CN103364548A - 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 the animal doctor and detect the reagent field, particularly a kind of haemophilus parasuis indirect hemagglutination detects 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 the Pasteurella section hemophilus, it is a kind of conditionality pathogen of pig, be worldwide distribution, infect the pig Ge Lazeshi that causes behind the piglet take polyserositis, arthritis and meningitis as feature (Glasser ' s) disease.The piglet in ages in main harm 2～8 week, the incidence of disease is generally 10%～15%, and mortality ratio can reach more than 50%.
Antigenicity and Virulence Difference are very large between the Hps bacterial strain, and serological typing is one of important method of difference different strains.At present, the serological typing method (KRG) based on the heat stable antigen immunodiffusion by propositions in 1992 such as Kielstein worldwide is accepted, the method is divided into 15 serotypes with Hps, yet has 15.2%～41% separated strain serotype can not determine.According to the seroepidemiological survey of Japan, Germany, the states such as the U.S. and Spain, with serotype 4,5 and 13 the most popular.Hps is generally popular in China, at present, and in Hebei, 12 provinces and cities such as Henan, Hubei, Hunan, Anhui, Shanghai, Guangdong, Jiangxi all have Haemophilus parasuis that the report of Hps occurs and isolates.
At present, the Haemophilus parasuis detection technique mainly contains and separates culture technique, round pcr and serology detection technique etc.Separate culture technique and round pcr mainly for the detection diagnosis of cause of disease, be presented as the infection conditions of Hps, relative merits are respectively arranged in actual applications.And the serology detection technique can not only reflect the popular infection conditions of Hps, can also react the immune level of swinery.Indirect hemagglutination test (IHA) is classical way during serology detects, and through after updating, has been widely used in the serum antibody that detects various cause of diseases, compares with ELISA, and the indirect hemagglutination test operating process is easy, save time, and does not need specific apparatus.Studies confirm that, detect Haemophilus parasuis serum antibody, the cross reaction of two of ubiquities and above serotype with the IHA test.At present, the applied IHA of each mechanism test is all only for the detection of some or several serotype haemophilus parasuises, 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 false negative in clinical detection, usually occurs or have undetected possibility.In addition, verify through inventor's test of many times, can not really detect the Haemophilus parasuis serum of all serotypes with the sensitization blood cell of certain two kinds of serological type strain antigen preparation, can only detect this serotype serum, and indivedual serotype serum of cross reaction are arranged.At present, do not form intellecture property about this series products yet.
This research detects the Haemophilus parasuis serum antibody of most serotypes as purpose take rapid sensitive, do not distinguish concrete certain serotype, according to the reactionogenicity of various antigen and the characteristic of type cross reactivity, 4 kinds of serotype haemophilus parasuises of Select to use whole bacterial protein antigen sensibilization dialdehyde sheep red blood cell (SRBC), and indirect hemagglutination diagnostic antigen and detection reagent production technology are optimized, set up a kind of easy, stable, susceptibility, the cause of disease high specificity, the a large amount of clinical samples that are used 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 that detects 15 kinds of serotypes and indefinite form Haemophilus parasuis antibody, provide simultaneously the preparation method of this reagent.
For achieving the above object, the invention provides a kind of Haemophilus parasuis indirect hemagglutination and detect reagent, preparing the antigen that this reagent place uses is the composition of the whole bacterial protein of serum 1 type, serum 4 types, Serotype 5 and serum 13 type haemophilus parasuises, described serum 1 type haemophilus parasuis adopts N4 strain (international Reference Strains, provided by the Beijing City Agriculture and Forestry Institute, referring to 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): the report among the 117-123), described serum 4 type haemophilus parasuises adopt the SW124 strain, and (international Reference Strains is provided by the Beijing City Agriculture and Forestry Institute, this world Reference Strains is referring to 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, the report among the 117-123) or FS0307 strain (Chinese pathogenic strain 118 (1-2):, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M2013094, preservation date: on March 21st, 2013), described Serotype 5 haemophilus parasuis adopts Nagasaki strain (international Reference Strains, provided by the Beijing City Agriculture and Forestry Institute, this world Reference Strains is referring to 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, the report of 118 (1-2): 117-123) or XX0306 strain (Chinese pathogenic strain, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M2013095, preservation date: on March 21st, 2013), described serum 13 type haemophilus parasuises adopt bacterium to adopt IA-84-17975 strain (international Reference Strains, provided by the Beijing City Agriculture and Forestry Institute, this world Reference Strains is referring to 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): the report among the 117-123).
Further, the total concentration of every kind of serotype haemophilus parasuis whole bacterial protein is 10～100 μ g/mL.
Further, every kind of serotype haemophilus parasuis whole bacterial protein all is 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 the serotype, and the angle of the characteristic of reference culture and popular bacterial strain is set out, 4 kinds of different serotypes haemophilus parasuises have been screened targetedly, the indirect hemagglutination of preparation detect reagent to the full extent with all serotype haemophilus parasuis antibody responses, greatly increase reaction range and susceptibility, avoided single antigen to the insensitivity of other serotype antibody.
(2) from stability and the standardization of reaction system, carry out same bacterial concentration and standardized program and extract whole bacterial protein, select optimal concentration, the equivalent packing as sensitization antigen, has at utmost kept detecting the stability of reagent after the mixed in equal amounts.
(3) to detect reagent susceptibility high for this haemophilus parasuis indirect hemagglutination, and good reproducibility is without obvious cross reaction, and easy and simple to handle, quick to other pathogenic autoantibodies, can be used for a large amount of clinical sample detections, Observation of immune antibody level and epidemiology survey.
This detection reagent can be used for a large amount of clinical sample detections, Observation of immune antibody level and the epidemiology survey of Haemophilus parasuis.Its serum antibody susceptibility that detects Haemophilus parasuis is high, and to other pathogenic autoantibodies without obvious cross reaction.
Embodiment
The present invention will be further described in detail below in conjunction with embodiment, and advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replace and all fall into protection scope of the present invention.
Embodiment 1
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strains, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, each strain antigens protein concentration is 20ug/mL, and preparation process is as follows:
1, haemophilus parasuis is cultivated
Get respectively respectively 1 of serum 1 type (N4 strain), 4 types (FS0307 strain), 5 types (Nagasaki strain) and 13 types (IA-84-17975 strain) haemophilus parasuis that freeze-drying preserves, add 0.5mL TSB basal medium, get 0.1mL bacterium liquid behind the mixing and evenly coat TSA solid medium (containing 5% serum and 0.01%NAD), 37 ℃ of constant temperature culture 24～48h.Then 3～5 single bacterium colonies of picking are inoculated in 5mL TSB fluid nutrient medium (containing 5% serum and 0.01%NAD), and 37 ℃, the 180rpm shaking table is cultivated 18～24h.Cultured liquid bacteria liquid is inoculated in 100mL TSB fluid nutrient medium (containing 5% serum and 0.01%NAD) further enlarges cultivation.After cultivating 18～24h, take out, utilize colony counting method to estimate the bacterium number of each bacterial strain bacterium liquid.
2, the preparation of sensitization antigen
According to TSA plate count result, adopt the centrifugal concentrating method that cultured serum 1 type (N4 strain), 4 types (FS0307 strain), 5 types (Nagasaki strain) and 13 types (IA-84-17975 strain) haemophilus parasuis are concentrated, all simmer down to 1 * 10
10CFU/mL.Then concentrated bacterium liquid is carried out ultrasonic processing, the whole bacterial protein supernatant of centrifugal each bacterial strain of extraction is as the antigen of sensitization blood cell.The UV spectrophotometer measuring protein concentration is 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 the sensitization blood cell, all the other-40 ℃ save backup.
3, the preparation of dialdehyde blood cell
The aseptic collection Sheep Blood is 200mL approximately, goes to leave standstill 2 days at 4 ℃ after the fiberization; Filter Sheep Blood with gauze at super-clean bench; Use 0.11M PB(pH=7.2) wash centrifugal 20 minutes of each 5000rpm 5 times; Be made into 10% red cell suspension with 0.11M PB afterwards.10% red cell suspension (500ml) of equivalent is mixed with 3% pyroracemic aldehyde solution (500ml), stirred 18 hours at 25 ℃; Use 0.11M PB(pH=7.2) wash centrifugal 5 minutes of each 4000rpm 5 times.3% pyroracemic aldehyde solution is changed to 3% formalin, repeats above-mentioned hydroformylation process.With 0.11M PB red blood cell is made into 10% red cell suspension, adds 2 ‰ formaldehyde and shake up anticorrosion.Packing, 4 ℃ 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 respectively serum 1 type (N4 strain), 4 types (FS0307 strain), 5 types (Nagasaki strain) and 13 types (IA-84-17975 strain) haemophilus parasuis packing and preserve each 1 part of antigen protein, mix, replenish and add acetate buffer solution to 0.8mL, mixing, room temperature effect 30 minutes.Then the dialdehyde blood cell suspension that adds 0.8mL10%, mixing, 37 ℃ of water-baths 1 hour are constantly stirred and are shaken up.With 0.11M PB washing 3 times, centrifugal 5 minutes of each 5000rpm abandons supernatant.Add 8mL TAP, the vibration mixing is various serotype haemophilus parasuis indirect hemagglutination and detects reagent.Being used for clinical sample detects and epidemiology survey.
5, yin and yang attribute sample survey
Adopt the haemophilus parasuis indirect hemagglutination detection reagent of above-mentioned preparation to test as follows to the yin and yang attribute sample of haemophilus parasuis:
Get the positive serum of negative serum, 15 kinds of serotypes and 2 kinds of indefinite forms that laboratory preparation preserves as sample, utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of preparation to detect reagent and detect.96 orifice plates, every hole add 20 μ L TAP, add 20 μ L blood serum samples at first, sample is made 2 times of doubling dilutions, and namely dilution ratio is 1:2～1:256, then discards 20 μ L, last each hole adds 20 μ L sensitization blood cells, oscillator plate mixing, concrete operations such as table 1.Leave standstill observations behind 1～2h, testing result such as table 2.
Table 1 application of sample grouping sheet
Table 2 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent detects the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
6, cause of disease specificity check
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test as follows:
Take 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, pig toxoplasmosis positive serum as sample, utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination detection reagent of preparation to detect.Set up simultaneously 3 groups of Haemophilus parasuis positive serum contrasts and 1 group of negative serum contrast.Specified operational procedure is with table 1.Leave standstill observations behind 1～2h, testing result such as table 3.The result shows, the yin and yang attribute contrast is all set up, the haemophilus parasuis indirect hemagglutination detects the agglutination titer≤1:2 of reagent and MPS, SS-2 and APP positive serum, and with CSF, PRRS and the not aggegation of pig toxoplasmosis positive serum, though illustrate that the haemophilus parasuis indirect hemagglutination of preparation detects reagent and with part cause of disease serum slight cross reaction arranged, do not affect it to the specific detection of Haemophilus parasuis positive serum.
The cause of disease specific test testing result of table 3 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
7, clinical sample detects
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect as follows:
Gather respectively the blood serum sample of morbidity pig and health pig 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 hemagglutination detection reagent of preparation to detect.Specified operational procedure is with table 1.Leave standstill observations behind 1～2h, testing result such as table 4.Result's demonstration, 6 pig farms all detect the positive serum sample of varying number, and are consistent with clinical observation result, illustrate that 6 pig farms all have haemophilus parasuis in various degree to infect; The haemophilus parasuis indirect hemagglutination that further specifies preparation detects reagent and can be used for a large amount of clinical sample detections, Observation of immune antibody level and epidemiology survey.
Table 4 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 2
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strains, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 40ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 6 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 6 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to trace routine identical among the embodiment 1, testing result such as table 7, and the result shows consistent with clinical observation result.
Table 7 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 3
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strains, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 20ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 9 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 9 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to embodiment 1 identical trace routine, testing result such as table 10, and the result shows consistent with clinical observation result.
Table 10 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 4
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis FS0307 strains, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 80ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 12 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 12 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to embodiment 1 identical trace routine, testing result such as table 13, and the result shows consistent with clinical observation result.
Table 13 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 5
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strains, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 10ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 15 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 15 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to embodiment 1 identical trace routine, testing result such as table 16, and the result shows consistent with clinical observation result.
Table 16 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 6
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strains, the Nagasaki strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 60ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 18 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 18 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to embodiment 1 identical trace routine, testing result such as table 19, and the result shows consistent with clinical observation result.
Table 19 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 7
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strains, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 20ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 21 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 21 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to embodiment 1 identical trace routine, testing result such as table 22, and the result shows consistent with clinical observation result.
Table 22 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Embodiment 8
The antigen that preparation haemophilus parasuis indirect hemagglutination detection reagent uses in the present embodiment is serum 1 type haemophilus parasuis N4 strain, serum 4 type haemophilus parasuis SW124 strains, the XX0306 strain of Serotype 5 haemophilus parasuis and serum 13 type haemophilus parasuis IA-84-17975 strains, and each strain antigens protein concentration is 90ug/mL; Haemophilus parasuis indirect hemagglutination detection preparation method of reagent thereof is identical with embodiment's 1.
Utilize above-mentioned batch of haemophilus parasuis blood clotting of the present embodiment preparation to detect reagent, according to the detection running program identical with embodiment 1, the positive serum sample 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 the 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 | - |
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out the cause of disease specific test.Test specimen is with the sample according to cause of disease specific test among the embodiment 1, and specified operational procedure carries out testing result such as table 24 according to embodiment 1 identical trace routine.
The cause of disease specific test testing result of table 24 more than serotype haemophilus parasuis indirect hemagglutination diagnostic reagent
Annotate: positive criterion is agglutination titer 〉=1:16, and-expression testing result is negative, and+expression testing result is positive.
Utilize above-mentioned batch of haemophilus parasuis indirect hemagglutination of the present embodiment preparation to detect reagent, carry out clinical sample and detect test.Clinical sample is with the pig farm sample that detects test according to clinical sample among the embodiment 1, and specified operational procedure carries out according to embodiment 1 identical trace routine, testing result such as table 25, and the result shows consistent with clinical observation result.
Table 25 pig farm sample detection result
Annotate: positive criterion is agglutination titer 〉=1:16.
Claims (3)
1. a haemophilus parasuis indirect hemagglutination detects reagent, it is characterized in that, preparing the antigen that this reagent place uses is serum 1 type, serum 4 types, the composition of the whole bacterial protein of Serotype 5 and serum 13 type haemophilus parasuises, described serum 1 type haemophilus parasuis adopts N4 strain (international Reference Strains, provided by the Beijing City Agriculture and Forestry Institute), described serum 4 type haemophilus parasuises adopt SW124 strain (international Reference Strains, provided by the Beijing City Agriculture and Forestry Institute) or FS0307 strain (Chinese pathogenic strain, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2013094, preservation date: on March 21st, 2013), described Serotype 5 haemophilus parasuis adopts Nagasaki strain (international Reference Strains, provided by the Beijing City Agriculture and Forestry Institute) or XX0306 strain (Chinese pathogenic strain, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2013095, preservation date: on March 21st, 2013), described serum 13 type haemophilus parasuises adopt IA-84-17975 strain (international Reference Strains is provided by the Beijing City Agriculture and Forestry Institute).
2. detect reagent by haemophilus parasuis indirect hemagglutination claimed in claim 1, it is characterized in that the concentration of every kind of serotype haemophilus parasuis whole bacterial protein is 10 ~ 100 μ g/mL.
3. detect reagent by claim 1 and 2 described haemophilus parasuis indirect hemagglutination, it is characterized in that every kind of serotype haemophilus parasuis whole bacterial protein all is mixed in equal amounts, the total protein concentration of described composition is 40 ~ 400 μ g/mL.
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