned on pEASY-T1 carrier, and 8 single bacteriums of random picking fall within ampicillin (content is 100 μ g/mL) resistance
After 15 hours of LB liquid medium culture, corresponding plasmid is extracted using the small extracts kit of rapid plasmid.Using PCR amplification
When primer and condition PCR identification is carried out to the plasmid of extraction, by the positive recombinant plasmid filtered out send raw work bioengineering (on
Sea) limited liability company carries out sequencing identification.Sequencing result is carried out BLAT analysis in NCBI row to verify, expected from Pass Test
Positive criteria product (T-TGEV) of the positive recombinant plasmid as real-time fluorescence quantitative PCR, is placed in -20 DEG C after packing and saves backup.
The optimization of 3.3 reaction conditions and the foundation of standard curve
The real-time glimmering of 20 μ L is configured according to TaqMan Fast qPCR Master Mix (High Rox) kit operation instructions
Fluorescent Quantitative PCR reaction system, in different annealing temperature (55,57,59,61 and 63 DEG C), primer concentration (0.1-0.5 μM) and probe
Real-time fluorescence quantitative PCR reaction is carried out under the conditions of concentration (1.5-20 μM), and reaction condition is optimized.With anomaly PEDV sun
Property standard items (T-PEDV) and TGEV positive criteria product (T-TGEV) be template, 520nm at selection the channel FAM, at 550nm
Select the channel VIC.It is expanded using the reaction condition after optimization, obtains amplification kinetic curve.With standard items starting copies
Several common logarithms (lgC) be abscissa, with cycle threshold (cycle threhold, Ct value) be ordinate, derive standard
Equation of linear regression (standard curve).
As a result: dual TaqMan real time fluorescence quantifying PCR method optimal reaction system (the 20 μ L) such as following table of final optimization pass
1。
Dual TaqMan real time fluorescence quantifying PCR method reaction system after the optimization of table 1
As a result: the dual TaqMan real time fluorescence quantifying PCR method reaction condition of optimization are as follows: 94 DEG C of 3 min；94 DEG C of 5s,
57 DEG C of 15s, 72 DEG C of 30s, 40 circulations.
The judgement of experimental result:
Real-time fluorescence quantitative PCR after reaction, analyzes test result, selects the channel FAM to see there is positive amplification letter at 520nm
Number, show that there are anomaly PEDV infection in test sample；It selects the channel VIC to see there is positive amplification signal at 550nm, shows
There are TGEV infection in test sample；If the selection channel VIC at the channel FAM and 550 is selected to see there is positive amplification type at 520nm
Number, show to exist simultaneously anomaly PEDV and TGEV infection in test sample.
3.4 anomaly PEDV sensitivity tests
Using the concentration of trace dna analyzer measurement positive criteria product (T-PEDV), measuring plasmid concentration is 71.89 ng/ μ L,
Calculating its copy number is 2.32 × 1010Copy/μ L, is diluted to 1.0 × 1010Copy/μ L carries out it as standard items
10 times of serial dilutions, the plasmid content for choosing serial dilution is respectively 1.0 × 106Copy/μ L, 1.0 × 105Copy/μ L, 1.0 ×
104Copy/μ L, 1.0 × 103Copy/μ L, 1.0 × 102Copy/μ L, 1.0 × 101Copy/μ L and 1.0 × 100Copy/μ L, point
Dress is placed on -20 DEG C and saves backup.Respectively using above-mentioned different dilution positive criteria products (T-PEDV) as template, after optimization
Reaction condition expanded, obtain amplification kinetic curve (see figure 1).With the common logarithm of standard items starting copy number
(lgC) it is abscissa, with recurring number threshold value (cycle threhold, Ct value) for ordinate, derives standard linear regression side
Journey (standard curve) (see figure 2), obtains its sensitivity tests data.
From table 2 it can be seen that the real time fluorescence quantifying PCR method lowest detection that the present invention establishes is limited to 10 copies/μ L.With
The common logarithm (lgC) of plasmid content (C) is abscissa in each standard items, with recurring number threshold value (cycle threhold, Ct
Value) be ordinate, obtain anomaly PEDV real-time fluorescence quantitative PCR standard curve (Fig. 2), obtained slope of standard curve for-
3.224, Y intercept 39.631, related coefficient 0.999, amplification efficiency 104.242%, meet experiment be expected.
2 real-time fluorescence quantitative PCR sensitivity technique of table
3.5 TGEV sensitivity tests
Using the concentration of trace dna analyzer measurement positive criteria product (T-TGEV), measuring plasmid concentration is 83.23 ng/ μ L,
Calculating its copy number is 2.71 × 1010Copy/μ L, is diluted to 1.0 × 1010Copy/μ L carries out it as standard items
10 times of serial dilutions, the plasmid content for choosing serial dilution is respectively 1.0 × 106Copy/μ L, 1.0 × 105Copy/μ L, 1.0 ×
104Copy/μ L, 1.0 × 103Copy/μ L, 1.0 × 102Copy/μ L, 1.0 × 101Copy/μ L and 1.0 × 100Copy/μ L, point
Dress is placed on -20 DEG C and saves backup.Respectively using above-mentioned different dilution positive criteria products (T-TGEV) as template, after optimization
Reaction condition expanded, obtain amplification kinetic curve (see figure 4).With the common logarithm of standard items starting copy number
(lgC) it is abscissa, with recurring number threshold value (cycle threhold, Ct value) for ordinate, derives standard linear regression side
Journey (standard curve) (see figure 5), obtains its sensitivity tests data.
From table 3 it can be seen that the real time fluorescence quantifying PCR method lowest detection that the present invention establishes is limited to 10 copies/μ L.With
The common logarithm (lgC) of plasmid content (C) is abscissa in each standard items, with recurring number threshold value (cycle threhold, Ct
Value) it is ordinate, it obtains TGEV real-time fluorescence quantitative PCR standard curve (Fig. 5), obtained slope of standard curve is -3.116, Y
Y-intercept is 37.772, related coefficient 0.999, amplification efficiency 109.395%, meets experiment and is expected.
3 real-time fluorescence quantitative PCR sensitivity technique of table
3.6 specific test
With the dual real-time fluorescence quantitative PCR condition after optimization respectively to other common pathogen of pig source, such as swine fever virus, indigo plant
Otopathy virus, Pseudorabies virus, porcine circovirus 2 type, encephalitis B virus and parvovirus carry out real-time fluorescence quantitative PCR detection,
Using anomaly PEDV and TGEV as positive control.The nucleic acid of corresponding virus is extracted with virus genom DNA/RNA extracts kit,
Reaction condition after the optimization of TaqMan Fast qPCR Master Mix (High Rox) kit specification is detected,
Evaluate the specificity for the real time fluorescence quantifying PCR method established.
It can be seen from figure 3 that with the dual real-time fluorescence quantitative PCR condition after optimization respectively to other encountered pathogenics of pig source, such as
It is fixed that swine fever virus, reproductive and respiratory syndrome virus, Pseudorabies virus, porcine circovirus 2 type, encephalitis B virus and parvovirus carry out real-time fluorescence
PCR detection is measured, rarely seen to have anomaly PEDV specific fluorescence signal from FAM fluorescence signal channel, which also has no TGEV sun
Property fluorescence signal.
As seen from Figure 6, with the dual real-time fluorescence quantitative PCR condition after optimization respectively to other encountered pathogenics of pig source, such as
It is fixed that swine fever virus, reproductive and respiratory syndrome virus, Pseudorabies virus, porcine circovirus 2 type, encephalitis B virus and parvovirus carry out real-time fluorescence
PCR detection is measured, rarely seen to have TGEV specific positive fluorescence signal from VIC fluorescence signal channel, which also has no anomaly
PEDV Positive fluorescence signal.
From fig.7, it can be seen that with the dual real-time fluorescence quantitative PCR condition after optimization respectively to other encountered pathogenics of pig source, such as
It is fixed that swine fever virus, reproductive and respiratory syndrome virus, Pseudorabies virus, porcine circovirus 2 type, encephalitis B virus and parvovirus carry out real-time fluorescence
PCR detection is measured, from FAM, VIC fluorescence signal channel, it is seen that there is anomaly PEDV, TGEV specific fluorescence signal.The above results
The dual real-time fluorescence quantitative PCR high specificity for showing foundation, to other encountered pathogenics of pig source, such as swine fever virus, blue otopathy disease
Poison, Pseudorabies virus, porcine circovirus 2 type, encephalitis B virus and parvovirus are showed no non-specific cross jamming.
The detection of 3.7 clinical samples
Anomaly PEDV and TGEV dual real-time fluorescence quantitative PCR detection are carried out to 65 parts of clinical inspection diarrhea pathological material of diseases, use commodity
Change nucleic acid extraction kit and extract corresponding nucleic acid, according to foundation dual real-time fluorescence quantitative PCR system and reaction condition into
Row detection.As a result, it has been found that selecting the channel FAM to see there are 30 parts of positive amplification signals at 520nm, show to deposit in 30 parts of test samples
It is infected in anomaly PEDV, positive rate 46.15%；It selects the channel VIC to see there are 5 parts of positive amplification signals at 550nm, shows 5
There are TGEV infection, positive rates 7.69% in part test sample；Wherein also want 2 parts of samples selected at 520nm the channel FAM and
It selects the channel VIC to have amplified signal at 550nm, shows that there are anomaly PEDV and TGEV mixed infection, sun in 2 parts of samples
Property rate be 3.08%.
By the product of relevant positive sample real-time fluorescence quantitative PCR after reaction, with Ago-Gel reclaim reagent
Box carries out gel extraction to specific target fragment.Connection kit is cloned according to pEASY-T1 Simple Cloning Kit
Corresponding genetic fragment (N genetic fragment, the S genetic fragment of TGEV of PEDV) is cloned on pEASY-T1 carrier by specification,
Random 8 single bacteriums of picking fall within 15 hours of LB liquid medium culture of ampicillin (content is 100 μ g/mL) resistance
Afterwards, corresponding plasmid is extracted using the small extracts kit of rapid plasmid.The matter of primer and condition when using PCR amplification to extraction
Grain carries out PCR identification, send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced the positive recombinant plasmid filtered out
Identification.Sequencing result is subjected to BLAT analysis verifying in NCBI row, is the N genetic fragment of anomaly PEDV and the S base of TGEV
Because of segment, coincidence rate 100%.
The foregoing is merely the preferred embodiment of invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations, all should belong to covering scope of the invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>for identifying the detection primer and probe of anomaly PEDV and TGEV
<130> 6
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
ctcccgagtg tagttgagat tg 22
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ctccacgacc ctggttattt 20
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<400> 3
caacccaaca cacctcctac ttcacg 26
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<400> 4
tgagggtgct ggctttgat 19
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
caagagtgac accacccgtt 20
<210> 6
<211> 25
<212> DNA
<213>artificial sequence
<400> 6
cactgtggca cccttacctg attgt 25
Claims (2)
1. one group for identifying the real-time fluorescence quantitative PCR detection primer and probe of anomaly PEDV and TGEV, it is characterised in that:
The primer and probe sequence is as follows:
For detecting the N gene of anomaly PEDV:
Upstream primer PEDV-F (5'- 3'): CTCCCGAGTGTAGTTGAGATTG,
Downstream primer PEDV-R (5'- 3'): CTCCACGACCCTGGTTATTT,
Fluorescence probe PEDV-P (5'- 3'): FAM-CAACCCAACACACCTCCTACTTCACG-BHQ1；
For detecting the S gene of TGEV:
Upstream primer TGEV-F (5'- 3'): TGAGGGTGCTGGCTTTGAT,
Downstream primer TGEV-R (5'- 3'): CAAGAGTGACACCACCCGTT,
Fluorescence probe TGEV-P (5'- 3'): VIC-CACTGTGGCACCCTTACCTGATTGT-BHQ1；
Wherein, the 5 ' ends of the fluorescence probe PEDV-P of anomaly PEDV are marked with fluorescent reporter group FAM, the fluorescence probe of TGEV
5 ' the ends of TGEV-P are marked with fluorescent reporter group VIC；3 ' the ends of the fluorescence probe PEDV-P of anomaly PEDV are glimmering with TGEV's
3 ' the ends of light probe TGEV-P mark quenching group BHQ1.
2. a kind of for identifying the reagent of anomaly PEDV and TGEV, it is characterised in that: the reagent includes claim 1 institute
The primer and probe stated.
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Application publication date: 20190402 |