CN103364548B - Haemophilus parasuis indirect hamagglutination detection reagent - Google Patents
Haemophilus parasuis indirect hamagglutination detection reagent Download PDFInfo
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Abstract
The invention relates to the field of veterinary detection reagents, and discloses a haemophilus parasuis indirect hamagglutination detection reagent. The haemophilus parasuis indirect hamagglutination detection reagent is prepared by taking multiple serotype haemophilus parasuis whole protein mixtures as antigens, and is capable of rapidly and sensitively detecting serum antibodies of all serotype haemophilus parasuis diseases. The production technology is optimized through balanced mixing of each whole protein antigen, and then sensibilization and double hydroformylation of mutton red cells; and the prepared haemophilus parasuis indirect hamagglutination detection reagent is stable, sensitive, good in specificity, long in storage life, simple in operation, and applicable to a large amount of clinical sample detection on the haemophilus parasuis diseases, immune level detection and epidemiology investigation.
Description
Technical field
The present invention relates to animal doctor and detect reagent field, particularly a kind of haemophilus parasuis indirect hamagglutination detection reagent, the invention still further relates to the preparation method of this reagent.
Background technology
Haemophilus parasuis (Haemophilus parasuis, Hps) be a kind of gram-negative coccobacillus in Pasteurella section hemophilus, it is a kind of conditionality pathogen of pig, in worldwide distribution, infect to cause after piglet with polyserositis, arthritis and the meningitis pig Ge Lazeshi that is feature (Glasser ' s) sick.The piglet in 2 ~ 8 week age of main harm, the incidence of disease is generally 10% ~ 15%, and mortality ratio can reach more than 50%.
Between Hps bacterial strain, antigenicity and Virulence Difference are very large, and serological typing is one of important method of difference different strains.At present, the serological typing method (KRG) based on heat stable antigen immunodiffusion proposed for 1992 by Kielstein etc. is worldwide accepted, Hps is divided into 15 serotypes by the method, but has the separated strain serotype of 15.2% ~ 41% can not determine.According to the seroepidemiological survey of the states such as Japan, Germany, the U.S. and Spain, the most popular with serotype 4,5 and 13.Hps is generally popular in China, and at present, in Hebei, Henan, Hubei, Hunan, Anhui, Shanghai, Guangdong, 12 provinces and cities such as Jiangxi all have Haemophilus parasuis to occur and isolate the report of Hps.
At present, Haemophilus parasuis detection technique mainly contains and is separated culture technique, round pcr and Serologic detection technology etc.Separation culture technique and round pcr, mainly for the checkout and diagnosis of cause of disease, are presented as the infection conditions of Hps, respectively have relative merits in actual applications.And Serologic detection technology can not only reflect the popular infection conditions of Hps, the immune level of swinery can also be reacted.Indirect hemagglutination test (IHA) is classical way in Serologic detection, after updating, has been widely used in the serum antibody detecting various cause of disease, and compared with ELISA, indirect hemagglutination test operating process is easy, save time, and does not need specific apparatus.Research confirms, with IHA testing inspection Haemophilus parasuis serum antibody, and the cross reaction of ubiquity two and above serotype.At present, the IHA test that each mechanism applies is all only for the detection of some or several serotype haemophilus parasuis, and the antigen that preparation sensitization blood cell uses is single certain serological type strain antigen [Shi Bi etc., Chinese veterinary science, 2007,37 (11); Liu Maojun etc., pig industry science, 2007,12] or two kinds of serological type strain antigens [Miniats et al., Can J Vet Res, 1991,55 (1); Tadjine et al., J Clin Microbiol, 2004,42 (2); Wei Zi tribute etc., Chinese veterinary science, 2006,36 (9)], therefore in clinical detection, usually occur false negative or there is undetected possibility.In addition, verify through inventor's test of many times, really can not detect the Haemophilus parasuis serum of all serotype with sensitization blood cell prepared by certain two kinds of serological type strain antigen, can only this serotyping sera be detected, and have the individual serotype serum of cross reaction.At present, the intellecture property about this series products is not formed yet.
This research detects for the purpose of the Haemophilus parasuis serum antibody of most serotype by rapid sensitive, do not distinguish certain serotype concrete, according to the reactionogenicity of various antigen and the characteristic of type cross reactivity, Select to use 4 kinds of serotype haemophilus parasuis whole bacterial protein antigen sensibilization dialdehyde sheep red blood cell (SRBC)s, and indirect hemagglutination diagnostic antigen and detection reagent production technology are optimized, set up a kind of easy, stable, susceptibility, cause of disease high specificity, a large amount of clinical samples for Haemophilus parasuis of long shelf-life detect, Observation of immune antibody level and epidemiology survey.
Summary of the invention
The object of the present invention is to provide a kind of reagent detecting 15 kinds of serotypes and indefinite form Haemophilus parasuis antibody, provide the preparation method of this reagent simultaneously.
For achieving the above object, the invention provides a kind of Haemophilus parasuis indirect hamagglutination detection reagent, the antigen preparing this reagent place is the composition of the whole bacterial protein of serum 1 type, serum 4 type, Serotype 5 and serum 13 type haemophilus parasuis, described serum 1 type haemophilus parasuis adopts N4 strain (International Reference strain, thered is provided by Beijing City Agriculture and Forestry Institute, see RafieeM et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, Vet Microbiol, 2005,106 (1-2): 145-151; Cai Xet al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): the report in 117-123), described serum 4 type haemophilus parasuis adopts SW124 strain, and (International Reference strain, is provided by Beijing City Agriculture and Forestry Institute, this International Reference strain is see Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, VetMicrobiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): the report in 117-123) or FS0307 strain (Chinese pathogenic strain, be preserved in China typical culture collection center CCTCC, deposit number: CCTCC NO:M2013094, preservation date: on March 21st, 2013), described Serotype 5 haemophilus parasuis adopts Nagasaki strain (International Reference strain, thered is provided by Beijing City Agriculture and Forestry Institute, this International Reference strain is see Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, VetMicrobiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006, the report of 118 (1-2): 117-123) or XX0306 strain (Chinese pathogenic strain, be preserved in China typical culture collection center CCTCC, deposit number: CCTCC NO:M2013095, preservation date: on March 21st, 2013), described serum 13 type haemophilus parasuis adopts bacterium to adopt IA-84-17975 strain (International Reference strain, thered is provided by Beijing City Agriculture and Forestry Institute, this International Reference strain is see Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, VetMicrobiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): the report in 117-123).
Further, the total concentration of often kind of serotype haemophilus parasuis whole bacterial protein is 10 ~ 100 μ g/mL.
Further, often kind of serotype haemophilus parasuis whole bacterial protein is all mixed in equal amounts, and the total protein concentration of described composition is 40 ~ 400 μ g/mL.
The invention has the beneficial effects as follows:
(1) from the scope of cross reaction between serotype, and the angle of the characteristic of reference culture and epidemic link is set out, 4 kinds of different serotypes haemophilus parasuises are screened targetedly, preparation indirect hamagglutination detection reagent to the full extent with all serotype haemophilus parasuis antibody responses, greatly add reaction range and susceptibility, avoid the insensitivity of single antigen to other serotype antibody.
(2) from stability and the standardization of reaction system, carry out same bacterial concentration and standardized program extraction whole bacterial protein, select optimal concentration, equivalent packing, as sensitising antigens after mixed in equal amounts, at utmost maintain the stability detecting reagent.
(3) this haemophilus parasuis indirect hamagglutination detection reagent susceptibility is high, reproducible, to other pathogenic autoantibodies without obvious cross reaction, and also easy and simple to handle, quick, can be used for the detection of a large amount of clinical sample, Observation of immune antibody level and epidemiology survey.
The a large amount of clinical samples that this detection reagent can be used for Haemophilus parasuis detect, Observation of immune antibody level and epidemiology survey.Its serum antibody susceptibility detecting Haemophilus parasuis is high, and to other pathogenic autoantibodies without obvious cross reaction.
Embodiment
Below in conjunction with embodiment, the present invention will be further described in detail, and advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall into protection scope of the present invention.
Embodiment 1
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strain, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, each strain antigens protein concentration is 20ug/mL, and preparation process is as follows:
1, haemophilus parasuis is cultivated
Get serum 1 type (N4 strain), 4 types (FS0307 strain), 5 types (Nagasaki strain) and each 1 of 13 types (IA-84-17975 strain) haemophilus parasuis that freeze-drying is preserved respectively, add 0.5mL TSB basal medium, get 0.1mL bacterium liquid after mixing and be spread evenly across TSA solid medium (containing 5% serum and 0.01%NAD), 37 DEG C of constant temperature culture 24 ~ 48h.Then picking 3 ~ 5 single bacterium colonies, are inoculated in 5mL TSB fluid nutrient medium (containing 5% serum and 0.01%NAD), 37 DEG C, and 18 ~ 24h cultivated by 180rpm shaking table.Cultured liquid bacteria liquid is inoculated in 100mL TSB fluid nutrient medium (containing 5% serum and 0.01%NAD) and expands cultivation further.Take out after cultivation 18 ~ 24h, utilize colony counting method to estimate the bacterium number of each bacterial strain bacterium liquid.
2, the preparation of sensitising antigens
According to TSA plate count result, centrifugal concentrating method is adopted to concentrate cultured serum 1 type (N4 strain), 4 types (FS0307 strain), 5 types (Nagasaki strain) and 13 types (IA-84-17975 strain) haemophilus parasuis, equal simmer down to 1 × 10
10cFU/mL.Then carry out ultrasonic process to concentrated bacterium liquid, the whole bacterial protein supernatant of each bacterial strain of centrifugal extraction, as the antigen of sensitization blood cell.UV spectrophotometer measuring protein concentration, according to the minimum final concentration of required each strain antigens albumen and system (8mL system) the packing albumen of preparation sensitization blood cell.Respectively get 1 part immediately for the preparation of sensitization blood cell, all the other-40 DEG C save backup.
3, the preparation of dialdehyde blood cell
Aseptic collection Sheep Blood is about 200mL, leaves standstill 2 days after going fiberization at 4 DEG C; On super-clean bench, Sheep Blood is filtered with gauze; Use 0.11M PB(pH=7.2) wash 5 times, centrifugal 20 minutes of each 5000rpm; 10% red cell suspension is made into afterwards with 0.11M PB.10% red cell suspension (500ml) of equivalent is mixed with 3% pyroracemic aldehyde solution (500ml), stirs 18 hours at 25 DEG C; Use 0.11M PB(pH=7.2) wash 5 times, centrifugal 5 minutes of each 4000rpm.3% pyroracemic aldehyde solution is changed to 3% formalin, repeats above-mentioned hydroformylation process.With 0.11M PB, red blood cell is made into the red cell suspension of 10%, the formaldehyde adding 2 ‰ shakes up anticorrosion.Packing, 4 DEG C save backup.
4, the preparation of sensitization blood cell
According to the 8mL system of preparation sensitization blood cell and the minimum final concentration of 20 μ g/mL of each serotype haemophilus parasuis whole bacterial protein, get serum 1 type (N4 strain), 4 types (FS0307 strain), 5 types (Nagasaki strain) and 13 types (IA-84-17975 strain) haemophilus parasuis packing respectively and preserve each 1 part of antigen protein, mixing, supplement and add acetate buffer solution to 0.8mL, mixing, room temperature effect 30 minutes.Then add the dialdehyde blood cell suspension of 0.8mL10%, mixing, 37 DEG C of water-baths 1 hour, constantly stir and shake up.Wash 3 times with 0.11M PB, centrifugal 5 minutes of each 5000rpm, abandons supernatant.Add 8mL TAP, vibration mixing, is various serotype haemophilus parasuis indirect hamagglutination detection reagent.Detect and epidemiology survey for clinical sample.
5, yin and yang attribute sample survey
The yin and yang attribute sample of the haemophilus parasuis indirect hamagglutination detection reagent of above-mentioned preparation to haemophilus parasuis is adopted to test as follows:
The positive serum getting negative serum, 15 kinds of serotypes and the 2 kinds of indefinite forms that laboratory preparation is preserved, as sample, utilizes above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent of preparation to detect.96 orifice plates, every hole adds 20 μ L TAP, adds 20 μ L blood serum samples at first, sample is made 2 times of doubling dilutions, namely dilution ratio is 1:2 ~ 1:256, then discards 20 μ L, last each hole adds 20 μ L sensitization blood cells, and oscillator plate mixes, and concrete operations are as table 1.Observations after standing 1 ~ 2h, testing result is as table 2.
Table 1 application of sample grouping sheet
Table 2 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:128 | 1:32 | 1:16 | 1:64 | 1:64 | 1:128 | 1:64 | 1:128 | 1:32 | 1:128 | 1:64 | 1:64 | 1:128 | 1:64 | 1:256 | 1:128 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
6, cause of disease specific assay
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, it is as follows to carry out cause of disease specific test:
With porcine mycoplasmal pneumonia (MPS) positive serum, 2 type streptococcosis (SS-2) positive serums, porcine contagious pleuropneumonia (APP) positive serum, swine fever (CSF) positive serum, pig blue-ear disease (PRRS) positive serum, toxplasmosis in pigs positive serum for sample, above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent of preparation is utilized to detect.Set up 3 groups of Haemophilus parasuis positive serum controls and 1 group of negative serum control simultaneously.Specified operational procedure is with table 1.Observations after standing 1 ~ 2h, testing result is as table 3.Result shows, yin and yang attribute contrast is all set up, agglutination titer≤the 1:2 of haemophilus parasuis indirect hamagglutination detection reagent and MPS, SS-2 and APP positive serum, and with CSF, PRRS and the not aggegation of toxplasmosis in pigs positive serum, though illustrate that the haemophilus parasuis indirect hamagglutination detection reagent of preparation has slight cross reaction with fraction of pathogens serum, do not affect its specific detection to Haemophilus parasuis positive serum.
The cause of disease specific test testing result of table 3 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
7, clinical sample detects
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carry out clinical sample and detect as follows:
Gather the blood serum sample of morbidity pig and health pig respectively from the pig farm of the ground generation Haemophilus parasuis such as Jiangning, Nanjing, Qixia, Nanjing, Hongze, Jiangsu, Jiangsu, Dongtai and Xuancheng Profile, anhui Province, utilize above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent of preparation to detect.Specified operational procedure is with table 1.Observations after standing 1 ~ 2h, testing result is as table 4.Result shows, and 6 pig farms all detect the positive serum samples of varying number, consistent with clinical observation result, illustrates that 6 pig farms all have haemophilus parasuis in various degree to infect; The haemophilus parasuis indirect hamagglutination detection reagent further illustrating preparation can be used for the detection of a large amount of clinical sample, Observation of immune antibody level and epidemiology survey.
Table 4 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 2
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strain, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 40ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 5 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:128 | 1:64 | 1:64 | 1:256 | 1:64 | 1:128 | 1:64 | 1:128 | 1:32 | 1:256 | 1:64 | 1:16 | 1:256 | 1:64 | 1:256 | 1:256 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 6.
The cause of disease specific test testing result of table 6 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to trace routine identical in embodiment 1, and testing result is as table 7, and result display is consistent with clinical observation result.
Table 7 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 3
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strain, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 20ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 8 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:128 | 1:64 | 1:64 | 1:128 | 1:128 | 1:128 | 1:64 | 1:128 | 1:64 | 1:256 | 1:64 | 1:16 | 1:256 | 1:64 | 1:256 | 1:128 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 9.
The cause of disease specific test testing result of table 9 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 10, and result display is consistent with clinical observation result.
Table 10 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 4
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strain, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 80ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 11 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:128 | 1:64 | 1:128 | 1:256 | 1:128 | 1:128 | 1:64 | 1:128 | 1:64 | 1:128 | 1:64 | 1:64 | 1:256 | 1:64 | 1:256 | 1:256 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 12.
The cause of disease specific test testing result of table 12 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 13, and result display is consistent with clinical observation result.
Table 13 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 5
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strain, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 10ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 14 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:128 | 1:128 | 1:64 | 1:256 | 1:256 | 1:128 | 1:64 | 1:128 | 1:32 | 1:256 | 1:128 | 1:64 | 1:256 | 1:128 | 1:256 | 1:256 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 15.
The cause of disease specific test testing result of table 15 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 16, and result display is consistent with clinical observation result.
Table 16 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 6
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strain, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 60ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 17 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:128 | 1:128 | 1:64 | 1:256 | 1:128 | 1:128 | 1:64 | 1:128 | 1:32 | 1:256 | 1:128 | 1:64 | 1:256 | 1:64 | 1:256 | 1:256 | 1:256 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 18.
The cause of disease specific test testing result of table 18 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 19, and result display is consistent with clinical observation result.
Table 19 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 7
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strain, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 20ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 20 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:256 | 1:64 | 1:64 | 1:128 | 1:64 | 1:128 | 1:128 | 1:128 | 1:32 | 1:256 | 1:64 | 1:32 | 1:128 | 1:64 | 1:256 | 1:256 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 21.
The cause of disease specific test testing result of table 21 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 22, and result display is consistent with clinical observation result.
Table 22 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Embodiment 8
The antigen preparing the use of haemophilus parasuis indirect hamagglutination detection reagent in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strain, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strain, and each strain antigens protein concentration is 90ug/mL; Haemophilus parasuis indirect hamagglutination detection reagent preparation method is identical with embodiment 1.
The above-mentioned batch of haemophilus parasuis Blood coagulation test reagent utilizing the present embodiment to prepare, according to the detection running program identical with embodiment 1, the positive serum samples of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that testing laboratory's preparation is preserved, testing result is as follows:
Table 23 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects yin and yang attribute sample result
| 1 type | 2 types | 3 types | 4 types | 5 types | 6 types | 7 types | 8 types | 9 types | 10 types | 11 types | 12 types | 13 types | 14 types | 15 types | Indefinite form 1. | Indefinite form 2. | Negative |
| 1:256 | 1:64 | 1:64 | 1:128 | 1:64 | 1:128 | 1:64 | 1:128 | 1:32 | 1:256 | 1:64 | 1:32 | 1:128 | 1:64 | 1:256 | 1:256 | 1:128 | - |
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out cause of disease specific test.Test specimen is with the sample according to cause of disease specific test in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 24.
The cause of disease specific test testing result of table 24 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Note: positive criterion is agglutination titer >=1:16 ,-represent that testing result is negative ,+represent that testing result is positive.
The above-mentioned batch of haemophilus parasuis indirect hamagglutination detection reagent utilizing the present embodiment to prepare, carries out clinical sample detection experiment.Clinical sample is with the pig farm sample according to clinical sample detection experiment in embodiment 1, and specified operational procedure carries out according to the trace routine that embodiment 1 is identical, and testing result is as table 25, and result display is consistent with clinical observation result.
Table 25 pig farm sample detection result
Note: positive criterion is agglutination titer >=1:16.
Claims (2)
1. a haemophilus parasuis indirect hamagglutination detection reagent, it is characterized in that, the antigen preparing this reagent place is the composition of the whole bacterial protein of serum 1 type, serum 4 type, Serotype 5 and serum 13 type haemophilus parasuis, described serum 1 type haemophilus parasuis adopts N4 strain (International Reference strain, Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, Vet Microbiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): 117-123), described serum 4 type haemophilus parasuis adopts SW124 strain (International Reference strain, Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, Vet Microbiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): 117-123) or FS0307 strain (Chinese pathogenic strain, be preserved in China typical culture collection center CCTCC, deposit number: CCTCC NO:M 2013094, preservation date: on March 21st, 2013), described Serotype 5 haemophilus parasuis adopts Nagasaki strain (International Reference strain, Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, Vet Microbiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): 117-123) or XX0306 strain (Chinese pathogenic strain, be preserved in China typical culture collection center CCTCC, deposit number: CCTCC NO:M 2013095, preservation date: on March 21st, 2013), described serum 13 type haemophilus parasuis adopts IA-84-17975 strain (International Reference strain, Rafiee M et al, Aust Vet J, 2000,78 (3): 172-174; Turni C et al, Vet Microbiol, 2005,106 (1-2): 145-151; Cai X et al, Vet Microbiol, 2005,111 (3-4): 231-236; Jin H et al, Vet Microbiol, 2006,118 (1-2): 117-123); The concentration of often kind of serotype haemophilus parasuis whole bacterial protein is 10 ~ 100 μ g/mL.
2., by haemophilus parasuis indirect hamagglutination detection reagent according to claim 1, it is characterized in that often kind of serotype haemophilus parasuis whole bacterial protein is all mixed in equal amounts, the total protein concentration of described composition is 40 ~ 400 μ g/mL.
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| 华东地区副猪嗜血杆菌的分离、血清型鉴定与药敏分析;周斌 等;《畜牧与兽医》;20091110;第41卷(第11期);摘要，第85-86页 * |
| 间接血凝方法检测副猪嗜血杆菌抗体;徐引弟 等;《中国畜牧兽医》;20110720;第38卷(第7期);摘要，第189页右栏至第191页左栏，表1 * |
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