CN113481321A - In-situ hybridization detection probe for carp edema virus and application thereof - Google Patents

Abstract

The invention discloses an in-situ hybridization detection probe for a carp edema virus and application thereof, belonging to the technical field of epidemic disease detection. The sequence of the probe is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the FAM-labeled probe can be used for detecting the carp edema virus and preparing a carp edema virus detection kit. The probe is specifically hybridized and combined with the CEV P4a gene in the tissue section, so that the detection is more sensitive and accurate compared with the conventional staining detection, and the CEV infection positive rate, infection part and degree can be analyzed according to the sample section.

Description

In-situ hybridization detection probe for carp edema virus and application thereof

The technical field is as follows:

the invention relates to the technical field of detection of epidemic diseases of aquatic animals in molecular biology and aquaculture industry, in particular to an in-situ hybridization detection probe for carp edema virus and application thereof.

Background art:

carp edema disease (CEVD), also known as Koi Sleep Disease (KSD), is a highly infectious epidemic caused by infection of Carp and koi with a pox virus, Carp Edema Virus (CEV). Diseased fish often has symptoms such as gill rot, fovea, lethargy and the like, the death rate caused by diseases is as high as 80-100%, and serious economic loss is caused to aquaculture industry. The disease has spread rapidly in the world, China reports the occurrence of CEVD in 2016 for the first time, and the disease is a new epidemic disease of aquatic animals in China.

At present, no effective treatment method for carp edema disease caused by CEV exists, and prevention is mainly performed. Therefore, the accurate and rapid in-situ detection method is established and is a guarantee for effectively preventing and controlling CEVD. Because CEV has no susceptible cell line, the detection method mainly depends on nested PCR, fluorescent PCR and other molecular biological methods. Although the PCR detection is simple and has high sensitivity, CEV cannot be visually observed and proved, and false positive is easy to occur due to template contamination during operation. The false positive rate of in situ hybridization is low, so that the comprehensive consideration of intuition, accuracy and sensitivity is a detection method which cannot be replaced by any other method, but the in situ hybridization detection method of CEV is not seen so far.

The invention content is as follows:

the invention aims to solve the technical problem of providing an in-situ hybridization detection probe for the carp edema virus and application thereof, wherein 3 DNA probes are designed and synthesized according to a conserved sequence of a carp edema virus coding P4a protein, can be used for in-situ hybridization detection of the carp edema virus, and have excellent intuitiveness, accuracy and sensitivity.

The invention designs 3 DNA probes according to the CEV P4a gene sequence, and prepares the in situ hybridization detection probe of the carp edema virus by marking with FAM.

The probe for detecting the carp edema virus by in situ hybridization is characterized by comprising 3 probes of the following nucleotide sequences:

1, probe 1: 5'-GCCCAAGAGTTTTCTTCTCATCGTTTGTTACC-3' SEQ ID NO 1

And (3) probe 2: 5'-GCAACTCCTTGAGGAATATGATCTAGAATTCC-3' SEQ ID NO 2

And 3, probe 3: 5'-GAACATAACATTTGCAATTTTAACTTGCTCTGG-3' SEQ ID NO 3

FAM is modified at the 5' end of the probe 1, the probe 2 and the probe 3.

The probe can be used for detecting the carp edema virus, and can also be used for preparing a carp edema virus detection kit, and the kit also comprises an in-situ hybridization detection reagent.

Specifically, the carp edema virus detection kit comprises: the in-situ hybridization detection probe, the proteinase K and the prehybridization solution. The detection method comprises the following steps:

the using method of the carp edema virus detection kit is characterized by comprising the following steps:

(1) boiling the slices for 15 min by heat restoration, adding protease K (20 μ l/ml) dropwise, digesting at room temperature for 20min, washing with distilled water once, and washing with PBS for 5min × 3 times;

(2) dripping the pre-hybridization solution to submerge, and placing in an incubator with the constant temperature of 42 ℃ for 2-3 h;

(3) after absorbing the redundant liquid, dripping the in-situ hybridization detection probe mixed liquid to submerge the slice, and keeping the temperature at 37 ℃ overnight; the in situ hybridization detection probe mixed solution is prepared by dissolving a probe 1, a probe 2 and a probe 3 in a soluble solution (preferably a prehybridization solution), wherein the molar ratio of the probe 1 to the probe 2 to the probe 3 is 1:1:1, and the total concentration of the three probes is 1.0 mu g/ml in work;

(4) washing with gradient SSC at 37 deg.C, i.e. 2 XSSC washing for 10min, 1 XSSC washing for 10min × 2 times, and 0.5 XSSC washing for 10min × 1 times;

(5) dripping DAPI, incubating for 5min in dark, staining nuclei, and washing redundant DAPI with PBS; finally, sealing by 50% of buffered glycerol (the mass ratio of the 50% of buffered glycerol to the PBS is 1: 1), and photographing and observing under a microscope;

(6) and (4) judging a result: after fluorescence excitation, CEV infected parts show green fluorescence, and cell nucleuses are dyed into blue fluorescence by DAPI.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the CEV pathogeny and histopathological changes caused by the CEV pathogeny can be intuitively combined, so that the infection position and the infection degree of the CEV are determined, and the pathogenicity of the CEV is known;

(2) the CEV can be efficiently and accurately detected, the CEV can be positioned at the same time, and the positive rate and the infection intensity of CEV infection can be analyzed according to a sample slice, which is a characteristic that other molecular biological detection methods do not have;

(3) because the hybridization of the probe and the CEV P4a gene is specific, the detection is more sensitive and accurate than the conventional staining detection;

(4) in this method, the sections can be stored for a long period in the absence of light.

Description of the drawings:

FIG. 1 shows in situ hybridization staining (100X) of CEV-infected Scirpus carpi heart, liver, spleen, kidney, brain, skin, gill tissue (arrows in the figure only indicate specific binding in gill tissue, others are not specifically indicated by arrows).

The specific implementation mode is as follows:

the present invention will be described in further detail with reference to the drawings and the detailed description, but the present invention is not limited to the following examples.

Materials, reagents and the like used in the following examples are conventional ones unless otherwise specified.

The experimental procedures used in the following examples are conventional unless otherwise specified.

The prehybridization solution in the following examples is from warrior, Inc.

DAPI in the following examples is a product of Kayki Biotech development, Inc. of Nanjing.

The probes in the following examples were synthesized by Shanghai Biotechnology engineering services, Inc.

1. Design of the Probe

3 specific probes (SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3) are designed according to a conserved sequence of the CEV P4a protein, and FAM is marked at the 5' end of the 3 probes.

In the probe mixed solution, the base sequences of the probes are shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, and the molar ratio is 1:1: 1. FAM was modified at the 5' end by 3 probes.

2. Tissue sample processing

And (4) performing on-site autopsy on the dying mirror carps suspected to be infected with the carp edema disease, and observing clinical symptoms. Selecting diseased fish with obvious clinical symptoms such as gill rot, eyeball depression, body surface erosion bleeding and the like. The heart, liver, spleen, kidney, brain, skin, gill were separately harvested and fixed with 4% paraformaldehyde. After fixation for 48h, conventional dehydration, clearing, waxing, embedding, and subsequent slicing into 5 μm thick serial sections. Tissue sections were routinely deparaffinized hydrated and washed 3 times for 5 minutes each in PBS.

CEV in situ hybridization assay

The sections were boiled for 15 minutes with heat-recovery, digested for 20min at room temperature with protease K (20. mu.l/ml) added dropwise, washed once with distilled water and 5min X3 times with PBS. 50 mu L of prehybridization solution is dripped into the culture box at the constant temperature of 42 ℃ for 2-3 h. After the excess liquid was aspirated, 100. mu.L of the in situ hybridization detection probe mixture was added dropwise, and incubated overnight at 37 ℃. Gradient SSC wash (2 XSSC wash 10min, 1 XSSC wash 10min X2 times, 0.5 XSSC wash 10min X1 times) at 37 ℃. Adding DAPI dropwise, incubating for 5min in dark, staining the specimen for nucleus, and washing the excessive DAPI with PBS. Finally, 50% glycerol was mounted and photographed under a microscope.

4. Analysis of results

The DNA probe marked by FAM is specifically combined with CEV in the tissue, CEV infected parts show green fluorescence after fluorescence excitation, and cell nucleuses are dyed into blue through DAPI. CEV was found to be expressed in large amounts mainly in the liver, spleen, kidney and especially gill by in situ hybridization. The observation dyeing result is consistent with the qPCR positioning result of the same part, and the reliability of the invention is further proved.

Claims (6)

1. The probe for detecting the carp edema virus by in situ hybridization is characterized by comprising 3 probes of the following nucleotide sequences:

1, probe 1: 5'-GCCCAAGAGTTTTCTTCTCATCGTTTGTTACC-3' SEQ ID NO 1

And (3) probe 2: 5'-GCAACTCCTTGAGGAATATGATCTAGAATTCC-3' SEQ ID NO 2

And 3, probe 3: 5'-GAACATAACATTTGCAATTTTAACTTGCTCTGG-3' SEQ ID NO. 3.

2. The probe for detecting the edema virus in carp in situ hybridization according to claim 1, wherein FAM is modified at the 5' end of the probe 1, the probe 2 and the probe 3.

3. A carp edema virus detection kit comprising the probe for detecting in situ hybridization of the carp edema virus according to any one of claims 1 to 2.

4. The carp edema virus detection kit according to claim 3, wherein the detection kit is an in situ hybridization detection kit, and comprises the in situ hybridization detection probe according to any one of claims 1 to 2, proteinase K, and a prehybridization solution.

5. The use method of the carp edema virus detection kit according to claim 4, characterized by comprising the following steps:

(1) boiling the slices for 15 minutes by heat restoration, adding protease K dropwise, digesting at room temperature for 20min, washing with distilled water once, and washing with PBS for 5min × 3 times;

(2) dripping the pre-hybridization solution to submerge, and placing in an incubator with the constant temperature of 42 ℃ for 2-3 h;

(3) after absorbing the redundant liquid, dripping the in-situ hybridization detection probe mixed liquid to submerge the slice, and keeping the temperature at 37 ℃ overnight; the in situ hybridization detection probe mixed solution is prepared by dissolving a probe 1, a probe 2 and a probe 3 in a soluble solution, wherein the molar ratio of the probe 1 to the probe 2 to the probe 3 is 1:1:1, and the total concentration of the three probes is 1.0 mu g/ml in work;

(4) washing with gradient SSC at 37 deg.C, i.e. 2 XSSC washing for 10min, 1 XSSC washing for 10min × 2 times, and 0.5 XSSC washing for 10min × 1 times;

(5) dripping DAPI, incubating for 5min in dark, staining nuclei, and washing redundant DAPI with PBS; finally, sealing the piece with 50% of glycerol, and taking a picture under a microscope for observation;

(6) and (4) judging a result: after fluorescence excitation, CEV infected parts show green fluorescence, and cell nucleuses are dyed into blue fluorescence by DAPI.

6. The method according to claim 5, wherein the solution in which probe 1, probe 2 and probe 3 are dissolved in step (3) is selected from a prehybridization solution.

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