CN1255555C - Diagnostic gene chip for pig virus disease and its use - Google Patents
Diagnostic gene chip for pig virus disease and its use Download PDFInfo
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- CN1255555C CN1255555C CN 200410078906 CN200410078906A CN1255555C CN 1255555 C CN1255555 C CN 1255555C CN 200410078906 CN200410078906 CN 200410078906 CN 200410078906 A CN200410078906 A CN 200410078906A CN 1255555 C CN1255555 C CN 1255555C
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Abstract
The present invention relates to a pig's viral disease diagnosing gene chip, a preparation method using the diagnostic gene chip and an application for detecting infectious diseases.
Description
The application is the mandate Chinese patent of submitting on December 12nd, 2,002 02155408.0, and denomination of invention is divided an application for " poultry and/or disease of domestic animals diagnostic gene chip and uses thereof ".
Invention field
The present invention relates to be used to diagnose the diagnostic gene chip of virus diseases of pigs, and the purposes of utilizing the preparation method of described diagnostic gene chip and being used to detect transmissible disease.
Background of invention
In recent years, China's livestock industry has obtained the high speed development.For a long time, hindering animal husbandry development and the subject matter that influences aquaculture production is the threat that the threat that described poultry and/or domestic animal suffer from eqpidemic disease, particularly chicken and pig suffer from transmissible disease.Owing to adopted the feeding manner of intensification, the transaction circulation of chicken, pig is accelerated, and makes the transmissible disease of pig and chicken become more complicated, usually shows as multiple infective pathogen polyinfection, causes the difficulty in the medical diagnosis on disease, the also corresponding greatly increase of difficulty of prevention and cure.It is reported, in the transmissible disease of 27 kinds of chickens of investigation, two or more transmissible disease of polyinfection, account for 65.1% of case sum, wherein the concurrent infection with infectious bursal disease is maximum, and usually concurrent newcastle disease of while also merges diseases such as intestinal bacteria, infectious laryngotracheitis sometimes.Also have report, part large scale of pig farm field, Beijing piglet mass mortality at the beginning of nineteen ninety-five, mortality ratio is that the mortality ratio of 63.21%, ten age in days piglet is 100%, exactly owing to transmissible gastroenteritis of swine, epidemic diarrhea and rotavirus concurrent infection cause.
At present, there are many difficulties in the detection for above-mentioned poultry and/or the common eqpidemic disease of domestic animal.To poultry and/or domestic animal, though particularly the diagnosis of pig and chicken transmissible disease has certain methods at present, suppress (HI) method, agar diffusion method and ELISA method etc. as isolation of virus, blood clotting (HA) method, blood clotting, there is more defective in above-mentioned traditional detection method.On the one hand, because domestic animal to be measured has generally all inoculated the vaccine of described disease, so can not diagnose with the serology that detects antibody.And viral isolating method commonly used generally needs the time in one to two week just to go out the result.Also have and detect all more complicated such as viral ELISA method, fluorescent antibody technique, need the time generally to take three days, specificity and susceptibility are not as gene chip.As for the PCR method is the comparison sensitivity, causes false positive, particularly RT-PCR to be difficult to operation but be easy to pollute, and is difficult for promoting.Above-mentioned all methods can only detect a kind of cause of disease at every turn, waste time and energy, and are unfavorable for the infection of the compound infective pathogen of mass detection simultaneously.
Biochip technology is a new and high technology that develops rapidly in life science in recent years, it mainly is the miniature organism chemical analysis system that makes up on the solid phase chip surface by micro-processing technology and microelectronics, with realize pair cell, protein, DNA and other multiple biotic components accurately, fast, the detection of bulk information.Biochip commonly used is divided into three major types: i.e. gene chip, protein chip and chip lab.The principal feature of biochip is a high-throughput, microminiaturization and automatization.
The inventor is applied to poultry and/or domestic animal with above-mentioned biochip technology first, the transmissible disease detection range of chicken and pig particularly, prepared the diagnostic gene chip that to diagnose multiple cause of disease by one-time detection, thereby solved the problem that existing eqpidemic disease diagnostic techniques can not single test detects multiple cause of disease simultaneously effectively, the transmissible disease that has realized required sample size is few, sensitive, special, quick, expense is cheap poultry and/or domestic animal, particularly pig and/or chicken detects.
Summary of the invention
One aspect of the present invention relates to a kind of gene chip that is used to diagnose the poultry and/or the disease of domestic animals, and it is selected from:
1) the sick diagnostic gene chip of pig common transmittable is used to diagnose swine fever, foot and mouth disease, pseudoabies and/or pig breeding and respiration syndrome;
2) virus diseases of pigs diagnostic gene chip is used to diagnose swine fever, foot and mouth disease, pseudoabies, transmissible gastroenteritis of swine, pig breeding and respiration syndrome, porcine influenza, porcine parvovirus, PRCV (Porcine Respiratory Coronavirus) disease, porcine rotavirus disease, epidemic encephalitis b of swine;
3) pig infectious diarrhea diagnostic gene chip is used to detect colibacillosis of pigs, swine dysentery, transmissible gastroenteritis of swine, porcine epizootic diarrhea, rotavirus disease;
4) pig breeding dysfunction syndrome gene diagnosis chip is used to detect rabies, parvovirus, epidemic encephalitis b of swine, pig breeding and respiration syndrome;
5) the sick diagnostic gene chip of chicken common transmittable is used to detect newcastle disease, bird flu, chicken Marek's disease, infectious bursal disease, chicken infectious bronchitis, infectious laryngotracheitis of chicken, fowl cholera, salmonellosis of chicken, colibacillosis;
6) chicken virus diagnostic gene chip is used to diagnose virus disease newcastle disease, bird flu, chicken infectious bronchitis, infectious bursal disease, infectious laryngotracheitis of chicken, chicken Marek's disease, egg-decreasing syndrome, fowl reticuloendotheliosis disease, avian encephalomyelitis, chicken infectious anemia disease;
7) chicken respiratory transmissible disease diagnostic gene chip is used to diagnose the chicken respiratory transmissible disease, newcastle disease epidemic disease, bird flu, chicken infectious bronchitis, infectious laryngotracheitis of chicken, chicken virus mycoplasma, synovia mycoplasma.
8) animal bacteria detection gene chip is used to measure corresponding infective pathogen.
Another aspect of the present invention relates to the method that a kind of gene chip of the present invention is used to detect domestic animal and/or poultry disease, comprises the steps:
1), selects at least a gene chip of the present invention for use according to individuality to be measured and disease type;
2) mark testing sample,
3) be suitable under the condition of hybridizing, adding through the testing sample of mark and make it to react time enough with selected gene chip; With
4) result of detection hybridization.
Detailed Description Of The Invention
The pig that livestock industry is had the greatest impact according to present China, a situation arises for the transmissible disease of chicken, the present invention has designed the diagnosis gene chip that is used to detect multiple infective pathogen, wherein said diagnosis is with having the dna fragmentation that is used to identify specific infective pathogen on the gene chip, it is fixed on the solid phase carrier that is suitable for detection reaction.
Solid phase carrier described in the present invention can be selected known those in this area for use, as long as described carrier is compatible with described reactant, can not influence detected result.Preferably, solid phase carrier of the present invention is nitrocellulose filter, nylon membrane, polystyrene, slide glass etc.
Diagnosis of the present invention can be included in the diagnostic kit with gene chip, wherein also comprises the corresponding reagent, damping fluid and the specification sheets that are used for diagnostic reaction.
One aspect of the present invention, disclose a kind of by Pestivirus suis, foot and mouth disease virus, pseudorabies virus, and/or the sick diagnostic gene chip of pig common transmittable of pig breeding and breath syndrome virus pathogenic genes characteristic fragment formation, be used to diagnose swine fever (Classical Swine Fever (CSF) or H0g Cholera (HC)), foot and mouth disease (Foot-and-Mouth Disease (FMD)), pseudoabies (Pseudorabies (PR) or Aujeszky ' s Disease), pig breeds and respiration syndrome (Porcine Reproductive and Respiratory Syndrome PRRS), wherein adopts the characteristic fragment of pathogenic genes as described below:
The cDNA fragment of Pestivirus suis (CSFV) (referring to people such as M.Harding, J.Clin.Microbi01. in 1994, the 31st volume 10 phase 2600-2602 pages or leaves, SEQ ID NO:1).Described swine fever virus characteristic fragment can detect Baker, the BAI strain of the U.S., Glentorf, Osterode, the V622 strain of Germany, Alfort, the Bas-rhin strain of France, the Classical strain of Britain, Canadian Windsor strain, the Henken strain of Holland, Belgian 1821 strains, Malay VRI strain, the EVI strain of Brazil.And other viruses and cytopathogenic effect and not cytopathogenic bovine viral diarrhea virus (BVDV) and border disease virus (BDV) are negative;
The cDNA fragment of foot and mouth disease virus (FMDV) (referring to, people such as M.C.H fner, J.Virol.Methods the 42nd volume 53-62 page or leaf in 1993, SEQ ID NO:2), described characteristic fragment can detect O, A, C, SAT 1, SAT 2, SAT 3 and each strain of Asia1 type of FMDV, and other viruses and swine vesicular disease virus (SVDV) and bovine enteroviruses (BEV 1 and BEV-2) are negative;
The characteristic dna fragmentation of pseudorabies virus (PRV) (referring to, people such as H.Hasebe, Vet.Microbiol. the 34th volume 221-231 page or leaf in 1993, SEQ ID NO:3), its be used for detecting as: strain PRV such as Becker, Iowa, Shope, 4892,5894, Bartha, Bukarest, and other various viruses are negative;
The characteristic cDNA fragment of pig breeding and breath syndrome virus (PRRSV) (referring to, people such as S.A.Gilbert, J.Clin.Microbiol. the 35th volume 264-267 page or leaf in 1997, SEQID NO:4), be used to detect strain PRRS such as Minnesota MN-1b, Quebec LHVA3, AlbertanPRRS B-9, H-5, U-28, European LV, and other various viruses are negative.
In one embodiment of the invention, described solid phase carrier is a nitrocellulose.
Another aspect of the present invention, disclose a kind of by Pestivirus suis, foot and mouth disease virus, pseudorabies virus, transmissible gastro-enteritis virus, pig breeding and breath syndrome virus, swine influenza virus, pig parvoviral, PRCV (Porcine Respiratory Coronavirus), porcine rotavirus, the virus diseases of pigs diagnostic gene chip that epidemic encephalitis b of swine virus causing disease gene expression characteristics fragment constitutes, it is used to diagnose swine fever, foot and mouth disease, pseudoabies, transmissible gastroenteritis of swine (Transmissible Gastroenteritis TGE), pig breeding and respiration syndrome, porcine influenza (Swine Influenza SI), porcine parvovirus (Porcine Parvovirus PP), PRCV (Porcine Respiratory Coronavirus) disease (Porcine RespiratoryCoronavirus PRC), porcine rotavirus disease (Porcine Rotavirus PRT), epidemic encephalitis b of swine (Japanese Encephalitis JE), wherein adopt the characteristic fragment of pathogenic genes as described below:
Swine fever virus characteristic fragment (the same, SEQ ID NO:1); Foot and mouth disease virus characteristic fragment (the same, SEQ ID NO:2); Pseudorabies virus characteristic fragment (the same, SEQ IDNO:3);
Transmissible gastro-enteritis virus characteristic cDNA fragment (referring to, J.Clin.Microbiol. in 1994, the 32nd volume 1809-1812 page or leaf, people such as E.M.Vaughn, SEQ IDNO:6), it can detect strains such as CHV, and the virus of the very approaching PRCV (Porcine Respiratory Coronavirus) (PRCV) of other and TGEV is negative;
Pig breeding and breath syndrome virus characteristic fragment (the same, SEQ ID NO:4);
Swine influenza virus (SIV) characteristic cDNA fragment (referring to, 1994, Am.J.Vet.Res., the 55th volume 952-956 page or leaf, people such as E.Schorr, SEQ ID NO:5), it can detect strains such as A/Sw/IN/1726/88, A/Sw/WI/1915/88, and other various viruses are negative;
Pig parvoviral (PPV) characteristic dna fragmentation (referring to, 1994, Am.J.Vet.Res., the 55th volume 344-347 page or leaf, people SEQ ID NO:8 such as C.M.Gradil), it can detect strains such as NADL-8, and other various viruses are negative;
PRCV (Porcine Respiratory Coronavirus) (PRCV) characteristic cDNA fragment (referring to, 1994, J.Clin.Microbiol., the 32nd volume 1678-1812 page or leaf, people such as E.M.Vaughn, SEQ IDNO:7), it can detect strains such as ISU-1,1894, LEEP, and transmissible gastro-enteritis virus and other various viruses are negative;
Porcine rotavirus (PRTV) characteristic cDNA fragment (referring to, 1994, J.Clin.Microbio1., the 32nd volume 1333-1337 page or leaf, people such as V.Gouvea, SEQ ID NO:9), it can detect strains such as OSU, Gottfried, and other various viruses are negative;
Epidemic encephalitis b of swine virus (JEV) characteristic cDNA fragment (referring to, 2000, Chinese animal doctor's journal, the 20th volume 10-14 page or leaf, people such as Sun Ming, SEQ ID NO:10), it can detect strains such as A2, JEV living vaccine, and other various viruses are negative.
Chip of the present invention can be used for detecting the virus of most of infected pigs, and having or not in detection has unique purposes aspect polyinfection, the secondary infection, also generally investigates applicable to eqpidemic disease.
Another aspect of the present invention, the pig infectious diarrhea diagnostic gene chip that is made of swine escherichia coli, transmissible gastroenteritis of swine, porcine epizootic diarrhea, porcine rotavirus pathogenic genes characteristic fragment is disclosed, it is used to detect colibacillosis of pigs (Colibacillosis), swine dysentery (SwineDysentery SD), transmissible gastroenteritis of swine, porcine epizootic diarrhea (Porcine EpidemicDiarrhoea PED), rotavirus disease, wherein adopts pathogenic genes characteristic fragment as described below:
Swine escherichia coli characteristic fragment (referring to, 1991, J.Bacteril., the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:26);
The snakelike spirochete characteristic of swine dysentery fragment (referring to, 1991, J.Bacteril. the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:27);
Porcine epidemic diarrhea virus (PEDV) characteristic cDNA fragment (referring to, 1999, J.VetMed.Sci., the 61st volume 827-830 page or leaf, people such as S.Kubota, SEQ ID NO:11), it can detect strains such as PEDV CV777, Korean, and close with it TGEV and hemagglutinating encephalomyelitis virus (HEV) and other various viruses are negative;
Porcine rotavirus characteristic fragment (the same, SEQ ID NO:9).
Because cause of disease more complicated in the pig complain of diarrhea is depended merely on clinical symptom and can not be made a definite diagnosis, do these several sick three weeks of diagnosis with existing diagnostic method and also can not finish, and can finish diagnosis in one day with gene chip of the present invention.
Another aspect of the invention, the pig breeding dysfunction syndrome gene diagnosis chip that is made of pseudorabies virus, pig parvoviral, epidemic encephalitis b of swine virus, pig breeding and breath syndrome virus pathogenic genes characteristic fragment is disclosed, be used to detect rabies, parvovirus, epidemic encephalitis b of swine, pig breeding and respiration syndrome, wherein adopt pathogenic genes characteristic fragment as described below:
Pseudorabies virus characteristic fragment (the same) referring to SEQ ID NO:3;
Pig parvoviral characteristic fragment (the same) referring to SEQ ID NO:8;
Epidemic encephalitis b of swine virus characteristic fragment (the same) referring to SEQ ID NO:10;
Pig breeding and breath syndrome virus characteristic fragment (the same) referring to SEQ ID NO:4.
Because above-mentioned cause of disease is the main pathogen that causes farrowing sow miscarriage, stillborn foetus, uses gene chip of the present invention and can comparatively fast find out the relevant eqpidemic disease that is specifically caused by one or more viruses.
Another aspect of the invention, disclose by newcastle disease virus, avian influenza virus, chicken Marek's disease virus, chicken infectivity bursa of Fabricius virus, avian infectious bronchitis virus, infectious laryngotracheitis of chicken (Infectious Laryngotracheitis ILT) virus, the fowl cholera bacillus, Salmonella gallinarum, the sick diagnostic gene chip of the chicken common transmittable that colibacillosis pathogenic genes characteristic fragment constitutes, be used to detect newcastle disease (Newcastle Disease ND), bird flu (Avian Influenza AI), chicken Marek's disease (Marek ' s Disease MD), infectious bursal disease (Infectious bursalDisease IBD), chicken infectious bronchitis (Infectious Bronchitis IB), infectious laryngotracheitis of chicken, fowl cholera (Avian Pasteurellosis), salmonellosis of chicken (Salmonelosis), colibacillosis (Avian Colibacillosis), wherein adopted pathogenic genes characteristic fragment as described below:
Newcastle disease virus (NDV) characteristic cDNA fragment (referring to, 1993, Avian Dis., the 37th volume 724-730 page or leaf, people such as Jarecki-Black.J.C, SEQ ID NO:12), it can detect the HE of NDV
2,, HE
3,, HE
11,, B
1,, strain such as I system, IV system, and avian influenza virus (AIV), infectious bursa of Fabricius virus (IBDV), infectious bronchitis virus (IBV) and other various viruses are negative;
Avian influenza virus characteristic cDNA fragment (referring to, 2000, J.Vet.Med.B, the 47th volume 295-301 page or leaf, people such as E.Starick, SEQ ID NO:13), it can detect H1-H13 type avian influenza virus (AIV), and virus (IBDV), infectious bronchitis virus (IBV) and other various viruses are negative and newcastle disease virus (NDV), infectivity Fa Shi are spoken in a low voice;
The characteristic dna fragmentation of chicken Marek's disease virus (MDV) (referring to, nineteen ninety-five, the 6th scientific seminar's collection of thesis of China animal and veterinary livestock epidemiology branch of association, the 261-264 page or leaf, people such as Lu Chun, SEQ ID NO:14), it can detect the strain such as capital-1, MD11/75 of MDV1, and MDV2, MDV3, SB-1, FC126 strain and other various viruses are negative;
The characteristic cDNA fragment of chicken infectivity bursa of Fabricius virus (IBDV) (referring to, 2000, Acta virologica, the 44th volume 259-263 page or leaf, people such as R.S.Kataria, SEQ IDNO:15), it can detect the strain such as WB1/93, JK1/96, CH1/97, UP1/97, KT1/98 of IBDV, other various viruses are negative, and vaccine strain Georgia, Im+, Lukert etc. also present feminine gender under the hybridization conditions of strictness simultaneously;
The characteristic cDNA fragment of avian infectious bronchitis virus (IBV) (referring to, 2000, Chinese Preventive Veterinary Medicine newspaper, the 22nd volume 64-66 page or leaf, people such as Wang Zelin, SEQ ID NO:16), it can detect the H of IBV
120, M
41, Conn, Gray, Holte, T, 118, suitable, go up, 12 strain strains such as HK, cloud, N, virus such as NDV, IBDV, EDS-76V and other various viruses are negative;
The characteristic dna fragmentation of infectious laryngotracheitis of chicken (ILTV) (referring to, 1996, Avian Dis., the 40th volume 56-62 page or leaf, people such as F.Abbas, SEQ ID NO:17), it can detect the 5 strain strains such as 88-1453,86-1169, Laryngo-Vac ILTV, 88-1059, LT-ivax ILTV of ILTV, and virus such as IBRV, FHV, EH1, EH2, PRV, BHV and other various viruses are negative;
Fowl cholera bacillus characteristic DNA (referring to, 1991, J.Bacteril., the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:24);
Salmonella gallinarum characteristic dna fragmentation (referring to, 1992, Mol.Cell Probes, the 6th volume 271-275 page or leaf, people such as Rahn K., SEQ ID NO:25), it can detect 99.4% Salmonellas, non-specific responding takes place in 15 kind of 33 strain bacterium of other various non-salmonella, but does not all hybridize with the invA gene probe of mark.
The DNA of colibacillosis bacterium (referring to, 1991, J.Bacteril., the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., the universal primer of 16s rRNA, SEQ ID NO:26).
Since adopt now high-density, extensive, concentrate the method for raising to raise chickens, so the transmissible disease of chicken is easy to take place especially, we can say anyly can't survive without the vaccine prevention transmissible disease once tame poulty house.And because the morbidity kind is very complicated, and main pathogen causes that eqpidemic disease constantly changes, also possibility polyinfection and secondary infection, this has brought difficulty just for the timely diagnosis and the control of eqpidemic disease.Adopt gene chip of the present invention, can be quick, efficient, special, in time complicated infective pathogen is made diagnosis delicately.
Another aspect of the invention, disclose a kind of by newcastle disease virus, avian influenza virus, avian infectious bronchitis virus, chicken infectivity bursa of Fabricius virus, avian infectious laryngotracheitis virus, chicken Marek's disease virus, egg-decreasing syndrome virus, fowl reticuloendotheliosis syndrome virus, avian encephalomyelitis virus, the chicken virus diagnostic gene chip that chicken infectious anemia syndrome virus pathogenic genes characteristic fragment constitutes, be used to diagnose the virus disease newcastle disease, bird flu, chicken infectious bronchitis, infectious bursal disease, infectious laryngotracheitis of chicken, chicken Marek's disease, egg-decreasing syndrome (EggDrop Syndrom-1976 EDS-76), fowl reticuloendotheliosis disease (AvianReticuloendotheliosis), avian encephalomyelitis (Avian Encephalomyelitis AE), chicken infectious anemia disease (Chicken Infectious Anemia CIA) is wherein adopted characteristic fragment as described below;
Newcastle disease virus characteristic fragment (the same, SEQ ID NO:12);
Avian influenza virus characteristic fragment (the same, SEQ ID NO:13);
Avian infectious bronchitis virus characteristic fragment (the same, SEQ ID NO:16);
Chicken infectivity bursa of Fabricius virus characteristic fragment (the same, SEQ ID NO:15);
Avian infectious laryngotracheitis virus characteristic fragment (the same, SEQ ID NO:17);
Chicken Marek's disease virus characteristic fragment (the same, SEQ ID NO:14);
Egg-decreasing syndrome virus (EDS-76V) characteristic dna fragmentation (referring to, nineteen ninety-five, 95 years national poultry diease molecular biology Conference Papers collection of China animal and veterinary association, the 116-125 page or leaf, open impartial now people, SEQ ID NO:18), it can detect strain isolateds such as Zhuhai, Shanghai and Nanjing, Shijiazhuang, Changchun, and virus such as NDV, IBV, ILTV, IBDV, FPV and other various viruses are negative;
The characteristic cDNA fragment of fowl reticuloendotheliosis syndrome virus (REV) (referring to, 1993, Avian Pathology, the 22nd volume 543-554 page or leaf, Aly, people such as M.M., SEQID NO:19), it can detect RE virus, and other various viruses are negative;
The characteristic cDNA fragment of avian encephalomyelitis virus (AEV) (referring to, 1999, AvianDis., the 43rd volume 219-226 page or leaf, people such as D.Todd, SEQ ID NO:20), it can detect the various strains of AEV, and other various viruses are negative;
The characteristic dna fragmentation of chicken infectious anemia syndrome virus (CIAV) (referring to, 1992, J.Clin.Microbiol., the 30th volume 1661-1666 page or leaf, people such as D.Todd, SEQ ID NO:21), it can detect 13 strain strains such as GIFU-1, TK5803,1/80,1/91, EF 88/78/276,87/10/44,87/11/52,89/3711,89/3713, IMP704, NI CAV-1, NI CAV-2, EF 90-1, and other various viruses are negative.
Chip of the present invention has comprised the most of virus that causes the chicken disease substantially, does generaI investigation with very suitable.
Another aspect of the invention, a kind of respiratory infectious disease diagnostic gene chip that is made of chicken newcastle disease virus, avian influenza virus, avian infectious bronchitis virus, avian infectious laryngotracheitis virus, chicken virus mycoplasma, synovia mycoplasmosis protogene characteristic fragment is disclosed, be used to diagnose newcastle disease epidemic disease, bird flu, chicken infectious bronchitis, infectious laryngotracheitis of chicken, chicken virus mycoplasma, synovia mycoplasma, wherein adopt pathogenic genes characteristic fragment as described below:
Newcastle disease virus characteristic fragment (the same, SEQ ID NO:12);
Avian influenza virus characteristic fragment (the same, SEQ ID NO:13);
Avian infectious bronchitis virus characteristic fragment (the same, SEQ ID NO:16);
Avian infectious laryngotracheitis virus characteristic fragment (the same, SEQ ID NO:17);
The characteristic dna fragmentation of chicken virus mycoplasma (MG) (referring to, 1991, Avian Dis., the 35th volume 62-69 page or leaf, people such as Elmiro R.Nascimento, SEQ ID NO:22), it can detect 16 strain mycoplasmas such as F-K810, F-F2F10, AAA, Fg38, F (g99), GM607, S6 (208), S6 (C4P8), PG31, A5969, GM747, K2101, K2101 (36P), R, V503, F.Conn, and other each mycoplasma species is negative;
The characteristic dna fragmentation of synovia mycoplasma (MS) (referring to, 1996, Avian Dis., the 40th volume 218-222 page or leaf, people such as R.M.Silveira, SEQ ID NO:23), it can detect the MS mycoplasma, and other each mycoplasma species is negative;
Another aspect of the invention, disclose a kind of by the rich animal bacteria detection gene chip that constitutes for Salmonella, actinobacillus pleuropneumoniae, streptococcus aureus, swine streptococcus, bacillus rhusiopathiae suis, Corynebacterium suis pathogenic genes characteristic fragment of jejunum knee bacterium, Brucella suis, segmental bronchus sepsis, be used to measure corresponding infective pathogen, wherein adopt characteristic fragment as described below:
Jejunum knee bacterium characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:28);
Brucella suis characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:29);
The segmental bronchus sepsis rich for Salmonella characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:30);
Actinobacillus pleuropneumoniae characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:31);
Streptococcus aureus characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:32);
Swine streptococcus characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:33);
Bacillus rhusiopathiae suis characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:34);
Corynebacterium suis characteristic dna fragmentation (referring to, J.Bacteril. in 1991, the 173rd volume 697-702 page or leaf, people such as Weiburg W.G., SEQ ID NO:35).
The specificity and the susceptibility that adopt the said gene chip to do the PCR detection of primer with 16s rRNA conserved sequence are 100%, and the susceptibility of biochemical analysis and microbial culture is respectively 86.4% and 22.7%.
Another aspect of the invention relates to a kind of method that is used to detect domestic animal and/or poultry disease, comprises the steps:
1), selects at least a gene chip of the present invention for use according to individuality to be measured and disease type;
2) mark testing sample,
3) be suitable under the condition of hybridizing, adding through the testing sample of mark and make it to react time enough with selected gene chip; With
4) result of detection hybridization.
Those skilled in the art know, and need testing sample is carried out mark in the method for above-mentioned detection transmissible disease.According to the difference of selected solid phase carrier, select different marks and corresponding detecting method for use.
In one embodiment of the invention, adopt nitrocellulose filter as solid phase carrier, fixedly have the selected kind infective pathogen of the present invention characteristic fragment on it, gene chip is used in the preparation diagnosis, simultaneously testing sample is carried out mark with vitamin H.Among the present invention, preferred vitamin H is photobiotin and vitamin H-dUTP.
Those skilled in the art know, can adopt 6-C photobiotin marking method fairly simple, it carries out mark (write Chinese agriculture press referring to veterinary microbiology Harbin Veterinary Medicine Inst., China Academy of Agriculture in 1998 and publish 691 pages) through excited by visible light to DNA, RNA.
For using fluorescein and vitamin H-dUTP mark testing sample following several method is arranged usually: mix during 1) with the reverse transcription of poly oligonucleoside (dT) primer fluorescein (cy3-dUTP) or vitamin H-dUTP (referring to, molecular cloning: the yellow training halls of chopsticks such as experiment guide Chinese third edition J. Sa in 2002 nurse Brooker wait translates the 753-757 page or leaf), this method is applicable to the array of forming from 3 ' terminal sequence; 2) mix during poly-6 (poly-9) random primer reverse transcription fluorescein (cy3-dUTP) or vitamin H-dUTP (referring to, molecular cloning: the yellow training halls of chopsticks such as experiment guide Chinese third edition J. Sa in 2002 nurse Brooker wait translates the 750-753 page or leaf); Do not mix fluorescein or vitamin H during 3) with above-mentioned primer reverse transcription, generate behind the cDNA again with nick-translation mix fluorescein or vitamin H (referring to, veterinary microbiology Harbin Veterinary Medicine Inst., China Academy of Agriculture in 1998 writes Chinese agriculture press and publishes 690 pages).
In another embodiment of the present invention, adopt the slide glass of modifying through aminosilane as solid phase carrier, on it, fixedly have the selected kind infective pathogen of the present invention characteristic fragment, fix through aquation with uviolizing again, obtain the diagnosis gene chip.With the nucleic acid of fluorescein-labelled testing sample, use the fluoroscopic examination results of hybridization simultaneously.
In the present invention, the extracting of testing sample nucleic acid can be adopted different sulphur nitrile acid guanidinesalt stabilization method, and is quantitative to RNA with spectrophotometer.In order to finish the mark of cDNA, need total RNA of 100ug at least.
Diagnosis of the present invention has been adopted strict hybridization condition with gene chip when diagnosing corresponding eqpidemic disease.According to the selected condition of the present invention, can realize making the most of reactants in a large amount of hybridizations to be in the optimum, thereby counterpart as much as possible is not omitted, promptly false negative is the least possible, there have the hybridization of mispairing to be reduced to be minimum, and promptly false positive is few as much as possible.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 294 bases on the gene chip of the sick diagnosis of described pig common transmittable, the hybridization conditions of described gene chip and testing sample is: for the testing sample (100ng/ml DNA or RNA) through mark, be chosen under about 41 ℃ of conditions and hybridized 5 hours, using methane amide concentration is 40%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 617 bases on the gene chip of diagnosis of described virus diseases of pigs, the hybridization conditions of described gene chip and testing sample is: for the testing sample (200ng/ml DNA or RNA) through mark, be chosen under about 43 ℃ of conditions and hybridized 10 hours, using methane amide concentration is 50%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 484 bases on the gene chip of diagnosis of described pig infectious diarrhea, the hybridization conditions of described gene chip and testing sample is: for the testing sample (150ng/ml DNA or RNA) through mark, be chosen under about 42 ℃ of conditions and hybridized 8 hours, using methane amide concentration is 45%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 251 bases on the gene chip of the syndromic diagnosis of described pig breeding dysfunction, the hybridization conditions of described gene chip and testing sample is: for the testing sample (100ng/ml DNA or RNA) through mark, be chosen under about 41 ℃ of conditions and hybridized 4 hours, using methane amide concentration is 40%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 439 bases on the gene chip of diagnosis of described chicken common transmittable disease, the hybridization conditions of described gene chip and testing sample is: for the testing sample (150ng/ml DNA or RNA) through mark, be chosen under about 42 ℃ of conditions and hybridized 7 hours, using methane amide concentration is 45%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 459 bases on the gene chip of diagnosis of described chicken virus, the hybridization conditions of described gene chip and testing sample is: for the testing sample (150ng/ml DNA or RNA) through mark, be chosen under about 42 ℃ of conditions and hybridized 6 hours, using methane amide concentration is 45%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 310 bases on the gene chip of diagnosis of described chicken respiratory transmissible disease, the hybridization conditions of described gene chip and testing sample is: for the testing sample (150ng/ml DNA or RNA) through mark, be chosen under about 43 ℃ of conditions and hybridized 7 hours, using methane amide concentration is 45%.
In a specific embodiments of the present invention, be used for that the segmental average polynucleotide sequence length of fixed pathogenic genes characteristic to be measured is 294 bases on the gene chip of described animal bacteria detection, the hybridization conditions of described gene chip and testing sample is: for the testing sample (100ng/ml DNA) through mark, be chosen under about 41 ℃ of conditions and hybridized 5 hours, using methane amide concentration is 40%.
In the present invention, adopt the known detection method in this area to detect results of hybridization.
In one embodiment of the invention, slide glass is made solid support use fluorescein-labeled results of hybridization simultaneously, (model Scan Array 3000 GSTLumonics Inc make to adopt the laser focusing scanner,) sweep and read, obtain the CY3 image file, determine the hybridization scope by punctuating, filter ground unrest, extract the fluorescence signal intensity value of hybridization, original extraction signal with fixed in advance internal reference gene pairs CY3 carries out equilibrium and correction, with ImaGene3.0 software analysis CY3 fluorescence signal intensity and ratio.
Concrete, hybridize the male standard with following two conditions as judging.The fluorescent signal mean value of CY3 positobe focus surpasses 3 times of negative some signal, and ratio range is at 2.7-3.3; After negative fluorescent signal value＜800, the positive fluorescent signal value of CY3 deduct negative fluorescent signal value＞400.
In another specific embodiments of the present invention, nitrocellulose filter is made the solid support results of hybridization of applicating biotin mark simultaneously, employing avidin-alkaline phosphatase enzyme method detection system (referring to, veterinary microbiology Harbin Veterinary Medicine Inst., China Academy of Agriculture in 1998 writes Chinese agriculture press and publishes 694 pages) detect, color reaction is observed under visible light, and purple dot is positive reaction.Choose wantonly, can use the digital camera record, described results of hybridization is analyzed with computer software.
Embodiment
The general policies of embodiment 1 gene chip preparation
About the preparation of gene chip of the present invention, can adopt general policies as detailed below
(1) based on the gene chip of slide glass as solid support:
1. slide glass is handled: get slide glass several pieces, place 70% ethanolic soln of 2mol/LNaOH to soak 2 hours, taking out the back washes for several times with pond, dry and be cooled to room temperature for 110 ℃, place 1% 3-aminopropyl triethoxysilane 95% acetone soln to soak 2 minutes slide glass, taking-up is washed 10 times with acetone, 110 ℃ of bakings 30 minutes and to be cooled to room temperature standby.
2. point sample: according to the particular type of the gene chip of the present invention that needs preparation, selected pathogenic genes characteristic dna fragmentation in the described gene chip is respectively got 17 μ tl, add 20 * SSC damping fluid 3ul mixing, be transferred in 96 orifice plates, plate is placed pixsys7500 DNAMicroarray system (cartesian Technologies Inc; Make) sample application platform on, be arranged on the slide glass of handling well with 3 * 10 and make gene chip.All comprise a positive control and a negative control in every part of gene chip at least.
The array that point is good was put in the wet box aquation 5 minutes, took out and put 100 ℃ of platform last 30 seconds, and facing down then places on the ultraviolet transilluminator, with power 0.5mW/cm
2Wavelength 230nm UV-irradiation was fixed in 18 minutes.Standby.
(2) based on the gene chip of nitrocellulose filter as solid support:
1. the processing of nitrocellulose filter: get nitrocellulose filter, place 2 * SSC solution (20 * SSC=NaCl 175.5g 3Mol/L, Trisodium Citrate C
6H
5O
7Na 2H
2O 88.2g 0.3Mol/L is dissolved in water, and transfers H to 7.0, and thin up becomes 1L, filtering, and this liquid dilution promptly gets 2 * SSC) for 10 times, and is moistening even, is placed on the filter paper, is clipped in two sheet glass, and 37 ℃ of oven dry get final product point sample.
2. point sample: according to the particular type of the gene chip of the present invention that needs preparation, selected pathogenic genes characteristic dna fragmentation in the described gene chip is respectively got 17 μ l, add 20 * SSC damping fluid 3ul mixing, array format point with 3 * 3 is on nitrocellulose filter, the array that point is good is placed on the filter paper, be clipped in two sheet glass, 80 ℃ were toasted 2 hours, made gene chip.All comprise a positive control and a negative control in every part of gene chip at least.
Embodiment 2 gene chips of the present invention detect the general policies of testing sample
(1) adopts biotin labeling sample and detection thereof
1. the mark of testing sample
According to the difference of institute's detected object, select concrete sample to be tested, extract nucleic acid samples according to following method, and gained testing sample nucleic acid is carried out biotin labeling:
I) by RNA extracting method (referring to molecular cloning: the yellow training halls of chopsticks such as experiment guide Chinese third edition J. Sa in 2002 nurse Brooker wait translates the 518-522 page or leaf); Nucleic acid to testing sample extracts, make template with the nucleic acid that extracts, adopt the RT-PCR method to carry out amplification label, in mixed solution (80ul), use random primer (production number: D3801 company: TaKaRa), dNTP (dTTP adds 0.5ul less), the Biotin-dUTP that other adds the 1mmol/L of 0.5ul carries out mark, amplified production PCR purification kit (production number D301, company's T aKaRa) purifying, product finally is dissolved in 5 an amount of * SSC, in the 0.2%SDS solution, final concentration is 50mg/L.
Ii) photobiotin mark
By RNA extracting method (translating the 518-522 page or leaf) referring to yellow training halls of chopsticks such as molecular cloning experiment guide Chinese third edition J. Sa in 2002 nurse Brooker etc.; Total RNA extracts to testing sample, make template with the nucleic acid that extracts, in mixed solution (100ul) according to the method described in (veterinary microbiology Harbin Veterinary Medicine Inst., China Academy of Agriculture in 1998 writes Chinese agriculture press and publishes 691 pages), the nucleic acid solution of need mark is added in the centrifuge tube, the 1 μ g/ μ l photobiotin (production number 2133222 that adds equivalent, the EZ-link of company) solution, fully mixing; Rayed 25-30 minute; After finishing, adding aqua sterilisa to cumulative volume is 100 μ l, adds 100 μ l sec-butyl alcohols, and vortex mixes, 3000 rev/mins centrifugal 1 minute, the careful suction gone upper strata butanols phase.The butyl alcohol extraction of above-mentioned for another example adding and solution equivalent is once inhaled and is removed butanols; In this mixed solution, add 5 μ l 3mol/L sodium-acetate (NaOOCCH
32.46g/10ml) thorough mixing.Add the cold dehydrated alcohol of 100 μ l, put and spent the night in-70 ℃ minutes or-20 ℃, 4 ℃ centrifugal 15 minutes.Abandon supernatant.Wash once with 80% cold ethanol, precipitate the drying of finding time; Precipitation is dissolved in 0.1mMol/LEDTA (C
10H
14N
2O
8Na
44H
2O, 45.2mg/L)-20 ℃ freezing preservation, or 4 ℃ of preservations after the freeze-drying are measured labeling nucleic acid content with ultraviolet method, and be standby.
2. the hybridization of gene chip and testing sample
Prepare following solution
1) 3%BSA confining liquid: 3gBSA is dissolved in the 70ml water, transfers PH to 3.0 with concentrated hydrochloric acid.Boil after 20 minutes to room temperature.Transfer PH to 7.5 with 10mM NaOH, add the following damping fluid of 10ml (1M Tris-HCl PH7.5; Add MgCl
2190mg, Triton X-100 0.05mlNaCl5.8g, dissolving adds water to 100ml).
2) 50 * Denhardt ' s solution: 1g Ficoll 400 ficolls, 1g PVP40,1g B is water-soluble, is made into 100ml.
3) preparation of calf thymus DNA: 100mg/10ml water logging maceration, stir, suck with needleless injector, load onto syringe needle and release, several times to the fracture of DNA long-chain, solution is thinning repeatedly.
4) 1M PBS/L:NaCl 18g KCl 0.2g Na
2HPO
41.44g KH
2PO
40.24g adding distil water 800ml is transferred to PH7.4 with HCl, adds water and supplies 1000ml.
5) 100mM/L EDTA:37.2g EDTA is dissolved in the 1000ml water.
6) face with preceding by every cm
2Amount preparation prehybridization and hybridization solution with 80ul.As with 5 * 10cm
2Need with 4ml prehybridization solution and hybridization solution.Be formulated as follows:
| Reagent | Prehybridization solution 4mlConsumption | Hybridization solution 4mlConsumption |
| Methane amide 20 * SSC 50 * Denhartd ' s calf thymus DNA *Cursor sample nucleic acid **1M PBS, PH6.4 100mM EDTA 20% sodium dextran sulfate | 2.0ml 1.0ml 0.4ml 0.2ml 0.2ml 0.2ml | 1.8ml 1.0ml 80ul 80ul 80-400ng 80ul 1.0ml |
*With preceding boiling sex change in 10 minutes, speed is put in the frozen water and is cooled off;
*Boiling sex change cooling in 10 minutes adds again
Prehybridization: have the film of DNA array to put into hybridization dish circular hole point, add prehybridization solution, covered, in 42 ℃ prehybridization 1-2 hour.
Hybridization: discard prehybridization solution, add the good sample nucleic acid of hybridization solution and mark according to quantity, covered, according to selected gene chip, the concrete hybridization conditions that partly discloses according to detailed Description Of The Invention is transferred to film in the hole that fills 0.1 * SSC solution in addition and to be washed film 3-4 time, prepares colour developing.
3. the detection of results of hybridization
1) for biotin labeled sample, colour developing:
1. with 42 ℃ of sealings of 3%BSA confining liquid 2-4 hour;
2. inhale deblocking liquid, add 4mlAV-AP enzyme connection liquid (damping fluid: 100mM Tris-HClpH7.5,1.0M NaCl, 2mM MgCl
2, 0.05% (v/v) TritonX-100; The 4ml damping fluid adds avidin-alkali Phosphoric acid esterase (AV-AP) 8-16U (1U/ul), lucifuge oscillatory reaction 15 minutes gently at room temperature.
3. diaphragm is moved on to and fill washings (Tris 1.21g NaCl 5.8g MgCl
219mg TritonX-1000.05ml adds 80ml water, transfers PH to 7.5 with HCl, adds water to 100ml) the hole in washing 4 times.
4. diaphragm is moved on to and fill substrate solution (100mM Tris-HCl pH9.5,100mMNaCl, 5mM MgCl
2), in the hole of BCIP (10mgBCIP is dissolved among the 200ul DMF), NBT (among the DMF of the molten 200ul 70% of 15mgNBT), lucifuge is placed several minutes to 1 hour, until going out to show blue purple dot, water termination reaction again.
5. observed and recorded or use the digital camera record is analyzed with computer software.
(2) adopt fluorescein-labelled sample and detection thereof
1. the mark of testing sample
By RNA extracting method (translating the 518-522 page or leaf) the total RNA of testing sample is extracted referring to yellow training halls of chopsticks such as molecular cloning experiment guide Chinese third edition J. Sa in 2002 nurse Brooker etc., make template with the nucleic acid that extracts, adopt the RT-PCR method to carry out amplification label, in (volume is 80ul) mixed solution, use random primer (production number D3801 company's T aKaRa), dNTP (dTTP adds 0.5ul less), the Cy3-dUTP that other adds the 1mmol/L of 0.5ul carries out mark, amplified production PCR purification kit (production number D301 company's T aKaRa) purifying, product finally is dissolved in 5 an amount of * SSC, in the 0.2%SDS solution, final concentration is 50mg/L.
2. the hybridization of gene chip and testing sample
Get the good testing sample solution of mark, add in advance through 100 ℃ and boil on 30 seconds denatured DNA arrays, covered places wet box, according to selected gene chip, the concrete hybridization conditions that discloses of part is described in detail in detail to specifications hybridizes, put then in 2 * SSC, the 0.1%SDS solution and washed 5 minutes, washed 5 minutes in 0.1 * SSC, the 0.1%SDS solution, swing in 0.1 * SSC solution wash several all over drying.
3. results of hybridization detects
The hybridization air dried DNA array that finishes is scanned under proper condition with Scan Array 3000 (GST LumonicsInc manufacturing) laser confocal scanning instrument and detects its hybridization signal.
Embodiment 3 gene chip detected results of the present invention
Adopt gene chip of the present invention, the sample to 100 routine sick pigs detects respectively, and the result is as follows: in 100 samples, detect 17 examples that have of viral CSFV, FMDV has 6 examples, and SIV has 4 examples, and PRV has 23 examples, PRRSV has 13 examples, TGEV has 7 examples, and PPV4 has 7 examples, and PRCV has 6 examples, PRTV has 14 examples, and JEV has 3 examples.Wherein 3 routine TGEV and PRTV polyinfection, 2 routine PRCV and SIV polyinfection, 5 routine CSFV and PRV polyinfection, 14 routine PRV and PPV polyinfection.
Above-mentioned detected result, with isolation of virus, the ELISA method of prior art, detecting above-mentioned viral coincidence rate is 96%.
Embodiment 4
Adopt gene chip of the present invention, the sample to every kind of disease 100 routine sick chickens detects respectively, and compares with blood clotting inhibition method (HI) of the prior art, isolation of virus (VI), PCR method, and the result is as shown in table 1 below.
Table 1
| HI | VI | PCR | Gene chips | |
| NDV | 42 | 35 | 60 | 54 |
| AIV | 31 | 22 | 45 | 43 |
| IBV | 24 | 18 | 51 | 47 |
| ILTV | 7 | 12 | 10 | |
| MDV | 9 | 23 | 20 | |
| EDS-76V | 30 | 21 | 45 | 43 |
| IBDV | 7 | 19 | 16 |
Can find out that from last table the gene chip detection sensitivity is higher than HI, AI, more lower slightly than PCR, but PCR once can only a kind of cause of disease, and gene chip once can detect multiple cause of disease.
Sequence table
＜110〉Shandong Aolan Biology Engineering Research Institute
＜120〉diagnostic gene chip for pig virus disease and uses thereof
<130>IDC020045
<160>35
<170>PatentIn version 3.1
<210>1
<211>508
<212>DNA
<213>Classical swine fever
<400>1
gctcctggtt ggtaacctcg ggaaatcttg ggacggaggt gggagatttg gaacaccttg 60
gctgggtgct tagagggcct gctgtttgca agaaggttac cgaacatgag aaatgcacca 120
catctataat ggataaattg actgcttttt tcggtgttat gccaaggggc accacaccta 180
gagcccctgt gagattcccc acttccctct taaagataag aagggggtta gaaactggct 240
gggcgtacac acaccaaggt ggcattagtt cagtggacca tgtcacttgt gggaaagact 300
tgctggtatg tgacactatg ggccggacaa gggtcgtttg ccaatcaaat aataagatga 360
cagacgagtc cgagtacgga gttaaaactg actccggatg cccggaagga gctaggtgtt 420
atgtgttcaa cccagaggca gttaacatat cagggactaa aggagccatg gtccacttac 480
agaagacttc acctgtgtga cagcatca 508
<210>2
<211>197
<212>DNA
<213>foot-and-mouth disease
<400>2
gaagggccca gggttggact caacattttg gacctcatgc agattccatc acacactttg 60
gtgggagcgc tcctacgtgc gtccacttac tacttctctg acttggagat agcagtaaaa 120
cacgagggag acctcacctg ggttccaaat ggagcgcctg aaaagggctt ggacaggagt 180
ccaaccctgg gcccttc 197
<210>3
<211>217
<212>DNA
<213>pseudorabies
<400>3
cacggaggac gagctggggc tcccacgtac cgcgcccacg tggcctggta ccgcatcgcg 60
gacgggtgcg cacacctgct gtactttatc gagtacgccg actgcgaccc caggcaggtc 120
tttgggcgct gccggcgccg caccacgccg atgtggtgga ccccgtccgc ggactacatg 180
ttccccacgg aggacgagct tcaacggggg cgtggac 217
<210>4
<211>255
<212>DNA
<213>procine reproductive and respiratory syndrome
<400>4
cctcctgtat gaacttgcaa actggcaaac tgtcacccct tgtactctta agaaacagta 60
ttgcgggaag aagaagacta ggaccatact cggcaccaat aacttcatcg cactagccca 120
ccgagcagtg ttgagtggtg ttacccaggg cttcatgaaa aaggcgttta actcgcccat 180
cgccctcgga aagaacaagt ttaaggagct acagactccg gtcctgggca ggtgccagct 240
caagttcgag gacct 255
<210>5
<211>591
<212>DNA
<213>Swine Influenza
<400>5
ggtgtgacgg cagcatgccc tcacagctca ggaattggac tgtataaaaa ttacacagca 60
ggttggtgta gaactcaacc tgtacctaac tgaattgact acagtattcg ggccacaaat 120
aacttcccct gccttaactc agctgactat ccaggcgctt tacaacctag ctggtggtaa 180
tatggattac ttgttgacta agttaggtgt agggaacaac caactcagct cattaattgg 240
tagcggcttg atcaccggca accctattct gtacgactca cagactcaac tcttgggtat 300
acaggtaact ttaccctcag tcggaaacct aaataatatg cgtgccacct acctggagac 360
cttgtctgta agcacaacca agggatttgc ctcagcactc gtcccaaaag tggtgacaca 420
ggtcggttct gtgatagaag aacttgacac ttcatactgt atagagaccg atttggattt 480
gtattgtaca agaatagtga cattccctat gtctcctggt atttattcct gtttgaacgg 540
caatacatcg gcttgcatgt attcaaagac ggtctgtttg gagccattgc t 591
<210>6
<211>570
<212>DNA
<213>Transmissible Gastroenteritis
<400>6
agaactatag gtaaccattg gccggttggc gcattcaagg cgcaagatgg gccccagatc 60
ttctaccagg atcccaatac ctctgatgtt gaccatccgg atcgcgctgg cactgagttg 120
tgtccatctg gcaagttctc ttgatggcag gcctcttgca gctgcaggga tcgtggtaac 180
aggggataaa cagtcaacaa tatacacctc atcccagaca gggtcaatca tagtcaagtt 240
actcccaaat atgcccaagg ataaagaggc gtgtgcaaaa gccccgttgg aggcatacaa 300
caggacactg actactttgc tcacccccct tggtgattct atccgcagga tacaagagtc 360
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 420
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 480
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 540
acatgaggtc atgcgtgcga ctacatagaa 570
<210>7
<211>429
<212>DNA
<213>Porcine Respiratory Coronavirus
<400>7
gggtaagttg ctcattagaa acaattgcaa gatgacaaca tgcagatgtg cagacccccc 60
gggtatcata tcgcaaaatt atggagaagc tgtgtctcta atagataggc actcatgcaa 120
tgtcttatcc ttggacggga taactttgag gctcagtggg gaatttgatg caacttatca 180
aaagaatatc tcaatactag attctcaagt aatagtgaca ggcaatctcg atatctcaac 240
tgagcttggg aatgtcaaca actcgataag taatgctttg gataagttag aggaaagcaa 300
cagcaaacta gacaaagtca atgtcaaact gaccagcaca tctgctctca ttacctatat 360
cgctttaact gccatatctc ttgtttgcgg tatacttagt ctggttcatg ataggactcg 420
tggcactgc 429
<210>8
<211>158
<212>DNA
<213>Porcine Parvovirus
<400>8
gcagtaccaa ttcatcttct ctctgatgtt gaccatccgg atcgcgctgg cactgagttg 60
tgtccatctg gcaagttctc ttgatggcag gcctcttgca gctgcaggga tcgtggtaac 120
aggggataaa gcagtcaacc taccacagaa ggagacca 158
<210>9
<211>887
<212>DNA
<213>Porcine Rotavirus
<400>9
tggcttcgct catttataga ccacccccct tggtgattct atccgcagga tacaagagtc 60
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 120
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 180
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 240
acatgaggtc actgacggat tatcacaact agcagtggca gttgggaaga tgcagcagtt 300
tgttaatgac cagtttaata acacagctca ggaattggac tgtataaaaa ttacacagca 360
ggttggtgta gaactcaacc tgtacctaac tgaattgact acagtattcg ggccacaaat 420
aacttcccct gccttaactc agctgactat ccaggcgctt tacaacctag ctggtggtaa 480
tatggattac ttgttgacta agttaggtgt agggaacaac caactcagct cattaattgg 540
tagcggcttg atcaccggca accctattct gtacgactca cagactcaac tcttgggtat 600
acaggtaact ttaccctcag tcggaaacct aaataatatg cgtgccacct acctggagac 660
cttgtctgta agcacaacca agggatttgc ctcagcactc gtcccaaaag tggtgacaca 720
ggtcggttct gtgatagaag aacttgacac ttcatactgt atagagaccg atttggattt 780
gtattgtaca agaatagtga cattccctat gtctcctggt atttattcct gtttgaacgg 840
caatacatcg gcttgcatgt attcaaaggt tataaatggt ccgaaat 887
<210>10
<211>374
<212>DNA
<213>Japanese Encephalitis
<400>10
cattccaagc gaagcaggag atcctggcag gcctcttgca gctgcaggga tcgtggtaac 60
aggggataaa gcagtcaaca tatacacctc atcccagaca gggtcaatca tagtcaagtt 120
actcccaaat atgcccaagg ataaagaggc gtgtgcaaaa gccccgttgg aggcatacaa 180
caggacactg actactttgc tcacccccct tggtgattct atccgcagga tacaagagtc 240
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 300
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga taccgacaaa 360
ccaacatgga cgtc 374
<210>11
<211>541
<212>DNA
<213>Porcine Epidemic Diarrhoea
<400>11
aggaacgtga cctcaaagac atcccagagg cgtgtgcaaa agccccgttg gaggcataca 60
acaggacact gactactttg ctcacccccc ttggtgattc tatccgcagg atacaagagt 120
ctgtgactac atccggagga aggagacaga aacgctttat aggtgctatt atcggcagtg 180
tagctcttgg ggttgcaaca gctgcacaga taacagcagc ctcggccctg atacaagcca 240
atcagaatgc tgccaacatc ctccggctta aggagagcat tactgcaacc aatgaagctg 300
tacatgaggt cactgacgga ttatcacaac tagcagtggc agttgggaag atgcagcagt 360
ttgttaatga ccagtttaat aacacagctc aggaattgga ctgtataaaa attacacagc 420
aggttggtgt agaactcaac ctgtacctaa ctgaattgac tacagtattc gggccacaaa 480
taacttcccc tgccttaact cagctgacta tccaggccaa tgttagaccg gcttatcctg 540
g 541
<210>12
<211>362
<212>DNA
<213>Newcastle Disease
<400>12
ttgatggcag gcctcttgcc tcacccccct tggtgattct atccgcagga tacaagagtc 60
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 120
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 180
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 240
acatgaggtc actgacggat tatcacaact agcagtggca gttgggaaga tgcagcagtt 300
tgttaatgac cagtttaata acacagctca ggaattggac tgtaatgctg ccaacatcct 360
cc 362
<210>13
<211>601
<212>DNA
<213>Avian Influenza
<400>13
agcgtagacg ctttgtccag ctgcacagat aacagcagcc tcggccctga tacaagccaa 60
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 120
acatgaggtc actgacggat tatcacaact agcagtggca gttgggaaga tgcagcagtt 180
tgttaatgac cagtttaata acacagctca ggaattggac tgtataaaaa ttacacagca 240
ggttggtgta gaactcaacc tgtacctaac tgaattgact acagtattcg ggccacaaat 300
aacttcccct gccttaactc agctgactat ccaggcgctt tacaacctag ctggtggtaa 360
tatggattac ttgttgacta agttaggtgt agggaacaac caactcagct cattaattgg 420
tagcggcttg atcaccggca accctattct gtacgactca cagactcaac tcttgggtat 480
acaggtaact ttaccctcag tcggaaacct aaataatatg cgtgccacct acctggagac 540
cttgtctgta agcacaacca agggatttgc ctcagcactc gtccgtggat tcttgatcgt 600
c 601
<210>14
<211>367
<212>DNA
<213>Marek’s Disease
<400>14
taacggcgcg cctttaactg aggtacctca ggaattggac tgtataaaaa ttacacagca 60
ggttggtgta gaactcaacc tgtacctaac tgaattgact acagtattcg ggccacaaat 120
aacttcccct gccttaactc agctgactat ccaggcgctt tacaacctag ctggtggtaa 180
tatggattac ttgttgacta agttaggtgt agggaacaac caactcagct cattaattgg 240
tagcggcttg atcaccggca accctattct gtacgactca cagactcaac tcttgggtat 300
acaggtaact ttaccctcag tcggaaacct aaataatatg ccgcgtatcg ctggtctcta 360
tggagtc 367
<210>15
<211>479
<212>DNA
<213>Infectious bursal Disease
<400>15
acccttgtgc tagaccagtg ggtcggttct gtgatagaag aacttgacac ttcatactgt 60
atagagaccg atttggattt gtattgtaca agaatagtga cattccctat gtctcctggt 120
atttattcct gtttgaacgg caatacatcg gcttgcatgt attcaaagac tgaaggcgca 180
cttactacgc catacatgac tctcaaaggc tcagttattg ccaattgcaa gatgacaaca 240
tgcagatgtg cagacccccc gggtatcata tcgcaaaatt atggagaagc tgtgtctcta 300
atagataggc actcatgcaa tgtcttatcc ttggacggga taactttgag gctcagtggg 360
gaatttgatg caacttatca aaagaatatc tcaatactag attctcaagt aatagtgaca 420
ggcaatctcg atatctcaac tgagcttggg aatgtcaaca actggaggca aaggggttc 479
<210>16
<211>600
<212>DNA
<213>Infectious Bronchitis
<400>16
cataactaac ataagggcaa ataaagaggc gtgtgcaaaa gccccgttgg aggcatacaa 60
caggacactg actactttgc tcacccccct tggtgattct atccgcagga tacaagagtc 120
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 180
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 240
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 300
acatgaggtc actgacggat tatcacaact agcagtggca gttgggaaga tgcagcagtt 360
tgttaatgac cagtttaata acacagctca ggaattggac tgtataaaaa ttacacagca 420
ggttggtgta gaactcaacc tgtacctaac tgaattgact acagtattcg ggccacaaat 480
aacttcccct gccttaactc agctgactat ccaggcgctt tacaacctag ctggtggtaa 540
tatggattac ttgttgacta agttaggtgt agggaacaac caacaccttg aagaggaccg 600
<210>17
<211>644
<212>DNA
<213>Infectious Laryngotracheitis
<400>17
acgatgactc cgactttcac ctctgatgtt gaccatccgg atcgcgctgg cactgagttg 60
tgtccatctg gcaagttctc ttgatggcag gcctcttgca gctgcaggga tcgtggtaac 120
aggggataaa gcagtcaaca tatacacctc atcccagaca gggtcaatca tagtcaagtt 180
actcccaaat atgcccaagg ataaagaggc gtgtgcaaaa gccccgttgg aggcatacaa 240
caggacactg actactttgc tcacccccct tggtgattct atccgcagga tacaagagtc 300
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 360
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 420
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 480
acatgaggtc actgacggat tatcacaact agcagtggca gttgggaaga tgcagcagtt 540
tgttaatgac cagtttaata acacagctca ggaattggac tgtataaaaa ttacacagca 600
ggttggtgta gaactcaacc tgtacctacc acctacctcc aacg 644
<210>18
<211>238
<212>DNA
<213>Egg Drop Syndrom-1976
<400>18
ttggcgtctt caaggcactg tcacccccct tggtgattct atccgcagga tacaagagtc 60
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 120
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 180
tcagaatgct gccaacatcc tccggcttaa ggagagcaca gtcagatgca gttgtgtg 238
<2t0>19
<211>291
<212>DNA
<213>Reticuloendotheliosis
<400>19
catactggag ccaatggttg tcggaaacct aaataatatg cgtgccacct acctggagac 60
cttgtctgta agcacaacca agggatttgc ctcagcactc gtcccaaaag tggtgacaca 120
ggtcggttct gtgatagaag aacttgacac ttcatactgt atagagaccg atttggattt 180
gtattgtaca agaatagtga cattccctat gtctcctggt atttattcct gtttgaacgg 240
caatacatcg gcttgcatgt attcaaagac tgagtacttc gatacaacat t 291
<210>20
<211>140
<212>DNA
<213>Avian Encephalomyelitis
<400>20
gtgagacagc taacttgatg atcccggttg gcgcattcaa ggcgcaagat gggccccaga 60
tcttctacca ggatcccaat acctctgatg ttgaccatcc ggatcgcgct ggcactgagt 120
cgacatcgat acgcgtggtc 140
<210>21
<211>675
<212>DNA
<213>Chicken Infectious Anemia
<400>21
gactgtaaga tggcaagacg agctcggcag gcctcttgca gctgcaggga tcgtggtaac 60
aggggataaa gcagtcaaca tatacacctc atcccagaca gggtcaatca tagtcaagtt 120
actcccaaat atgcccaagg ataaagaggc gtgtgcaaaa gccccgttgg aggcatacaa 180
caggacactg actactttgc tcacccccct tggtgattct atccgcagga tacaagagtc 240
tgtgactaca tccggaggaa ggagacagaa acgctttata ggtgctatta tcggcagtgt 300
agctcttggg gttgcaacag ctgcacagat aacagcagcc tcggccctga tacaagccaa 360
tcagaatgct gccaacatcc tccggcttaa ggagagcatt actgcaacca atgaagctgt 420
acatgaggtc actgacggat tatcacaact agcagtggca gttgggaaga tgcagcagtt 480
tgttaatgac cagtttaata acacagctca ggaattggac tgtataaaaa ttacacagca 540
ggttggtgta gaactcaacc tgtacctaac tgaattgact acagtattcg ggccacaaat 600
aacttcccct gccttaactc agctgactat ccaggcgctt atcgcgctgg tacaagaatg 660
agggatcctt cagcc 675
<210>22
<211>730
<212>DNA
<213>Mycoplasma Gallisepticum
<400>22
ggatcccatc cgaccacgaa aaaccccttg gtgattctat ccgcaggata caagagtctg 60
tgactacatc cggaggaagg agacagaaac gctttatagg tgctattatc ggcagtgtag 120
ctcttggggt tgcaacagct gcacagataa cagcagcctc ggccctgata caagccaatc 180
agaatgctgc caacatcctc cggcttaagg agagcattac tgcaaccaat gaagctgtac 240
atgaggtcac tgacggatta tcacaactag cagtggcagt tgggaagatg cagcagtttg 300
ttaatgacca gtttaataac acagctcagg aattggactg tataaaaatt acacagcagg 360
ttggtgtaga actcaacctg tacctaactg aattgactac agtattcggg ccacaaataa 420
cttcccctgc cttaactcag ctgactatcc aggcgcttta caacctagct ggtggtaata 480
tggattactt gttgactaag ttaggtgtag ggaacaacca actcagctca ttaattggta 540
gcggcttgat caccggcaac cctattctgt acgactcaca gactcaactc ttgggtatac 600
aggtaacttt accctcagtc ggaaacctaa ataatatgcg tgccacctac ctggagacct 660
tgtctgtaag cacaaccaag ggatttgcct cagcactcgt cccaatcatc agttactcac 720
tgattgaaag 730
<210>23
<211>206
<212>DNA
<213>Mycoplasma Synoviae
<400>23
gaagcaaata tgatatcaaa cacagctcag gaattggact gtataaaaat tacacagcag 60
gttggtgtag aactcaacct gtacctaact gaattgacta cagtattcgg gccacaaata 120
acttcccctg ccttaactca gctgactatc caggcgcttt acaacctagc tggtggtaat 180
atggattttg ttaacttcga gacgac 206
<210>24
<211>558
<212>DNA
<213>Avian Pasteurellosis
<400>24
agagtttgat ctggctcagc caattgcaag atgacaacat gcagatgtgc agaccccccg 60
ggtatcatat cgcaaaatta tggagaagct gtgtctctaa tagataggca ctcatgcaat 120
gtcttatcct tggacgggat aactttgagg ctcagtgggg aatttgatgc aacttatcaa 180
aagaatatct caatactaga ttctcaagta atagtgacag gcaatctcga tatctcaact 240
gagcttggga atgtcaacaa ctcgataagt aatgctttgg ataagttaga ggaaagcaac 300
agcaaactag acaaagtcaa tgtcaaactg accagcacat ctgctctcat tacctatatc 360
gctttaactg ccatatctct tgtttgcggt atacttagtc tggttctagc atgctaccta 420
atgtacaagc aaaaggcgca acaaaagacc ttgttatggc ttgggaataa taccctgggt 480
cagatgagag ccactacaaa aatgtgaatg cagatgagag gcggaggtat ccccaatagc 540
aggctggatc acctcctt 558
<210>25
<211>282
<212>DNA
<213>Salmonelosis
<400>25
gtgaaattac gccacgttcg gcaaacactt catactgtat agagaccgat ttggatttgt 60
attgtacaag aatagtgaca ttccctatgt ctcctggtat ttattcctgt ttgaacggca 120
atacatcggc ttgcatgtat tcaaagactg aaggcgcact tactacgcca tacatgactc 180
tcaaaggctc agttattgcc aattgcaaga tgacaacatg cagatgtgca gaccccccgg 240
gtatcatatc gcaaaattat ggttcctttg acggtgcgat ga 282
<210>26
<211>324
<212>DNA
<213>Colibacillosis
<400>26
agagtttgat ctggctcagc ccggttggcg cattcaaggc gcaagatggg ccccagatct 60
tctaccagga tcccaatacc tctgatgttg accatccgga tcgcgctggc actgagttgt 120
gtccatctgg caagttctct tgatggcagg cctcttgcag ctgcagggat cgtggtaaca 180
ggggataaag cagtcaacat atacacctca tcccagacag ggtcaatcat agtcaagtta 240
ctcccaaata tgcccaagga taaagaggcg tgtgcaaaag ccccgttgga ggcatacaac 300
aggacactgc tggatcacct cctt 324
<210>27
<211>515
<212>DNA
<213>Serpulina hyodysenteriae
<400>27
agagtttgat ctggctcaga cacagctcag gaattggact gtataaaaat tacacagcag 60
gttggtgtag aactcaacct gtacctaact gaattgacta cagtattcgg gccacaaata 120
acttcccctg ccttaactca gctgactatc caggcgcttt acaacctagc tggtggtaat 180
atggattact tgttgactaa gttaggtgta gggaacaacc aactcagctc attaattggt 240
agcggcttga tcaccggcaa ccctattctg tacgactcac agactcaact cttgggtata 300
caggtaactt taccctcagt cggaaaccta aataatatgc gtgccaccta cctggagacc 360
ttgtctgtaa gcacaaccaa gggatttgcc tcagcactcg tcccaaaagt ggtgacacag 420
gtcggttctg tgatagaaga acttgacact tcatactgta tagagaccga tttggatttg 480
tattgtacaa gaatagtgag ctggatcacc tcctt 515
<210>28
<211>356
<212>DNA
<213>Campylobacter jejuni
<400>28
agagtttgat ctggctcaga gctgactatc caggcgcttt acaacctagc tggtggtaat 60
atggattact tgttgactaa gttaggtgta gggaacaacc aactcagctc attaattggt 120
agcggcttga tcaccggcaa ccctattctg tacgactcac agactcaact cttgggtata 180
caggtaactt taccctcagt cggaaaccta aataatatgc gtgccaccta cctggagacc 240
ttgtctgtaa gcacaaccaa gggatttgcc tcagcactcg tcccaaaagt ggtgacacag 300
gtcggttctg tgatagaaga acttgacact tcatactgtg gctggatcac ctcctt 356
<210>29
<211>796
<212>DNA
<213>Brucella suis
<400>29
agagtttgat ctggctcaga gggatttgcc tcagcactcg tcccaaaagt ggtgacacag 60
gtcggttctg tgatagaaga acttgacact tcatactgta tagagaccga tttggatttg 120
tattgtacaa gaatagtgac attccctatg tctcctggta tttattcctg tttgaacggc 180
aatacatcgg cttgcatgta ttcaaagact gaaggcgcac ttactacgcc atacatgact 240
ctcaaaggct cagttattgc caattgcaag atgacaacat gcagatgtgc agaccccccg 300
ggtatcatat cgcaaaatta tggagaagct gtgtctctaa tagataggca ctcatgcaat 360
gtcttatcct tggacgggat aactttgagg ctcagtgggg aatttgatgc aacttatcaa 420
aagaatatct caatactaga ttctcaagta atagtgacag gcaatctcga tatctcaact 480
gagcttggga atgtcaacaa ctcgataagt aatgctttgg ataagttaga ggaaagcaac 540
agcaaactag acaaagtcaa tgtcaaactg accagcacat ctgctctcat tacctatatc 600
gctttaactg ccatatctct tgtttgcggt atacttagtc tggttctagc atgctaccta 660
atgtacaagc aaaaggcgca acaaaagacc ttgttatggc ttgggaataa taccctgggt 720
cagatgagag ccactacaaa aatgtgaatg cagatgagag gcggaggtat ccccaatagg 780
gctggatcac ctcctt 796
<210>30
<211>626
<212>DNA
<213>Bordetella bronchiseptica
<400>30
agagtttgat ctggctcagc attccctatg tctcctggta tttattcctg tttgaacggc 60
aatacatcgg cttgcatgta ttcaaagact gaaggcgcac ttactacgcc atacatgact 120
ctcaaaggct cagttattgc caattgcaag atgacaacat gcagatgtgc agaccccccg 180
ggtatcatat cgcaaaatta tggagaagct gtgtctctaa tagataggca ctcatgcaat 240
gtcttatcct tggacgggat aactttgagg ctcagtgggg aatttgatgc aacttatcaa 300
aagaatatct caatactaga ttctcaagta atagtgacag gcaatctcga tatctcaact 360
gagcttggga atgtcaacaa ctcgataagt aatgctttgg ataagttaga ggaaagcaac 420
agcaaactag acaaagtcaa tgtcaaactg accagcacat ctgctctcat tacctatatc 480
gctttaactg ccatatctct tgtttgcggt atacttagtc tggttctagc atgctaccta 540
atgtacaagc aaaaggcgca acaaaagacc ttgttatggc ttgggaataa taccctgggt 600
cagatgagag gctggatcac ctcctt 626
<210>31
<211>586
<212>DNA
<213>Actinobaci1lus pleuropneumoniae
<400>31
agagtttgat ctggctcagt cacccccctt ggtgattcta tccgcaggat acaagagtct 60
gtgactacat ccggaggaag gagacagaaa cgctttatag gtgctattat cggcagtgta 120
gctcttgggg ttgcaacagc tgcacagata acagcagcct cggccctgat acaagccaat 180
cagaatgctg ccaacatcct ccggcttaag gagagcatta ctgcaaccaa tgaagctgta 240
catgaggtca ctgacggatt atcacaacta gcagtggcag ttgggaagat gcagcagttt 300
gttaatgacc agtttaataa cacagctcag gaattggact gtataaaaat tacacagcag 360
gttggtgtag aactcaacct gtacctaact gaattgacta cagtattcgg gccacaaata 420
acttcccctg ccttaactca gctgactatc caggcgcttt acaacctagc tggtggtaat 480
atggattact tgttgactaa gttaggtgta gggaacaacc aactcagctc attaattggt 540
agcggcttga tcaccggcaa ccctattctg gctggatcac ctcctt 586
<210>32
<211>586
<212>DNA
<213>Staphylococcus aureus
<400>32
agagtttgat ctggctcagc tgcacagata acagcagcct cggccctgat acaagccaat 60
cagaatgctg ccaacatcct ccggcttaag gagagcatta ctgcaaccaa tgaagctgta 120
catgaggtca ctgacggatt atcacaacta gcagtggcag ttgggaagat gcagcagttt 180
gttaatgacc agtttaataa cacagctcag gaattggact gtataaaaat tacacagcag 240
gttggtgtag aactcaacct gtacctaact gaattgacta cagtattcgg gccacaaata 300
acttcccctg ccttaactca gctgactatc caggcgcttt acaacctagc tggtggtaat 360
atggattact tgttgactaa gttaggtgta gggaacaacc aactcagctc attaattggt 420
agcggcttga tcaccggcaa ccctattctg tacgactcac agactcaact cttgggtata 480
caggtaactt taccctcagt cggaaaccta aataatatgc gtgccaccta cctggagacc 540
ttgtctgtaa gcacaaccaa gggatttgcg gctggatcac ctcctt 586
<210>33
<211>796
<212>DNA
<213>Streptococcus suis
<400>33
agagtttgat ctggctcaga gggatttgcc tcagcactcg tcccaaaagt ggtgacacag 60
gtcggttctg tgatagaaga acttgacact tcatactgta tagagaccga tttggatttg 120
tattgtacaa gaatagtgac attccctatg tctcctggta tttattcctg tttgaacggc 180
aatacatcgg cttgcatgta ttcaaagact gaaggcgcac ttactacgcc atacatgact 240
ctcaaaggct cagttattgc caattgcaag atgacaacat gcagatgtgc agaccccccg 300
ggtatcatat cgcaaaatta tggagaagct gtgtctctaa tagataggca ctcatgcaat 360
gtcttatcct tggacgggat aactttgagg ctcagtgggg aatttgatgc aacttatcaa 420
aagaatatct caatactaga ttctcaagta atagtgacag gcaatctcga tatctcaact 480
gagcttggga atgtcaacaa ctcgataagt aatgctttgg ataagttaga ggaaagcaac 540
agcaaactag acaaagtcaa tgtcaaactg accagcacat ctgctctcat tacctatatc 600
gctttaactg ccatatctct tgtttgcggt atacttagtc tggttctagc atgctaccta 660
atgtacaagc aaaaggcgca acaaaagacc ttgttatggc ttgggaataa taccctgggt 720
cagatgagag ccactacaaa aatgtgaatg cagatgagag gcggaggtat ccccaatagg 780
gctggatcac ctcctt 796
<210>34
<211>586
<212>DNA
<213>Bacillus rhusiopathiae suis
<400>34
agagtttgat ctggctcagt cacccccctt ggtgattcta tccgcaggat acaagagtct 60
gtgactacat ccggaggaag gagacagaaa cgctttatag gtgctattat cggcagtgta 120
gctcttgggg ttgcaacagc tgcacagata acagcagcct cggccctgat acaagccaat 180
cagaatgctg ccaacatcct ccggcttaag gagagcatta ctgcaaccaa tgaagctgta 240
catgaggtca ctgacggatt atcacaacta gcagtggcag ttgggaagat gcagcagttt 300
gttaatgacc agtttaataa cacagctcag gaattggact gtataaaaat tacacagcag 360
gttggtgtag aactcaacct gtacctaact gaattgacta cagtattcgg gccacaaata 420
acttcccctg ccttaactca gctgactatc caggcgcttt acaacctagc tggtggtaat 480
atggattact tgttgactaa gttaggtgta gggaacaacc aactcagctc attaattggt 540
agcggcttga tcaccggcaa ccctattctg gctggatcac ctcctt 586
<210>35
<211>586
<212>DNA
<213>Corynebacterium suis
<400>35
agagtttgat ctggctcagt cacccccctt ggtgattcta tccgcaggat acaagagtct 60
gtgactacat ccggaggaag gagacagaaa cgctttatag gtgctattat cggcagtgta 120
gctcttgggg ttgcaacagc tgcacagata acagcagcct cggccctgat acaagccaat 180
cagaatgctg ccaacatcct ccggcttaag gagagcatta ctgcaaccaa tgaagctgta 240
catgaggtca ctgacggatt atcacaacta gcagtggcag ttgggaagat gcagcagttt 300
gttaatgacc agtttaataa cacagctcag gaattggact gtataaaaat tacacagcag 360
gttggtgtag aactcaacct gtacctaact gaattgacta cagtattcgg gccacaaata 420
acttcccctg ccttaactca gctgactatc caggcgcttt acaacctagc tggtggtaat 480
atggattact tgttgactaa gttaggtgta gggaacaacc aactcagctc attaattggt 540
agcggcttga tcaccggcaa ccctattctg gctggatcac ctcctt 586
Claims (6)
1. gene chip that is used to diagnose virus diseases of pigs, comprising: be fixed on the solid phase carrier with corresponding infective pathogen characteristic DNA to be measured of pathogen species to be checked and/or cDNA fragment, wherein the characteristic fragment of Cai Yonging is by Pestivirus suis, foot and mouth disease virus, pseudorabies virus, transmissible gastro-enteritis virus, pig breeding and breath syndrome virus, swine influenza virus, pig parvoviral, PRCV (Porcine Respiratory Coronavirus), porcine rotavirus, epidemic encephalitis b of swine virus causing disease gene expression characteristics fragment is formed, and it is respectively corresponding to SEQ ID NO:1,2,3,6,4,5,8,7,9 and 10 sequence.
2. the described gene chip of claim 1, wherein said solid phase carrier is selected from: nitrocellulose filter, nylon membrane.
3. a method that is used to detect Pestivirus suis, foot and mouth disease virus, pseudorabies virus, transmissible gastro-enteritis virus, pig breeding and breath syndrome virus, swine influenza virus, pig parvoviral, PRCV (Porcine Respiratory Coronavirus), porcine rotavirus, epidemic encephalitis b of swine virus comprises the steps:
1) selects the described gene chip of claim 1 for use;
2) mark testing sample;
3) be suitable under the condition of hybridizing, adding through the testing sample of mark and make it to react time enough with selected gene chip; With
4) result of detection hybridization.
4. the described method of claim 3, wherein the mark of testing sample adopts vitamin H.
5. the described method of claim 4, wherein said vitamin H is selected from: photobiotin and vitamin H-dUTP.
6. the described method of claim 3, wherein with hybridization conditions be: for 200ng/mlDNA or RNA through the testing sample of mark for being fixed with the segmental gene chip of cause of disease characteristic to be measured, its average polynucleotide sequence length is 617 bases, hybridization is 10 hours under 43 ℃ of conditions, and using methane amide concentration is 50%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410078906 CN1255555C (en) | 2002-12-12 | 2002-12-12 | Diagnostic gene chip for pig virus disease and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410078906 CN1255555C (en) | 2002-12-12 | 2002-12-12 | Diagnostic gene chip for pig virus disease and its use |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021554080A Division CN1181210C (en) | 2002-12-12 | 2002-12-12 | Gene chip for epidemic disease of fowl and/or livestock and its usage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1616682A CN1616682A (en) | 2005-05-18 |
| CN1255555C true CN1255555C (en) | 2006-05-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410078906 Expired - Fee Related CN1255555C (en) | 2002-12-12 | 2002-12-12 | Diagnostic gene chip for pig virus disease and its use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1255555C (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100412205C (en) * | 2005-10-26 | 2008-08-20 | 山东省医药生物技术研究中心 | Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its prepn and application |
| CN100447253C (en) * | 2005-10-26 | 2008-12-31 | 山东省医药生物技术研究中心 | Glass gene chip for simultaneous detection of Group A, B and C human rotaviruses and the prepn and application |
| CN102234693B (en) * | 2011-03-11 | 2013-03-06 | 重庆理工大学 | Gene chip and method for detecting classical swine fever virus (CSFV), porcine circovirus virus type 2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN102154516B (en) * | 2011-03-22 | 2013-05-01 | 上海交通大学 | Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof |
| CN102605099B (en) * | 2011-12-31 | 2014-07-23 | 北京农学院 | Pig virus gene chip and detection method thereof |
| CN104232799B (en) * | 2014-09-29 | 2016-05-25 | 四川农业大学 | Detect genetic chip and the kit of transmissible gastro-enteritis virus and/or porcine rotavirus |
| CN104232801B (en) * | 2014-09-29 | 2016-08-24 | 四川农业大学 | Detection Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and the kit of porcine rotavirus |
| CN104263856B (en) * | 2014-09-29 | 2016-05-25 | 四川农业大学 | Detect genetic chip and the kit of Porcine epidemic diarrhea virus and/or transmissible gastro-enteritis virus |
| CN104293977B (en) * | 2014-09-29 | 2016-11-23 | 四川农业大学 | The genetic chip of detection Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and porcine rotavirus and kit |
| CN104263855B (en) * | 2014-09-29 | 2016-05-25 | 四川农业大学 | Detect genetic chip and the kit of Porcine epidemic diarrhea virus and/or porcine rotavirus |
| CN104730248A (en) * | 2015-03-17 | 2015-06-24 | 吉林吉和迅生物技术有限公司 | Rapid diagnosis and detection test paper card for detecting swine fever as well as preparation and application method of rapid diagnosis and detection test paper card |
| CN114317816B (en) * | 2021-10-28 | 2023-09-01 | 台州市药品检验研究院 | Kit for simultaneously detecting six common viruses of pigs in compound koto raw materials and products thereof |
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2002
- 2002-12-12 CN CN 200410078906 patent/CN1255555C/en not_active Expired - Fee Related
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