Immunodetection kit and detection method for shrimp enterocytozoon hepatopenaei

Abstract

The invention relates to the technical field of agricultural technologies, in particular to an enterocytozoon hepatopenaei immunodetection kit and a detection method.The enterocytozoon hepatopenaei immunodetection kit comprises a lysis buffer solution, a neutralization buffer solution, an enterocytozoon hepatopenaei immunodetection test strip, disposable tweezers, a sample tube, a disposable grinding rod and a pipette, an operator collects an enterocytozoon hepatopenaei sample, and a collected sample is obtained; preprocessing the collected sample to obtain a ground sample; carrying out cracking treatment on the ground sample based on a cracking buffer solution to obtain a treated sample; carrying out neutralization treatment on the treated sample based on a neutralization buffer solution to obtain a final sample; the prawn enterocytozoon hepatopenaei immunodetection test strip is used for determining the final sample, so that the infection degree of the sample is judged, by using the kit and the detection method, the submission process is omitted, prawn enterocytozoon hepatopenaei can be detected on a culture site, the detection is rapid and accurate, and the problems that the traditional submission time of the prawn enterocytozoon hepatopenaei is long and the detection time is short are solved. And improper storage in the transportation process affects the detection result.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural technology, and in particular to a shrimp hepatoenterocystis immune detection kit and a detection method. Background Art

[0002] Enterocytozoon hepatopenaei (EHP), also known as enterocytosis, is a microsporidia found in shrimp in recent years. Shrimp enterocytosis is a major disease affecting the aquaculture industry, which has a serious impact on shrimp farming. It mainly infects important farmed shrimps such as Litopenaeus vannamei and Penaeus monodon.

[0003] At present, the detection of shrimp hepatocellular carcinoma mainly relies on fluorescent quantitative PCR. Although this method is accurate, it requires the samples to be sent to a third-party technical service testing center or aquatic technology station with testing qualifications. This is not only time-consuming, but also the improper storage of samples during transportation may affect the accuracy of the test results.

[0004] Therefore, there is an urgent need for a method for on-site detection of shrimp hepatoenteroides that is both convenient and quick for farmers to solve the above problems. Summary of the invention

[0005] The present invention aims to provide a shrimp hepatoenteric cytidylcholine immune detection kit and a detection method, aiming to solve the problem that traditional shrimp hepatoenteric cytidylcholine detection takes a long time and is improperly stored during transportation, which affects the detection results.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a shrimp hepatoenteric cytidylcholine immunoassay kit, comprising a lysis buffer, a neutralization buffer, a shrimp hepatoenteric cytidylcholine immunoassay test strip, disposable tweezers, a sample tube, a disposable grinding rod and a pipette.

[0007] In a second aspect, the present invention further provides a shrimp hepatoenteric cytidylcholine immunoassay kit detection method, which is applied to the shrimp hepatoenteric cytidylcholine immunoassay kit as described in the first aspect above, and is characterized in that it comprises the following steps:

[0008] The operator collects the shrimp hepatocystis sample and obtains the collected sample;

[0009] Pre-processing the collected sample to obtain a ground sample;

[0010] Performing a lysis treatment on the ground sample based on the lysis buffer to obtain a processed sample;

[0011] Performing a neutralization treatment on the treated sample based on the neutralization buffer to obtain a final sample;

[0012] The final sample is tested using the shrimp hepatocellular carcinoma immune detection test strip to determine the infection degree of the sample.

[0013] The operator collects shrimp hepatocystis samples and obtains the specific method of collecting samples:

[0014] The operator wears disposable gloves to catch the live shrimps from the farm to be tested, and obtains the shrimps to be tested;

[0015] The cephalothorax of the shrimp to be tested is opened, and the hepatopancreas sample is clamped with the disposable tweezers to obtain a collected sample.

[0016] The specific method of pre-processing the collected sample to obtain the ground sample is as follows:

[0017] Cleaning and disinfecting the sample tube, and placing the collected sample in the disinfected sample tube for storage;

[0018] The collected sample in the sample tube is ground using the disposable grinding rod to obtain a ground sample.

[0019] The specific method of performing lysis treatment on the ground sample based on the lysis buffer to obtain the processed sample is as follows:

[0020] Using the pipette to introduce the lysis buffer into the sample tube;

[0021] The sample tube is shaken to mix the lysis buffer and the ground sample for lysis to obtain a processed sample.

[0022] Wherein, the specific method of neutralizing the treated sample based on the neutralization buffer to obtain the final sample is:

[0023] Using the pipette to introduce the neutralization buffer into the sample tube;

[0024] The sample tube is shaken to mix the neutralization buffer and the treated sample, and the reaction environment is adjusted to obtain a final sample.

[0025] The specific method of using the shrimp hepatocellular carcinoma immune detection test strip to measure the final sample to determine the infection degree of the sample is:

[0026] Using the pipette, the final sample is evenly dripped onto the shrimp hepatocellular carcinoma immune detection test strip;

[0027] The shrimp hepatocellular carcinoma immune detection test strip is left to stand for 10-15 minutes, and the reaction lines are observed to obtain the number and position of the lines;

[0028] Based on the number and position of the observed lines, it is determined whether the shrimps in the farm are infected with Enterocytosis hepatocellular carcinoma and the extent of the infection.

[0029] The invention discloses a shrimp hepatoenteric cytid immune detection kit and a detection method. The lysis buffer is used to destroy the cell structure in the sample and release the shrimp hepatoenteric cytid antigen. The neutralization buffer is used to neutralize the lysis reaction and ensure the reaction environment of the shrimp hepatoenteric cytid immune detection test strip. The disposable tweezers, the sample tube, the disposable grinding rod and the pipette are used for sample collection, processing and transfer operations. The shrimp hepatoenteric cytid immune detection test strip is used to quickly detect shrimp hepatoenteric cytid through specific antibody-antigen reaction. The operator collects the shrimp hepatoenteric cytid sample to obtain the collected sample. The sample is collected for pretreatment to obtain a ground sample; the ground sample is lysed based on the lysis buffer to obtain a treated sample; the treated sample is neutralized based on the neutralization buffer to obtain a final sample; the final sample is measured using the shrimp hepatoenteric cytosis immune test strip to determine the infection degree of the sample. The kit and the detection method save the inspection process, and the shrimp hepatoenteric cytosis can be detected on the breeding site. The detection is fast and accurate, and the problem of the traditional shrimp hepatoenteric cytosis inspection taking a long time and improper storage during transportation affecting the detection result is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The present invention provides a flow chart of a shrimp hepatocellular carcinoma immune detection kit detection method.

[0032] Figure 2 The operator collects shrimp hepatocystis samples and obtains the flow chart of sample collection.

[0033] Figure 3 This is a flow chart of preprocessing the collected sample to obtain a ground sample.

[0034] Figure 4 The present invention is a flow chart of performing lysis treatment on the ground sample based on the lysis buffer to obtain a processed sample.

[0035] Figure 5 It is a flow chart of neutralizing the treated sample based on the neutralization buffer to obtain the final sample.

[0036] Figure 6 The present invention is a flow chart for using the shrimp hepatocellular carcinoma immune detection test strip to measure the final sample, thereby determining the infection degree of the sample. DETAILED DESCRIPTION

[0037] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0038] In a first aspect, the present invention provides a shrimp hepatocystis immune detection kit.

[0039] Includes lysis buffer, neutralization buffer, shrimp hepatocellular carcinoma immunoassay test strips, disposable tweezers, sample tubes, disposable grinding rods and pipettes.

[0040] In this embodiment, the lysis buffer is used to destroy the cell structure in the sample and release the shrimp hepatoenteric cytozoon antigen, and its components are 1% NaOH and 1% Triton X-100. The neutralization buffer is used to neutralize the lysis reaction and ensure the reaction environment of the shrimp hepatoenteric cytozoon immunodetection test strip, and its components are 2% Tris-HCl and 0.56% phosphoric acid. The disposable tweezers, the sample tube, the disposable grinding rod and the pipette are used for sample collection, processing and transfer operations, and the shrimp hepatoenteric cytozoon immunodetection test strip is used to quickly detect shrimp hepatoenteric cytozoon through specific antibody-antigen reaction.

[0041] See also Figures 1 to 6 In a second aspect, the present invention further provides a shrimp hepatoenteric cytidylcholine immune detection kit detection method, which is applied to the shrimp hepatoenteric cytidylcholine immune detection kit as described in the first aspect above, and is characterized in that it comprises the following steps:

[0042] Operator S1 collects shrimp hepatocystis samples and obtains the collected samples;

[0043] In an embodiment of the present invention:

[0044] S11 The operator wears disposable gloves to catch live shrimps from the farm to be tested to obtain the shrimps to be tested;

[0045] Specifically, operators need to wear disposable gloves to ensure aseptic operation during the sampling process and avoid sample contamination.

[0046] S12: opening the cephalothorax of the shrimp to be tested, and using the disposable tweezers to clamp the hepatopancreas sample to obtain a collected sample.

[0047] Specifically, the cephalothorax of the shrimp to be tested is opened, and about 30 μg of hepatopancreas sample is clamped with the disposable tweezers to ensure the sufficiency and representativeness of the collected sample to improve the detection accuracy.

[0048] S2 pre-processes the collected sample to obtain a ground sample;

[0049] In an embodiment of the present invention:

[0050] S21: Clean and disinfect the sample tube, and place the collected sample in the disinfected sample tube for storage;

[0051] Specifically, the sample tube is cleaned and disinfected to ensure that there are no pollutants, thereby improving the accuracy of the detection.

[0052] S22 uses the disposable grinding rod to grind the collected sample in the sample tube to obtain a ground sample.

[0053] Specifically, the purpose of grinding is to ensure that the shrimp hepatocellular carcinoma antigens in the collected sample are fully released.

[0054] S3 performs lysis treatment on the ground sample based on the lysis buffer to obtain a treated sample;

[0055] In an embodiment of the present invention:

[0056] S31 introduces the lysis buffer into the sample tube using the pipette;

[0057] Specifically, 200 μL of the lysis buffer was added to the sample tube using a pipette.

[0058] S32 shakes the sample tube to mix the lysis buffer and the ground sample for lysis to obtain a processed sample.

[0059] Specifically, the lysis buffer comprises 1% NaOH and 1% Triton X-100, and is used to destroy the cell structure in the sample and release the shrimp hepatoenteric cytozoon antigens. The sample is fully mixed with the ground sample and lysed for 5 minutes at room temperature to ensure that the shrimp hepatoenteric cytozoon antigens in the sample are fully released.

[0060] S4 neutralizes the treated sample based on the neutralization buffer to obtain a final sample;

[0061] In an embodiment of the present invention:

[0062] S41 introduces the neutralization buffer into the sample tube using the pipette;

[0063] Specifically, 200 μL of the neutralization buffer solution was added to the sample tube using the pipette.

[0064] S42 shakes the sample tube to mix the neutralization buffer and the treated sample, adjusts the reaction environment, and obtains a final sample.

[0065] Specifically, the neutralization buffer comprises 2% Tris-HCl and 0.56% phosphoric acid, and is used to neutralize the lysis reaction, ensure the reaction environment of the shrimp hepatocellular carcinoma immunoassay test strip, and fully mix with the lysed sample to perform a neutralization reaction to adjust the reaction environment and prepare for the detection reaction.

[0066] S5 uses the shrimp hepatocellular carcinoma immune detection test strip to test the final sample, so as to determine the infection degree of the sample.

[0067] In an embodiment of the present invention:

[0068] S51 uses the pipette to evenly drop the final sample onto the shrimp hepatocellular carcinoma immune detection test strip;

[0069] Specifically, 70 μL of the final sample is dripped onto the shrimp hepatoenteric cytidylcholine immunoassay test strip using the pipette to ensure uniform dripping of the sample to improve the accuracy and repeatability of the test.

[0070] S52: leaving the shrimp hepatocellular carcinoma immune detection test strip to stand for 10-15 minutes, observing the reaction lines, and obtaining the number and position of the lines;

[0071] S53 determines whether the shrimps in the farm are infected with enterocytosis and the extent of the infection based on the number and position of the observed lines.

[0072] The use of this test kit and detection method eliminates the process of sending shrimp for inspection, and the shrimp hepatoenteric cytosis can be detected at the breeding site. The detection is fast and accurate, which significantly improves the farmers' ability to prevent and control shrimp hepatoenteric cytosis. It is of great significance to promote the sustainable development of the aquaculture industry, and solves the problem that the traditional shrimp hepatoenteric cytosis inspection takes a long time and is improperly stored during transportation, which affects the test results.

[0073] In order to better understand the present technical solution, the following embodiments are provided for further explanation:

[0074] A farmer collects shrimp samples on-site at his farm, processes and tests them according to the above method, and it only takes 20 minutes to quickly understand whether the farmed shrimp are infected with shrimp hepatoenteroides, so that appropriate prevention and control measures can be taken in time to effectively reduce the spread of the disease and losses.

[0075] The above disclosure is only a preferred embodiment of a shrimp hepatocellular carcinoma immune detection kit and a detection method of the present invention. Of course, this cannot be used to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A shrimp hepatocellular carcinoma immune detection kit, characterized in that: Includes lysis buffer, neutralization buffer, shrimp hepatocellular carcinoma immunoassay test strips, disposable tweezers, sample tubes, disposable grinding rods and pipettes.

2. A shrimp hepatoenteric cytidylcholine immunoassay kit detection method, applied to the shrimp hepatoenteric cytidylcholine immunoassay kit according to claim 1, characterized in that: The following steps are involved: The operator collects the shrimp hepatocystis sample and obtains the collected sample; Pre-processing the collected sample to obtain a ground sample; Performing a lysis treatment on the ground sample based on the lysis buffer to obtain a processed sample; Performing a neutralization treatment on the treated sample based on the neutralization buffer to obtain a final sample; The final sample is tested using the shrimp hepatocellular carcinoma immune detection test strip to determine the infection degree of the sample.

3. The shrimp hepatocellular carcinoma immune detection kit detection method according to claim 2, characterized in that: The operator collects shrimp hepatocystis samples and obtains the specific method of collecting samples: The operator wears disposable gloves to catch the live shrimps from the farm to be tested, and obtains the shrimps to be tested; The cephalothorax of the shrimp to be tested is opened, and the hepatopancreas sample is clamped with the disposable tweezers to obtain a collected sample.

4. The shrimp hepatocellular carcinoma immune detection kit detection method according to claim 2, characterized in that: The specific method of pre-processing the collected sample to obtain the ground sample is: Cleaning and disinfecting the sample tube, and placing the collected sample in the disinfected sample tube for storage; The collected sample in the sample tube is ground using the disposable grinding rod to obtain a ground sample.

5. The shrimp hepatocellular carcinoma immune detection kit detection method according to claim 2, characterized in that: The specific method of performing lysis treatment on the ground sample based on the lysis buffer to obtain the processed sample is: Using the pipette to introduce the lysis buffer into the sample tube; The sample tube is shaken to mix the lysis buffer and the ground sample for lysis to obtain a processed sample.

6. The shrimp hepatocellular carcinoma immune detection kit detection method according to claim 2, characterized in that: The specific method of neutralizing the treated sample based on the neutralization buffer to obtain the final sample is: Using the pipette to introduce the neutralization buffer into the sample tube; The sample tube is shaken to mix the neutralization buffer and the treated sample, and the reaction environment is adjusted to obtain a final sample.

7. The shrimp hepatocellular carcinoma immune detection kit detection method according to claim 2, characterized in that: The specific method of using the shrimp hepatocellular carcinoma immune detection test strip to measure the final sample to determine the infection degree of the sample is: Using the pipette, the final sample is evenly dripped onto the shrimp hepatocellular carcinoma immune detection test strip; The shrimp hepatocellular carcinoma immune detection test strip is left to stand for 10-15 minutes, and the reaction lines are observed to obtain the number and position of the lines; Based on the number and position of the observed lines, it is determined whether the shrimps in the farm are infected with Enterocytosis hepatocellular carcinoma and the extent of the infection.

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