Simple long-term preservation method for small insect immersion specimens used for DNA molecular detection

Abstract

This invention discloses a simple long-term preservation method for small insect specimens used in DNA molecular detection, comprising the following steps: Freshly collected insect specimens are placed in centrifuge tubes filled with anhydrous ethanol, which is added to 4 / 5 of the tube's height, and a label is written on the tube before placement; insects of the same species from the same collection site are classified under a dissecting microscope and placed in the same centrifuge tube; after classification, centrifuge tubes from the same collection site are placed in the same sealed container, and a magnesium sheet wrapped in a water-permeable material is placed in the container, with anhydrous ethanol added until the magnesium sheet is submerged; the sealed container is then stored away from light, and anhydrous ethanol is added periodically. This invention solves the problem of preservation solution evaporation due to poor sealing of centrifuge tubes, and also reduces the water content of the anhydrous ethanol in the outer container, enabling long-term or permanent preservation of insect specimens, and allowing for quick identification based on label markings.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of insect specimen preservation, and in particular to a simple long-term preservation method for small insect immersion specimens for DNA molecular detection. BACKGROUND

[0002] Currently, the preservation of insect immersion specimens mainly uses 2 ml or 5 ml centrifuge tubes sold on the market for preservation, and the preservation liquid uses 75% alcohol solution or 95% alcohol solution. However, for DNA molecular detection, water solution can cause DNA molecular hydrolysis, affecting the detection result, so anhydrous ethanol should be used as the preservation liquid. However, the sealing performance of the centrifuge tubes sold on the market is poor, and ethanol is a very volatile solvent. In practice, it is found that the preservation liquid in a 2 ml centrifuge tube is significantly reduced or evaporated after one year, and it is necessary to re-open the cover and add the preservation liquid, which is time-consuming and laborious, and can easily damage the specimen. In addition, a large number of centrifuge tubes are stored in the container, and it is difficult to find the specimen. If a completely sealed centrifuge tube is developed, the cost is high. In addition, when the centrifuge tube containing the specimen is taken out, the container needs to be opened, and the moisture in the air will enter the anhydrous ethanol in the container, which may affect the purity of the anhydrous ethanol in the centrifuge tube.

[0003] In summary, the existing problems of insect immersion specimen preservation are: 75% alcohol solution or 95% alcohol solution cannot be used for specimen preparation in DNA molecular detection; the insect immersion specimen is stored in the 2 ml and 5 ml centrifuge tubes sold on the market, and due to the poor sealing performance of the centrifuge tube, the preservation liquid (anhydrous ethanol) therein is easily evaporated, and the moisture enters the ethanol during the process of taking out and putting in the specimen, which leads to the decrease of the ethanol concentration, and the specimen cannot be stored for a long time or permanently, and it is difficult to find the specimen. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides a simple long-term preservation method for small insect immersion specimens for DNA molecular detection, which comprises the following steps:

[0005] Step 1: Clean the collected fresh small insect specimen with anhydrous ethanol;

[0006] Step 2: Put the small insect specimen cleaned in step 1 into a centrifuge tube, add anhydrous ethanol to 4 / 5 of the height of the centrifuge tube, and write a label into the centrifuge tube;

[0007] Step three: insert the centrifuge tube of step two into the round hole on the tray, the tray is uniformly distributed with several round holes for inserting the centrifuge tube, the inner diameter of the round hole matches the outer diameter of the inserted centrifuge tube; the tray is placed in a container with a cover, the inner wall of the container is provided with one or more steps for placing the tray, and the shape of the step matches the shape of the periphery of the tray, a groove is formed in the center of the bottom of the container for placing the magnesium sheet packaging wrapped with water permeable material, and anhydrous ethanol is added into the container until the magnesium sheet packaging is submerged;

[0008] Step four: tightly seal the container cover and store it in the dark;

[0009] Step five: check the volatilization of anhydrous ethanol in the centrifuge tube and the container at regular intervals, and if it is found that the anhydrous ethanol is almost exhausted or significantly reduced, add anhydrous ethanol again.

[0010] Further, in step one, the small insect specimens are placed in centrifuge tubes containing anhydrous ethanol and stored in a-20℃ low temperature refrigerator, and are taken out in batches for cleaning with anhydrous ethanol.

[0011] Further, in step two, the cleaned small insect specimens are classified under a dissection microscope, the insect specimens in the same centrifuge tube are the same species of insects collected from the same place, and if the number of insect specimens of the same species collected from the same place is large, they are divided and packaged into multiple centrifuge tubes; insect specimens of different species collected from the same place are packaged into different centrifuge tubes for easy searching, but insect specimens from the same nest are packaged into the same centrifuge tube.

[0012] Further, in step three, the inner diameter of the container gradually decreases from top to bottom; the edge of the tray is also provided with symmetrical semicircular holes for taking and placing the tray when the centrifuge tubes are full, and for flowing into the bottom of the container when anhydrous ethanol is supplemented.

[0013] Further, the centrifuge tube is selected to be 2ml in size, and a label written with the collection site and the content of the insect specimen is placed in the centrifuge tube. The container is pasted with a label.

[0014] The present application utilizes the principle of evaporation to solve the problem of volatilization of the preservation liquid in the centrifuge tube due to poor sealing, and can also reduce the water content of anhydrous ethanol in the outer container, so that the insect immersion specimen can be well preserved for a long or permanent period of time; according to the label identification, it can be quickly searched, which can greatly save time compared with the existing method. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The centrifuge tube of the classified insect specimens in embodiment one of the present application is a physical object;

[0016] Figure 2 The plastic mineral water bottle for preserving the insect specimens in embodiment one of the present application is a physical object;

[0017] Figure 3 Figure 1 is a physical object of the arrangement for preserving insect specimens in Embodiment One of the present application;

[0018] Figure 4 Figure 2 is a schematic diagram of the arrangement for preserving insect specimens designed according to the principle of the present application;

[0019] Figure 5 Figure 3 is a schematic diagram of the container in Figure 4 ;

[0020] Figure 6 Figure 4 is a schematic diagram of the container cover in Figure 4 ;

[0021] Figure 7 Figure 5 is a schematic diagram of the tray in Figure 4 ;

[0022] In the figure, 1 is a centrifuge tube, 3 is a container, 2 is a cover, 4 is a tray, 5 is a groove for placing the packaging containing magnesium sheets, and 6 is a step for placing the tray. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. If no specific technology or condition is specified in the embodiments, the technology or condition described in the literature or according to the product manual is used. If the raw materials, reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased.

[0024] Embodiment One: A simple long-term preservation method for small insect immersion specimens for DNA molecule detection is performed according to the following steps, as shown in Figures 1-3 :

[0025] Step One: The fresh ant specimens collected are immediately placed in a 2 ml centrifuge tube containing anhydrous ethanol (≥99.5%) and taken back to the laboratory, and then stored in a low-temperature refrigerator at -20°C, and then taken out in batches for cleaning with anhydrous ethanol;

[0026] Step Two: The cleaned ant specimens are classified under a dissection microscope, the same ant specimens of the same collection site are placed in the same centrifuge tube 1, anhydrous ethanol is added to 4 / 5 of the height of the centrifuge tube, the same nest ant specimens are placed in the same centrifuge tube separately, and a label is written and put into the numbered record, as shown in Figure 1 ;

[0027] Step three: Collect discarded plastic mineral water bottles, clean them with water and reserve them; Put the centrifuge tubes marked with the same collection site into the plastic mineral water bottles, place the encapsulated magnesium pieces wrapped in water-permeable material in the plastic mineral water bottles, then add anhydrous ethanol to submerge all the centrifuge tubes, write the label and put it in or externally paste it, number it for easy searching, tightly cover the lid, see Figure 2 .

[0028] Step four: Place the tightly covered plastic mineral water bottles in the sorting box to avoid light, and externally paste the label, as shown in Figure 3 .

[0029] Step five: Check the evaporation of anhydrous ethanol in the centrifuge tubes and plastic mineral water bottles every 3-5 years. If it is found that the anhydrous ethanol in them has been completely or significantly reduced, only need to add anhydrous ethanol, which can be stored for a long time or permanently.

[0030] Example two, a simple long-term preservation method for small insect immersion specimens for DNA molecule detection, is carried out according to the following steps, and the preservation device used is shown in Figures 4-7 .

[0031] Step one: Immediately place the collected fresh ant specimens into 2 ml centrifuge tubes containing anhydrous ethanol (≥99.5%) and bring them back to the laboratory, then store them in a -20℃ low-temperature refrigerator, and then take them out in batches for cleaning with anhydrous ethanol;

[0032] Step two: Classify the cleaned ant specimens under a dissection microscope, place the same species of ants collected from the same site into the same centrifuge tube 1, add anhydrous ethanol to 4 / 5 of the height of the centrifuge tube, and place the ants from the same nest into the same centrifuge tube separately, and write the label and put it in, number it for easy searching;

[0033] Step three: Insert the centrifuge tube into the round hole on the tray 4, the inner diameter of the round hole matches the outer diameter of the inserted centrifuge tube, place the tray 4 into the container 3 with the container lid 2, the inner wall of the container is provided with one or more steps 6 for placing the tray 4, and the shape of the step matches the shape of the periphery of the tray, and the inner diameter of the container gradually decreases from top to bottom. A groove 5 is provided in the center of the bottom of the container for placing the encapsulated magnesium pieces wrapped in water-permeable material, add anhydrous ethanol until the encapsulated magnesium pieces are submerged, write the label and externally paste it on the container;

[0034] The tray is uniformly distributed with a plurality of round holes for inserting the centrifuge tubes, and the edges of the tray have symmetrical semicircular holes for taking and placing the tray from the step 6 when the centrifuge tubes are full, and for supplementing anhydrous alcohol when the alcohol flows into the bottom of the container.

[0035] Step four: After tightly sealing the container lid 2 in step three, store it in the dark.

[0036] Step five: check the evaporation of ethanol in the centrifuge tube and container every 3-5 years, if the ethanol is found to be almost exhausted or significantly reduced, only need to add ethanol for long-term or permanent preservation.

[0037] The embodiment one or the embodiment two can solve the problem of evaporation of the preservation solution caused by poor sealing of the centrifuge tube, so that the insect immersion specimen is well preserved for a long time or permanently, and can be quickly found according to the label record, compared with the existing method, the time can be greatly saved.

[0038] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A simple long-term preservation method for small insect immersion specimens for DNA molecule detection, characterized by, The method comprises the following steps: Step 1: clean the collected fresh small insect specimens with anhydrous ethanol; Step 2: place the cleaned small insect specimens in a centrifuge tube, add anhydrous ethanol to 4 / 5 of the height of the centrifuge tube, and write a label into the centrifuge tube; Step 3: insert the centrifuge tube of step 2 into a round hole on a tray, the tray is uniformly distributed with a plurality of round holes for inserting the centrifuge tube, the inner diameter of the round hole matches the outer diameter of the inserted centrifuge tube; the tray is placed in a container with a cover, the inner wall of the container is provided with one or more steps for placing the tray, and the shape of the step matches the shape of the periphery of the tray; a groove is formed in the center of the bottom of the container, a magnesium sheet package wrapped with water-permeable material is placed in the groove, and anhydrous ethanol is added into the container until the magnesium sheet package is submerged; Step 4: tightly seal the container and store it in the dark; Step 5: check the volatilization of anhydrous ethanol in the centrifuge tube and the container at regular intervals, and add anhydrous ethanol if the anhydrous ethanol is found to be almost exhausted or significantly reduced.

2. The simple long-term preservation method according to claim 1, characterized in that, In step 1, the small insect specimens are placed in a centrifuge tube containing anhydrous ethanol and stored in a -20℃ low-temperature refrigerator, and are taken out in batches for cleaning with anhydrous ethanol.

3. The simple long-term preservation method according to claim 1, characterized in that, In step 2, the cleaned small insect specimens are classified under a dissection microscope, the insect specimens in the same centrifuge tube are the same species of insects collected from the same place, and if the number of the same species of insect specimens collected from the same place is large, they are divided and placed in multiple centrifuge tubes; different species of insect specimens collected from the same place are placed in different centrifuge tubes for easy searching, but insect specimens from the same nest are placed in the same centrifuge tube.

4. The simple long-term preservation method according to claim 1, characterized by, In step 3, the inner diameter of the container gradually decreases from top to bottom.

5. The simple long-term preservation method according to claim 1, characterized in that, In step 3, the edge of the tray is also provided with symmetrical semicircular holes for taking and placing the tray when the centrifuge tubes are full, and for flowing into the bottom of the container when anhydrous ethanol is supplemented.

6. The simple long-term preservation method according to claim 1, characterized in that, The centrifuge tube is selected to be 2 ml, and a label written with the collection site and the content of the insect specimens is placed in the centrifuge tube.

7. The simple long-term preservation method according to claim 1 or 4, characterized in that, A label is attached to the outside of the container.

Images