CN106119421A - Fluorescent quantitation detection primer, probe and the test kit of pig blue-ear disease QYYZ strain - Google Patents
Fluorescent quantitation detection primer, probe and the test kit of pig blue-ear disease QYYZ strain Download PDFInfo
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Abstract
The invention discloses fluorescent quantitation detection primer, probe and the test kit of a kind of pig blue-ear disease QYYZ strain.Described primer is as shown in SEQ ID NO:1 and SEQ ID NO:2, and described probe is as shown in SEQ ID NO:3.Described test kit comprises described primer and probe.Described fluorescence quantitative detecting method is: through reverse transcription, the RNA of detected sample is obtained DNA profiling, utilizes described primer and probe to carry out fluorescent quantitative PCR, and sentence read result.The fluorescent quantitation detection system of the QYYZ strain that the present invention provides, specificity and sensitivity are the highest, simple operation, practicality, the QYYZ strain of Porcine reproductive and respiratory syndrome variation strain and other strain can carry out quick discriminating accurately and distinguish.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to the fluorescent quantitation detection of a kind of pig blue-ear disease QYYZ strain
Primer, probe and test kit.
Background technology
Porcine reproductive and respiratory syndrome (PRRS), also known as reproductive and respiratory syndrome, is a kind of breeding difficulty with in-pig and piglet
The viral infectious that is characterized of respiratory tract disease, this disease has become one of main epidemic disease of harm whole world pig industry.In recent years
Coming, the variation phenomenon of PRRSV attracts attention, and external research shows, deposits between the isolated strain of same gene type
At obvious sequence difference, the variation of PRRSV is to cause one of unmanageable major reason of primary disease.China's outburst in 2006
High-pathogenicity porcine reproductive and respiration syndrome, cause the biggest loss to China's pig industry, and its cause of disease is exactly PRRSV variation strain,
There is deletion mutation at NSP2 genome in this strain, and former classical strain vaccine is poor to variation strain's protectiveness, is the outburst of this disease
Main cause.In recent years, there is the reproductive and respiratory syndrome with in-pig miscarriage as cardinal symptom in a lot of pig farms, and the vaccine of immunity is to this at present
Reproductive and respiratory syndrome protectiveness is poor.Being found by nucleotide sequence analysis, cause of disease is Lan Er variation strain QYYZ strain (Qingyuan City's strain).At present
Vaccine limited to this virus control effect, it is therefore necessary to grope the preventing control method for this cause of disease.
Detection and Epidemiological study are the bases of preventing control method research, and the most conventional method is sequencing, is with core
Based on the clone of thuja acid and Sequence Detection technology, concrete operation step is first to use the special of primer amplified sample nucleic
Fragment gene, enters PMD-18T carrier by genes of interest fragment purification rear clone, then the carrier cloned is sent to order-checking company enters
Row order-checking, finally will record sequence and compare, with QYYZ with respiration syndrome vaccine poison QYYZ strain with high-pathogenicity porcine reproductive
Strain sequence homology is higher, and has special insertion sequence, then be judged to QYYZ strain, it is generally required to 7～10 day time.
The major defect of sequencing is that the detection cycle is longer, relatively costly, sensitivity is low.The reason of detection cycle length is real
Testing step more, order-checking inherently needs the long period；Cost height is because needing more reagent and the instrument, general order-checking are all
It is sent to the paying detection of outer company；Sensitivity is low to be because checking order and is first intended to expand this strain sequence with pair of primers,
And whether can amplify sequence, determined by the own efficiency of design of primers and PCR method, general PCR method relatively quantitative fluorescent PCR side
Method sensitivity is low.
As can be seen here, prior art could be improved in detection and qualification QYYZ strain.
Summary of the invention
In view of this, it is necessary to for above-mentioned problem, it is provided that the fluorescent quantitation detection of a kind of pig blue-ear disease QYYZ strain is drawn
Thing, probe and test kit, primer, probe and test kit that the present invention provides are highly sensitive, react quick, easy and simple to handle.
The present invention is by the following technical solutions:
The fluorescent quantitation detection primer of pig blue-ear disease QYYZ strain, the gene order of described primer is:
Forward primer: 5 '-TCACATGAGATTGGCCTTGCT-3 ' (SEQ ID NO:1)；
Downstream primer: 5 '-CGCAGGTGCCGTCGTTC-3 ' (SEQ ID NO:2).
The fluorescent quantitation detection probe of pig blue-ear disease QYYZ strain, described probe gene order is:
Probe: 5 '-AAGGAGATGAGCAGCCCTTCGTG-3 ' (SEQ ID NO:3).
Further, 5 ' end mark fluorescent reporter groups of described probe；3 ' end mark fluorescent cancellation bases of described probe
Group.
Further, the group of 5 ' end labellings of described probe is FAM；The group of 3 ' end labellings of described probe is BQ1.
Further, the fluorescence quantitative detection kit of pig blue-ear disease QYYZ strain, consist predominantly of described primer and probe；
Primer sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2, and probe sequence is as shown in SEQ ID NO:3.
Further, the fluorescence quantitative detection kit of described pig blue-ear disease QYYZ strain, specifically include:
(1) reverse transcription part:
RT premixed liquid 1: random primer and dNTP mix；
2:5 times of reverse transcription buffer of RT premixed liquid, RNase inhibitor, reverse transcription, without the distilled water of RNase；
(2) quantitative PCR part:
Quantitative PCR premixed liquid: quantitative PCR buffer, Taq archaeal dna polymerase, primer SEQ ID NO:1, SEQ ID NO:2
With probe SEQ ID NO:3, distilled water.
Further, the fluorescent quantitation detection primer of pig blue-ear disease QYYZ strain, probe application in fluorescence quantitative detecting method,
Concrete operations are: extract the RNA in detected sample, use after reverse transcription primer SEQ ID NO:1, SEQ ID NO:2 and
Probe SEQ ID NO:3 carries out real-time fluorescence quantitative PCR amplification；
Reaction condition: wherein the program of reverse transcription is 42 DEG C, 15 minutes；95 DEG C, 2 minutes.Quantitative fluorescent PCR program is 95
DEG C, 5 seconds；60 DEG C, 30 seconds, do 40 circulations by this reaction condition.
Result and interpretation:
The QYYZ strain of reporter group FAM has that amplification curve occurs Ji Shi reproductive and respiratory syndrome variation strain, if nothing
Amplification curve produces, then without the QYYZ strain of reproductive and respiratory syndrome variation strain in explanation sample.Wherein amplification curve CT value
≤ 35 be judged to the positive；CT value ＞ 35 and≤37 be judged to is suspicious, need to repeat test, CT value still sentencing in this scope for the second time
For the positive；If CT value ＞ 37, beyond the detection range of this method, it is determined that for feminine gender.
Compared with prior art, the method have the advantages that
The invention provides primer and the probe of one group of quantitative fluorescent PCR, this primer specificity and sensitivity are the highest, and
It is capable of detecting when the reproductive and respiratory syndrome variation strain QYYZ strain of variation, various clinical samples can be detected, reflect
Not, such that it is able to the reproductive and respiratory syndrome variation strain QYYZ strain strain of pop makes a distinction efficiently, simple to operate,
Practical.
Compared with sequence measurement, the present invention has that the time is short, simple to operate and several respects advantage such as sensitivity is high.Time
Short, simple to operate being because fluorescence quantifying PCR method, operating procedure is few, saves the plenty of time, and highly sensitive to be because fluorescence fixed
Amount PCR method uses the technical combinations that fluorescent labelling techniques, laser technology, digital visualization techniques are integrated, and ratio is in sequence measurement
Conventional PCR method is sensitive.
Accompanying drawing explanation
Fig. 1 specificity experiments of the present invention result: the curve to different strain nucleic acid samples amplifications, wherein A is for breeding and exhaling
Inhaling the QYYZ strain of syndrome variation strain, B is JXA1 variant, and C is CH-1a classics strain, and D is class NADC30 strain, and E is hog cholera
Poison nucleic acid amplification curve.
Fig. 2: susceptiveness assay of the present invention: 1,2,3,4,5,6,7 copy numbers represented are respectively 1.92 × 106、
1.92×105、1.92×104、1.92×103、1.92×102、1.92×101, 1.92,8 be negative control.
Fig. 3: Repeatability checking result of the present invention: wherein A is variant, B and C is 3 replica tests of same sample, D
For negative control.
Detailed description of the invention
Primer, the design of probe:
The porcine reproductive and respiratory syndrome virus gene order logged according to NCBI: porcine reproductive and respiratory syndrome virus
QYYZ strain (accession number: JQ308798), highly pathogenic PRRSV JXA1 strain (accession number
EF112445.1), highly pathogenic PRRSV HuN (accession number: EF517962.1), PRRSVBJ-4 (steps on
Record number: AF331831.1), PRRSV-HB-1 (accession number AY150312.1), PRRSVNADC30 (accession number JN654459.1) carry
The information of confession, through comparison analysis, devises primer and the probe of above quantitative PCR, is respectively as follows:
Forward primer: 5 '-TCACATGAGATTGGCCTTGCT-3 ' (SEQ ID NO:1)
Downstream primer: 5 '-CGCAGGTGCCGTCGTTC-3 ' (SEQ ID NO:2)
Probe: 5 '-AAGGAGATGAGCAGCCCTTCGTG-3 ' (SEQ ID NO:3)
Detection kit:
The fluorescence quantitative detection kit of pig blue-ear disease QYYZ strain, including:
(1) reverse transcription part:
RT premixed liquid 1: random primer 25 μm and dNTP mix 5mm, totally 60 μ L；
2:5 times of reverse transcription buffer of RT premixed liquid, RNase inhibitor 5U/ μ L, reverse transcription 25U/ μ L, totally 110 μ L；
Distilled water without RNase: 300 μ L；
(2) quantitative PCR part:
Quantitative PCR premixed liquid: 2 times of quantitative PCR buffer, Taq archaeal dna polymerase 6U/ μ L, shown in primer SEQ ID NO:1
0.8pm, totally 700 μ L shown in 1pm shown in 1pm, SEQ ID NO:2 and probe SEQ ID NO:3；
Distilled water 700 μ L.
(3) yin and yang attribute control section: negative control 1.5mL；Positive control 1.5mL.
Detection method:
1, extracting the RNA in detected sample, concrete operations are:
Blood sample to be checked is centrifuged 5 minutes through 8000g, takes upper serum standby；Histoorgan sample needs with 1:5's
Mass volume ratio mixes with sterilizing distilled water, and after grinding, 8000g is centrifuged 5 minutes, takes supernatant standby.
Serum or tissue supernatant AxyGen body fluid viral DNA/RNA pillar method extraction agent box in a small amount (also can use other
Method for extracting nucleic acid) extract nucleic acid, save backup.
2, being cDNA by the RNA reverse transcription of extraction, concrete operations are
(1) reverse transcription reaction system (every part):
RT premixed liquid 1:1 μ L, RT premixed liquid 2:2 μ L, without the distilled water of RNase: 5 μ L, adds and extracts RNA:2 μ L, altogether
10μL。
(2) reverse transcription reaction program:
It it is 42 DEG C, 15 minutes；95 DEG C, 2 minutes.
3, cDNA after reverse transcription being carried out fluorescent quantitative PCR, concrete operations are:
(1) quantitative PCR reaction system (every part):
Quantitative PCR premixed liquid: 12.5 μ L, distilled water: 7.5 μ L.
In above-mentioned PCR reaction system, add 5 μ L invert the cDNA liquid recorded.
(2) fluorescent quantitative PCR program:
95 DEG C, 5 seconds；60 DEG C, 30 seconds, 40 circulations, collect fluorescence signal for wherein 60 DEG C, 30 seconds.
More than operation gained amplified production sequence is as follows:
5-TCACATGAGATTGGCCTTGCTAAAGGAGATGAGCAGCCCTTCGTGCCGAACGACGGCACCTGCG-3
(SEQ ID NO:4)
Quickly the QYYZ strain of Porcine reproductive and respiratory syndrome variation strain and other strain are carried out fast by amplification curve
Discriminating distinguish, concrete operations are:
1, prepared by sample
(1) sample collecting: die of illness or catch and kill pig, takes lung or lymph node；Live hog to be checked, take a blood sample 1mL, and 2～8 DEG C of preservations are sent
Test in laboratory.
(2) sample treatment: die of illness or catch and kill the tissue sample of pig, taking about 1g sample from every part of three diverse locations, use hands
Art takes 0.2g after shredding mixing, adds 1mL normal saline and grinds, and after being homogenized, 8000g is centrifuged 5 minutes, takes supernatant standby；
The blood sample of live hog to be measured is centrifuged 5 minutes through 8000g, takes upper serum standby.
2, the extraction of viral RNA
The sample of preparation, respectively takes 200 μ L together with negative control and positive control, with AxyGen body fluid viral DNA/RNA post
Sub-method extraction agent box (also can use other method for extracting nucleic acid) in a small amount extracts nucleic acid, saves backup.
3, reverse transcription operation
Often pipe adds 1 μ L RT premixed liquid 1,2 μ L RT premixed liquid 2, distilled water 5 μ L without RNase, adds extraction nucleic acid
Liquid 2 μ L, after mixing, by 42 DEG C, 15 minutes；95 DEG C, 2 minutes reaction conditions react in PCR instrument.
4, real-time fluorescence PCR operation
Every tube reaction liquid, quantitative PCR premixed liquid: 12.5 μ L, distilled water: 7.5 μ L, the cDNA liquid that reversion records: 5 μ L,
The enterprising performing PCR of quantitative real time PCR Instrument reacts, and reaction condition is: 95 DEG C, 5 seconds；60 DEG C, 30 seconds, do 40 by this reaction condition and follow
Ring, collects fluorescence signal for wherein 60 DEG C, 30 seconds.
Whether testing result: after Fluorescence PCR terminates, have amplification curve and Ct according to positive control and negative control
Whether value judges whether experimental result is set up, if experimental result is set up, have amplification curve and Ct value to judge sample according to test sample
Whether product contain reproductive and respiratory syndrome variation strain QYYZ strain strain.
Interpretation of result: positive control Ct value≤25 also specific amplification curve occur, and negative control without Ct value, then tests knot
Fruit is set up；Also there is specific amplification curve in test sample Ct value≤35, it is determined that for reproductive and respiratory syndrome variation QYYZ strain
The positive, CT value ＞ 35 and≤37 be judged to is suspicious, test need to be repeated, CT value is still judged to the positive in this scope for the second time；If
CT value ＞ 37, beyond the detection range of this method, it is determined that for feminine gender；Specific amplification curve do not occurs for some, but background
Higher sample, it is determined that for feminine gender.
Test a specificity experiments
Take known containing Porcine reproductive and respiratory syndrome QYYZ strain, JXA1 variant, CH-1a classics strain, the strain of class NADC30
Sample, take 200 μ L AxyGen body fluid viral DNAs/RNA pillar method extraction agent box in a small amount after process and extract nucleic acid, then
Carry out reverse transcription and fluorescent PCR operation, obtain a result as it is shown in figure 1, contain the sample of Porcine reproductive and respiratory syndrome QYYZ strain
Amplifying A curve in Fig. 1, Ct value is 15.26, and result is judged to the positive；Containing JXA1 variant, CH-1a classics strain, class
The sample of NADC30 strain does not amplifies specific curve, it is determined that for feminine gender.Thus result understands the present invention program good spy
The opposite sex.
Test two susceptiveness inspections
It is 1.92 × 10 by concentration known9The PMD-18T plasmid of copy/mL (has been cloned into sequence 5-
TCACATGAGATTGGCCTTGCTAAAGGAGATGAGCAGCCCTTCGTGCCGAACGACGG CACCTGCG-3) carry out 10 times and be
Row dilution, is diluted to 1.92 × 103Copy/mL, each dilution factor takes 1 μ L, addition quantitative PCR premixed liquid: 12.5 μ L, double steamings
Water: 11.5 μ L, carries out quantitative PCR reaction on quantitative real time PCR Instrument, result as shown in Figure 2, curve 1,2,3,4,5,6,
7Ct value is 17.28,20.37,27.25,23.75,30.78,33.59,37.12 respectively, and corresponding plasmid copy number is respectively
1.92×106、1.92×105、1.92×104、1.92×103、1.92×102、1.92×101, 1.92, thus result understand,
The solution of the present invention has very great number sensitivity.
Test three Repeatability checkings
Take the known sample containing Porcine reproductive and respiratory syndrome QYYZ strain and be divided into 3 parts, after process, take 200 μ L AxyGen
Body fluid viral DNA/RNA pillar method extraction agent box in a small amount extracts nucleic acid, then carries out reverse transcription and fluorescent PCR operation, draws
As shown in Figure 3, curve A, B, C in Fig. 3, corresponding Ct value is for 16.57,16.39 and 16.89 to result respectively, and result judges
For the positive；Thus result understands the present invention good repeatability.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (7)
1. the fluorescent quantitation detection primer of a pig blue-ear disease QYYZ strain, it is characterised in that the gene order of described primer is SEQ
ID NO:1 and SEQ ID NO:2.
2. the fluorescent quantitation detection probe of a pig blue-ear disease QYYZ strain, it is characterised in that described probe gene order is SEQ
ID NO:3。
Probe the most according to claim 2, it is characterised in that 5 ' end mark fluorescent reporter groups of described probe；Described
3 ' end mark fluorescent quenching groups of probe.
Probe the most according to claim 3, it is characterised in that described fluorescent reporter group is FAM；Described fluorescent quenching base
Group is BQ1.
5. the fluorescence quantitative detection kit of a pig blue-ear disease QYYZ strain, it is characterised in that comprise the pig described in claim 1
Pig blue-ear disease QYYZ strain described in the fluorescent quantitation detection primer of reproductive and respiratory syndrome QYYZ strain and any one of claim 2～4 glimmering
Light detection by quantitative probe.
Detection kit the most according to claim 5, it is characterised in that including:
(1) reverse transcription part:
RT premixed liquid 1: random primer and dNTP mix；
2:5 times of reverse transcription buffer of RT premixed liquid, RNase inhibitor, reverse transcription, without the distilled water of RNase；
(2) quantitative PCR part:
Quantitative PCR premixed liquid: quantitative PCR buffer, Taq archaeal dna polymerase；Primer SEQ ID NO:1, SEQ ID NO:2 and spy
Pin SEQ ID NO:3；Distilled water.
7. the fluorescent quantitation detection primer of pig blue-ear disease QYYZ strain, probe application in fluorescence quantitative detecting method, its feature
It is, comprises the following steps:
(1) product of the RNA reverse transcription of detected sample is used primer SEQ ID NO:1, SEQ ID NO:2 and probe SEQ
ID NO:3 carries out real-time fluorescence quantitative PCR amplification；
The response procedures of quantitative fluorescent PCR is 95 DEG C, 5 seconds；60 DEG C, 35 seconds, do 40 circulations by this reaction condition, wherein 60
DEG C, 30 seconds collect fluorescence signal；
(2) result interpretation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106435039A (en) * | 2016-12-26 | 2017-02-22 | 广东温氏食品集团股份有限公司 | Primers, probe, kit and method for detecting PRRS (porcine reproductive and respiratory syndrome) NADC-30 strain |
| CN114410843A (en) * | 2022-01-29 | 2022-04-29 | 龙岩学院 | Fluorescent quantitative detection primer and probe set for simultaneously identifying four types of strains |
| CN116004920A (en) * | 2022-12-30 | 2023-04-25 | 北京亿森宝生物科技有限公司 | Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome |
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| CN103725794A (en) * | 2013-09-17 | 2014-04-16 | 广西壮族自治区动物疫病预防控制中心 | Fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) primers, probes and method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) |
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| CN103725794A (en) * | 2013-09-17 | 2014-04-16 | 广西壮族自治区动物疫病预防控制中心 | Fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) primers, probes and method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106435039A (en) * | 2016-12-26 | 2017-02-22 | 广东温氏食品集团股份有限公司 | Primers, probe, kit and method for detecting PRRS (porcine reproductive and respiratory syndrome) NADC-30 strain |
| CN114410843A (en) * | 2022-01-29 | 2022-04-29 | 龙岩学院 | Fluorescent quantitative detection primer and probe set for simultaneously identifying four types of strains |
| CN116004920A (en) * | 2022-12-30 | 2023-04-25 | 北京亿森宝生物科技有限公司 | Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome |
| CN116004920B (en) * | 2022-12-30 | 2023-11-17 | 北京亿森宝生物科技有限公司 | Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome |
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| CN106119421B (en) | 2019-12-03 |
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