阅读说明：本技术 一种显微染色鉴别肝肠胞虫的方法及其专用装置 (Method for identifying liver enterocytozoon by microscopic staining and special device thereof ) 是由 乔毅 沈辉 万夕和 蒋葛 王李宝 黎慧 史文军 成婕 孙瑞健 秦亚丽 李浩澜 于 2021-01-18 设计创作，主要内容包括：本发明公开了一种显微染色鉴别肝肠胞虫的方法及其专用装置,属于肝肠胞虫的检测领域。该方法包括以下步骤：在普通显微镜的镜座上设置激发波长为360-400nm的紫外光源；取待测样品置于载玻片上,取等体积的染色液与待测样品混合均匀,再取等体积的背景降低液混合均匀,盖上盖玻片,染色2-4min,置于改造后的显微镜载物台上,开启紫外光源,在显微镜下观察直径大小为0.75-1.50μm的圆形或椭圆形的亮蓝色物质,即为肝肠胞虫。该方法能够降低检测成本,提高检测效率,且对设备和操作人员的技术依赖性低,能够在养殖现场进行肝肠胞虫的检查,具有快捷、高效、低成本、易操作等优点,有助于对虾养殖过程中肝肠胞虫的防控。(The invention discloses a method for identifying liver enterocytozoon by microscopic staining and a special device thereof, belonging to the field of detection of liver enterocytozoon. The method comprises the following steps: an ultraviolet light source with the excitation wavelength of 360-400nm is arranged on a microscope base of a common microscope; placing a sample to be detected on a glass slide, taking an equal volume of staining solution to be uniformly mixed with the sample to be detected, then taking an equal volume of background reducing solution to be uniformly mixed, covering the glass slide, staining for 2-4min, placing on a modified microscope stage, starting an ultraviolet light source, and observing a circular or elliptical brilliant blue substance with the diameter of 0.75-1.50 mu m under a microscope, namely the liver enterocytozoon. The method can reduce the detection cost, improve the detection efficiency, has low technical dependence on equipment and operators, can be used for detecting the enterocytozoon in the culture site, has the advantages of rapidness, high efficiency, low cost, easy operation and the like, and is beneficial to the prevention and control of the enterocytozoon in the prawn culture process.)

1. A method for identifying liver enterocytozoon by microscopic staining is characterized by comprising the following steps:

1) the light source of the ordinary optical biomicroscope is modified, an ultraviolet light source with the excitation wavelength of 360-400nm replaces the ordinary light source, and the intensity of the ultraviolet light source can be continuously adjusted;

2) dissolving Fluorogenic Brightene 28 with PBS buffer solution to a final concentration of 1% w/v Fluorogenic Brightene 28, and adding Evan's Blue dye to a final concentration of 0.1% w/v Evan's Blue as a staining solution;

3) placing a sample to be detected on a glass slide, uniformly mixing an equal volume of staining solution with the sample to be detected, uniformly mixing an equal volume of background reducing solution, covering the glass slide, staining for 2-4min, placing the prepared glass slide on a modified microscope stage, starting an ultraviolet light source, and observing a circular or elliptical brilliant blue substance with the diameter of 0.75-1.50 mu m under a microscope, namely the liver enterocytozoon.

2. The method for identifying enterocytozoon hepatica by microscopic staining according to claim 1, wherein in step 2), the pH value of the PBS buffer is 7.2-7.4.

3. The method for identifying enterocytozoon hepatica by microscopic staining according to claim 1, wherein in the step 3), the background reducing solution is KOH solution.

4. The method for identifying enterocytozoon hepatica by microscopic staining according to claim 3, wherein the concentration of the KOH solution in the step 3) is 10% w/v.

5. The method for identifying enterocytozoon hepatica by microscopic staining according to claim 1, wherein in the step 3), the sample to be tested is prepared by: taking shrimp liver pancreas tissue, and homogenizing the tissue to prepare the shrimp liver pancreas tissue.

6. The method for identifying enterocytozoon hepatica by microscopic staining according to claim 1, wherein in step 3), staining is performed for 3 min.

7. A special device for identifying liver and intestinal cytozoon by microscopic staining comprises an ocular lens (1), a lens cone (2), a focusing screw (3), a lens arm (4), a converter (5), an objective lens (6), an objective table (7), a lens holder (8) and a base (9), and is characterized in that a groove for placing a light source is formed in the lens holder (8), and the light source is an ultraviolet light source (10) with the excitation wavelength of 360 and 400 nm.

Technical Field

The invention belongs to the field of detection of liver enterocytozoon, relates to a method for identifying liver enterocytozoon by microscopic dyeing and a special device thereof, and particularly relates to an improved observation method of liver enterocytozoon based on a common microscope and an on-site inspection application of inspection equipment thereof in the process of prawn culture.

Background

The Enterocytozoon hepatopenaei (EHP for short) belongs to the family of microsporidae and the genus of enterozoon, and is an obligate intracellular parasite capable of infecting various eukaryotes. Mature spore is oval, length is 0.75-1.50um, rear end has a vacuole, cell contains a cell nucleus, 5-6 polar filament circles, 1 anchor disc connected with polar filament, and 1 layer of electron dense spore wall.

The shrimp enterocytozoon is an obligate intracellular parasite capable of infecting various eukaryotes, is found and separated in Thai in 2009 for the first time, and EHP is found in 2013 for the first time in China, and is reported to infect the hepatopancreatic part of the shrimp, so that the shrimp infected with the shrimp enterocytozoon does not generally have the acute and large-scale death phenomenon caused by pathogens such as white spot syndrome (WSD) and the like, but causes the food intake of the shrimp to be reduced, the growth of the shrimp to be slow or even stopped, and the specifications of the shrimp to be uneven.

Although the molecular means is mainly used for detecting the enterocytozoon hepatica at present, the quantity and morphological characteristics of the enterocytozoon hepatica can be visually observed through microscope observation, and the infection severity of the enterocytozoon hepatica can be better judged. The existing fluorescence microscope observation technology of the enterocytozoon mainly utilizes the fact that a special structure of a spore wall is combined with a special dye to show bright blue fluorescence under the excitation of fluorescence, and therefore whether the penaeus vannamei infects the enterocytozoon is judged.

However, the fluorescence microscope is expensive, the instrument operation is complex, the detection method is mostly carried out in a laboratory, and the method cannot be applied to the prawn culture production field.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for identifying the enterocytozoon through microscopic dyeing, and the invention aims to provide a special device for identifying the enterocytozoon through microscopic dyeing, which is convenient to be directly applied to a prawn culture production field.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for identifying liver enterocytozoon by microscopic staining comprises the following steps:

1) the light source of the ordinary optical biomicroscope is modified, and the ultraviolet light source with the excitation wavelength of 360-400nm replaces the ordinary light source, so that the intensity of the ultraviolet light source can be continuously adjusted;

2) dissolving Fluorogenic Brightene 28 with PBS buffer solution to a final concentration of 1% w/v Fluorogenic Brightene 28, and adding Evan's Blue dye to a final concentration of 0.1% w/v Evan's Blue dye as a staining solution;

3) placing a sample to be detected on a glass slide, uniformly mixing an equal volume of staining solution with the sample to be detected, uniformly mixing an equal volume of background reducing solution, covering the glass slide, staining for 2-4min, placing the prepared glass slide on a modified microscope stage, starting an ultraviolet light source, and observing a circular or elliptical brilliant blue substance with the diameter of 0.75-1.50 mu m under a microscope, namely the liver enterocytozoon.

Further, in step 2), the pH value of the PBS buffer solution is 7.2-7.4.

Further, in step 3), the background reducing solution is a KOH solution.

Further, in step 3), the concentration of the KOH solution is 10% w/v.

Further, in step 3), the preparation method of the sample to be tested comprises: taking shrimp liver pancreas tissues, homogenizing the tissues and preparing the shrimp liver pancreas tissues.

Further, in step 3), staining was performed for 3 min.

A special device for identifying liver enterocytozoon by microscopic staining is formed by modifying a common optical microscope, wherein a microscope base of the common optical biomicroscope is provided with a groove for placing a light source, and the light source is an ultraviolet light source with the excitation wavelength of 360-400 nm.

Has the advantages that: compared with the prior art, the invention has the advantages that:

1) according to the invention, the structure of a common microscope is improved, the dyeing operation method is simplified, the improved common microscope is used for inspecting the enterocytozoon hepatica, the severity of prawn infection with enterocytozoon hepatica can be judged according to the quantity of enterocytozoon hepatica in the visual field, the breeding production is guided, and the economic loss is reduced;

2) the invention can greatly reduce the detection cost, improve the detection efficiency, has no influence on the detection quality and precision, reduces the technical dependence on equipment and operators, can be popularized and applied in prawn culture production, and has great significance for preventing and controlling the liver enterocytozoon in the prawn culture process.

Drawings

FIG. 1 is a diagram of a special device for identifying liver enterocytozoon by microscopic staining according to the application;

FIG. 2 is a microscopic image of enterocytozoon hepatica detected by the method of the present invention;

FIG. 3 is a microscopic image of enterocytozoon hepatica detected by the method of the present invention; the scale bar is 5 μm;

FIG. 4 is a microscopic image of enterocytozoon hepatica detected by the method of the present invention; the scale bar is 5 μm.

Detailed Description

The invention is further described with reference to specific examples.

Example 1

An improved device for identifying liver enterocytozoon by microscopic staining is shown in figure 1 and comprises an ocular lens 1, a lens cone 2, a focusing screw 3, a lens arm 4, a converter 5, an objective lens 6, an objective table 7, a lens base 8 and a base 9; the lens base 8 is provided with a groove for placing a light source, the light source is an ultraviolet light source 10 with the excitation wavelength of 360 and 400nm, and the light source intensity can be continuously adjusted.

The method for identifying the liver enterocytozoon by using the improved microscope comprises the following steps:

s1 reagent preparation

Preparing a dyeing solution: an appropriate amount of Fluorescent Brightene 28 reagent (from Sigma) was taken, the Fluorescent Brightene 28 was dissolved in PBS (pH 7.2) to a final concentration of 1% w/v, and an appropriate amount of Evan's Blue dye (from Sigma) was added to a final concentration of 0.1% w/v, and the prepared dye solution was stored in low temperature and dark place.

Preparing a background reducing solution: taking a proper amount of KOH to prepare the mixture to the final concentration of 10% w/v, and storing the mixture at normal temperature for later use.

S2 sample preparation

Taking hepatopancreas tissues (0.05-0.2g) of Penaeus vannamei Boone in a 1.5mL EP tube, grinding with a disposable grinding rod, and homogenizing the hepatopancreas tissues of multiple prawn seedlings if the prawn seedlings are used.

S3 sample staining

And (3) taking 5 mu L of the prepared sample on a glass slide by using a micropipette, uniformly mixing 1% of staining solution with the same volume with the sample, then taking 5 mu L of 10% KOH solution for mixing, covering a cover glass, and staining for 3 min.

S4, sample observation

Placing the prepared glass slide on an improved microscope stage, starting an ultraviolet light source, and observing a circular or elliptical bright blue substance with the diameter of 0.75-1.50 μm under a microscope as shown in figures 2-4, wherein the substance is the liver enterocytozoon.

Comparative example 1

The procedure is as in example 1 except for the following technical parameters.

Microscope using Nikon Ni fluorescence microscope, the excitation wavelength was varied and the results of comparison for the detection of the identified enterohepatica were as shown in table 1.

TABLE 1 comparison of the detection of the modified microscope and the Nikon Ni fluorescence microscope for the identification of the liver enterocytozoon

Comparative example 2

Common parasitic diseases of penaeus vannamei boone include sessile ciliate diseases (such as bella, polypiperus and wireworm, which usually parasitize on the body surface and sometimes cause serious harm to a host together with other protozoa and filamentous bacteria) and microsporidiosis (such as enterobacter hepatica), and the specific method is the same as that in example 1.

TABLE 2 specificity of the improved microscopic examination for the identification of Enteromorpha hepatica

Improved microscope	Bell-shaped insect	Insect polycondensation	Twig-accumulating insects	Liver and intestine cytozoon
					Test results (with/without)	Is free of	Is free of	Is free of	Is provided with
Size and shape of the product	Is free of	Is free of	Is free of	Circular ellipse with diameter of 0.75-1.5 μm

Comparative example 3

The procedure is as in example 1 except for the following technical parameters.

The concentration of the separated and purified known liver enterocytozoon is 10⁷Diluting the tissue/mg into 10 and 10 respectively with hepatopancreas tissue of healthy Penaeus vannamei²、10³、10⁴、10⁵、10⁶、10⁷Doubling the tissue homogenate, then performing the operating step of staining the S3 sample: and (3) taking 5 mu L of the prepared sample on a glass slide by using a micropipette, uniformly mixing 1% of staining solution with the same volume with the sample, then taking 5 mu L of 10% KOH solution for mixing, covering a cover glass, and staining for 3 min. And (3) observing a sample: placing the prepared glass slide on an improved microscope stage, and turning on ultravioletAs shown in FIGS. 2 to 4, a light source is used to observe a circular or elliptical bright blue substance with a diameter of 0.75 to 1.50 μm under a microscope, which is a liver enterocytozoon.

As a result, the sensitivity of detecting and identifying the liver enterocytozoon is high (the lowest detection limit can reach 10)³One per mg tissue).

It is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.