CN101319253B - Method for detecting high-pathogenicity blue ear disease - Google Patents
Method for detecting high-pathogenicity blue ear disease Download PDFInfo
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- CN101319253B CN101319253B CN 200810133893 CN200810133893A CN101319253B CN 101319253 B CN101319253 B CN 101319253B CN 200810133893 CN200810133893 CN 200810133893 CN 200810133893 A CN200810133893 A CN 200810133893A CN 101319253 B CN101319253 B CN 101319253B
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Abstract
The invention discloses a loop-mediated isothermal amplification quick detection technology for a highly pathogenic blue ear disease. By using a special primer and utilizing a special region of a loop-mediated isothermal amplification technology (RT-LAMP) platform amplification target sequence, a highly pathogenic blue ear virus nucleic acid is detected from molecular level with the assistance of a series of quality control, negative contrast and positive contrast. The method has the characteristics of simplicity, convenience, economization, quickness, sensitivity and specificity, and has broad application prospect.
Description
Technical field
The invention belongs to a kind of detection technique, is a kind of method whether pig suffers from high-pathogenicity blue ear disease that detects exactly.
Background technology
Highly pathogenic PRRS is a kind of acute high lethality eqpidemic disease that is caused by the porcine reproductive and respiratory syndrome virus variant.The piglet sickness rate can reach 100%, mortality ratio can reach more than 50%, and the sow abortion ratio can reach more than 30%, the growing and fattening pigs death of also can falling ill.Its pathological characters is that the infarct kitchen range appears in spleen edge or surface, and microscopically is seen hemorrhagic infarct; Kidney is khaki color, visible needle point to the small rice grain profuse bleeding stigma in surface, subcutaneous, tonsilla, heart, bladder, liver and all visible blutpunkte of enteron aisle and blood spots.Microscopically is seen the kidney interstitial inflammation, heart, liver and pathologies such as cystorrhagia property, exudative inflammation; The visible gastrointestinal hemorrhage of some cases, ulcer, necrosis.Pig is unique natural reservoir (of bird flu viruses) of PRRSV, and the pig under various kinds, age and the various raising condition only all can infect.Sustainable 2-4 of acute outbreak period month, acute phase, transferred to chronic or subclinical infection later.After finding first clinically, the efficient infection is exactly the sign of PRRS.Owing to lacking effectively control or vaccine, can not stop the discharge of virus, also can not eliminate its infection to carrying animal, the method for prevention and control PRRSV must be based upon the thorough assurance to spread path.Pig can oral, in the nose, muscle, peritonaeum and vaginal infection PRRSV.Virus has hyperinfection property, gets final product infection morbidity through muscle or 10 of intranasal vaccinations or virus particle still less, is nearly 1 virus particle through abdominal cavity or its Minimum Infective Dose of tail cartilage injection.On the contrary, through mucomembranous surface inoculation as eye, vagina or oral cavity route, minimal infecting dose is 10
3.3-10
5.3Individual infected seed, its dosage depends on the specific region of anatomy.
The pig that infects PRRSV is at clinical symptom disappearance toxin expelling still after 8 weeks, and PRRSV can survive about time more than 5 months at the pig upper respiratory tract and tonsilla, is viral main source with malicious pig therefore.Piglet can become the nature carrier.Virus existence in swinery, circulation and propagation once more.Cause and infect and the not direct or indirect contact transmission between the infected pigs.Especially introducing toxemic in spite of illness reserve boar is its main route of transmission with the parallel and vertical transmission of former swinery, and different swinerys also can be propagated through infecting boar semen.Containing movements such as the viral excrement of PRRS, urine also is the potential source of pollution.Airborne transmission is the another important route of transmission of PRRS, particularly in short range (＜3km) propagate the important effect that has more.With morbidity point is initial point, and 500 meters of radiuses are with interior zone, has 45% pig farm to infect, and apart from the zone of breaking out a 1-2km only 2% pig farm infect.In addition, virus is survival easily under the low temperature and moisture condition, so temperature is low, and humidity is high, and degradation all can be accelerated the propagation of this disease under the big and ultraviolet irradiation intensity of wind speed.
The notable feature of PRRS clinical manifestation is to produce miscarriage to take place the last week or give a birth to shift to an earlier date, and produces the decline of making outstanding achievements.After this disease of outburst stops in same pig farm, also be prone to break out once again, its sickness rate significantly increases.Have very big-difference because of the feeding environment difference latent period, and be 2-4 days piglet this sick latent period, and pregnant pig is 4-7 days.The pathogenic difference of different strains perhaps can cause different latent period.The general popular phase is 70-100 days, and length can reach 4-6 month.Young pig infects comparatively gentleness of back symptom, and sow and piglet symptom are more serious, and the mortality ratio of sow is lower, and the mortality ratio of sucking pig is very high.This disease is more easier than the propagation between the adult pig in the propagation between the piglet.Popular back inapparent infection case increases, and the pig of no clinical symptom also can be propagated this disease, and the lasting several months.
The serious harm that high-pathogenicity blue ear disease brings to pig industry in the wide-scale distribution of China, countries in the world have given great attention to this disease, and have dropped into lot of manpower and material resources it is studied.In view of the biological characteristics and the epidemiologic feature of high-pathogenicity blue ear disease, eliminate and should disease almost can't accomplish high-pathogenicity blue ear disease popular countries and regions, in the long run, this also will be a very difficult task.
Detect the pig high-pathogenicity blue ear disease in the prior art and adopt ELISA method or RT-PCR method more, but these methods detect and take longlyer, it is higher to detect the instrument condition that requires, and the sensitivity that detects is relatively low.
Summary of the invention
The present invention provides a kind of prior art deficiency that overcomes, and has the method that quick, easy, sensitive and economic being used to detects the pig high-pathogenicity blue ear disease, and this method not only both had been applicable to and has detected dead pig, was applicable to that also live hog detects.
Detection method of the present invention is brain or intestinal contents or lung or kidney or intestines or spleen or the liver or the heart tissue of gathering seized dead pig; Perhaps gather blood or mouthful swab or the ight soil of seized live hog; From institute's sample thief, extract RNA; RNA to extract is a template, carries out the RT-LAMP reaction with upstream and downstream, outside primer and inboard upstream and downstream primer, and the RT-LAMP reaction finishes back sampling carrying out agarose gel electrophoresis and detects.
In the detection method of the present invention, used upstream and downstream, outside primer and inboard upstream and downstream primer are respectively:
Primer title primer sequence
F 5′-GCTCCGCGCAGGAAGGTCA-3
B 5′-GTGCGTCAGCGTTGTTGTC-3
FIP 5′-GGATGGTGTCGGAAAATTG?TTTTT?CCTAACGGTTCGGAAGAAA-3′
BIP 5′-CGTCGCGACGTGTCCCCAA?TTTTT?CCACTCAAAGGTGTCATCA-3′
The present invention utilizes loop-mediated isothermal amplification technique, i.e. the RT-LAMP technology.This technology is from the swine disease material, to extract nucleic acid, utilizes institute's designed primer to carry out amplified reaction, and whether the detection reaction product contains specific scalariform band, with the Fast Detection Technique of confirming whether pig infects high-pathogenicity blue ear disease.
The detection that method of the present invention is used for high-pathogenicity blue ear disease 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 RT-PCR.
2.RT-LAMP technology has higher sensitivity than the RT-PCR of the existing employing of Duoing, but required equipment is very simple, only needing one can provide the water-bath about 60 ℃ to get final product, and its experimentation cost can greatly reduce.
3.RT-LAMP technology can be accomplished in general one hour, saves time than RT-PCR.RT-PCR detects that take will be above 2 hours generally speaking.
The concrete steps of the inventive method are following:
(1) test samples is extracted and nucleic acid extraction
Aseptic disease pig blood, mouthful swab or the ight soil got; The organ-tissue of perhaps dead pig, preferably organ-tissues such as the brain of pig, spleen and lung grind to form emulsion suspension liquid with sterile saline; And every milliliter add blue or green, each 2 000IU of Streptomycin sulphate, and it is subsequent use to divide the bottle of packing into to post label-80 ℃ preservation; Adopt Trizol method or MiniBEST viral RNA to extract test kit according to the test kit explanation respectively, from blood, mouthful swab and tissue, extract the full geneome RNA that comprises virogene; Simultaneously, the RNA in the healthy brush,throat of aseptic extraction is to be used as negative control.
(2) carry out the RT-LAMP reaction
The RT-LAMP reaction system is following: final concentration is respectively FIP and the BIP primer of 2.0 μ M, the F of 0.2 μ M and B primer, 1.0mM dNTP, the Bst archaeal dna polymerase of 8U (New England Biolabs), 10 * buffer (containing 2mM of MgSO
4, 0.8M betaine) and template cDNA that and 1 μ l extracts, the reaction final volume is 50 μ l, is reflected in the 0.2ml PCR pipe to carry out.
The RT-LAMP response procedures is: react 45min under 63 ℃ of conditions of thermostat water bath, then 80 ℃ of heating 10min termination reactions.
The RT-LAMP reaction product detects and the result judges: reaction product is used 2.0% agarose gel electrophoresis, 10V/cm, and bromination second pyridine dyeing back 260nm wavelength is observed after 30 minutes.
Description of drawings
Fig. 1 is the RT-LAMP of high-pathogenicity blue ear disease and the susceptibility electrophorogram relatively of RT-PCR detection method.Among the figure: (A) be followed successively by from left to right: M, dna molecular amount standard DL-2000; 1 swimming lane, negative control; The 2-6 swimming lane, the RT-PCR reaction that the template of different copy numbers is carried out is respectively every pipe 1,10,10
2, 10
3, 10
4With 10
5Copy.(B) contain the template of different copy numbers in the 1-6 swimming lane, RT-LAMP reaction in every pipe, be respectively every pipe 1,10,10
2, 10
3, 10
4With 10
5Copy; 7 swimming lanes, negative control.Visible by figure, RT-PCR is limited to 100 copies to detecting of high-pathogenicity blue ear disease, and the method for detecting of RT-LAMP is 10 copies.
Embodiment
Embodiment of the invention separated into two parts is accomplished, and promptly embodiment one: the foundation of high-pathogenicity blue ear disease RT-LAMP method; Embodiment two: the specificity and the sensitivity test of high-pathogenicity blue ear disease RT-LAMP method, the experimental technique among the following embodiment if no special instructions, is ordinary method.
The foundation of embodiment <>high-pathogenicity blue ear disease RT-LAMP method
1, the nucleic acid extraction of high-pathogenicity blue ear disease
Aseptic collection infects the pig peripheral blood of high-pathogenicity blue ear disease, and the porcine tissue sample is added a small amount of pH 7.4, and the phosphate buffered saline buffer of 0.05M (PBS) is then with organizing the kibbler homogenized.The viral RNA that sample after the processing provides according to precious biotechnology (Dalian) ltd extracts test kit and extracts viral RNA.Seized pig also can add an amount of antithrombotics after getting blood in blood,
2, the RT-LAMP of high-pathogenicity blue ear disease reaction
Sequence with reference to the viral NSP2 gene of the high-pathogenicity blue ear disease of GenBank login designs two pairs of primers.The present invention adopts the sequence of primer following:
Outside primer:
Upper reaches F:5 '-GCTCCGCGCAGGAAGGTCA-3
Downstream B:5 '-GTGCGTCAGCGTTGTTGTC-3
Inboard primer:
FIP：5′-GGATGGTGTCGGAAAATTG?TTTTTCCTAACGGTTCGGAAGAAA-3′
BIP：5′-CGTCGCGACGTGTCCCCAA?TTTTTCCACTCAAAGGTGTCATCA-3′
RNA with the high-pathogenicity blue ear disease that extracts is a template; The RNA 1 μ L that in 50 μ L reaction systems, adds high-pathogenicity blue ear disease, 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/L dNTPs 2 μ L; Bst archaeal dna polymerase 8U, 10x damping fluid 5 μ L add ddH
2O to 50 μ L.The RT-LAMP response procedures is following: 63 ℃ of 45min, 80 ℃ of 10min then.
The result detects: RT-LAMP finishes back sampling carrying out 2.5% agarose gel electrophoresis and detects, and deposition condition is 7V/cm, and 30 minutes, the special scalariform band of RT-LAMP reaction appearred in the result, sees Fig. 1.
The specificity and the sensitivity test of embodiment < two>high-pathogenicity blue ear disease RT-LAMP method
1, the sensitivity test of the RT-LAMP of high-pathogenicity blue ear disease
1.1 confirming of the quantitative and different extent of dilution templates of high-pathogenicity blue ear disease virus.
According to the concentration measuring and calculating of the viral RNA of genomic size of high-pathogenicity blue ear disease and extraction, according to RT-LAMP and the every pipe 1,10,10 of RT-PCR
2, 10
3, 10
4, 10
5Copy number dilutes.
1.2 the RT-LAMP method detection limit of high-pathogenicity blue ear disease and the contrast of RT-PCR method
The RT-LAMP reaction of high-pathogenicity blue ear disease forms and response procedures carries out according to embodiment < 1 >, and the RT-PCR reaction of high-pathogenicity blue ear disease is formed as follows: comprise the dNTP of 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 cDNA. amplification programs 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.RT-PCR is reflected in the MJ Research Minicycler amplification appearance and carries out.
1.3 the result detects:
RT-PCR product and RT-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 appearance and analysis; Electrophoresis result is seen Fig. 1; As can be seen from the figure the detection of high-pathogenicity blue ear disease RT-LAMP method is limited to 10 copies of each reaction, and detecting of RT-PCR reaction is limited to each reaction 100 copy, and the RT-LAMP reaction is higher 10 times than the susceptibility of RT-PCR reaction.
2, the specificity of high-pathogenicity blue ear disease RT-LAMP test
2.1 the extraction of FMDV, PCV2, PRX, JEV and CSFV viral nucleic acid and RT-LAMP reaction
Respectively with behind the RNA that extracts among the field strain isolated FMDV, PCV2, PRX, JEV and the CSF that identified through RT-PCR, the template of the high-pathogenicity blue ear disease RT-LAMP of RNA conduct reaction, with the RNA of extraction in the health pig tissue as the negative control template.React according to the RT-LAMP reaction system and the condition of high-pathogenicity blue ear disease among the embodiment <>then, reaction product detects with agarose gel electrophoresis.
2.2 specific reaction interpretation of result
FMDV, PCV2, PRX, JEV and CSF all do not have the intercrossing reaction with high-pathogenicity blue ear disease RT-LAMP method.It is also negative that the RNA that extracts in the health pig tissue makes the RT-LAMP reaction result of template.The above results show high-pathogenicity blue ear disease RT-LAMP detection method that this test sets up with above-mentioned four kinds at the similar viral no cross reaction of clinical symptom performance.
3, the clinical sample of the RT-LAMP of high-pathogenicity blue ear disease detects
3.1 the preparation of clinical sample
Formed by peripheral blood, mouthful swab and lung by high-pathogenicity blue ear disease virus-positive clinical sample by RT-PCR and order-checking or viral isolation diagnostic respectively.
3.2 RT-LAMP detected result
Use the RT-LAMP method of being set up to detect to above-mentioned high-pathogenicity blue ear disease virus-positive clinical sample, detected result is seen table 2, from table, can find out, the recall rate than RT-PCR is high generally to the recall rate of clinical sample for the RT-LAMP method.Wherein brush,throat and whole blood sample are fit to prenatal diagnosis.
Table 2.RT-LAMP and RT-PCR method are to the analysis of high-pathogenicity blue ear disease clinical sample
4, conclusion
The high-pathogenicity blue ear disease RT-LAMP method 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 high-pathogenicity blue ear disease virus.
Claims (1)
1. the detection method of high-pathogenicity blue ear disease; It is characterized in that gathering brain or intestinal contents or lung or kidney or intestines or spleen or liver or the heart tissue of seized dead pig; From the tissue of being gathered, extracting RNA, is template with the RNA that extracts, and carries out the RT-LAMP reaction with upstream and downstream, outside primer and inboard upstream and downstream primer; The RT-LAMP reaction finishes back sampling carrying out agarose gel electrophoresis and detects, and employed outside upstream and downstream primer and inboard upstream and downstream primer are respectively:
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| CN102277445A (en) * | 2010-12-10 | 2011-12-14 | 中华人民共和国珠海出入境检验检疫局 | PRRS RT-LAMP detection method and detection kit |
| CN104651536A (en) * | 2015-03-04 | 2015-05-27 | 广西壮族自治区兽医研究所 | Reverse transcription loop-mediated isothermal amplification kit for PRRSV (Porcine Reproductive And Respiratory Syndrome Virus) and application thereof |
| CN105821161A (en) * | 2016-04-26 | 2016-08-03 | 武汉璟泓万方堂医药科技股份有限公司 | Primer, kit and method for detecting porcine reproductive and respiratory syndrome virus |
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