Method for detecting agglutination titer of yolk antibody against different serotypes of vibrio parahaemolyticus and application thereof

Abstract

The application discloses a detection method for agglutination titer of yolk antibody of Vibrio parahaemolyticus of different serotypes and application thereof. The application determines the optimal conditions of agglutination reaction through screening of sample diluent, screening of bacterial liquid diluent and screening of reaction temperature; determines that there is no cross agglutination of yolk antibody of Vibrio parahaemolyticus of different serotypes through detection of cross reaction; evaluates the growth and decline law of titer of yolk antibody of Vibrio parahaemolyticus; and the detection method is simple in operation, is helpful to solve the problem of effect evaluation of yolk antibody of Vibrio parahaemolyticus, and lays a foundation for popularization and application of yolk antibody of Vibrio parahaemolyticus.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of disease prevention and control of aquaculture, and particularly relates to establishment of a Vibrio parahaemolyticus yolk antibody agglutination titer detection method and application of the method in Vibrio parahaemolyticus yolk antibody titer growth and decline rules. BACKGROUND

[0002] Penaeus vannamei, also known as South American white shrimp, is a major farmed shrimp in China, and the farmed yield shows a trend of increasing year by year. The China Fishery Statistical Yearbook shows that from 2020 to 2021, the yield of farmed South American white shrimp in seawater increased by 75897 tons, with an increase of 6.34%; the yield of farmed South American white shrimp in freshwater increased by 38565 tons, with an increase of 5.80%.

[0003] Vibrio parahaemolyticus is one of the main pathogenic bacteria that harm farmed South American white shrimp. Penaeus vannamei infected with Vibrio parahaemolyticus can cause acute hepatopancreas necrosis in shrimp, and the diseased shrimp has symptoms such as empty intestine, empty stomach, and white feces, and can cause 100% mortality of shrimp in severe cases. The infection process of Vibrio parahaemolyticus is divided into early infection, acute infection and late infection. In the early infection, the hepatopancreas small tube epithelial cells extend into the lumen, and the cell vacuoles decrease, at which time the blood lymph can quickly respond to the immune response; a rapid inflammatory response is observed in the acute phase; in the late stage, the hepatopancreas atrophies and completely necroses, and there is obvious bacterial proliferation in the hepatopancreas small tube lumen. According to the related conclusions of the pathogenicity research of Vibrio parahaemolyticus, the early shrimp has the ability to eliminate the strain, and no bacterial colonization is detected in the hepatopancreas at this time. It is speculated that after the strain colonizes in the digestive organ, it secretes toxins, causing hepatopancreas to gradually atrophy and become diseased, breaking through the immune defense system of the shrimp, and leading to the final necrosis of the hepatopancreas, accompanied by secondary infection.

[0004] Yolk antibody refers to the specific antibodies produced by birds (mainly chickens) after immunization with specific antigens, which are transported and stored in the yolk, and then high-yield specific polyclonal antibodies are obtained from the yolk through specific extraction techniques. Studies have shown that yolk antibodies extracted from immunized hens against Vibrio parahaemolyticus or yolk antibodies extracted from immunized hens against virulent proteins can significantly improve the survival rate of shrimp infected with Vibrio parahaemolyticus. Based on the advantages of yolk antibodies, such as low cost, high yield, and green and healthy, yolk antibodies have the potential to replace antibiotics to prevent and control diseases.

[0005] In the development process of Vibrio parahaemolyticus yolk antibody product, the effect evaluation of the product is a key link, and the commonly used evaluation method is to detect the ELISA antibody titer in the egg yolk. There are more than 70 serotypes of Vibrio parahaemolyticus, and the serotypes of epidemic strains are different. Due to the cross reaction of the traditional ELISA method, it cannot meet the effect evaluation requirement of multi-link Vibrio parahaemolyticus yolk antibody, and it is necessary to construct an evaluation method which can distinguish the effect of yolk antibody of different serotypes of Vibrio parahaemolyticus. The method can accurately quantify the titer of yolk antibody of various serotypes of Vibrio parahaemolyticus on the basis of high sensitivity and strong stability. SUMMARY

[0006] The purpose of the present application is to provide a kit and a detection method for distinguishing the effect of yolk antibody of different serotypes of Vibrio parahaemolyticus.

[0007] The technical scheme adopted by the present application is:

[0008] In a first aspect of the present application, a kit is provided, which comprises: Vibrio parahaemolyticus bacterial liquid, Vibrio parahaemolyticus yolk antibody, strain diluent, yolk antibody diluent.

[0009] Preferably, the serotypes of the Vibrio parahaemolyticus are O1:KUT and OUT:KUT.

[0010] Preferably, the strain diluent is 5-7% (w / v) sodium chloride solution.

[0011] Preferably, the strain diluent is 6% (w / v) sodium chloride solution.

[0012] Preferably, the yolk antibody diluent is PBS buffer.

[0013] In a second aspect of the present application, the kit of the first aspect of the present application is applied to detect the agglutination titer of Vibrio parahaemolyticus yolk antibody or prepare a product for detecting the agglutination titer of Vibrio parahaemolyticus yolk antibody.

[0014] Preferably, the serotypes of the Vibrio parahaemolyticus are O1:KUT and OUT:KUT.

[0015] In a third aspect of the present application, a detection method for the agglutination titer of Vibrio parahaemolyticus yolk antibody is provided, which comprises the following steps:

[0016] S1: diluting the Vibrio parahaemolyticus bacterial liquid with the bacterial liquid diluent;

[0017] S2: diluting the Vibrio parahaemolyticus yolk antibody to be detected by the yolk antibody diluent;

[0018] S3: mixing the yolk antibody and the bacterial liquid to prepare an agglutination reaction system for detecting the antibody titer of the Vibrio parahaemolyticus yolk antibody to be detected.

[0019] Preferably, the concentration of the diluted Vibrio parahaemolyticus bacterial solution in step S1 is 1.0*10^5 cfu / mL-1.0*10^6 cfu / mL.

[0020] Preferably, the concentration of the diluted Vibrio parahaemolyticus bacterial solution in step S1 is 1.0*10^6 cfu / mL.

[0021] Preferably, the dilution ratio of the dilution in step S2 is 1:(1-256), and the dilution gradient is 7-10.

[0022] Preferably, the dilution ratio of the dilution is 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256, respectively.

[0023] Preferably, the antibody titer in step S3 is the highest dilution of the antibody when the agglutination reaction system appears agglutination.

[0024] Preferably, the volume ratio of the egg yolk antibody and the bacterial solution in step S3 is 1:(0.5-1.5).

[0025] Preferably, the volume ratio of the egg yolk antibody and the bacterial solution in step S3 is 1:1.

[0026] Preferably, the reaction condition of the reaction system in step S3 is 26-30℃ standing for 14-18h.

[0027] Preferably, the incubation temperature of the reaction system in step S3 is 28℃ standing for 16h.

[0028] Preferably, the preparation method of the Vibrio parahaemolyticus egg yolk antibody comprises the following steps:

[0029] (1) Inactivated Vibrio parahaemolyticus is used as an antigen to immunize laying hens;

[0030] (2) After immunization, egg samples are collected, egg yolk is separated, and egg yolk antibody is purified.

[0031] The beneficial effects of the present application are:

[0032] The present application provides a detection method for the agglutination titer of Vibrio parahaemolyticus egg yolk antibody, which determines the optimal conditions of the agglutination reaction through the screening of sample diluent, bacterial solution diluent and reaction temperature; determines that there is no cross agglutination of egg yolk antibody of different serotypes of Vibrio parahaemolyticus by detecting cross reaction, and evaluates the titer growth and decline rule of Vibrio parahaemolyticus egg yolk antibody; and the detection method is simple to operate, which helps to solve the problem of evaluating the effect of Vibrio parahaemolyticus egg yolk antibody, and lays a foundation for the popularization and application of Vibrio parahaemolyticus egg yolk antibody. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Agglutination titer of the yolk antibody against Vibrio parahaemolyticus. DETAILED DESCRIPTION

[0034] The concept and technical effects of the present application will be described below in combination with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0035] Experimental materials

[0036] 1. Egg-laying hens, strains

[0037] The egg-laying hens used for preparing the yolk antibody were 200-day-old Hyline gray egg-laying hens, and the Vibrio parahaemolyticus (V.p. 20:O1:K25, V.p. 24:O1:K33, V.p. 25:OUT:KUT, V.p. 26:O1:K36, V.p. 28:O1:K68, V.p. 27 and V.p. 29:O1:KUT) of 6 serotypes were isolated from the sick pond of Litopenaeus vannamei in Zhuhai.

[0038] 2. Reagents and consumables

[0039] The tryptone soy broth and TCBS agar medium were purchased from Guangdong Huanke Company, sodium chloride, anhydrous sodium acetate and glacial acetic acid were purchased from the National Pharmaceutical Group, n-octanoic acid was purchased from Komiyu, PBS buffer was purchased from Biosharp, Marcol 52 white oil adjuvant was purchased from Mobil, and Tween 80 was purchased from Shengong.

[0040] Example 1: A method for detecting agglutination titer of yolk antibody against different serotypes of Vibrio parahaemolyticus

[0041] The method comprises the following steps:

[0042] 1. Preparation of positive and negative yolk antibodies against different serotypes of Vibrio parahaemolyticus

[0043] 1.1 Preparation of Vibrio parahaemolyticus inactivated vaccine and immunization of egg-laying hens

[0044] Vibrio parahaemolyticus (V.p. 25) with serotype OUT:KUT and Vibrio parahaemolyticus (V.p. 27 and V.p. 29) with serotype O1:KUT were selected, recovered and cultured in large scale. The formaldehyde inactivated bacteria solution was coated on the plate to detect the inactivation effect. The inactivated bacteria solution was mixed with 40% sterilized glycerol, and stored at -20℃ for standby.

[0045] The inactivated Vibrio parahaemolyticus was mixed with Tween-80 to prepare the water phase antigen. The Marcol 52 white oil adjuvant was slowly added to the water phase antigen, and the oil emulsion vaccine was prepared by emulsification with a dispersion machine. The prepared vaccine was injected into the pectoral muscle of laying hens by multiple point injection method, 20 hens were injected with each vaccine, and one booster immunization was performed after three basic immunizations. The negative control group was only injected with white oil adjuvant. The egg samples were collected every 10 days after 3 and 4 immunizations for yolk antibody extraction.

[0046] 1.2 Extraction of Vibrio parahaemolyticus yolk antibody by octanoic acid method

[0047] The egg samples were taken, the egg yolk was separated and weighed, 2.5 times the volume of pure water was measured, anhydrous sodium acetate and glacial acetic acid were added, and the pH was adjusted to 4.8. The above acidified aqueous solution was slowly added to the egg yolk solution, stirred in a 48°C water bath for 20 min, then n-octanoic acid was added, and stirred in a 48°C water bath for 1 h. The yolk antibody was filtered with a polyethylene filter cloth, the pH was adjusted to 7.0, and then filtered with a 0.22 μm filter and stored at 4°C (Table 1).

[0048] Table 1 Vibrio parahaemolyticus yolk antibody samples extracted by octanoic acid method

[0049]

[0050]

[0051] 2. Selection of bacterial solution concentration in yolk antibody agglutination reaction

[0052] 2.1 Quantification of bacterial solution

[0053] According to the bacterial solution concentration in the "Guidelines for Bacteriostatic Agent Efficacy Test" and "GB 15981-1995 Evaluation Method and Standard for Disinfection and Sterilization Effect", the effect of bacterial solution concentration on agglutination titer was determined.

[0054] The preserved V.p. 27 glycerol bacteria were taken out, inoculated into 5 mL TSB medium after recovery, and cultured for 4 h. The OD600 of the bacterial solution was measured, and the concentration of the bacterial strain was calculated using the Vibrio parahaemolyticus bacterial concentration-absorbance value curve (y = 8.5209 + 1.1045x). The bacterial concentration was adjusted to 1.0*10^7 cfu / mL, 1.0*10^6 cfu / mL, and 1.0*10^5 cfu / mL using 1.5% NaCl (w / v) solution for standby.

[0055] 2.2 Dilution of yolk antibody

[0056] Take 9 test tubes, numbered and marked in turn, except for the first tube, each of the remaining test tubes added 1 mL of 1.5% NaCl solution; take V.p.27 egg yolk antibody 3 immune 20 days sample 1 mL added to the first tube and the second tube, from the second tube, using vortex mixer to mix after shaking 1 mL added to the third tube, so continuous dilution to the ninth tube, mixed and discarded after 1 mL. At this time, the dilution of each tube sample is: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256; according to the same method to prepare the control group gradient dilution sample.

[0057] 2.3 bacteria and egg yolk antibody agglutination reaction

[0058] The diluted bacteria solution in step 2.1 was taken 1 mL and added to each dilution sample (bacteria solution: sample = 1 mL: 1 mL) to prepare 2 mL agglutination reaction system, vortex mixer to mix; the above sample and bacteria solution mixture was placed at 28℃ and left overnight, and the next day the agglutination phenomenon was observed. The results showed that the agglutination titer was inversely proportional to the concentration of bacteria solution, that is, the higher the concentration of bacteria solution, the lower the agglutination titer, and the agglutination titer of 1.0*10^7 cfu / mL bacteria solution was <1.0*10^6 cfu / mL bacteria solution <1.0*10^5 cfu / mL bacteria solution (Table 2). The recommended bacteria solution concentration in the “Guidelines for Antibacterial Agent Efficacy Test” and “GB15981-1995 Evaluation Method and Standard for Disinfection and Sterilization Effect” is 1.0*10^5 cfu / mL-1.0*10^6 cfu / mL, and finally 1.0*10^6 cfu / mL of bacteria solution concentration is selected as the bacteria solution concentration for agglutination reaction.

[0059] Table 2 Effect of bacteria solution concentration on agglutination titer

[0060]

[0061]

[0062] 3, determination of egg yolk antibody agglutination reaction temperature

[0063] 3.1 bacteria quantification

[0064] Take out the preserved V.p.27 glycerol bacteria, inoculate into 5 mL TSB medium after recovery and culture for 4 h. Measure the OD600 of the bacteria solution, calculate the strain concentration according to the bacteria concentration-absorbance value curve (y = 8.5209 + 1.1045x), and adjust to 1.0*10^6 cfu / mL with 1.5% NaCl (w / v) solution for standby.

[0065] 3.2 dilution of Vibrio parahaemolyticus egg yolk antibody

[0066] Take 3 test tubes, numbered and labeled in turn, and add 1 mL of 1.5% (w / v) NaCl solution to each test tube. Take 1 mL of V. p. 27 yolk antibody 3#20d sample and add it to the first test tube, shake it well with a vortex mixer, then take 1 mL and add it to the second test tube, mix it well, then take 1 mL and add it to the third test tube, mix it well, and then discard 1 mL. Prepare the negative control group and unrelated protein (BSA) control group gradient dilution samples in the same way. The blank control group is the mixture of the diluted bacterial solution and 1.5% NaCl solution.

[0067] 3.3 Agglutination of yolk antibodies with strains

[0068] Diluted bacterial solution, 1 mL each, was added to each dilution sample (bacterial solution: sample = 1 mL: 1 mL) to prepare a 2 mL agglutination reaction system, which was mixed well with a vortex mixer. The mixture of the above samples and bacterial solution was incubated at 4°C for 16 hours, and the next day the agglutination phenomenon was observed and the samples were plated for counting. The results showed that the blank control group, unrelated protein group, negative yolk antibody group and positive antibody group all had no agglutination phenomenon. After plating and counting, no colonies grew in each group. Incubation at 4°C is not suitable for detecting agglutination effect and bacteriostatic level (Table 3).

[0069] Table 3 Agglutination results and plating count results of 4°C incubation

[0070]

[0071] 4. Determination of sodium chloride concentration in yolk antibody agglutination reaction system

[0072] 4.1 Dilution of bacterial solution with different concentrations of sodium chloride

[0073] Take the preserved V. p. 27 glycerol bacteria, inoculate 5 mL of TSB medium after recovery, and incubate for 4 hours. Measure the OD600 of the bacterial solution using a spectrophotometer. According to the relationship curve between the concentration of the bacterial solution and the absorbance value (y = 8.5209 + 1.1045x), calculate the concentration of the bacterial solution. According to the following Table 4, dilute the bacterial solution to 1.0*10^6 cfu / mL using 0.9%, 1.5%, 3%, 6%, 12% (w / v) sodium chloride solution and PBS buffer.

[0074] Table 4 Screening of sodium chloride concentration in agglutination reaction system

[0075]

[0076] 4.2 Dilution of yolk antibodies

[0077] Take 3 test tubes, numbered and marked in turn, and add 1 mL of PBS buffer to each test tube according to the grouping in Table 4 above. Take 1 mL of V. p. 27 yolk antibody 3 sample 20d and add it to the first test tube, mix well with vortex, then take 1 mL and add it to the second test tube, mix well, then take 1 mL and add it to the third test tube, mix well, then take 1 mL and discard.

[0078] Prepare the negative yolk antibody control group and irrelevant protein (BSA) control group gradient dilution samples in the same way. In the blank control group, use different concentrations of sodium chloride solution instead of the sample, i.e. the blank control group only contains diluted bacterial solution.

[0079] 4.3 Agglutination reaction of bacterial solution and yolk antibody

[0080] Dilute the bacterial solution with different concentrations of sodium chloride, and take 1 mL of each dilution and add it to the corresponding sample (bacterial solution: sample = 1 mL: 1 mL) to prepare a 2 mL agglutination reaction system. Mix well with vortex. Place the mixture of the above samples and bacterial solution at 28°C and incubate for 16 hours. Observe the agglutination phenomenon the next day.

[0081] The results show that when the bacterial solution is 0.9%, 1.5%, 3%, and 6% (w / v) sodium chloride solution and the sample dilution is PBS, there is no agglutination phenomenon in the blank control group, irrelevant protein group, and negative control group, but the positive yolk antibody group can observe agglutination particles. Among them, when the bacterial solution dilution is 6% sodium chloride solution, the agglutination phenomenon is very significant (Table 5). When the bacterial solution and the sample are diluted with 12% sodium chloride solution or PBS, there is agglutination, but the agglutination particles are smaller.

[0082] Table 5 Agglutination results under different sodium chloride concentrations

[0083]

[0084] 4.4 Coating count of agglutinated samples

[0085] Take 6 1.5 mL centrifuge tubes, and add 0.9 mL of PBS buffer to each centrifuge tube. Take 0.1 mL of the agglutinated sample and add it to the first tube, mix well with vortex, then take 0.1 mL and add it to the second tube, and so on until the sixth tube. At this time, the dilution multiples of the samples in each tube are 1:10^1, 1:10^2, 1:10^3, 1:10^4, 1:10^5, and 1:10^6, respectively.

[0086] Take 0.1 mL of 1:10^4, 1:10^5, 1:10^6 dilution samples, respectively, and inject them into TCBS solid plates. Use 4 mm glass beads to roll the plates up and down and left and right to ensure that the samples are fully absorbed by the plates. Place the coated plates in an inverted position at 28°C and incubate overnight; count the number of colonies the next day.

[0087] The dilution and coating count of each sample in Groups 1 to 6 showed (Table 6) that the number of colonies in the negative control group was higher than that in the positive sample group. When the bacteria solution was diluted with 0.9%, 1.5%, and 12% sodium chloride, no colonies grew in the blank group, and the number of colonies in the irrelevant protein group was significantly lower than that in the negative control group. When the bacteria solution was diluted with PBS, the number of colonies in the negative control group was less than that in the positive sample group. When the bacteria solution was diluted with 3% sodium chloride, the number of colonies in the blank group and the irrelevant control group was significantly lower than that in the negative yolk antibody group. When the bacteria solution was diluted with 6% sodium chloride, the number of colonies in the negative control group was consistent with that in the irrelevant control group and the blank control group, and the number of colonies in the positive antibody group was significantly lower than that in the negative control group. Based on the agglutination effect and coating count results, 6% sodium chloride diluted bacteria solution and PBS diluted samples were selected for agglutination reaction at 28°C for 16 h.

[0088] Table 6 Coating count results of agglutinated samples

[0089]

[0090]

[0091] 5. Establishment of a detection method for the agglutination titer of Vibrio parahaemolyticus yolk antibody

[0092] 5.1 Detection of Vibrio parahaemolyticus yolk antibody samples

[0093] Using the above-mentioned conditions of the agglutination reaction system, the negative and positive yolk antibody samples isolated at different time points were detected to evaluate the sensitivity and stability of the detection method.

[0094] Take V.p.25, V.p.27, and V.p.29 yolk antibody samples from 3 pre-immune to 3 post-immune 50 days and 4 pre-immune to 4 post-immune 50 days for agglutination titer detection. Adjust the concentration of the bacteria solution to 1.0*10^6 cfu / mL, dilute the yolk antibody samples at different time points by 2 times from the original solution in 8 gradients, mix 1:1 with 1.0*10^6 cfu bacteria solution to prepare 2 mL of agglutination reaction system, and incubate at 28°C for 16 h to detect the agglutination titer.

[0095] The results showed that the three strains of Vibrio parahaemolyticus yolk antibodies all appeared peak titer at 20 days after the third immunization, and then the agglutination titer began to decrease; the agglutination titer began to rise after the fourth immunization, and the titer entered the plateau period at 20 days after the fourth immunization. From the agglutination titer in the plateau period, V.p.27 yolk antibody = V.p.29 yolk antibody > V.p.25 yolk antibody. Figure 1

[0096] 5.2 Cross agglutination reaction of Vibrio parahaemolyticus yolk antibodies

[0097] Using the samples of V.p.25, V.p.27 and V.p.29 yolk antibodies at 10 days after the fourth immunization and at 20 days after the fourth immunization, the cross agglutination against different serotypes of Vibrio parahaemolyticus was detected.

[0098] ① Cross agglutination of V.p.25 yolk antibodies against different serotypes of Vibrio parahaemolyticus

[0099] The samples of V.p.25 yolk antibodies at 10 days after the fourth immunization and at 20 days after the fourth immunization were diluted by 2 times in 8 gradients from the original solution, and then mixed with 1.0*10^6 cfu of different serotypes of Vibrio parahaemolyticus (V.p.20O1:K25; V.p.24O1:K33; V.p.26O1:K36; V.p.27O1:KUT; V.p.28O1:K68; V.p.29O1:KUT) at 1:1 to prepare 2 mL agglutination reaction system, which was incubated at 28℃ for 16 h to detect the agglutination titer. The agglutination reaction of V.p.25 bacteria solution with the sample was used as a positive control, and the samples of the negative group at 10 days after the fourth immunization and at 20 days after the fourth immunization were used as negative controls.

[0100] The agglutination results are shown in Table 7. The agglutination titers of the samples of V.p.25 yolk antibodies at 10 days after the fourth immunization and at 20 days after the fourth immunization against V.p.25 strain were 1:4 and 1:8, respectively, and there was no agglutination effect against V.p.20, V.p.24, V.p.26, V.p.27, V.p.28 and V.p.29 strains, and there was no agglutination phenomenon in the negative control samples.

[0101] Table 7 Cross agglutination of V.p.25 yolk antibodies against different serotypes of Vibrio parahaemolyticus

[0102]

[0103]

[0104] ② Cross agglutination of V.p.27 yolk antibodies against different serotypes of Vibrio parahaemolyticus

[0105] ​The V.p.27 yolk antibody 4 immunized 10 days and 4 immunized 20 days samples were diluted 8 times from the original solution by 2 times of dilution ratio, mixed with 1.0*10^6 cfu of different serotypes of Vibrio parahaemolyticus (V.p.20O1:K25; V.p.24O1:K33; V.p.25OUT:KUT; V.p.26O1:K36; V.p.28O1:K68) 1:1 to prepare a 2 mL agglutination reaction system, and incubated at 28°C for 16 h to detect the agglutination titer. The agglutination reaction of V.p.27 bacteria solution with the sample was used as a positive control, and the 4 immunized 10 days and 4 immunized 20 days negative group samples were used as negative controls.

[0106] The agglutination results are shown in Table 8. The agglutination titers of V.p.27 yolk antibody 4 immunized 10 days and 4 immunized 20 days yolk antibody samples against V.p.27 strain were 1:8 and 1:64, respectively, and there was no agglutination effect on V.p.20, V.p.24, V.p.25, V.p.26 and V.p.28 strains. The negative control samples had no agglutination phenomenon.

[0107] Table 8 Cross agglutination of V.p.27 yolk antibody against different serotypes of Vibrio parahaemolyticus

[0108]

[0109] ③Cross agglutination of V.p.29 yolk antibody against different serotypes of Vibrio parahaemolyticus

[0110] The V.p.29 yolk antibody 4 immunized 10 days and 4 immunized 20 days samples were diluted 8 times from the original solution by 2 times of dilution ratio, mixed with 1.0*10^6 cfu of different serotypes of Vibrio parahaemolyticus (V.p.20O1:K25; V.p.24O1:K33; V.p.25OUT:KUT; V.p.26O1:K36; V.p.28O1:K68) 1:1 to prepare a 2 mL agglutination reaction system, and incubated at 28°C for 16 h to detect the agglutination titer. The agglutination reaction of V.p.29 bacteria solution with the sample was used as a positive control, and the 4 immunized 10 days and 4 immunized 20 days negative group samples were used as negative controls.

[0111] The agglutination results are shown in Table 9. The agglutination titers of V.p.29 yolk antibody 4 immunized 10 days and 4 immunized 20 days yolk antibody samples against V.p.29 strain were 1:32 and 1:64, respectively, and there was no agglutination effect on V.p.20, V.p.24, V.p.25, V.p.26 and V.p.28 strains. The negative control samples had no agglutination phenomenon.

[0112] Table 9 Cross agglutination of V.p.27 yolk antibody against different serotypes of Vibrio parahaemolyticus

[0113]

[0114] IV. Cross agglutination of the same serotype of V. parahaemolyticus yolk antibody

[0115] The V.p.27 yolk antibody 4 immunized for 30 days sample was diluted 8 times from the original solution, mixed with 1.0*10^6 cfu of the same serotype of V. parahaemolyticus V.p.29 1:1 to prepare a 2 mL agglutination reaction system, and incubated at 28°C for 16 h to detect the agglutination titer. The V.p.29 yolk antibody 4 immunized for 30 days sample was diluted 8 times from the original solution, mixed with 1.0*10^6 cfu of the same serotype of V. parahaemolyticus V.p.27 1:1 to prepare a 2 mL agglutination reaction system, and incubated at 28°C for 16 h to detect the agglutination titer. The agglutination reaction of the sample with the bacterial solution itself was used as a positive control, and the negative group sample immunized for 30 days was used as a negative control.

[0116] The agglutination results are shown in Table 10. The V.p.27 yolk antibody 4 immunized for 30 days sample had an agglutination titer of 1:64 for V.p.27 and V.p.29 strains, and the V.p.29 yolk antibody 4 immunized for 30 days sample had an agglutination titer of 1:64 for V.p.27 and V.p.29 strains. The negative control had no agglutination phenomenon. Cross agglutination can be detected for strains of the same serotype, and the agglutination titer is the same as that of the strain itself.

[0117] Table 10 Cross agglutination of the same serotype of V. parahaemolyticus

[0118]

[0119] The above detailed description of the present application, but the present application is not limited to the above examples, within the scope of knowledge possessed by those skilled in the art, can also be made various changes without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for detecting the agglutination titer of Vibrio parahaemolyticus egg yolk antibodies, comprising the following steps: S1: Dilute Vibrio parahaemolyticus bacterial suspension with strain dilution solution; S2: Serially dilute the Vibrio parahaemolyticus egg yolk antibody to be tested using egg yolk antibody dilution buffer; S3: Prepare an agglutination reaction system by mixing egg yolk antibodies with bacterial culture, and then detect the antibody titer of the Vibrio parahaemolyticus egg yolk antibodies to be tested; The serotypes of Vibrio parahaemolyticus are O1:KUT and OUT:KUT; The strain dilution was a 4-7% (w / v) sodium chloride solution; The egg yolk antibody dilution solution was PBS buffer.

2. The detection method according to claim 1, characterized in that, The concentration of the diluted Vibrio parahaemolyticus bacterial solution in step S1 is 1.0*10⁻⁶. ^5 cfu / mL 1.0*10 ^6 cfu / mL.

3. The detection method according to claim 1, characterized in that, The dilution factor in step S2 is 1:(1-256), with 7-10 dilution gradients.

4. The detection method according to claim 1, characterized in that, The volume ratio of egg yolk antibody to bacterial culture in step S3 is 1:(0.5-1).

5. The detection method according to claim 1, characterized in that, The reaction conditions for the reaction system in step S3 are 26–30°C and standing for 14–18 hours.

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