CN101343671A - Fast detecting reagent kit for porcine type 2 circular ring virus PCR - Google Patents
Fast detecting reagent kit for porcine type 2 circular ring virus PCR Download PDFInfo
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- CN101343671A CN101343671A CNA2008100321420A CN200810032142A CN101343671A CN 101343671 A CN101343671 A CN 101343671A CN A2008100321420 A CNA2008100321420 A CN A2008100321420A CN 200810032142 A CN200810032142 A CN 200810032142A CN 101343671 A CN101343671 A CN 101343671A
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Abstract
The invention discloses a mature PCR quick detection reagent kit made through steps that a pair of pig 2 type circular ring virus specificity amplification primers are designed according to a sequence issued on a pig 2 type circular ring virus net, advanced virus nucleic acid extractive technique and highly efficient PCR commodity reagent are combined, and the primers are optimized and combined under experimental conditions. The reagent kit can directly detect tissues to be detected, the detection process is simple, and the contamination resistance is strong. The process from specimen treatment to result acquisition only requires 4 hours, the result is accurate, the sensitivity is high, and the specificity is good, so the detection reagent kit is the best replacing method to the existing detection methods for the pig 2 type circular ring virus.
Description
Technical field
The present invention relates to the diagnostic techniques in Vet Biotechnology field, specifically is a kind of diagnostic method and dedicated kit that detects pig 2 type PCV-II.
Background technology
Pig circular ring virus (Porcine Circovirus PCV) is the animal virus of the present minimum of finding, the about 17nm of virus particle diameter, and for covalence closed, ring-type, Single-stranded DNA virus, PCV have two kinds of genotype: PCV1 and PCV2.PCV1 no pathogenicity, but extensively be present in the pig body and pig source continuous cell line, PCV2 has pathogenic to pig, mainly cause multisystemic exhaustion syndrome behind the weaned piglet (post-weaning multisystemic wasting syndrome, PMWS).PMWS is a kind of new transmissible disease, finds in Canada first in 1991, mainly betide 5～12 age in week weanling pig, show as progressive emaciation, expiratory dyspnea, ochrodermia, diarrhoea, jaundice.Subsequently, the U.S., Britain, Germany, France, Ireland, Czech, Spain, TaiWan, China, Holland, Switzerland, Argentina, Mexico, Japan, Denmark, Italy, Korea S, Hungary, Thailand etc. have reported in succession should disease, epidemiology survey shows, this interior syndrome state also is widely current, and causes suitable serious economy loss.PCV2 is except causing PMWS, also can cause growing and fattening pigs dermatitis and nephrotic syndrome (porcine dermatitis andnephropathy syndrome, PNDS), and with the breeding difficulty of farrowing sow, the congenital of newborn piglet trembled, and newborn piglet diarrhoea and hyperplasia necrotizing pneumonia are in close relations.Epidemiology, clinical symptom and dissection pathological change according to PMWS can be made tentative diagnosis to the PCV2 infection, still need be with laboratory diagnosis technology for detection PCV2 antigen but make a definite diagnosis.
At present, the PCV2 detection technique that adopts mainly contains both at home and abroad: viral separation and Culture, indirect immunofluorescence (IFA), immunoenzyme monolayer assay (IMPA), in situ hybridization (ISH), enzyme linked immune assay (ELISA) etc., these methods all exist complicated operation, requirement for experiment condition height, not strong, the consuming time length of specificity, be not suitable for deficiency such as extensive detection.Therefore, develop quick, the easy PCV2 detection reagent be applicable to common lab, realize the quick diagnosis of PCV2, the prevention and the control of eqpidemic disease is had great significance.
Summary of the invention
The present invention is according to the online reported sequence of pig 2 type PCV-II, design a pair of pig 2 type PCV-II specificity amplification primers, in conjunction with advanced viral nucleic acid extractive technique and High Efficiency PC R commercially available reagent, a kind of sophisticated PCR quick detection kit that forms through experiment condition optimum combination.This test kit can directly detect tissue to be checked, and testing process is simple, and resistance to crocking is strong.Handle the acquisition result from pathological material of disease and only need 4 hours, the result is accurate and highly sensitive, specificity is good, is that present pig 2 type PCV-II detect extraordinary a kind of alternative method.
Main technical schemes of the present invention has:
1. design of primers: according to the online reported sequence of pig 2 type PCV-II, design a pair of pig 2 type PCV-II specificity amplification primers, primer is in detail:
Primer?P1：5’-CCGCGGGCTGGCTGAACTT-3’
Primer?P2：5’-ACCCCCGCCACCGCTACC-3’
Wherein Primer P1/P2 primer goes out the product of 1154bp to specific amplification in pig 2 type PCV-II nucleic acid.
2. the nucleic acid extraction of pathological material of disease to be checked: adopt the DNA rapid extracting method to extract the DNA nucleic acid of tissue sample to be checked.
3.PCR system: the PCR reaction system is 25 μ L, comprise 10 * PCR buffer (containing Mg2+), 2.5 μ L, concentration is respectively the dNTPs 2 μ L of 2.5mmol/L, 5U/ μ L TaqDNA polysaccharase 0.5 μ L, each 1 μ L of 20 μ mol/L Primer P1/P2, DNA extraction solution 10 μ L supply 25 μ L with distilled water.
4.PCR amplification program: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 68 ℃ are extended 8min.4 ℃ of preservations.
5. electrophoresis detection: get 8 μ LPCR products, mix 1 μ L sample-loading buffer, 1～1.5% agarose gel electrophoresis is analyzed the PCR product, is reference with dna molecular amount Marker.
6. the result judges: do not have band at negative control, positive control has under the prerequisite of obvious 1154bp band, sample the 1154bp size strip occurs and is decided to be the Pestivirus suis nucleic acid positive, otherwise is judged to feminine gender.
Composition of this test kit and preservation
Damping fluid GA 1.5mL * 1 pipe room temperature storage
Damping fluid GB 3mL * 1 pipe room temperature storage
Protein liquid removal GD 6mL * 1 pipe room temperature storage
Rinsing liquid PW 25mL * 1 pipe room temperature storage
Dehydrated alcohol 3mL * 1 pipe room temperature storage
Elution buffer TE 2mL * 1 pipe room temperature storage
Proteinase K 0.3mL *-20 ℃ of storages of 1 pipe avoids multigelation.
12 room temperature storage of adsorption column CB3 are avoided multigelation.
12 room temperature storage of 2mL collection tube
36 room temperature storage of sterilization centrifuge tube
PCR system 15 μ L *-20 ℃ of storages of 12 pipes avoid multigelation.
Positive control 1mL *-20 ℃ of storages of 1 pipe avoids multigelation.
Sterilization H
2O 1mL *-20 ℃ of storages of 1 pipe
This test kit validity period 6 months.
Advantage of the present invention
1. simple to operate, resistance to crocking is strong, weak point consuming time.Present method has adopted advanced viral nucleic acid extractive technique, and is simple to operate, and better pollution resistance is arranged, and improved the accuracy of detected result; Simultaneously, the High Efficiency PC R commercially available reagent of employing has improved detection sensitivity.
2. high specificity, good reproducibility, susceptibility height.This test kit only has amplified production to the nucleic acid of pig 2 type PCV-II, and the nucleic acid of Pestivirus suis, porcine reproductive and respiratory syndrome virus and pseudorabies virus and healthy animal tissue is not had amplified production; Detected result repeatability 100% to pig 2 type PCV-II standard strains and the positive pathological material of disease of pig 2 type PCV-II; Nucleic acid extraction liquid through the clinical positive pathological material of disease of 106 times of dilutions there is stable and accurate detected result.
Description of drawings
The sequence of Fig. 1, primer Primer P1, Primer P2
Fig. 2, this test kit detect positive products order-checking back sequence and standard strain sequence alignment result
Fig. 3, this test kit are to the detected result figure of sample segment
The concrete operations mode
1. specimen preparation
1.1 tissue sample: the 0.5g of clip parenchymal tissue to be checked (removing lymphoglandula, spleen, the kidney of manadesma) puts and shreds in the mill and grind, adding 1ml PBS (pH7.2) grinds, with ground tissue juice to be checked, be transferred in the 1.5ml sterilization centrifuge tube, the centrifugal 1min of 6000rpm gets in the new 1.5ml sterilization centrifuge tube of supernatant 100 μ L to, adds 100 μ L damping fluid GA, mixing carries out nucleic acid extraction then.
1.2 positive control: get 100 μ L positive controls and 100 μ L damping fluid GA carry out nucleic acid extraction.
1.3 negative control: get 100 μ L aqua sterilisas and 100 μ L damping fluid GA carry out nucleic acid extraction.
2. operation steps
2.1 in the sample that 200 μ L handle well, add 20 μ L Proteinase K solution (20mg/mL), mixing is placed 15～30min in 56 ℃ of water-baths, clarifies to solution.
2.2 add 220 μ L damping fluid GB, fully put upside down mixing, 10min, the centrifugal 30s of 10000rpm are placed in 70 ℃ of water-baths.
2.3 add 220 μ L dehydrated alcohols, the mixing 15 seconds of fully vibrating, the centrifugal 30s of 10000rpm.
2.4 all solution are transferred among the adsorption column CB3 (adsorption column is put into collection tube), and the centrifugal 30s of 12000rpm outwells waste liquid, adsorption column CB3 puts back in the collection tube.
2.5 add 500 μ L protein liquid removal GD in adsorption column CB3, the centrifugal 30s of 12000rpm outwells waste liquid, adsorption column CB3 puts back in the collection tube.
2.6 add 700 μ L rinsing liquid PW in adsorption column CB3, the centrifugal 30s of 12000rpm outwells waste liquid, adsorption column CB3 puts back in the collection tube.
2.7 add 500 μ L rinsing liquid PW in adsorption column CB3, the centrifugal 30s of 12000rpm outwells waste liquid.
2.8 adsorption column CB3 is put back in the collection tube the centrifugal 2min of 12000rpm.
2.9 adsorption column is moved in the new sterilization centrifuge tube, and at the elution buffer TE of unsettled Dropwise 5 0～200 μ L of film central authorities through 65～70 ℃ of water-bath preheatings, room temperature leaves standstill 5～10min, the centrifugal 2min of 12000rpm obtains sample total DNA.
2.10 get 10 μ L sample DNAs, be added in the PCR system mixing, the centrifugal 30s of 6000rpm.
2.11 place the PCR instrument, carry out following program: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 72 ℃ are extended 8min.4 ℃ of preservations.
2.12 get 8 μ LPCR products, mix 1 μ L sample-loading buffer, 1～1.5% agarose gel electrophoresis is analyzed the PCR product, is reference with dna molecular amount Marker.
2.13 do not have product at negative control, positive control has under the prerequisite of obvious 1154bp band, sample the 1154bp size strip occurs and is decided to be the PCV-2 nucleic acid positive, otherwise is judged to feminine gender.
Claims (2)
1. a pair of pig 2 type PCV-II specific detection primer Primer P1/P2, its sequence is:
Primer?P1：5’-CCGCGGGCTGGCTGAACTT-3’
Primer?P2：5’-ACCCCCGCCACCGCTACC-3’
Wherein Primer P1/P2 primer goes out the product of 1154bp to specific amplification in pig 2 type PCV-II nucleic acid.
2. the pig 2 type PCV-II quick detection kit of utilizing the described primer of claim 1 to form is characterized by:
Adopt the DNA rapid extracting method to extract the DNA of tissue sample to be checked, by following PCR reaction system and amplification program, with Primer P1/P2 primer to carrying out the RT-PCR augmentation detection of pig 2 type PCV-II.
The PCR system: the PCR reaction system is 25 μ L, comprise 10 * PCR buffer (containing Mg2+), 2.5 μ L, concentration is respectively the dNTPs 2 μ L of 2.5mmol/L, 5U/ μ L TaqDNA polysaccharase 0.5 μ L, each 1 μ L of 20 μ mol/L Primer P1/P2, DNA extraction solution 10 μ L supply 25 μ L with distilled water.
Pcr amplification program: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 68 ℃ are extended 8min.4 ℃ of preservations.
Electrophoresis detection: get 8 μ LPCR products, mix 1 μ L sample-loading buffer, 1～1.5% agarose gel electrophoresis is analyzed the PCR product, is reference with dna molecular amount Marker.
The result judges: do not have band at negative control, positive control has under the prerequisite of obvious 1154bp band, sample the 1154bp size strip occurs and is decided to be the pig 2 type PCV-II nucleic acid positives, otherwise is judged to feminine gender.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100321420A CN101343671A (en) | 2008-08-25 | 2008-08-25 | Fast detecting reagent kit for porcine type 2 circular ring virus PCR |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008100321420A CN101343671A (en) | 2008-08-25 | 2008-08-25 | Fast detecting reagent kit for porcine type 2 circular ring virus PCR |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012083837A1 (en) * | 2010-12-22 | 2012-06-28 | 福又达生物科技股份有限公司 | Porcine circovirus type 2, immune composition containing the same, assay kit, and use thereof |
| CN106929606A (en) * | 2017-04-14 | 2017-07-07 | 华南农业大学 | A kind of Testing and appraisal non-typical swine fever virus（APPV）PCR primer and detection method and detection kit |
| CN109825642A (en) * | 2019-02-25 | 2019-05-31 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | For detecting real-time fluorescence quantitative PCR primer, probe and its application of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement |
-
2008
- 2008-08-25 CN CNA2008100321420A patent/CN101343671A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012083837A1 (en) * | 2010-12-22 | 2012-06-28 | 福又达生物科技股份有限公司 | Porcine circovirus type 2, immune composition containing the same, assay kit, and use thereof |
| CN103314103A (en) * | 2010-12-22 | 2013-09-18 | 施怀哲维克有限公司 | Porcine circovirus type 2, immune composition containing the same, assay kit, and use thereof |
| CN103314103B (en) * | 2010-12-22 | 2015-06-24 | 施怀哲维克有限公司 | Porcine circovirus type 2, immune composition containing the same, assay kit, and use thereof |
| EA028376B1 (en) * | 2010-12-22 | 2017-11-30 | Сбк Вирбак Лимитед | Porcine circovirus type 2 (pcv2), immunogenic composition containing the same, test kit, and application thereof |
| CN106929606A (en) * | 2017-04-14 | 2017-07-07 | 华南农业大学 | A kind of Testing and appraisal non-typical swine fever virus（APPV）PCR primer and detection method and detection kit |
| CN109825642A (en) * | 2019-02-25 | 2019-05-31 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | For detecting real-time fluorescence quantitative PCR primer, probe and its application of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement |
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Open date: 20090114 |