CN103952500B - The fluorescence quantitative PCR detection primer of PRV (Pseudorabies virus) street strain, probe and test kit - Google Patents
The fluorescence quantitative PCR detection primer of PRV (Pseudorabies virus) street strain, probe and test kit Download PDFInfo
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Abstract
Does the present invention disclose the fluorescence quantitative PCR detection primer of a kind of PRV (Pseudorabies virus) street strain, probe and test kit, and the sequence of described primer is such as SEQ ID NO:1 and SEQ ID is the sequence of described probe such as SEQ shown in NO:2 ID shown in NO:3. Does the fluorescent quantificationally PCR detecting kit of described PRV (Pseudorabies virus) street strain comprise SEQ ID NO:1 and SEQ ID primer shown in NO:2 and SEQ ID probe shown in NO:3. This primer specificity and sensitivity are all higher, it is possible to detect out PRV street strain, it is possible to detected by various clinical sample, such that it is able to quickly PRV (Pseudorabies virus) street strain is carried out etiological diagnosis, simple to operate, practical.
Description
Technical field
The invention belongs to field of molecular biotechnology, it is specifically related to the fluorescence quantitative PCR detection primer of PRV (Pseudorabies virus) street strain, probe and test kit.
Background technology
Pseudoabies (Pseudorabies, PR) Ao Yeziji disease (Aujeszky'sdisease is had another name called, AD), it is by hcrpesviruses section (Herpesviridae) �� hcrpesviruses subfamily Pseudorabies virus (PseudorabiesVirus, what PRV) cause comprises ox, sheep, rabbit, dog, cavy, mouse etc. interior multiple domestic animal and wildlife to generate heat, very itch and encephalomyelitis is a kind of height contact of main symptom, acute infectious disease (RoizmornB.DesrosiersR, FleebensteinB, LoPezC, MinsonAdandstuddertMJ.TheFamilyHerpesviridae, anupdate.ArchVirol, 1992,123:425-449). pig is storage person and the contagium of this cause of disease, but pig infects this does not show the unusual symptom itched after being ill, and the pig of different days infects this and shows different clinical symptom after being ill: newborn piglet a large amount of death in 1 week after birth, weanling pig shows diarrhoea and nervous symptoms, growing-finishing pigs is slow, efficiency of feed utilization reduction etc., pregnant sow is miscarried, and produces the phenomenon such as stillborn foetus, mummy tire, sow returns feelings rate height, and Repeat breeding (Shuai Zi state. express structure and the immunity test research of the recombinant pseudorabies virus of Pseudorabies virus glycoprotein. [Ph D dissertation]. Jilin University, 2008).
Pseudoabies is found in the cows of the U.S. the earliest, symptom was mainly described as " very itching " at that time, itself and rabies differentiation are come by Hungary rabies scientist Aujeszky by animal experiment, people were called Aujes ' sdisease afterwards, the cause of disease Pseudorabies virus of this disease is first successfully separated by doctor schmiedhoffer and obtains (CharlesEcetal.AdvancesinveterinaryScienceandCompartiveMe dieine:SeletedAnimalHerPesvirus.Newconceptsandtechnologi es1985,35:4786-4792). The clinical symptom that initial pig occurs this disease to show is not remarkable, until the eighties in last century, the number of times that this disease breaks out in pig farm sharply rises, the harm caused is more and more serious, just cause worldwide extensive concern (Zhang Luan. the isolation identification of Xinjiang porcine pseudorabies epidemiology survey and virus strain. [Master's thesis]. Xibei Univ. of Agricultural & Forest Science & Technology, 2005).
At present swinery is carried out vaccine inoculation, the production sow eliminating pig farm band poison and strict bio-security by pseudoabies the most effective prevention and control measure beyond doubt. Currently, the pseudorabies disease vaccine being applied to large-scale pig farm mainly contains swine pseudorabies vaccine and gene-deleted vaccine two kinds, and wherein the application of gE gene-deleted vaccine is the most general. The vaccine of gE genetically deficient is not only very effective to kind of a pig, piglet, and newborn piglet pre-suckling vaocination is also fool proof, has strong protection, also has good urgent immunity control effect for morbidity pig; And by serology (ELISA) method, immunity swinery and wild virus infection swinery can be distinguished, the effect of swinery purification can be reached by a series of measures.
At home, but, PRV (Pseudorabies virus) street strain is still popular to a certain extent, and there is the possibility of outburst. Specificity and the susceptibility of the primer of existing detection PRV (Pseudorabies virus) street strain, probe and test kit are lower, can not PRV street strain be detected fast.
Summary of the invention
It is an object of the invention to for the problems referred to above of the prior art, by the analysis to PRV (Pseudorabies virus) gE gene, design primer and probe, it is possible to quick, special increases to the malicious street strain of pseudo-swine rabies, diagnose; And this primer is become with probe preparation test kit or is applied in the reagent of detection PRV (Pseudorabies virus) street strain.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The fluorescence quantitative PCR detection primer of PRV (Pseudorabies virus) street strain, the sequence of described primer is as shown in SEQIDNO:1 and SEQIDNO:2.
The fluorescence quantitative PCR detection probe of PRV (Pseudorabies virus) street strain, the sequence of described probe is as shown in SEQIDNO:3.
Preferably, 3 ' end combined with fluorescent quenching group of described probe, 5 ' end combined with fluorescent reporter group.
More preferably, the fluorescent reporter group of described probe is FAM, and fluorescent quenching group is TAMRA.
A fluorescent quantificationally PCR detecting kit for PRV (Pseudorabies virus) street strain, comprises the primer shown in SEQIDNO:1 and SEQIDNO:2 and the probe shown in SEQIDNO:3.
Preferably, the fluorescent quantificationally PCR detecting kit of described PRV (Pseudorabies virus) street strain, described test kit comprises quantitative PCR premixed liquid, described quantitative PCR premixed liquid is composed of the following components: pre-mixed enzyme system 10 �� L, primer 0.2 �� L shown in SEQIDNO:1, the primer 0.2 �� L shown in SEQIDNO:2, the probe 0.1 �� L shown in SEQIDNO:3, ROX0.04 �� L, and aseptic double-distilled water 7.46 �� L.
Compared with prior art, the present invention has following useful effect: the fluorescence quantitative PCR detection primer of PRV (Pseudorabies virus) street strain of the present invention, probe and test kit, this primer and probe specificity and sensitivity are all higher, and PRV street strain can be detected, various clinical sample can be detected, such that it is able to quickly the wild poison of PRV is diagnosed, simple to operate, practical.
Accompanying drawing explanation
Fig. 1 is the amplification curve of several samples in embodiment 1, and wherein curve 1 is tonsilla pathological material of disease PRV street strain amplification curve, and curve 2 is lymphoglandula pathological material of disease PRV street strain amplification curve.
Fig. 2 is embodiment 3 specific detection result, and wherein curve 1 is the amplification curve of the positive samples of PRV street strain; Curve 2-7 is respectively the amplification curve of transmissible gastro-enteritis virus, porcine rotavirus, pig breeding and disordered breathing syndrome virus, pig circular ring virus, Pestivirus suis and pseudorabies disease vaccine.
Fig. 3 is embodiment 4 sensitivity technique result, and the copy number that curve 1-8 represents respectively is respectively 6.27 �� 109��6.27��108��6.27��107��6.27��106��6.27��105��6.27��104��6.27��103��6.27��102copies/��L��
Embodiment
The present invention being explained further below in conjunction with specific embodiment, so that those skilled in the art can understand the present invention better and can be implemented, but embodiment is not as a limitation of the invention.
Embodiment 1 fluorescence quantifying PCR method detects PRV (Pseudorabies virus) street strain
According to the PRV (Pseudorabies virus) gE genome sequence that NCBI logs in, accession number: the information that AY170318, AF403049, EF552427, KC415026, KC415029, KC415028, KC415027EU561349, JF797219 provide, through compare of analysis, devise primer and the probe of quantitative fluorescent PCR, the sequence of described primer is as shown in SEQIDNO:1 and SEQIDNO:2, and the sequence of described probe is as shown in SEQIDNO:3.
SEQIDNO:1:AACCGGAAGTGACGAATGGA;
SEQIDNO:2:CGGTTCTCCCGGTATTTAAGC;
SEQIDNO:3:CCAACCGCCTGTTGATGTCCCG.
Use above-mentioned primer and probe to carry out fluorescent quantitative PCR, differentiate PRV (Pseudorabies virus) street strain fast and accurately by amplification curve. Concrete fluorescence quantifying PCR method is:
(1) the tonsilla pathological material of disease of sample DNA to be checked: 100mg and 100mg lymphoglandula and 200 �� LDMEM cell culture mediums is extracted as sample to be checked. Wherein, DMEM cell culture medium, not containing the wild poison of PRV, is negative control. The operation of concrete extraction sample DNA is as follows:
In sample to be checked, add 1mLPBS damping fluid fully grind ,-20 DEG C of multigelations 3 times, the centrifugal 10min of 8000rpm, get supernatant liquor 200 �� L, carrying out extracting with commercialization DNA extraction agent box, the DNA of extraction is stored in-20 DEG C, as the DNA profiling of subsequent experimental.
(2) amplification system: 2 �� LDNA templates joined in quantitative PCR premixed liquid, prepares 20 �� L reaction systems, and mixes. Wherein quantitative PCR premixed liquid is:
1.. mixed enzyme system in advance: THUNDERBIRDProbeqPCRMix10 �� L, purchased from TOYOBO company;
2.. each 0.2 �� L (being 20pmol) of upstream primer F and downstream R; The sequence of described upstream primer F is shown in SEQIDNO:1; The sequence of described downstream primer R is shown in SEQIDNO:2; By the synthesis of the precious biotechnology company limited in Dalian;
3. .ROX0.04 �� L;
4.. probe P (20pmol) 0.1 �� L, the sequence of probe P is shown in SEQIDNO:3, by the synthesis of upper sea base health Bioisystech Co., Ltd, the 5 ' end of probe P and 3 ' end flag F AM and TAMRA respectively;
5.. self-control aseptic double-distilled water 7.46 �� L.
(3) amplification condition: being placed on ABI company 7500Fast quantitative PCR instrument by the PCR pipe of step (2) and carry out amplified reaction, amplification condition is: 50 DEG C, keeps 2min; 95 DEG C, keep 10min; 95 DEG C, 15s, 60 DEG C, 1min, totally 40 circulations.
(4) result judges: have amplification curve occur and CT value be less than 35 be namely the wild poison positive of PRV. The judgement that wherein amplification curve CT value is less than 35 for positive, CT value be greater than 35 exceed present method sensitivity, be judged to feminine gender. Detected result is in table 1.
Table 1 sample detection result to be checked
| Sample to be checked | Amplification curve | Amplification judges |
| Tonsilla pathological material of disease | Have | Street strain |
| Lymphoglandula pathological material of disease | Have | Street strain |
| DMEM cell culture medium | Without amplification curve | Without street strain |
The fluorescent quantificationally PCR detecting kit of embodiment 2 PRV (Pseudorabies virus) street strain
A fluorescent quantificationally PCR detecting kit for PRV (Pseudorabies virus) street strain, comprises the primer shown in SEQIDNO:1 and SEQIDNO:2 and the probe shown in SEQIDNO:3. Described test kit also comprises quantitative PCR premixed liquid, described quantitative PCR premixed liquid is composed of the following components: pre-mixed enzyme system 10 �� L, primer 0.2 �� L shown in SEQIDNO:1, primer 0.2 �� L shown in SEQIDNO:2, probe 0.1 �� L shown in SEQIDNO:3, ROX0.04 �� L, and aseptic double-distilled water 7.46 �� L.
Embodiment 3 specific assay
Using positive pathological material of disease lapping liquid as positive control, to transmissible gastro-enteritis virus, porcine rotavirus, pig breeding and disordered breathing syndrome virus, pig circular ring virus, Pestivirus suis, pseudorabies disease vaccine 6 kinds of viruses, adopt the primer described in embodiment 1 and probe, utilize the fluorescence quantifying PCR method described in embodiment 1 to carry out specific detection. Transmissible gastro-enteritis virus, porcine rotavirus and pig circular ring virus are positive pathological material of disease, pig breeding all purchases the commercialized vaccine on market with disordered breathing syndrome virus, Pestivirus suis and porcine pseudorabies, wherein pig breeding and disordered breathing syndrome virus are big China agriculture JXA1-R vaccine, Pestivirus suis is Yongshun, Guangdong C-strain cell vaccine poison, and porcine pseudorabies is big China agriculture Bartha-K61 (pseudo-blue prestige) vaccine. As shown in Figure 2, result shows all not have amplification curve except positive control to detected result, and specificity is good.
Embodiment 4 sensitivity test
Taking the DNA of extraction from positive pathological material of disease as template, taking SEQIDNO:1��2 as primer, increase according to the fluorescence quantifying PCR method described in embodiment 1, obtain 86bp amplified production (sequence of amplified production is as shown in SEQIDNO:4), this amplified production is connected to cloning vector pMD18-T (the precious biotechnology company limited in Dalian) and prepares the standard positive plasmid (pMD-gE) containing gE gene. After measured, pMD-gE concentration is 6.27 �� 1010copies/��L��
The sterilizing distilled water 10 configured by positive plasmid pMD-gE doubly increases progressively dilution as template, each extent of dilution respectively gets 2 �� L as template, detect by the fluorescence quantifying PCR method described in embodiment 1, observe amplification curve, calculate its susceptibility so that the highest extent of dilution of the template used amount of positive expecting curve to occur. As shown in Figure 3, result shows that minimum detected level is 6.27 �� 10 to detected result2Copies/ �� L, sensitivity is good.
The above embodiment only have expressed several enforcement modes of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to patent scope of the present invention. , it is also possible to make some distortion and improvement, it should be appreciated that for the person of ordinary skill of the art, without departing from the inventive concept of the premise these all belong to protection scope of the present invention. Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. the fluorescence quantitative PCR detection primer of PRV (Pseudorabies virus) street strain, it is characterised in that, the sequence of described primer is as shown in SEQIDNO:1 and SEQIDNO:2.
2. the fluorescence quantitative PCR detection probe of PRV (Pseudorabies virus) street strain, it is characterised in that, the sequence of described probe is as shown in SEQIDNO:3.
3. the fluorescence quantitative PCR detection probe of PRV (Pseudorabies virus) street strain according to claim 2, it is characterised in that, 3 ' end combined with fluorescent quenching group of described probe, 5 ' end combined with fluorescent reporter group.
4. the fluorescence quantitative PCR detection probe of PRV (Pseudorabies virus) street strain according to claim 3, it is characterised in that, described fluorescent reporter group is FAM, and described fluorescent quenching group is TAMRA.
5. the fluorescent quantificationally PCR detecting kit of a PRV (Pseudorabies virus) street strain, it is characterised in that, comprise the primer shown in SEQIDNO:1 and SEQIDNO:2 and the probe shown in SEQIDNO:3.
6. the fluorescent quantificationally PCR detecting kit of PRV (Pseudorabies virus) street strain according to claim 5, it is characterised in that, described test kit comprises quantitative PCR premixed liquid, and described quantitative PCR premixed liquid is composed of the following components:
Pre-mixed enzyme system 10 �� L,
Primer 0.2 �� L shown in SEQIDNO:1,
Primer 0.2 �� L shown in SEQIDNO:2,
Probe 0.1 �� L shown in SEQIDNO:3,
ROX0.04 �� L,
With aseptic double-distilled water 7.46 �� L.
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| CN104388594B (en) * | 2014-12-09 | 2016-06-29 | 中国农业科学院兰州兽医研究所 | A kind of Taqman Real-time PCR kit for detecting PRV (Pseudorabies virus) |
| CN106591490B (en) * | 2016-12-15 | 2020-06-30 | 畜科生物工程有限公司 | Nucleic acid combination for detecting pseudorabies virus, kit and application |
| CN107502682A (en) * | 2017-10-17 | 2017-12-22 | 河南牧业经济学院 | A kind of preparation and its application process for being used to detect the genetic chip of the pseudo- mad sick dog of pig |
| CN111560465A (en) * | 2019-12-25 | 2020-08-21 | 龙岩学院 | qPCR (quantitative polymerase chain reaction) kit and method for pseudorabies wild virus and swine hepatitis E virus |
| CN112522446A (en) * | 2020-12-25 | 2021-03-19 | 河南宏信检测技术有限公司 | Detection primer pair and kit for wild strain of porcine pseudorabies virus |
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