阅读说明：本技术 一种检测昆虫中微粒子原虫孢子的试剂盒及方法 (Kit and method for detecting corpuscular protozoan spores in insects ) 是由 朱方容 黄深惠 黄旭华 李标 李韬 王霞 罗梅兰 蒋满贵 于 2020-08-07 设计创作，主要内容包括：本发明公开了一种利用流式细胞仪机械检测昆虫中微粒子原虫孢子的试剂盒,包括试剂盒和如下使用步骤：将荧光粉末溶解后得到荧光染色液置于试剂盒,试剂盒有昆虫微粒子原虫孢子染色液、还有浓度为标准孢子液、还带有能过滤40um物质的细胞过滤器；将样本研磨或稀释、加温使蛋白质沉淀、过滤,得到样本液；将样本液与荧光染色液混合后加入多孔板,并采用流式细胞仪检测；将样本液的检测结果与标准物检测结果比较,判定样本中是否含有微粒子原虫孢子。其具有检测准确度高,操作简单,效率高,改人工看镜为机械检测,材料和人工成本低的优势。(The invention discloses a kit for mechanically detecting corpuscular protozoan spores in insects by using a flow cytometer, which comprises the kit and the following use steps: dissolving the fluorescent powder to obtain a fluorescent staining solution, and placing the fluorescent staining solution into a kit, wherein the kit contains an insect microparticle protozoan spore staining solution, a standard spore solution and a cell filter capable of filtering 40um substances; grinding or diluting the sample, heating to precipitate protein, and filtering to obtain sample solution; mixing the sample solution and the fluorescent staining solution, adding the mixture into a porous plate, and detecting by using a flow cytometer; and comparing the detection result of the sample liquid with the detection result of the standard substance to judge whether the sample contains the corpuscular protozoan spores or not. The method has the advantages of high detection accuracy, simple operation, high efficiency, mechanical detection by changing manual observation, and low material and labor cost.)

1. A kit for detecting corpuscular protozoan spores in insects comprises a box body and a box cover hinged with the box body, and is characterized in that a fluorescent staining liquid bottle and a standard spore liquid bottle are fixed in the box body through a bottle rack, and a cell filter is fixed in the box body; the pore size of the filter screen of the cell filter is 400 meshes.

2. A method for detecting corpuscular protozoan spores in insects is characterized by comprising the following steps:

(1) dissolving fluorescent powder to obtain a fluorescent staining solution, filtering, and adding the fluorescent staining solution into the kit of claim 1;

(2) grinding or diluting a sample, then performing heating treatment, and filtering to obtain a sample solution;

(3) adding the sample solution into the kit, mixing uniformly, placing in the dark, and detecting by adopting a flow cytometer;

(4) and comparing the detection result of the sample liquid with the detection result of the standard substance to judge whether the sample contains the corpuscular protozoan spores or not.

3. The method according to claim 1, wherein the fluorescent powder in step (1) is isothiocyanate, the fluorescent powder is dissolved in advance by using a phosphate buffer solution with a pH of 7.8 to 9.0, and the concentration of the fluorescent staining solution is 0.1mg/mL to 0.5 mg/mL.

4. The method according to claim 1, wherein the step (2) comprises adding KOH or NaOH solution with a concentration of 0.5% by mass to the sample, and diluting or grinding; the heating temperature is 65-70 ℃, and the time is 10-30 min.

5. The method according to claim 1, wherein the filtering in steps (1) to (2) is performed by using a 35-45 μm filter or pipette tip filter.

6. The method according to claim 1, wherein the ratio of the volume of the sample liquid to the fluorescent staining solution in the step (3) is 4: 1, the treatment time under dark condition is 25-30 min.

7. The method of claim 6, wherein the excitation light wavelength detected in step (3) is 488nm and the emission maximum wavelength is 530 nm.

8. The method according to claim 1, wherein the detection result of the standard substance in the step (4) is a result of a positive control using 6-fold solution of the standard substance or a result of a computer-stored flow cytometer of the same type.

9. The method according to claim 8, wherein in the step (4), the standard substance is diluted to obtain a 6-fold solution of the standard substance, and then the solution is mixed with the fluorescent staining solution according to a volume ratio of 4: 1, treating for 25min in the dark after mixing, adding a porous plate, and automatically detecting by adopting a flow cytometer; the wavelength of the detected excitation light is 488nm, and the maximum emission wavelength is 530 nm.

Technical Field

The invention relates to the technical field of microbial molecule detection and quarantine, in particular to a kit and a method for detecting nosema spores in insects.

Background

Insects can infect nosema bombycis diseases, such as nosema bombycis, and nosema ricini. The corpuscular protozoa is a unicellular organism parasitized in cells, and the infection route mainly includes a food-down infection caused by contamination of host excreta and corpses, etc., and an infection route to the next generation by host embryos is called an embryo infection. Because insect corpuscular protozoal diseases are mostly infected by embryo, whether the insect corpuscular protozoal diseases are carried or not is determined during reproduction, and the corpuscular protozoal diseases are prevented from the source through quarantine.

In order to detect whether an insect sample carries the corpuscular protozoan disease or is at risk of infecting the corpuscular protozoan disease, the currently adopted mode is mainly a method for detecting the corpuscular protozoan disease of silkworms created by Pasteur more than 100 years ago, and the method mainly depends on a microscope to artificially detect whether the sample has mature corpuscular protozoan spores or not, and has the problems of low artificial observation efficiency, poor specificity and easy erroneous judgment by the microscope. In addition, the DNA detection technology is simple, the problem of misjudgment can be accurately solved, but the problem of mechanical detection is not solved, and the method still has the defects of low efficiency, complex operation and high material and labor cost.

Therefore, it is an urgent need to solve the problems of the art to provide a new kit and method for mechanically detecting the spores of the protozoa microparticles in insects.

Disclosure of Invention

In view of the above, the invention provides a kit and a method for detecting nosema spores in insects, which have the advantages of high detection accuracy, simple operation, high efficiency and low material and labor cost.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a kit for detecting corpuscular protozoan spores in insects, which comprises a box body and a box cover hinged with the box body, wherein a fluorescent staining liquid bottle and a standard spore liquid bottle are fixed in the box body through a bottle frame, and a cell filter is fixed in the box body; the pore size of the filter screen of the cell filter is 400 meshes.

Preferably, the top surface of the bottle rack is inwards provided with a groove, a fluorescent staining liquid bottle and a standard spore liquid bottle are arranged in the groove,

preferably, the cell filter includes cell filter top skeleton, cell filter bottom skeleton and set up in cell filter top skeleton with a plurality of cell filter spliced poles between the cell filter bottom skeleton, cell filter top skeleton and cell filter bottom skeleton are the annular structure, between the cell filter spliced pole with cell filter bottom skeleton passes through the cell filter screen involution.

The kit provided by the invention has the beneficial effects that: the preparation kit solves the problem of difficult solubility of the fluorescent powder, has simple and convenient preparation operation, saves materials, provides professional filtering equipment, has simpler operation and is easy to popularize and apply.

The invention discloses a method for detecting corpuscular protozoan spores in insects, which is characterized by comprising the following steps:

(1) dissolving fluorescent powder to obtain a fluorescent staining solution, filtering, and adding the fluorescent staining solution into the kit of claim 1;

(2) grinding or diluting a sample, then heating to precipitate protein impurities in the sample, and then filtering to obtain a sample solution;

(3) adding the sample solution into the kit, mixing uniformly, placing in the dark, and detecting by adopting a flow cytometer;

(4) and comparing the detection result of the sample liquid with the detection result of the standard substance to judge whether the sample contains the corpuscular protozoan spores or not.

The beneficial effects of the preferred technical scheme are as follows: the operation method disclosed by the invention is simple, has high automation degree, can realize mechanical detection, and has high detection result accuracy.

Preferably, the fluorescent powder in the step (1) is isothiocyanate, the fluorescent powder is dissolved by using a phosphate buffer solution with the pH value of 7.8-9.0, and the concentration of the fluorescent staining solution is 0.1-0.5 mg/mL.

Preferably, in the step (2), a KOH or NaOH solution with the mass concentration of 0.5% is added into the sample for dilution or grinding; the heating temperature is 65-70 ℃, and the time is 10-30 min.

The beneficial effects of the preferred technical scheme are as follows: according to the invention, KOH or NaOH solution is added for grinding, so that fat globules can be removed, and the fat globules are prevented from interfering the detection result; proteins can be denatured by heating and precipitated, and thus can be removed by filtration.

Preferably, the filtration is performed by using a 35-45 μm filter screen or a pipette tip filter element, so that impurities, such as bacteria and the like, with non-microparticle spores smaller than the pore size of the filter screen can be removed.

Preferably, the volume ratio of the sample liquid to the fluorescent staining liquid in the step (3) is 4: 1, the treatment time in the dark after mixing is 25-30 min.

Preferably, the excitation light wavelength detected in step (3) is 488nm and the maximum emission wavelength is 530 nm. Wherein forward angle scattering (FSC), side angle scattering (SSC) and fluorescence emission information are detected and analyzed using a flow cytometer to obtain qualitative and quantitative results. The accuracy is ensured by setting a gate of a scatter diagram of the comparison standard substance containing information such as appearance, long diameter, short diameter, internal polar filament, green fluorescence and the like.

Preferably, the detection result of the standard substance in the step (4) is the detection result by using 6 times of the liquid of the standard substance as a positive control or the detection result of a computer-stored flow cytometer of the same model.

The linear relation of the graph obtained by the flow cytometer is reflected by adjusting the gate, and the edge bias phenomenon is reduced by debugging the peak value, so that the accuracy of the experiment is improved; the sample shows a peak similar to that of the standard control, and then the sample can be judged to have microspores.

Preferably, in the step (4), the standard substance is diluted to obtain 6 times of the standard substance, and then the volume ratio of the standard substance to the fluorescent staining solution is 4: 1, treating for 25min in the dark after mixing, adding a porous plate, and automatically detecting by adopting a flow cytometer; the wavelength of the detected excitation light is 488nm, and the maximum emission wavelength is 530 nm.

According to the technical scheme, compared with the prior art, the invention discloses a kit and a method for detecting the corpuscular protozoan spores in insects, and the kit and the method have the following beneficial effects:

(1) the invention uses the flow cytometry for detection, has simple operation, realizes mechanized detection and has high detection efficiency;

(2) the problem of misjudgment can be effectively solved, and the accuracy of the obtained detection result is high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic structural diagram of a kit for detecting protozoan spores of microparticles in insects according to example 1 of the present invention;

FIG. 2 is a schematic diagram of a cell filter according to embodiment 1 of the present invention

FIG. 3 is a graph showing the results of detection of a sample 31 provided in example 2 of the present invention;

FIG. 4 is a graph showing the results of detection of a specimen 14 provided in example 3 of the present invention;

FIG. 5 is a graph showing the results of detection of a sample 17 provided in example 4 of the present invention;

FIG. 6 is a graph showing the results of detection of a sample 8 provided in example 5 of the present invention;

FIG. 7 is a graph showing the results of detection of the standard substance provided in example 2 of the present invention.

In the figure:

2 is a box body, 3 is a box cover, 4 is a fluorescent staining solution bottle, 5 is a standard spore solution bottle (the concentration of the standard spore solution is 1.0 × 10)⁶Granule/ml), 6 is 400 mesh cell filter, 7 is bottle holder, 8 is cell filter top skeleton, 9 is cell filter bottom skeleton, 10 is cell filter connecting post, 11 is cell filter screen.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention relates to a method for detecting corpuscular protozoan spores in insects, which specifically comprises the following steps:

(1) dissolving the fluorescent powder to obtain fluorescent staining solution, filtering and adding the fluorescent staining solution into the kit; wherein the fluorescent powder is isothiocyanate, the fluorescent powder is dissolved by adopting a phosphate buffer solution with the pH value of 7.8-9.0, and the concentration of the fluorescent staining solution is 0.1-0.5 mg/m

(2) Grinding or diluting a sample, then heating to precipitate protein impurities in the sample, and then filtering to obtain a sample solution; adding KOH or NaOH solution with the mass concentration of 0.5% into the sample for dilution or grinding; the heating temperature is 65-70 ℃, and the time is 10-30 min;

(3) according to the volume ratio of the sample liquid to the fluorescent staining solution of 4: 1, uniformly mixing, placing in the dark, standing for 25-30min, and detecting by using a flow cytometer, wherein the wavelength of detected excitation light is 488nm, and the maximum emission wavelength is 530 nm;

(4) comparing the detection result of the sample liquid with the detection result of the standard substance, and judging whether the sample contains the corpuscular protozoan spores or not; specifically, the standard substance can be diluted to obtain 6 times of solution of the standard substance, and then the solution is mixed with the fluorescent staining solution according to the volume ratio of 4: 1, treating for 25min in the dark after mixing, adding a porous plate, and automatically detecting by adopting a flow cytometer; the wavelength of the detected excitation light is 488nm, and the maximum emission wavelength is 530 nm.

Wherein, the filtration in the steps (1) to (2) adopts a 35-45 μm filter screen or a pipette tip filter core for filtration.

Forward angle scatter (FSC), side angle scatter (SSC) and fluorescence emission information were detected and analyzed by flow cytometry to obtain qualitative and quantitative results.

Preferably, the detection result of the standard substance in the step (4) is the detection result by using 6 times of the liquid of the standard substance as a positive control or the detection result of a computer-stored flow cytometer of the same model.

The linear relation of the gate of a graph obtained by adjusting a flow cytometer fluorescence scatter diagram is reflected, the peak value is debugged to reduce the edge phenomenon, and the accuracy of the experiment is improved; the sample shows a peak similar to that of the standard control, and then the sample can be judged to have microspores.