CN101307367B - Technology for rapidly detecting porcine circovirus type2 - Google Patents
Technology for rapidly detecting porcine circovirus type2 Download PDFInfo
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- CN101307367B CN101307367B CN2008100822091A CN200810082209A CN101307367B CN 101307367 B CN101307367 B CN 101307367B CN 2008100822091 A CN2008100822091 A CN 2008100822091A CN 200810082209 A CN200810082209 A CN 200810082209A CN 101307367 B CN101307367 B CN 101307367B
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Abstract
The invention discloses a loop-mediated isothermal amplification fast detection method for porcine circovirus type 2, which belongs to the animal medical field. By using a special primer and utilizing a specific region of a platform amplification target sequence of a loop-mediated isothermal amplification (LAMP) technology, virus nucleic acid containing the porcine circovirus type 2 is detected according to molecular level with the assistance of a series of quality control as well as negative contrast and positive contrast. The method has the characteristics of simplicity, convenience, economization, sensitivity and specificity, and has broad application prospect.
Description
Technical field
The invention belongs to the animal medicine field, relate to a kind of method that from blood or tissue, detects virus, and the purposes of this method.More exactly, the present invention relates to utilize loop-mediated isothermal amplification technique and the method for porcine circovirus 2 type (PCV2) of detecting from blood or in the tissue researched and developed.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) be the member that PCV-II section PCV-II belongs to, belong to sub-thread cyclic DNA virus, it is one of animal virus of known minimum, and this virus does not have cyst membrane, 20 bodies of tool, mean diameter is 17nm, does not possess hemagglutination activity, can resist the sour environment that pH equals 3, can in pig source cell and vero cell, grow, but not produce cytopathy.Pathogenic, nucleotide sequence and antigenicity according to PCV can be divided into PCV two serotypes, i.e. PCV1 and PCV2.PCV1 and PCV2 have 80% homology approximately at nucleotide level.PCV1 no pathogenicity, PCV2 then are to cause multisystemic exhaustion syndrome (Post-weaning Multisystemic Wasting Syndrome, a kind of main virus PMWS) behind the weaned piglet.Some scholars such as Ellis report that pig parvoviral (PPV), PRV (Pseudorabies virus) (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) also can produce typical PMWS symptom, therefore be necessary when the PMWS symptom takes place swinery, to detect PCV2, and do further differential diagnosis with PCV1, PPV, PRV, PRRSV.Can carry out tentative diagnosis to PCV2 according to clinical symptom and pathological change, virus is the disconnected main laboratory diagnosis technology that relies on of diagnosis really.
The clinical classical symptom of PCV2 comprises progressive emaciation, ochrodermia, and expiratory dyspnea, apocleisis, spirit is depressed, is emitted fluffy and disorderly.Dissect to change and mainly comprise mesentery and the unusual enlargement of inguinal lymph nodes, lung's beige inflammatory disorders, if any concurrent or secondary infection, then pathology lung lesion such as seen hemorrhage, the sex change of its lymphoglandula or suppuration is also apparent in view, as lung swelling, sex change, surface many rubber shape lump not of uniform size etc. is arranged.Its liver surface adularescent hyperplasia decorative pattern of the case that has, what have then presents icterohepatitis.The cortex of lymphoglandula has monocyte or macrophages infiltration, also is studded with a large amount of multinuclear scavenger cells sometimes.Some case lymphopenia, lymphoglandula matrix hyperplasia occur or has eosinophil to soak into.The lymph follicle central part has cellular necrosis, forms by histocyte, epithelioid cell giant cells, polykaryocyte, lymphocyte and eosinophil leucocyte accumulative inflammatory granuloma around it.Lung has slight many kitchen ranges property or height diffuse interstitial pneumonia, because it is significantly plump that alveolar epithelial cells enlargement, hyperplasia, oedema and cellular infiltration often cause alveolus wall, causing has monocyte, neutrophil and eosinophil leucocyte to ooze out in the alveolar, lymphocyte and histiocytic infiltrate also take place in the part lung around segmental bronchus and blood vessel, and occur with syncyte once in a while.
The genome of PCV2 is made up of two main open reading frame (ORF), and wherein ORF1 is called the rep gene again, the albumen of a 35.7ku relevant with virus replication of coding; ORF2 is called the cap gene again, and the size of encoding is a 27.8ku antigen capsid protein.
The laboratory diagnosis technology of PCV2 comprise virus isolation identification and at PCV2 antigen and detection of antibodies technology.It mainly is to separate by inoculation experiments animal or sensitive cells that virus is separated, PCV2 separates laboratory animal commonly used and is generally the SPF pig, cell commonly used is porcine kidney cell or vero cell, take disease pig lung, liver, spleen, lymphoglandula, tissues such as tonsilla, make tissue homogenate, handle to remove the virus of cyst membrane with chloroform, and inoculate virus-free and porcine kidney cell mycoplasma contamination, utilize the glucosamine short period of time to handle cells infected simultaneously, to promote the propagation of virus. because the PCV2 cells infected does not produce pathology, therefore, usually determine existing of virus with round pcr, sometimes also available immunofluorescence technique detects the growing state of virus, or observes the PCV2 virus particle by electron microscopy and ultrathin sectioning.Confirm that now PCV2 is many on the PK15 cell could effectively to be bred for blind passage.Laboratory diagnostic method at virus antigen generally comprises polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry technique etc., wherein hybridization in situ technique can detect the virus antigen in the fixing organization, and immunohistochemistry technique can be used for the virus antigen localized observation of growing in tissue culture and body cell.Round pcr then with its sensitivity, special, advantage becomes one of the most frequently used technology of the diagnosis of present virusology, molecular biology experiment fast and accurately, it also be cause of disease at PCV2 detect and typing in the most frequently used technology.Detection technique at antiviral antibody mainly comprises indirect immunofluorescence (IFA), enzyme linked immunosorbent assay (EL ISA) and antigen capture EL ISA detection technique etc.
Summary of the invention
One of purpose of the present invention provides the loop-mediated isothermal amplification method that a kind of quick, easy, sensitive and economic being used for detects pig blood or tissue PCV2.
The present invention utilizes loop-mediated isothermal amplification technique, from pig blood or tissue, extract nucleic acid, utilize designed primer to carry out amplified reaction, whether the detection reaction product contains specific scalariform band, to determine the whether Fast Detection Technique of infected pigs's circovurus type 2 of pig.
The present invention is with pig blood or to organize nucleic acid extractive be template, and employed four Auele Specific Primers are:
Implementation method of the present invention is as follows: by using Auele Specific Primer, utilize the specific region on the LAMP technology amplification PCV2 genome, whether the formation by detecting specific product, from molecular level the PCV2 nucleic acid blood or the tissue sample carried out examination.
The LAMP detection method of PCV2 of the present invention is: with blood of tested pig or the DNA in the tissue is template, according to the PCV2 genome sequence, designs two pairs of Auele Specific Primers at the cap gene of PCV2 and carries out isothermal amplification.
Further, in the LAMP detection method of PCV2 of the present invention, introduced positive control, positive control is the recombinant vectors of the cap gene of the PCV2 that is connected with pMD18T.Employed detection method is 2% agarose electrophoresis detection.
(Loop-mediated isothermal amplification LAMP) is rapid, easy, accurate, nucleic acid recognizing technology that cost is low by a kind of alternative PCR of Japanese scientist Notomi invention to loop-mediated isothermal amplification technique.The detection that the LAMP technology is used for PCV2 has following advantage:
1. 4 kinds of primers are set at 6 positions of target gene, utilized strand replacement reaction under constant temperature, target gene efficiently to be increased, because its reaction is by the common startup of a plurality of primers, so more special than PCR.
2.LAMP technology has identical sensitivity with PCR, but required equipment is very simple, only needs the water-bath or the hot piece that can provide 60 ℃-65 ℃, can greatly reduce experimentation cost.
3.LAMP technology can be finished in general one hour, saves time than PCR.
Employed reagent comprises among the present invention: viral DNA extracts reagent and LAMP nucleic acid amplification reagent.The step of diagnostic method is as follows:
(1) nucleic acid extraction
MiniBEST viral RNA/DNA extraction the test kit that uses precious biotechnology (Dalian) company limited to provide can extract the DNA of PCV2 according to following method.
This operation designs for example with purified virus RNA/DNA from 250 μ l samples, when using the sample of other volume, the usage quantity of its SolutionA, Solution B, DB Buffer needs to adjust in proportion, but the usage quantity of Rinse A, Rinse B need not change.
The detailed step of operating process is as follows.
1) gets 250 μ l samples, add in the 1.5ml centrifuge tube.
2) SolutionA of adding 500 μ l, after thermal agitation was mixed, room temperature was placed 5 minutes.
3) add the Solution B of 125 μ l and the DB Buffer of 125 μ l, behind the uniform mixing, 12, centrifugal 5 minutes of 000rpm.
4) the Spin Column in the test kit is placed on the 2ml collection tube.
5) the supernatant liquor 850 μ l with aforesaid operations 3 are transferred among the Spin Column, and 12, centrifugal 1 minute of 000rpm (as among the Spin Column liquid residue being arranged, can suitably improve centrifugal speed, centrifugal again 1 minute) abandons filtrate.
6) the Rinse A with 500 μ l is added among the Spin Column, and 12, centrifugal 1 minute of 000rpm abandons filtrate.
7) the Rinse B with 500 μ l is added among the Spin Column, and 12, centrifugal 1 minute of 000rpm abandons filtrate.
8) repetitive operation step 7.
9) Spin Column is placed on the centrifuge tube of new 1.5ml, the sterile purified water or the Elution Buffer (no RNA enzyme) that add 30 μ l in the centre of Spin Column film, room temperature leaves standstill [to be annotated: for preventing the decomposition of RNA in 1 minute, can add RNase inhibitor (TaKaRa Code:D2310) in Elution Buffer, additional proportion is: the RNase inhibitor that adds 1 μ among per 50 μ l Elution Buffer].
10) 12, centrifugal 1 minute wash-out viral RNA/DNA of 000rpm.
(2) LAMP reaction
The LAMP reaction system is as follows: final concentration is respectively FIP and the BIP primer of 2.0 μ M, the F of 0.2 μ M and B primer, 1.0 mM dNTP, the Bst archaeal dna polymerase of 8U (New England Biolabs), 10 * buffer (containing 2 mM of MgSO
4, 0.8M betaine) and template DNA or cDNA that and 1 μ l extracts, the reaction final volume is 50 μ l, is reflected in the 0.2ml PCR pipe and carries out.
The LAMP response procedures is: react 60min under 64 ℃ of conditions of thermostat water bath, then 80 ℃ of heating 10min termination reactions.
The LAMP reaction product detects and the result judges: reaction product 2.0% agarose gel electrophoresis, and 10V/cm, bromination second pyridine dyeing back 260nm wavelength is observed after 30 minutes.
Description of drawings
Fig. 1 is the LAMP of porcine circovirus 2 type and the susceptibility electrophorogram relatively of PCR detection method.Be followed successively by from left to right: M, dna molecular amount standard DL-2000; The 1-6 swimming lane, the PCR reaction that the template of different copy numbers is carried out is respectively every pipe 1,5,25,125,625,3125 copies.Contain the template of different copy numbers in the 7-12 swimming lane, LAMP reaction in every pipe, be respectively every pipe 1,5,25,125,625,3125.PCR is limited to 25 copies to detecting of porcine circovirus 2 type, and the method for detecting of LAMP is 5 copies.
That Fig. 2 shows is pig circular ring virus LAMP and PCV1, PPV, the intercrossing reaction detection electrophoresis result of PRV and PRRSV virus.Be followed successively by from left to right: M, dna molecular amount standard DL2000, swimming lane 1, the DNA of PCV2-BJ are template; Swimming lane 2, the DNA of PCV1; Swimming lane 3, the DNA of PPV; Swimming lane 4, the DNA of PRV; Swimming lane 5, the cDNA of PRRSV; Swimming lane 6, the DNA of health pig tissue.
Embodiment
Embodiment of the invention separated into two parts is finished, and promptly embodiment one: the foundation of porcine circovirus 2 type LAMP method; Embodiment two: the specificity and the sensitivity test of porcine circovirus 2 type LAMP method, the experimental technique among the following embodiment if no special instructions, is ordinary method.
Embodiment＜one〉foundation of porcine circovirus 2 type LAMP method
The nucleic acid extraction of 1 porcine circovirus 2 type
The peripheral blood of the pig of aseptic collection infected pigs circovurus type 2 adds an amount of antithrombotics, and tissues such as lung, liver,spleen,kidney are added a small amount of pH 7.4, and the phosphate buffered saline buffer of 0.05M (PBS) is then with organizing the pulverizer homogenized.The viral RNA that sample after the processing provides according to precious biotechnology (Dalian) company limited/DNA extraction test kit extracts viral DNA.
The LAMP reaction of 2PCV2
Sequence with reference to the cap gene of the PCV2 of GenBank login designs two pairs of primers.The present invention adopts the sequence of primer as follows:
Outside primer:
Upstream F:5 '-ATGGGCTGCTAATTTTGCAGAC-3 '
Downstream B:5 '-TCAATAGGAAATTCAGGGCATG-3 '
Inboard primer:
FIP：5′-GTACAGTTCCACCTTTAGTCTC
TTTTGGTTACCATGGTGAAGAAGTGG-3′
BIP：
5′-CAACTGCTGTCCCAGCTGTAGATTTTTCCTCCGTGGATTGTTCTGTAG-3′
DNA with the PCV2 that extracts is a template, adds the DNA 1 μ L of PCV2 in 50 μ L reaction systems, upstream and downstream, the 10 μ mol/L outside each 1 μ l of primer, the inboard upstream and downstream of 1 μ mol/L each 1 μ l of primer, 2.5mmol/LdNTPs 2 μ L, Bst archaeal dna polymerase 8U, 10x damping fluid 5 μ L add ddH
2O to 50 μ L.The LAMP response procedures is as follows: 65 ℃ of 60min, 85 ℃ of 10min then.
The result detects: PCR finishes back sampling carrying out 2.0% agarose gel electrophoresis and detects, and deposition condition is 7V/cm, and 30 minutes, the special scalariform band of LAMP reaction appearred in the result, sees Fig. 1.
Embodiment＜two〉specificity and the sensitivity test of porcine circovirus 2 type LAMP method
1, the sensitivity test of the LAMP of PCV2
1.1PCV2 determining of the quantitative and different extent of dilution templates of virus.
According to the concentration measuring and calculating of the viral DNA of genomic size of PCV2-BJ and extraction, according to LAMP and the every pipe 1,5,25,625 of PCR, 3125 copy numbers dilute.
1.2PCV2 LAMP method detection limit and the contrast of PCR method
The LAMP reaction composition of PCV2 and response procedures are according to embodiment＜1〉carry out, the PCR reaction of PCV2 is composed as follows: the dNTP that comprises 1.5mM in the 50 μ l reaction systems, 10 * buffer of 5 μ l, the Taq polymerase of 5U (Nippon Gene), 1 μ M upstream and downstream primers F and B, 1.0 μ l template DNA or cDNA. amplification program are 94 ℃, 5min is 94 ℃ of sex change 1min of cycling program then, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations.Last 72 ℃ are extended 5min.PCR is reflected in the MJ Research Minicycler amplification instrument and carries out.
1.3 the result detects:
PCR product and LAMP reaction product are carried out electrophoresis in the sepharose on same.Carry out bromination second pyridine dyeing then, take a picture in the BIO-RAD gel imaging instrument and analysis, electrophoresis result is seen Fig. 2, as can be seen from the figure the detection of the LAMP method of PCV2 is limited to 5 copies of each reaction, and detecting of PCR reaction is limited to each reaction 25 copy, and the LAMP reaction is higher 5 times than the susceptibility of PCR reaction.
2, the specificity of the LAMP of PCV2 test
2.1PRRSV, the extraction and the LAMP reaction of PPV, PRV and PCV1 viral nucleic acid
The template of reacting as the LAMP of PCV2 respectively with the DNA that extracts among the field strain isolated PCV1 that identified through PCR or RT-PCR, PPV, the PRV and the cDNA among the PRRSV, with the DNA of extraction in the health pig tissue as the negative control template.Then according to embodiment＜one〉in LAMP reaction system and the condition of PCV2 react, reaction product detects with agarose gel electrophoresis.
2.2 specific reaction interpretation of result
The LAMP method intercrossing reaction result of PCV1, PPV, PRV and PRRSV and PCV2 is seen Fig. 1.As can be seen from the figure above-mentioned four kinds of viruses all do not have the intercrossing reaction with the LAMP method of PCV2.It is also negative that the DNA that extracts in the health pig tissue makes the LAMP reaction result of template.The LAMP detection method that The above results shows the PCV2 that this test is set up to above-mentioned four kinds at the similar viral no cross reaction of clinical symptom performance.
3, the clinical sample of the LAMP of PCV2 detects
3.1 the preparation of clinical sample
Formed by peripheral blood, Lymphoid tissue, lung, liver, kidney, the heart and spleen by 86 clinical samples of PCV2 male by PCR and order-checking or viral isolation diagnostic respectively.Wherein 78 samples identify that by PCR and order-checking other 8 samples are by viral isolation identification.
3.2LAMP detected result
Use the LAMP method of being set up to detect to above-mentioned 86 PCV2 male clinical samples, detected result sees Table 2, and as can be seen from the table, the LAMP method is 96.5% to the recall rate of 86 clinical samples, generally than the recall rate height of PCR.Wherein the recall rate to the PCV2 of the PCR of lung, kidney and the heart and LAMP is 100%, and is slightly higher than PCR method to the recall rate LAMP method of the PCV2 of blood, Lymphoid tissue, kidney and spleen.
4. conclusion
The LAMP method of the PCV2 that above-mentioned evidence is set up has susceptibility height, high specificity, quick, characteristics such as required equipment is simple, processing ease, is suitable for laboratory and the field quick diagnosis to PCV2.
Table 2.LAMP and PCR method are to the analysis of the positive clinical sample of 86 PCV2
Primer title primer sequence
Outside primer upstream F:5 '-ATGGGCTGCTAATTTTGCAGAC-3 '
Outside primer downstream B:5 '-TCAATAGGAAATTCAGGGCATG-3 '
Inboard primer upstream FIP:
5′-GTACAGTTCCACCTTTAGTCTCTTTTGGTTACCATGGTGAAGAAGTGG-3′
Inboard primer downstream BIP:
5′-CAACTGCTGTCCCAGCTGTAGATTTTTCCTCCGTGGATTGTTCTGTAG-3′
Claims (1)
1. the quick detection kit of a porcine circovirus 2 type is characterized in that also having four Auele Specific Primers in the test kit, and these four Auele Specific Primers are:
Outside primer:
Upstream F:5 '-ATGGGCTGCTAATTTTGCAGAC-3 '
Downstream B:5 '-TCAATAGGAAATTCAGGGCATG-3 '
Inboard primer:
FIP：5′-GTACAGTTCCACCTTTAGTCTC
TTTTGGTTACCATGGTGAAGAAGTGG-3′
BIP：
5′-CAACTGCTGTCCCAGCTGTAGATTTTTCCTCCGTGGATTGTTCTGTAG-3′。
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586169B (en) * | 2009-06-30 | 2012-02-08 | 中国兽医药品监察所 | Porcine circovirus 2 LAMP detection kit and detecting method |
| CN103225001B (en) * | 2013-05-06 | 2014-12-31 | 杨毅 | Porcine circovirus type 2 rapid typing detection kit |
| CN103820580B (en) * | 2014-03-16 | 2016-06-08 | 贵州省畜牧兽医研究所 | Porcine circovirus 2 type LAMP diagnostic kit |
| CN104894293A (en) * | 2015-04-30 | 2015-09-09 | 陕西溯源农业发展有限公司 | Porcine circovirus type 2 isothermal PCR on-site rapid detection kit |
| CN106435016B (en) * | 2016-08-30 | 2019-07-26 | 中国农业科学院兰州兽医研究所 | A kind of LAMP kit of quick colour-developing one-step method detection porcine circovirus 2 type |
| CN107287350A (en) * | 2017-06-27 | 2017-10-24 | 广东温氏食品集团股份有限公司 | A kind of primer, kit and method for detecting the type of pig circular ring virus 3 |
| CN109055612A (en) * | 2018-08-24 | 2018-12-21 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection porcine circovirus 2 type |
| 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 |
| CN111850173A (en) * | 2020-08-07 | 2020-10-30 | 宁波爱基因科技有限公司 | Primer and kit for efficiently detecting porcine circovirus type 2 |
| CN116574848A (en) * | 2023-06-20 | 2023-08-11 | 中国农业科学院兰州兽医研究所 | Primer and detection method for detecting porcine circovirus type 2 based on LAMP-CRISPRCas12a |
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