CN202404102U - Test paper for rapidly detecting viral hemorrhagic septicemia of fishes - Google Patents

Abstract

A test paper for rapid detection of fish viral hemorrhagic septicemia virus, comprising a self-adhesive carrier plate (1) of appropriate size, and a monoclonal antibody E coated with fish viral hemorrhagic sepsis virus is arranged in the middle The detection layer (2) made of the nitrocellulose membrane of the detection line (6) and the quality control line (7) of goat anti-mouse IgG, and the two ends continuous with this layer are provided with absorbent paper, which respectively constitute the sample loading end. layer (3) and the hand-held end water-absorbing layer (4); at the far end of the detection line (6) of the detection layer (2) and the junction of the sample-loading end water-absorbing layer (3), a gold-labeled fish viral virus As for the glass fiber membrane (5) of the monoclonal antibody A of hemorrhagic sepsis virus, one end of the membrane is arranged under the water-absorbing layer (3) at the sample loading end, and the other end is arranged on the detection layer (2). The test paper has good stability and repeatability, and has the characteristics of simplicity, speed, accuracy and high sensitivity and specificity.

Description

Rapid detection test strip for fish viral hemorrhagic sepsis virus

technical field

The utility model relates to the improvement of a breeding disease pathogen detection technology, in particular to a fish virus hemorrhagic septicaemia virus (Viral haemorhagic septicaemia virus, VHSV) rapid detection test paper, which belongs to the technical field of detection test paper structure.

Background technique

Fish viral hemorrhagic septicemia (VHS) is caused by viral hemorrhagic septicemia virus (VHSV), which belongs to the Rhabdoviridae RNA virus. The disease can attack wild seawater and freshwater fish, resulting in the death of a large number of wild fish. The invasion of farmed fish is more serious. In recent years, the number of fish species detected by VHSV in the world has continued to rise, reaching more than 50 species of fish. VHS is an emerging infectious disease of marine fish. The disease has caused serious harm to European countries such as Denmark, Sweden, and Finland for marine rainbow trout; it has caused serious damage to European countries such as Germany, the United Kingdom, and Ireland for turbot; Farming of Atlantic salmon has caused certain losses; in recent years, it has caused serious damage to Japanese flounder and red sea bream, and the North American genotype VHSV is widely seen in Japanese and Korean cultured flounder, Japanese cultured unprepared scorpionfish and Pacific scorpionfish, and is widely seen in Japan Coastal wild flounder and Pacific flounder; and the North American genotype and European genotype VHSV isolated from Japanese cultured flounder diseased fish are also harmful to China's marine cultured fish such as Xu's flatfish, red-spotted grouper, black sea bream, etc. sick. Therefore, for the domestic marine fish farming industry, especially for turbot, flounder, red sea bream, black sea bream, red-spotted grouper, and Xu's flat carp, it is necessary to prevent the occurrence of fish VHS and strengthen the fish VHS. Epidemic surveillance is very important.

VHS is the first type of quarantine object at the fish port, and it is also a fish disease that must be reported to the World Organization for Animal Health (OIE). In view of the fact that VHS is a common, highly contagious and serious loss of freshwater, marine fish and wild fish, At present, there are only a few entry-exit inspection and quarantine departments in the country that can do this kind of testing. The testing methods are complicated, limited to laboratory testing, require equipment, and the testing time is long. The reagents used are all imported and expensive. Thereby, it is imperative to develop a method that is applicable to entry-exit inspection and quarantine departments all over the country and can be used conveniently, quickly, easily and accurately at the port site, so as to prevent the introduction of the disease in time.

Utility model content

The utility model aims at the present situation of fish viral hemorrhagic septicemia harm, and aims at the physiological characteristics of aquatic organisms. Viral hemorrhagic sepsis virus rapid test strips

The technical scheme of the utility model is realized as follows

Fish viral hemorrhagic septicemia virus rapid detection test paper, which includes: an appropriate size self-adhesive carrier plate (1), in the middle of the carrier plate is provided with fish viral hemorrhagic septicemia virus monoclonal antibody E The detection layer (2) made of the nitrocellulose membrane of the detection line (6) and the quality control line (7) of goat anti-mouse IgG, and the two ends continuous with this layer are provided with absorbent paper, which respectively constitute the sample loading end. layer (3) and the hand-held end water-absorbing layer (4); at the far end of the detection line (6) of the detection layer (2) and the junction of the sample-loading end water-absorbing layer (3), a gold-labeled fish viral virus As for the glass fiber membrane (5) of the monoclonal antibody A of hemorrhagic sepsis virus, one end of the membrane is arranged under the water-absorbing layer (3) at the sample loading end, and the other end is arranged on the detection layer (2).

The detection principle of the quick detection test paper for fish viral hemorrhagic septicemia virus of the utility model: insert the water-absorbing layer of the sampling end of the quick detection test paper for fish viral hemorrhagic septicemia virus or drop the phosphate buffer solution from the pounding layer on it. The test sample containing virus particles was extracted from the crushed fish kidney. According to the principle of sandwich immunochromatography, the gold-labeled monoclonal antibody A reacted with the antigen in the test sample liquid to form a gold-labeled monoclonal antibody A-fish viral hemorrhagic sepsis Virus complex, when the complex is chromatographed to the monoclonal antibody E pre-coated on the detection line on the nitrocellulose membrane, the antibody here will recognize the antigen in the complex, and the result of the reaction will form a gold standard The sandwich structure of monoclonal antibody A + fish viral hemorrhagic sepsis virus antigen + monoclonal antibody E, and finally the colloidal gold particles labeled on monoclonal antibody A are immobilized and accumulated here, and a red line visible to the naked eye appears, unreacted gold-labeled monoclonal antibody A still continues to chromatographically. When the arrival point is pre-coated at the quality control line goat anti-mouse IgG, the gold-labeled monoclonal antibody A with the antibody type of IgG is bound by it, so that colloidal gold also appears here. The particles are fixed and accumulated to show a red line visible to the naked eye. The result is: if the detection line shows red, it means that the fish virus hemorrhagic septicemia virus is positive; The sample contained no or very low levels of FIHSV. If the quality control line shows red, it means that the test paper is valid; if the quality control line does not show red, it means that the test paper is invalid, that is, the gold-labeled antibody A or goat anti-mouse IgG is inactivated, and the test result is invalid.

This new type of use has the following advantages:

1. The rapid detection test paper for fish viral hemorrhagic septicemia virus of the utility model does not require special facilities and operating techniques when used, and is suitable for on-site detection of samples. It has the characteristics of fast detection, accuracy, sensitivity, and high specificity, and has high Stability and repeatability;

2. In the method of using the rapid detection test paper for fish viral hemorrhagic septicemia virus of the present utility model, no special lysate is added; only the fish kidneys need to be crushed. Compared with traditional virus extraction, grinding, centrifugation and other methods, This method is convenient and simple, and can be quickly extracted for virus detection.

Description of drawings

Embodiments of the utility model are further described as follows in conjunction with the accompanying drawings:

Fig. 1 schematic diagram of rapid detection test paper for fish viral hemorrhagic septicemia virus;

Figure 2 Schematic diagram of the detection results of the rapid detection test paper for fish viral hemorrhagic sepsis virus.

Detailed ways

Embodiment 1. Referring to Fig. 1, 2 quick detection test papers of fish viral hemorrhagic septicemia virus of the present utility model, it comprises: the anti-fish viral hemorrhagic septicemia virus monoclonal antibody A (being called for short: gold standard sheet of colloidal gold mark) anti-A); anti-fish viral hemorrhagic sepsis virus monoclonal antibody E (abbreviation: monoclonal antibody E) and goat anti-mouse IgG; coated with monoclonal antibody E and goat anti-mouse IgG nitrocellulose membrane 2; Glass fiber membrane 5 loaded with gold-labeled monoclonal antibody A; carrier plate 1 with self-adhesive. In the middle of the carrier plate 1 with self-adhesive, paste the nitrocellulose membrane coated with the detection line 6 of monoclonal antibody E and the quality control line 7 of goat anti-mouse IgG as the detection layer 2; continue with this layer Absorbent paper is arranged at both ends of the sample loading end absorbent layer 3 and the handheld end absorbent layer 4 respectively; at the junction of the far end of the detection line 6 of the detection layer 2 and the sample loading end absorbent layer 3, a gold-labeled monoclonal antibody is provided. For the glass fiber membrane 5 of A, one end of the membrane is set under the water-absorbing layer 3 at the sample loading end, and the other end is set on the detection layer 2 .

Example 2. The method of using the rapid detection test paper for fish viral hemorrhagic septicemia virus, that is, the detection steps of fish viral hemorrhagic septicemia virus: take the fish kidney and put it into a centrifuge tube to fully mash it, and press the mass/volume ratio of 1 : (5-10) (g/ml) Add 0.01mol/L phosphate buffer solution with pH 6.0-8.0 to prepare the test sample. Then, drop the sample on the water-absorbing layer 3 of the sampling end, and read the result within 5 minutes.

Positive result: Two red detection lines 6 and quality control lines 7 appear on the top and bottom of the detection layer 2, indicating that there is fish viral hemorrhagic sepsis virus infection;

Negative result: a red quality control line 7 appears on the upper end of the detection layer 2, indicating that there is no fish viral hemorrhagic sepsis virus infection or its virus content is extremely low;

The test result is invalid: 10 minutes after adding the sample, there is no red line at the quality control line 7 on the upper end of the test layer 2, indicating that the test strip is invalid and the test result is invalid. (See Figure 2)

Embodiment 3. The determination of the minimum amount of virus detected by the fish virus hemorrhagic septicemia virus rapid detection test paper of the utility model is as follows: the results are shown in Table 2.

Table 2 The results of the determination of the lowest amount of virus (TAIE ₅₀ ＝ ^10-5.3 /0.1ml)

Note: ++ means that the test line shows purple red, which is a strong positive; + means that the test line shows red, which is positive; - means that the test line is colorless, which is negative.

Example 4. The stability measurement of the fish virus hemorrhagic septicemia virus rapid detection test paper of the utility model is as follows: the test paper stored at 4° C. has a validity period of 12 months. The test strip is valid for 8 months when stored at room temperature.

Instruments and reagents used in the utility model: fluorescent inverted phase contrast microscope (purchased from LEICA company); marking machine (purchased from U.S. BIODOT company); chloroauric acid (purchased from China Shanghai Reagent No. 1 Factory, analytically pure); 1640 (purchased from GIBCO company); fetal bovine serum (purchased from PAA company); HAT (purchased from GIBCO company); goat anti-mouse IgG antibody (purchased from SIGMA company); bovine serum albumin (purchased from SIGMA company); nitrocellulose membrane (purchased from MILLIPORE);

Those skilled in the art will understand that within the protection scope of the present utility model, it is possible to modify, add and replace the above-mentioned embodiments, which do not exceed the protection scope of the present utility model.

Claims (1)

1. A quick detection test paper for fish viral hemorrhagic septicemia virus, which comprises: a self-adhesive carrier plate (1) of appropriate size, which is provided with a fish viral hemorrhagic septicemia virus coated in the middle part of the carrier plate The detection layer (2) made of the nitrocellulose membrane of the detection line (6) of monoclonal antibody E and the quality control line (7) of goat anti-mouse IgG, and the two ends continuous with this layer are provided with absorbent paper, respectively constitute The water-absorbing layer (3) at the sample loading end and the water-absorbing layer (4) at the hand-held end; at the junction of the far end of the detection line (6) of the detection layer (2) and the water-absorbing layer (3) at the sample loading end, a gold label is provided. A glass fiber membrane (5) of the monoclonal antibody A of fish viral hemorrhagic sepsis virus, one end of the membrane is arranged under the water-absorbing layer (3) at the sample loading end, and the other end is arranged under the detection layer (2) superior.

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