CN106967845A - A kind of kit detected for pig Delta coronavirus and detection method - Google Patents
A kind of kit detected for pig Delta coronavirus and detection method Download PDFInfo
- Publication number
- CN106967845A CN106967845A CN201710261995.0A CN201710261995A CN106967845A CN 106967845 A CN106967845 A CN 106967845A CN 201710261995 A CN201710261995 A CN 201710261995A CN 106967845 A CN106967845 A CN 106967845A
- Authority
- CN
- China
- Prior art keywords
- pcr
- probe
- kit
- seq
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of kit and detection method for being used to detect pig Delta coronavirus.Kit of the present invention includes RT PCR reaction systems, it includes primed probe mixed liquor, the primed probe mixed liquor includes a primer pair and a probe, the nucleotide sequence of the primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.3.This method uses Taqman sonde methods, and amplified reaction and product detection are carried out under stopped pipe state, it is to avoid amplified production pollution and the false positive that causes；Probe hybrid specificities are stronger, sensitivity is higher；Without subsequent treatment after PCR, more simple and quick, safety non-pollution is operated；It is good to the Detection results of the genomes of DCoV containing trace P in sample, test in laboratory and Molecule Epidemiology Investigation available for PDCoV.
Description
Technical field
It is more particularly to a kind of to be used for pig Delta coronavirus (PDCoV) detection the invention belongs to gene engineering technology field
Kit and detection method.
Background technology
Pig Delta coronavirus (porcine deltacoronavirus, PDCoV) belongs to coronaviridae
(Coronaviridae) coronavirus subfamily (Coronavirinae) fourth type coronavirus genus, at first by the Woo of Hong Kong University
The positive sample of more than 50 coronavirus was detected from 7140 samples that dead birds, chicken, mammal obtain equal to 2012
This, wherein seven kinds are newfound fourth type coronavirus (Woo, Patrick C.Y.et al. " Discovery of Seven
Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus
Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and
Betacoronavirus and Avian Coronaviruses as the Gene Source of
Gammacoronavirus and Deltacoronavirus.”Journal of Virology 86.7(2012):3995–
4008.PMC.Web.10Apr.2017.).PDCoV is a kind of unique fourth type coronavirus for infecting non-birds.Hereafter, the U.S.,
Many countries such as South Korea, China's Mainland are found that PDCoV in succession.PDCoV can cause the intestines problem of pig, cause to suffer from diarrhoea, vomit
Tuhe is dehydrated, and morbidity and mortality may be up to 50%~100%, is fallen ill especially with nursery phase piglet the most serious.
Although Woo et al. just detected PDCoV in 2012 from clinical sample, virus is not separated to, do not had yet
Have and large-scale epidemiology survey is carried out to the virus.The 1-2 months in 2014, during U.S.'s outburst grice diarrhoea, study people
The pig farm that member suffers from diarrhoea from Ohio, Iowa, Illinois detects PDCoV, and the pig stream on these pig farms
The several frequently seen virus for causing diarrhoea such as row diarrhea virus, transmissible gastro-enteritis virus, porcine rotavirus is feminine gender,
PDCoV is pointed out to have stronger pathogenic.Complete genome sequencing discovery, the two plants of PDCoV detected with Hong Kong in 2012
(HUK15-44 and HUK15-155) homology is up to 99%.Examine in 9 states such as Minnesota, South Dakota then, in the U.S.
Measure PDCoV infection.At present, the U.S. has nearly 19 states to occur PDCoV infection, shows that PDCoV is general in the main hog area in the U.S.
All over popular.
In April, 2014, PDCoV is also detected that in the piglet excrement that South Korea, Canada suffer from diarrhoea, with U.S.'s separation strains
Homology is up to more than 99.7% (Lee S, Lee C.Complete Genome Characterization of Korean
Porcine Deltacoronavirus Strain KOR/KNU14-04/2014.Genome announcements.2014,
2.).Xiao Shao ripples seminar of Hua Zhong Agriculture University is picking up from the provinces and cities such as Hubei, Jiangsu, Guangdong, Henan, Anhui 2013-2014
258 parts of fecal specimens of large-scale pig farm are detected, 21 parts of positives are detected altogether, and positive rate is 14.3%, is demonstrate,proved first
Real PDCoV exists in China pig farm.Whole genome analysis is carried out to some positive sample of detection, China mainland pig farm is found
In HUK15-155 plants of the PDCoV that are equally detected with Hong Kong in 2012 of PDCoV have higher homology (Dong N,
Fang L,Zeng S,et al.Porcine Deltacoronavirus in Mainland China[J].Emerging
Infectious Diseases,2015,21(12):2254-5.).Recently, domestic scholars Song etc. is using the nido RT- set up
PCR have detected the 356 parts of clinical samples gathered between 2012-2015 from Jiangxi Province, and PDCoV positive rate is up to 33.71%
(Song D,Zhou X,Peng Q,et al.Newly Emerged Porcine Deltacoronavirus Associated
With Diarrhoea in Swine in China:Identification,Prevalence and Full-Length
Genome Sequence Analysis.[J].Transboundary and emerging diseases,2015,62(6):
575.).So far, 25 have been included in GenBank altogether and has derived from the U.S., China, the PDCoV whole genome sequences of South Korea,
Dendrogram analysis shows that the homology of these strains is very high.
Because clinical symptoms caused by other pig enteric coronavirus virus infection such as PDCoV and PEDV, TGEV are closely similar simultaneously
And there is mixed infection, to this, viral diagnosis relies primarily on laboratory diagnosis.PDCoV is the virus just found in recent years, is arrived
Less, PDCoV conventional RT-PCRs, RT-Nested PCR for having built up at present etc., its sensitivity phase are reported in research so far
To relatively low.The tender grade of Pang phoenix (spoil, Zhang Baimeng, what Kong Wang, waits the foundation of pig δ coronavirus M gene RT-PCR detection methods by Pang phoenix
And apply [J] herdings and animal doctor, 2016,48 (9):50-53.) specific primer is designed for PDCoV M GFPs fragment
The RT-PCR method of foundation, it is minimum can detected level be 6.33 × 104Copy/μ L.The country not yet has fluorescence RT-PCR to be applied to
The report of PDCoV detections.Although each research group has established different PDCoV test in laboratory methods, also successfully obtain
The PDCoV popularity data in Countries area, but up to the present stable commercialization detection kit is not available for
Use, large-scale clinical detection is still had any problem, and in addition to China, the U.S., South Korea, other countries PDCoV popularity according to
It is so unknown.Based on this, it is necessary to set up a kind of quick, sensitive, high specificity fluorescent quantitative PCR detection method, and become
An important means for grasping PDCoV epidemiologic datas seems especially necessary.
The content of the invention
The technical problems to be solved by the invention are all to use common RT- substantially to PDCoV detection at present to overcome
Sensitivity that PCR, RT-Nested PCR method have is low, reaction time length defect, and it is coronal to provide a kind of detection pig Delta
Kit, primer pair and the detection method of virus.This method uses Taqman sonde methods, carries out expanding instead under stopped pipe state
Should and product detection, it is to avoid amplified production pollution and the false positive that causes；Probe hybrid specificities are stronger, sensitivity is higher；PCR
Afterwards without subsequent treatment, more simple and quick, safety non-pollution is operated；To the Detection results of the genomes of DCoV containing trace P in sample
Well, the test in laboratory and Molecule Epidemiology Investigation available for PDCoV.
The general principle of fluorescence TaqMan technologies is the 5 ' 5 prime excision enzyme activities using Taq enzyme, one energy of synthesis and PCR primer
The probe of hybridization, 5 ' end mark fluorescent reporter groups (R) of the probe, 3 ' end mark fluorescent quenching groups.Probe is without special
Property PCR when occurring, 3 ' end fluorescent quenching groups can absorb or suppress 5 ' end fluorescent reporter groups (R) fluorescence for sending, fluorescence
Signal does not change；When there is specific PCR generation, probe can during PCR by the 5 ' of Taq enzyme->3 ' 5 prime excision enzyme activity
Effect cut-out (nick translation effect), the inhibitory action of quenching group disappears, so as to cause the growth of reporter group fluorescence signal.
The fluorescent reporter group and the number of PCR primer cut out is man-to-man, and with the increase of amplification cycles number, what is discharged is glimmering
Light group is constantly accumulated.Whole PCR processes are monitored in real time using fluorescence signal accumulation, finally by standard curve to unknown template
The method for carrying out quantitative analysis.In general, the remolding sensitivity conventional RT-PCR of quantitative real-time PCR is high 100 times.This hair
In bright, inventor creatively devises the extremely strong primer and probe of specificity, with reference to RT-PCR advantage, will detect virus
Sensitivity is greatly improved compared with prior art medium sensitivity.
One of technical scheme that the present invention is provided is：One kind is used for the reagent for detecting pig Delta coronavirus (PDCoV)
Box, it includes RT-PCR reaction systems, and the RT-PCR reaction systems include primed probe mixed liquor, the primed probe mixing
Liquid include a primer pair and a probe, the nucleotide sequence of the primer pair as shown in SEQ ID NO.1 and SEQ ID NO.2,
The nucleotide sequence of the probe is as shown in SEQ ID NO.3.
The concentration of the primer is preferably 200~400nM, is more preferably 200nM.The concentration of the probe is preferably
200~400nM, is more preferably 400nM.
It is preferred that the RT-PCR reaction systems also include RT-PCR reaction solutions and enzyme mixation, the RT-PCR reactions
Liquid includes Mg2+, dNTP and PCR reaction buffers, the enzyme mixation by reverse transcriptase (RT enzymes), hot start Taq polymerase and
RNase inhibitor (RNasin) is constituted；
Wherein, the Mg2+Concentration be preferably 1~2.5mmol/L, more preferably 2.0mmol/L；The concentration of the dNTP
Preferably 0.1~0.25mmol/L, more preferably 0.2mmol/L；The PCR buffer solutions are preferably that 2 × One step RT-PCR delays
Fliud flushing (One Step RT-PCR Buffer) (no Mg2+)；The consumption of the reverse transcriptase is preferably 0.5~2U/ reactions, described
The consumption of hot start Taq polymerase is preferably 0.1~0.5U/ reactions, and the consumption of the RNase inhibitor is preferably 1~3U/ reactions.
More preferably, the kit also includes positive control and negative control, and the positive control is preferably containing PDCoV
The recombinant plasmid of M gene orders, the PCR primer that plasmid is expanded by upstream and downstream primer is built-up by commercially available carrier cloning,
Used upstream and downstream primer sequence SEQ ID NO.1 and SEQ ID NO.2, the concentration of the positive control is preferably 2.6 ×
106Copy/μ L；The negative control is preferably deionized water, more preferably DEPC-H2O。
Kit of the present invention is preferably real-time fluorescence quantitative PCR (FQ-PCR) kit.
The two of the technical scheme that the present invention is provided are：A kind of primer pair for being used to detect pig Delta coronavirus, it is described to draw
One of thing pair is the nucleic acid of the sequence as shown in SEQ ID NO.1, another be the sequence as shown in SEQ ID NO.2 nucleic acid.
The three of the technical scheme that the present invention is provided are：A kind of probe for detecting pig Delta coronavirus, the core of the probe
Nucleotide sequence is as shown in SEQ ID NO.3.
It is preferred that above-mentioned probe is the fluorescence probe for being applicable real-time fluorescence quantitative PCR, the two ends connection of its above-mentioned sequence
It is conventional fluorescent reporter group and quenching group.Wherein, fluorescent reporter group is preferably FAM, and quenching group is preferably
TAMRA。
The four of the technical scheme that the present invention is provided are：Described primer or described probe are preparing detection pig Delta
Application in the kit of coronavirus.It is preferred that the kit is real-time fluorescence quantitative PCR kit.
The five of the technical scheme that the present invention is provided are：A kind of method of the detection pig Delta coronavirus of non-diagnostic purpose,
It comprises the following steps：
(1) total serum IgE in measuring samples is extracted using RNA extracts reagents；
(2) total serum IgE using step (1) extraction utilizes the RT- in any one of Claims 1 to 44 kit as template
PCR reaction systems carry out RT-PCR reactions.
(3) testing result is analyzed.
Wherein, described RNA extracts reagents can be the reagent of the conventional extraction animal virus total serum IgE in this area, preferably
Ground, described RNA extracts reagents include sample dissociation liquid, chloroform, DEPC- ethanol, isopropanol and DEPC-H2One kind in O or
It is a variety of；The described preferred Trizol of sample dissociation liquid, the DEPC- second that described DEPC- ethanol preferred mass percentage is 75%
Alcohol.
It is preferred that the reaction condition of the reactions of RT-PCR described in step (2) is：
1. 42~50 DEG C of 10~15min of reverse transcription；2. 94~95 DEG C of 10~15min；3. 94~95 DEG C of 10~15s；④55
~60 DEG C of 35~60s；3. -4. circulate 40~45 times；
Described in step (2) fluorescence RT-PCR reaction course of reaction be：Take respectively the μ L of primed probe mixed liquor 1~3,
The μ L of RT-PCR reaction solutions 14~16, the μ L of enzyme mixation (Enzyme Mix) 2~4 are configured to 17~22 μ L reaction systems, add 2
RNA obtained by~8 μ L steps (1).It regard PDCoV positive plasmids as positive control sample, DEPC-H2O is used as negative control sample
Product, fluorescence RT-PCR reaction is carried out with measuring samples RNA simultaneously；
The operating process of analysis testing result described in step (3) is that this area is conventional, it is preferred that described analysis detection
As a result operating process is as follows：After reaction terminates, when positive control is in typical S types amplification curve, negative control is bent without amplification
During line, judge that detection reaction is set up；If S types amplification curve or amplification curve, which also occurs, in detection sample obvious Exponential growth stage,
Then it is determined as that detection is positive, is otherwise determined as feminine gender.
The present invention a preferred embodiments be：A kind of kit for being used to detect PDCoV, it includes：
(1) RNA extracts reagents, including：
Sample dissociation liquid pipe：Trizol 750μL；Chloroform pipe：The μ L of chloroform 200；75% ethanol pipe：75%DEPC- ethanol
800μL；Isopropanol pipe：The μ L of isopropanol 600；DEPC-H2O is managed：11μL；It is above the reagent for the total serum IgE for being adapted to extract 1 sample
Consumption；Described RNA extracts reagents need 4 DEG C of preservations；
(2) RT-PCR reaction systems, including：
Primed probe mixes liquid pipe：10 μM of upstream and downstream primers each 0.5 μ L, 10 μM of μ L of probe 1, add up to 2 μ L；
RT-PCR reacts liquid pipe：2 × One step RT-PCR buffer solution (One Step RT-PCR Buffer) (no Mg2+)10
μ L, 25mM MgCl22 μ L, 10mM dNTP 1 μ L, DEPC-H2The μ L of O 2, add up to 15 μ L；
Enzyme mixation (Enzyme Mix) is managed：The μ of 11 μ L, 5U/ μ L Taq enzymes of μ L, 25U/ μ L RT enzymes of 40U/ μ L RNasin 1
L, adds up to 3 μ L；
The reagent dosage reacted above for single, described reaction system needs -20 DEG C of preservations；
(3) positive control pipe：Concentration is 2.6 × 106Copy/μ L μ the L of PDCoV positive plasmids 5；
(4) negative control pipe：DEPC-H2O 5μL。
In above-mentioned preferred embodiments, described 2 × One step RT-PCR buffer solution (One Step RT-PCR Buffer) (nothing
Mg2+)、25mM MgCl2, 2.5mM dNTP mixtures and Taq enzyme can also use the conventional reagent kit product in this area, such as may be used
To substitute above-mentioned several reagents using one-step method fluorescence RT-PCR kit (One Step Real Time RT-PCR Kit),
It is preferred that TaKaRa Products one-step method fluorescence RT-PCR kits (One Step PrimeScriptTM RT-PCR Kit
(Perfect Real Time))。
The present invention a preferred embodiments be：A kind of method for detecting PDCoV of non-diagnostic purpose, it is a kind of use
The method that fluorescence quantitative RT-RCR detects PDCoV, it comprises the following steps：
(1) extraction of sample total serum IgE；
(2) PCR reactions and interpretation of result：The μ L of 2 μ L, RT-PCR reaction solution of primed probe mixed liquor 15, enzyme mixing are taken respectively
The μ L of liquid (Enzyme Mix) 3 are configured to reaction system.
Each 5 μ L of nucleic acid of negative control, sample and positive control are taken, is separately added into reaction system and uses commercially available fluorescence
Quantitative PCR apparatus (such as ABI7500) enters performing PCR amplification.PCR reaction conditions are：1. 42 DEG C of reverse transcription 10min；②94℃15min；
③95℃15s；4. 60 DEG C of 45s, 3. -4. circulate 40 times (collection fluorescence signal).
Reaction preserves detection data file after terminating.According to the curve obtained by PCR amplifications, experimental result is analyzed.
As amplification curve has obvious Exponential growth stage, then it is determined as the positive, is otherwise feminine gender.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and produce each preferable reality of the present invention
Example.
Agents useful for same and raw material of the present invention are commercially available.
The positive effect of the present invention is：The method for the detection PDCoV that the present invention is provided uses Taqman sonde methods,
Amplified reaction and product detection are carried out under stopped pipe state, it is to avoid amplified production pollution and the false positive that causes, probe hybridization
Specific stronger, sensitivity is higher, and minimum detection limit is about 1.3 × 101Copy/μ L, its sensitivity is that prior art (can be detected
Measure as 6.33 × 104Copy/μ L) 4869 times, it is good to the Detection results of the genomes of DCoV containing trace P in sample；While its
Simple to operate, cost is low and result is easy to judge, is adjusted available for PDCoV test in laboratory and large-scale molecular epidemiology
Look into research.Therefore, kit of the invention, primer pair and detection method have good application prospect.
Brief description of the drawings
Fig. 1 is, with the amplification curve result of the fluorescence RT-PCR of primer (F1 and R1) and probe the P1 combination 1 combined, to use
DEPC-H2O does 10 times to plasmid standard and is serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3
×106、1.3×105、1.3×104、1.3×103There is typical S types amplification curve, exponential region in copy/μ L after being expanded
It is more apparent.
Fig. 2 is, with the standard curve result of the fluorescence RT-PCR of primer (F1 and R1) and probe the P1 combination 1 combined, to use
DEPC-H2O does 10 times to plasmid standard and is serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3
×106、1.3×105、1.3×104、1.3×103Copy/μ L are expanded, 3 repetitions, standard items initial template concentration and Ct
Good linear relationship, slope close -3.04, correlation coefficient r is presented in value2For 0.994.
Fig. 3 is, with the amplification curve result of the fluorescence RT-PCR of primer (F2 and R2) and probe the P2 combination 2 combined, to use
DEPC-H2O does 10 times to plasmid standard and is serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3
×106、1.3×105、1.3×104、1.3×103There is typical S types amplification curve, exponential region in copy/μ L after being expanded
It is more apparent.
Fig. 4 is, with the standard curve result of the fluorescence RT-PCR of primer (F2 and R2) and probe the P2 combination 2 combined, to use
DEPC-H2O does 10 times to plasmid standard and is serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3
×106、1.3×105、1.3×104、1.3×103Copy/μ L are expanded, 3 repetitions, standard items initial template concentration and Ct
Preferable linear relationship, slope close -2.95, correlation coefficient r is presented in value2For 0.983.
Fig. 5 is, with the amplification curve result of the fluorescence RT-PCR of primer (F3 and R3) and probe the P3 combination 3 combined, to use
DEPC-H2O does 10 times to plasmid standard and is serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3
×106、1.3×105、1.3×104、1.3×103There is typical S types amplification curve, exponential region in copy/μ L after being expanded
It is more apparent.
Fig. 6 is, with the standard curve result of the fluorescence RT-PCR of primer (F3 and R3) and probe the P3 combination 3 combined, to use
DEPC-H2O does 10 times to plasmid standard and is serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3
×106、1.3×105、1.3×104、1.3×103Copy/μ L are expanded, 3 repetitions, standard items initial template concentration and Ct
Good linear relationship, slope close -2.96, correlation coefficient r is presented in value2For 0.985.
Fig. 7 is combined for the present invention in the specific test result of 1 fluorescence RT-PCR, 7 parts of specific reference materials, only PDCoV
The amplification curve of positive plasmid is S type amplification curves, the amplification curves of other 6 parts specific reference materials for it is straight or obliquely and
With baseline without intersecting, without Ct values, it can clearly be determined as feminine gender；6 parts of specific reference materials are respectively PEDV vaccine strains, TGEV epidemic diseases
Miao Zhu, Rotavirus Vaccine strain, prompt Shen virus, Kobu virus-positives sample, Escherichia coli (reference culture ATCC 25922) etc.
Other pigs easily cause the pathogen of diarrhoea.
Fig. 8 is the specific test result of the present invention 2 fluorescence RT-PCRs of combination, 4 parts of specific reference material (rotavirus epidemic diseases
Miao Zhu, prompt Shen virus, Kobu virus-positives sample, Escherichia coli reference culture ATCC 25922) amplification curve it is straight or oblique
Downwards and with baseline without intersecting, without Ct values, it can clearly be determined as feminine gender；Wherein there are 3 parts of specific reference material respectively PDCoV
There is amplification curve and intersected with baseline in positive plasmid, PEDV vaccine strains and TGEV vaccine strains, have Ct values, can be judged to the positive.
Fig. 9 is the specific test result of the present invention 3 fluorescence RT-PCRs of combination, 5 parts of specific reference material (TGEV vaccines
Strain, Rotavirus Vaccine strain, prompt Shen virus, Kobu virus-positives sample, Escherichia coli reference culture ATCC 25922) amplification
Curve is straight or obliquely and with baseline without intersecting, without Ct values, can clearly be determined as feminine gender；Wherein there are 2 parts of specificity references
Product are PDCoV positive plasmids, PEDV vaccine strains amplification curve occur and intersected with baseline, have Ct values, can be judged to the positive.
Figure 10 combines concentration in the sensitivity tests result of 1 fluorescence RT-PCR, 8 parts of sensitivity reference materials for the present invention
1.3×100Copy/μ L sample amplification curve is straight or obliquely and with baseline without intersecting, without Ct values, can clearly be determined as
It is negative.Concentration is 1.3 × 10130≤Ct≤35 of copy/μ L sample amplification curve, are judged to suspicious, and Ct values are still after reinspection
30≤Ct≤35, are judged to the positive；Remaining sample standard deviation can clearly be determined as the positive.
Figure 11 combines concentration in the sensitivity tests result of 2 fluorescence RT-PCRs, 8 parts of sensitivity reference materials for the present invention
1.3×101Copy/μ L and 1.3 × 100Copy/μ L sample amplification curve is straight or obliquely and with baseline without intersecting, does not have
Ct values, can clearly be determined as feminine gender；Remaining sample standard deviation can clearly be determined as the positive.
Figure 12 combines concentration in the sensitivity tests result of 3 fluorescence RT-PCRs, 8 parts of sensitivity reference materials for the present invention
1.3×101Copy/μ L and 1.3 × 100Copy/μ L sample amplification curve is straight and Ct values>35, it can determine that as feminine gender；Remaining
Sample standard deviation can clearly be determined as the positive.
Figure 13 is that certain pig farm PDCoV fluorescence RT-PCRs detect figure, wherein, P is positive control；N is negative control；S1~S3
For 3 parts of pig intestinal samples.
Figure 14 is certain pig farm PDCoV conventional RT-PCR electrophoretograms, wherein, M is DL2000Marker；P is positive control；N
For negative control；S1~S3 is 3 parts of pig intestinal samples.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification is selected.
The primer is synthesized by Invitrogen (Shanghai) Trading Co., Ltd. in following embodiments.
Portion of reagent and instrument source are as follows：
2 × One step RT-PCR buffer solution (One Step RT-PCR Buffer) (no Mg2+)、25mM MgCl2、10mM
DNTP mixtures, 40U/ μ L RNase inhibitors (RNasin), 5U/ μ L Taq enzymes, 25U/ μ L RT enzymes, PrimeScriptTM
One Step RT-PCR Kit Ver 2.0 (Dye Plus) and one-step method fluorescence RT-PCR kit (One Step
PrimeScriptTMRT-PCR Kit (Perfect Real Time)) it is purchased from precious bioengineering (Dalian) Co., Ltd；
ABI7500 is Applied biosystems' product；
PEDV vaccine strains, TGEV vaccine strains, Rotavirus Vaccine strain are purchased from Zhongmu Industry Co., Ltd.
The design of the specific primer of embodiment 1 and probe
1 pair of specific primer and probe are designed for the conserved sequence of PDCoV in GenBank, it is as shown in table 1 below, according to
Following sequence, synthesis specificity fluorescent RT-PCR primer and probe.With DEPC-H2O dilutes primer to 10 μM, and -20 DEG C of preservations are standby
With.
The primer and probe of table 1 is combined
Wherein F1 nucleotide sequence is as shown in SEQ ID NO.1 in sequence table, in R1 nucleotide sequence such as sequence table
Shown in SEQ ID NO.2, P1 nucleotide sequence is as shown in SEQ ID NO.3 in sequence table.
The foundation and optimization of the reaction system of embodiment 2
1st, the preparation of sample：Build the positive control that 1 plasmid containing purposeful amplification region is detected as PDCoV；With
PEDV vaccine strains, TGEV vaccine strains, Rotavirus Vaccine strain, prompt Shen virus, Kobu virus-positives sample, Escherichia coli (standard
Strains A TCC 25922) (being derived from Shanghai Municipal Center for Animal Disease Control ＆ Prevention Shanghai veterinary conditions diagnostic center laboratory) conduct
Specific reference material；Using deionized water as negative control, extract above-mentioned positive control with Trizol respectively and referred to specificity
The RNA of product, negative control is stand-by.
2nd, the screening of primed probe：With the primer pair probe designed in embodiment 1 detect respectively above-mentioned positive control with
The RNA of negative control, through repetition test, filters out specificity, sensitivity and reproducible optimal primer combination of probe (such as
Shown in SEQ ID NO.1~SEQ ID NO.3).
3rd, the optimization of primed probe concentration：In the case that other components are constant in reaction system, use respectively from
100nM/, which is reacted to the primer and probe of 400nM/ reaction gradients, enters performing PCR reaction, and primer and spy are found through experiment is repeated several times
Pin concentration 200~400nM/ reaction between, wherein optimal primer concentration be 200nM/ reaction, concentration and probe concentration be
400nM/ reacts.
4th, the optimization of magnesium ion concentration：In the case that other components are constant in reaction system, use respectively from 1mmol/L
Magnesium ion to 2.5mmol/L concentration gradients enters performing PCR reaction, is tested through being repeated several times, finally determines that optimal magnesium ion is dense
Spend for 2.0mmol/L.
5th, the optimization of dNTP concentration：In the case that other components are constant in reaction system, use respectively from 0.1mmol/L
DNTPs to 0.25mmol/L concentration gradients enters performing PCR reaction, is tested through being repeated several times, finally determines optimal dNTP concentration
For 0.2mmol/L.
6th, the optimization of hot start Taq polymerase consumption：In the case that other components are constant in 25 μ L reaction systems, use respectively
From 1U (enzyme unit) to the enzyme dosage of 8U concentration gradients/react into performing PCR reaction, tested through being repeated several times, it is final determine it is optimal
Hot start Taq polymerase consumption be 0.1~0.5U/ reaction.
7th, the optimization of RT enzyme dosages：In the case that other components are constant in 25 μ L reaction systems, use respectively from 1U (enzymes
Unit) to the enzyme dosage/react of 8U concentration gradients into performing PCR reaction, tested through being repeated several times, finally determine that optimal RT enzymes are used
Measure and reacted for 0.5~2U/.
8th, the optimization of RNasin consumptions：In the case that other components are constant in 25 μ L reaction systems, use respectively from 5U
(enzyme unit) is tested to the enzyme dosage/react of 40U concentration gradients into performing PCR reaction through being repeated several times, it is final determine it is optimal
RNasin consumptions react for 1~3U/.
9th, the optimization of reaction temperature：According to the activity and the length of target polynucleotide of enzyme, mainly to annealing temperature and extension
Time is optimized, and is tested through being repeated several times, final to determine that PCR reaction conditions are：(1) 50 DEG C of reverse transcription 10min；(2)94
℃15min；(3)95℃15s；Circulate 45 times (collection fluorescence signal) in (4) 55~60 DEG C of 45s, (3)-(4).
The foundation of the standard curve of embodiment 3
By PDCoV positive plasmids DNA with after spectrophotometric determination concentration, DEPC-H is used2O does 10 times to plasmid standard
It is serially diluted, it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption7、1.3×106、1.3×105、1.3×104、
1.3×103Copy/μ L, sets 3 repetitions, is expanded, after reaction terminates, and analyzes soft using ABI7500 quantitative fluorescent PCRs
Part automatically derives standard curve.As a result show：
, is there are typical S types and expanded in the fluorescence RT-PCR method set up with primer (F1 and R1) and probe the P1 combination 1 combined
Increasing curve, exponential region is more apparent, standard items initial template concentration is presented with Ct values has the good range of linearity, slope is close-
3.04, correlation coefficient r2For 0.994 (see Fig. 1,2).
Accurate quantitative analysis can be carried out to sample according to Ct values and standard curve.
The specificity of embodiment 4 and sensitivity tests
(1) specific test
Using the system and amplification condition optimized described in embodiment 2, it is respectively PEDV vaccines to take 7 parts of specific reference materials
Strain, the strain of TGEV vaccine strains, Rotavirus Vaccine, PDCoV positive plasmids, prompt Shen virus, viral (PKV) positives of pig Kobu,
7 samples such as Escherichia coli (reference culture ATCC 25922) positive, extract RNA, and reagent is carried out using fluorescence RT-PCR
The specific test of box.
Wherein, the step of sample Total RNAs extraction is as follows：1. the μ of testing sample 200 is added in 750 μ L Trizol lysates
L, is mixed, 15~25 DEG C of standing 5min；2. 200 μ L chloroforms are added, vibration is mixed, in 4 DEG C, 12000r/min centrifugations 15min；③
Water intaking phase supernatant, adds 600 μ L in the isopropanol of -20 DEG C of precoolings, after mixing, 12000r/min centrifugations 10min；4. supernatant is abandoned,
Add 800 μ L 75%DEPC- ethanol, 12000r/min centrifugations 10min；5. abandon supernatant and add 11 μ after being dried in 15~25 DEG C
L DEPC-H2O dissolves, and obtains sample RNA, standby.
Fluorescence RT-PCR reaction system：The μ L of 2 μ L, RT-PCR reaction solution of primed probe mixed liquor 15 and enzyme mixation
(Enzyme Mix)3μL。
The reaction condition of fluorescence RT-PCR is as follows：
(1) 42 DEG C of reverse transcription 10min；(2)94℃15min；(3)95℃15s；(4)60℃45s；(3)-(4) circulate 40 times
(collection fluorescence signal).
As a result show：
The fluorescence RT-PCR result of combination 1 shows that only PDCoV positive plasmids amplification curve is S type amplification curves.And PEDV
Vaccine strain, TGEV vaccine strains, Rotavirus Vaccine strain, prompt Shen virus, viral (PKV) positives of pig Kobu, Escherichia coli (mark
Quasi- strains A TCC 25922) positive do not occur specific amplification curve (see Fig. 7), illustrates the specificity of this combination very
It is high.
(2) sensitivity tests
3 PDCoV positive plasmids DNA are used into DEPC-H with after spectrophotometric determination concentration respectively2O is to plasmid control
Product do 10 times and are serially diluted, and it is respectively 1.3 × 10 to make the copy number in every 5 μ L detections consumption6、1.3×105、1.3×104、1.3
×103、1.3×102、1.3×101、1.3×100Copy/μ L carry out fluorescence RT-PCR amplification.Fluorescence RT-PCR reaction system with
And reaction condition is with shown in (1) specific test.
As a result show：
The minimum detection limit of the fluorescence RT-PCR method of combination 1 is about 1.3 × 101Copy/μ L (see Figure 10).And Pang phoenix is tender
(Pang phoenix is spoilt, Zhang Baimeng, what Kong Wang, wait the foundation and application [J] herdings of pig δ coronavirus M gene RT-PCR detection methods with
Animal doctor, 2016,48 (9):50-53.) etc. the RT-PCR side that specific primer is set up is designed for PDCoV M GFPs fragment
Method, it is minimum can detected level be 6.33 × 104Copy/μ L.And combine 1 sensitiveness be prior art (can detected level be 6.33 ×
104Copy/μ L) 4869 times.It is of the invention equally to design a pair of more special primers and spy for PDCoV M GFPs fragment
Pin, by optimum organization, substantially increases the sensitiveness of experiment.As can be seen here, sensitiveness of the invention is significantly larger than common RT-
PCR。
The assembling of the kit of embodiment 5
The preparation for being partially completed reagent constituents according to Total RNAs extraction and fluorescence RT-PCR two, data presented below
And reagent set turns into reagent needed for the detection of single Nest RT-PCR：
(1) Total RNAs extraction (4 DEG C of preservations)
Sample dissociation liquid pipe：Trizol 750μL；
Chloroform pipe：The μ L of chloroform 200；
75% ethanol pipe：The μ L of 75%DEPC- ethanol 800；
Isopropanol pipe：The μ L of isopropanol 600；
DEPC-H2O is managed：11μL；
(2) fluorescence RT-PCR (- 20 DEG C of preservations)
Primed probe mixes liquid pipe：10 μM of upstream and downstream primers each 0.5 μ L, 10 μM of μ L of probe 1, add up to 2 μ L；
RT-PCR reacts liquid pipe：
2 × One step RT-PCR buffer solution (One Step RT-PCR Buffer) (no Mg2+)10μL；25mM MgCl2 2
μL；10mM dNTP 1μL；DEPC-H2O 2μL；Total 15 μ L；
Enzyme mixation (Enzyme Mix) is managed：The μ of 11 μ L, 5U/ μ L Taq enzymes of μ L, 25U/ μ L RT enzymes of 40U/ μ L RNasin 1
L；
Positive control pipe：Concentration is 2.6 × 106Copy/μ L μ the L of PDCoV positive plasmids 5；
Wherein, the preparation method of PDCoV positive plasmids is：Positive plasmid is by Shanghai You Long bio tech ltd structure
Build, specially synthesis is containing upstream and downstream primer sequence (being above-mentioned primers F 1, R1, as shown in SEQ ID NO.1~2)
PDCoV M genes, and carrier pUC57-Amp is cloned into, inverted, screening, identification obtain PDCoV positive plasmids.
Negative control pipe：DEPC-H2O 5μL。
It is a packaging according to 48 parts of detection dosage, the above-mentioned reagent prepared is respectively charged into 10 without RNase enzymes
In bottle or pipe, and transport is preserved according to corresponding storage temperature.
The application of the kit of embodiment 6
It is young to 3 parts of diarrhoea of Shanghai pig farm censorship using the PDCoV fluorescence quantitative RT-PCR detecting kits of the present invention
The intestinal samples (sample S1, S2 and S3) of pig are detected.
(1) pretreatment of sample
It is fully ground with sterile scissors and tweezers clip measuring samples 1.0g in mortar, then adds 1mL PBS to mix, so
Tissue suspension is transferred in 1.5mL sterile centrifugation tubes afterwards, 3000r/min centrifugation 10min take supernatant to be transferred to another sterile centrifugation tube
In, number standby.
(2) RNA extraction
1. the μ L of testing sample 200 are added in 750 μ L Trizol lysates, mixes, is stored at room temperature 5min；2. 200 are added
μ L chloroforms, vibration is mixed, in 4 DEG C of 12000r/min centrifugations 15min；3. fetch water phase supernatant, adding 600 μ L isopropanols, (- 20 DEG C pre-
It is cold), after mixing, 12000r/min centrifugations 10min；4. abandon supernatant, add 800 μ L 75%DEPC- ethanol, 12000r/min from
Heart 10min；5. supernatant is abandoned and in adding 11 μ L DEPC-H after drying at room temperature2O dissolves, and obtains sample RNA, standby.
(3) fluorescence RT-PCR reaction and interpretation of result
The μ L of 2 μ L, RT-PCR reaction solution of primed probe mixed liquor 15, the μ L of enzyme mixation (Enzyme Mix) 3 is taken to prepare respectively
Into reaction system.
Each 5 μ L of nucleic acid of negative control (N), sample (S1, S2, S3) and positive control (P) are taken, reaction system is separately added into
It is middle to enter performing PCR amplification using commercially available quantitative real time PCR Instrument (such as ABI7500).PCR reaction conditions：(1) 42 DEG C of reverse transcription
10min；(2)94℃15min；(3)95℃15s；(4)60℃45s；(3) 40 times (collection fluorescence signal)-(4) is circulated, it is whole anti-
85min should be needed altogether.
Reaction preserves detection data file after terminating.According to the curve obtained by PCR amplifications, experimental result is analyzed,
As a result it is as shown in figure 13.Testing result shows that the control of setting is all set up, and the Ct values of S1, S2, S3 parts of samples are respectively
14.86th, 18.12,21.53, respectively less than 30, and there is typical S types amplification curve, show there are 3 parts to there is sun in detection sample
Property amplification.
(4) with conventional RT-PCR Comparison between detecting methods
Reference literature (Wang L, Byrum B, Zhang Y.Detection and genetic
characterization of deltacoronavirus in pigs,Ohio,USA,2014.Emerg Infect
Dis2014,20:1227-1230) primer of synthesis PDCoV N genes, sense primer 41F (5 '-
TTTCAGGTGCTCAAAGCTCA-3 ') and anti-sense primer 735R (5 '-GCGAAAAGCATTTCCTGAAC-3 ').
Reaction system：
2×PrimeScriptTMOne Step Buffer (Dye Plus) 25 μ L, PrimeScriptTM Enzyme
The μ L of Mix 2, each 1 μ L of upstream and downstream primer, use DEPC-H2O complements to 50 μ L.
PCR reaction conditions：(1) 50 DEG C of reverse transcription 30min；(2)95℃15min；(3)95℃15s；(4)55℃45s；(5)
72℃1min；(3)-(5) circulate 40 times；(6)72℃7min.
5 μ L amplified productions are taken, electrophoresis detection, uviol lamp are carried out under the conditions of 110V 30min with 1.2% Ago-Gel
Lower observation amplification, pcr amplification reaction and electrophoresis detection need 132min altogether.
As a result it is as shown in figure 14.Testing result shows that the control of setting is all set up, and has 3 parts to there is sun in detection sample
Property amplification, i.e. S1, S2 and S3.It is consistent with fluorescence RT-PCR testing result.
As can be seen here, the time is greatlyd save using fluorescence RT-PCR, saves labour, operate simpler, safety non-pollution,
Suitable for the PDCoV quick diagnosis infected and large-scale epidemiological investigation, there is extraordinary application prospect.
Comparative example
(1) design of specific primer and probe
2 pairs of specific primers for being different from above-described embodiment and spy are designed for the conserved sequence of PDCoV in GenBank
Pin, it is as shown in table 2 below, according to following sequence, synthesis specificity fluorescent RT-PCR primer and probe.With DEPC-H2O dilutes primer
To 10 μM, -20 DEG C save backup.
The primer and probe of table 2 is combined
SEQ ID NO in above-mentioned F2~P3 nucleotide sequence such as sequence table:Shown in 4~9.
(2) foundation of standard curve
The system set up and optimized using embodiment 2 carries out the foundation of standard curve, method for building up be the same as Example 3.As a result
Display：
, is there are typical S types and expanded in the fluorescence RT-PCR method set up with primer (F2 and R2) and probe the P2 combination 2 combined
Increasing curve, exponential region is more apparent, standard items initial template concentration is presented with Ct values has the preferable range of linearity, slope is close-
2.95, correlation coefficient r2For 0.983 (see Fig. 3,4).
, is there are typical S types and expanded in the fluorescence RT-PCR method set up with primer (F3 and R3) and probe the P3 combination 3 combined
Increasing curve, exponential region is more apparent, standard items initial template concentration is presented with Ct values has the preferable range of linearity, slope is close-
2.96, correlation coefficient r2For 0.985 (see Fig. 5,6).
Accurate quantitative analysis can be carried out to sample according to Ct values and standard curve.
(3) specificity and sensitivity tests
1. specific test
Concrete operation step be the same as Example 4, as a result shows：
The fluorescence RT-PCR PDCoV positive plasmids amplification curve of combination 2 is S type amplification curves, PEDV vaccine strains and TGEV
Vaccine strain also has amplification curve, non-specific amplification occurs；And Rotavirus Vaccine strain, prompt Shen virus, pig Kobu viruses
(PKV) positive, Escherichia coli (reference culture ATCC25922) positive do not occur specific amplification curve (see
Fig. 8), the specific bad of this combination is illustrated.
The fluorescence RT-PCR result of combination 3 shows that PDCoV positive plasmids amplification curve is S type amplification curves, PEDV vaccines
Strain has amplification curve, non-specific amplification occurs.And the strain of TGEV vaccine strains, Rotavirus Vaccine, prompt Shen virus, pig Kobu viruses
(PKV) positive, Escherichia coli (reference culture ATCC 25922) positive do not occur specific amplification curve (see
Fig. 9), the specific bad of this combination is illustrated.
As can be seen here, the fluorescence RT-PCR specificity of combination 2 and combination 3 is worse than the fluorescence RT-PCR specificity of combination 1.
2. sensitivity tests
Concrete operation step be the same as Example 4, as a result shows：
The minimum detection limit of the fluorescence RT-PCR method of combination 2 is about 1.3 × 102Copy/μ L (see Figure 11), combination 3
The minimum detection limit of fluorescence RT-PCR method is about 1.3 × 102Copy/μ L (see Figure 12)；As can be seen here, combination 2 and combination 3
The test limit of fluorescence RT-PCR method is far above combination 1.
SEQUENCE LISTING
<110>Shanghai Municipal Center for Animal Disease Control ＆ Prevention
<120>A kind of kit detected for pig Delta coronavirus and detection method
<130> P1710073C
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223>Primers F 1
<400> 1
gagagtagac tccttgcagg gattat 26
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer R1
<400> 2
gcttgccatg cttaacgact g 21
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe P1
<400> 3
aatgcacctc catgtacc 18
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Primers F 2
<400> 4
cgaccacatg gctccaatt 19
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer R2
<400> 5
cagctcttgc ccatgtagc 19
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe P2
<400> 6
cacaccagtc gttaagcatg gca 23
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Primers F 3
<400> 7
tgagagtaga ctccttgcag g 21
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer R3
<400> 8
gagaattgga gccatgtgg 19
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe P3
<400> 9
tgtacccatt ggatccataa 20
Claims (10)
1. a kind of kit for being used to detect pig Delta coronavirus, it includes RT-PCR reaction systems, it is characterised in that institute
Stating RT-PCR reaction systems includes primed probe mixed liquor, and the primed probe mixed liquor includes a primer pair and a probe, institute
The nucleotide sequence of primer pair is stated as shown in SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence such as SEQ of the probe
Shown in ID NO.3.
2. kit as claimed in claim 1, it is characterised in that the concentration of the primer is 200~400nM, preferably
200nM；The concentration of the probe is 200~400nM, preferably 400nM.
3. kit as claimed in claim 1, it is characterised in that the RT-PCR reaction systems also include RT-PCR reaction solutions
And enzyme mixation, the RT-PCR reaction solutions include Mg2+, dNTP and PCR reaction buffers, the enzyme mixation is by reverse transcription
Enzyme, hot start Taq polymerase and RNase inhibitor composition；
It is preferred that the Mg2+Concentration be 1~2.5mmol/L, be more preferably 2.0mmol/L；
And/or, the concentration of the dNTP is 0.1~0.25mmol/L, is more preferably 0.2mmol/L；
And/or, the PCR buffer solutions are 2 × One step RT-PCR buffer solution；
And/or, the consumption of the reverse transcriptase reacts for 0.5~2U/, and the consumption of the hot start Taq polymerase is 0.1~0.5U/
Reaction, the consumption of the RNase inhibitor reacts for 1~3U/.
4. kit as claimed in claim 3, it is characterised in that the primed probe mixed liquor is by 10 μM of 0.5 μ L upstream
The probe composition of 10 μM of primer and 0.5 μ L 10 μM of anti-sense primer and 1 μ L, the RT-PCR reaction solutions are by 10 μ L 2 × mono-
The MgCl of footwork RT-PCR buffer solutions, 2 μ L 25mM2, 1 μ L10mM dNTP and 2 μ L DEPC-H2O is constituted, the enzyme mixing
Liquid is made up of the Taq enzyme of 1 μ L 40U/ μ L RNase inhibitor, 1 μ L 25U/ μ L reverse transcriptase and 1 μ L 5U/ μ L.
5. the kit as described in any one of Claims 1 to 4, it is characterised in that the kit is real-time fluorescence quantitative PCR
Kit；And/or,
The kit also includes positive control and negative control, and the positive control is the restructuring containing PDCoVM gene orders
Plasmid, it is preferred that the concentration of the positive control is 2.6 × 106Copy/μ L；The negative control is deionized water, preferably
For DEPC-H2O。
6. a kind of primer pair for detecting pig Delta coronavirus, it is characterised in that one of the primer pair is such as SEQ ID
The nucleic acid of sequence shown in NO.1, another be the sequence as shown in SEQ ID NO.2 nucleic acid.
7. a kind of probe for detecting pig Delta coronavirus, it is characterised in that the nucleotide sequence of the probe such as SEQ ID
Shown in NO.3.
8. primer as claimed in claim 6 or probe as claimed in claim 7 are preparing detection pig Delta coronavirus
Kit in application；It is preferred that the kit is real-time fluorescence quantitative PCR kit.
9. the method for the detection pig Delta coronavirus of a kind of non-diagnostic purpose, it is characterised in that it comprises the following steps：
(1) total serum IgE in measuring samples is extracted using RNA extracts reagents；
(2) total serum IgE using step (1) extraction utilizes the RT-PCR in any one of Claims 1 to 55 kit as template
Reaction system carries out RT-PCR reactions.
(3) testing result is analyzed.
10. method as claimed in claim 9, it is characterised in that RNA extracts reagents described in step (1) include sample dissociation
Liquid, chloroform, DEPC- ethanol, isopropanol and DEPC-H2One or more in O；It is preferred that the sample dissociation liquid is
Trizol, the DEPC- ethanol is the DEPC- ethanol that mass percent is 75%；
And/or, the reaction condition of the reactions of RT-PCR described in step (2) is：
(1) 42~50 DEG C of 10~15min of reverse transcription；(2) 94~95 DEG C of 10~15min；(3) 94~95 DEG C of 10~15s；(4)55
~60 DEG C of 35~60s；(3)-(4) circulate 40~45 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710261995.0A CN106967845A (en) | 2017-04-20 | 2017-04-20 | A kind of kit detected for pig Delta coronavirus and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710261995.0A CN106967845A (en) | 2017-04-20 | 2017-04-20 | A kind of kit detected for pig Delta coronavirus and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106967845A true CN106967845A (en) | 2017-07-21 |
Family
ID=59333710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710261995.0A Pending CN106967845A (en) | 2017-04-20 | 2017-04-20 | A kind of kit detected for pig Delta coronavirus and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106967845A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557497A (en) * | 2017-10-20 | 2018-01-09 | 中国农业科学院兰州兽医研究所 | Pig Delta coronavirus TaqMan PCR kit for fluorescence quantitative and detection method |
| CN107630109A (en) * | 2017-10-27 | 2018-01-26 | 华南农业大学 | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus |
| CN108842001A (en) * | 2018-07-10 | 2018-11-20 | 山东新希望六和集团有限公司 | For detecting the primer and probe, PCR kit for fluorescence quantitative and methods and applications of pig Delta coronavirus |
| CN109097490A (en) * | 2018-07-10 | 2018-12-28 | 广西壮族自治区兽医研究所 | A kind of the duplex RT-PCR detection primer and kit of quick differentiation PDCoV and PReoV |
| CN109536642A (en) * | 2018-12-19 | 2019-03-29 | 长江大学 | A kind of universal pig fourth type coronavirus RT-Nested PCR detection method |
| CN113981149A (en) * | 2021-11-23 | 2022-01-28 | 广东省农业科学院动物卫生研究所 | Porcine delta coronavirus detection primer group, probe, kit and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886134A (en) * | 2009-05-12 | 2010-11-17 | 中山大学达安基因股份有限公司 | Swine flu virus real-time fluorescent RT-PCR typing kit |
| CN105925729A (en) * | 2016-06-29 | 2016-09-07 | 广东温氏食品集团股份有限公司 | Primer, probe, kit and method for fluorogenic quantitative PCR detection on pig delta coronavirus |
-
2017
- 2017-04-20 CN CN201710261995.0A patent/CN106967845A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886134A (en) * | 2009-05-12 | 2010-11-17 | 中山大学达安基因股份有限公司 | Swine flu virus real-time fluorescent RT-PCR typing kit |
| CN105925729A (en) * | 2016-06-29 | 2016-09-07 | 广东温氏食品集团股份有限公司 | Primer, probe, kit and method for fluorogenic quantitative PCR detection on pig delta coronavirus |
Non-Patent Citations (3)
| Title |
|---|
| JIANQIANG ZHANG等: "Evaluation of two singleplex reverse transcription-Insulatedisothermal PCR tests and a duplex real-time RT-PCR test for thedetection of porcine epidemic diarrhea virus and porcinedeltacoronavirus", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| TSUNEYUKI MASUDA等: "Development of one-step real-time reverse transcriptase-PCR-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea", 《JAPANESE JOURNAL OF VETERINARY RESEARCH》 * |
| 刘玲玲等: "新发PDCoV和TGEV双重RT-PCR检测方法的建立及初步应用", 《农业生物技术学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557497A (en) * | 2017-10-20 | 2018-01-09 | 中国农业科学院兰州兽医研究所 | Pig Delta coronavirus TaqMan PCR kit for fluorescence quantitative and detection method |
| CN107630109A (en) * | 2017-10-27 | 2018-01-26 | 华南农业大学 | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus |
| CN108842001A (en) * | 2018-07-10 | 2018-11-20 | 山东新希望六和集团有限公司 | For detecting the primer and probe, PCR kit for fluorescence quantitative and methods and applications of pig Delta coronavirus |
| CN109097490A (en) * | 2018-07-10 | 2018-12-28 | 广西壮族自治区兽医研究所 | A kind of the duplex RT-PCR detection primer and kit of quick differentiation PDCoV and PReoV |
| CN109536642A (en) * | 2018-12-19 | 2019-03-29 | 长江大学 | A kind of universal pig fourth type coronavirus RT-Nested PCR detection method |
| CN113981149A (en) * | 2021-11-23 | 2022-01-28 | 广东省农业科学院动物卫生研究所 | Porcine delta coronavirus detection primer group, probe, kit and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| CN106967845A (en) | A kind of kit detected for pig Delta coronavirus and detection method | |
| CN111363860A (en) | Nucleic acid composition for detecting novel coronavirus COVID-19 and application | |
| CN105695634B (en) | Detect PCR primer, kit and its application of African swine fever virus | |
| CN107630109A (en) | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus | |
| Hu et al. | A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3 | |
| CN110512027B (en) | Quintuple RT-PCR detection method for pig viral diarrhea pathogen | |
| CN108950068A (en) | A kind of avian infectious bronchitis virus QX type strain identification detection kit | |
| CN105886667A (en) | Detection kit for porcine epidemic diarrhea virus and detection method thereof | |
| CN104774953A (en) | Fluorescent PCR (polymerase chain reaction) detection reagent for African swine fever virus (ASFV) CP530R gene, and preparation method and application thereof | |
| CN105112558B (en) | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I | |
| CN103146846A (en) | Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit | |
| CN107937610A (en) | For differentiating the triple real-time fluorescence PCR primers of highly pathogenic H7N9 influenza viruses and probe | |
| CN102534052B (en) | Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV) | |
| CN101985664B (en) | Nucleotide sequence and kit for detecting type-A influenza virus | |
| Li et al. | Fast detection of duck circovirus by real-time fluorescence-based recombinase-aided amplification | |
| CN110643740A (en) | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for Palimam serogroup virus | |
| CN102912038B (en) | RT-HDA kit and primer for detecting avian influenza virus | |
| CN105925729A (en) | Primer, probe, kit and method for fluorogenic quantitative PCR detection on pig delta coronavirus | |
| CN106521038B (en) | A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit | |
| CN108676922A (en) | Primer and probe for detecting Porcine epidemic diarrhea virus street strain and TaqMan real time fluorescence quantifying PCR methods | |
| CN108130385A (en) | A kind of human cytomegalovirus kit for detecting nucleic acid | |
| CN111647683B (en) | New coronavirus 2019-nCoV nucleic acid detection kit and application thereof | |
| CN107227380A (en) | The primer sequence and method of a kind of synchronous detection PCV2 and PRV infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170721 |
|
| RJ01 | Rejection of invention patent application after publication |