Digestion extraction method of diatom in dead body viscera in water

Abstract

The invention discloses a digestion extraction method for diatom in dead body visceral organs in water. The method comprises the following steps: (1) taking organ visceral organ tissues and putting the organ visceral organ tissues in a disposable plastic digestion test tube; (2) adding 1-10mL of strong acid and 2-5mL of analytically pure hydrogen peroxide solution; (3) adopting a special test tube cover as a cover of the disposable plastic digestion test tube, and screwing the cover when in use; (4) graphite digestion; (5) alternately carrying out underwater microwave digestion and ultraviolet digestion; and (6) ending digestion, taking out the test tube from the water tank, placing the test tube in a fuming cupboard, unscrewing a test tube cover, and adding pure water to dilute the sample solution. According to the method, a normal-pressure microwave digestion mode is adopted for the first time, low-cost disposable digestion consumables are used, and the problem of false positive caused by diatom residues is solved; by using the microwave oven, the equipment cost is reduced; the graphite digestion-underwater microwave ultraviolet digestion combined technology is adopted for the first time, and the effects of complete digestion of organic matters, high diatom extraction rate, pollution prevention, safety and environmental protection can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of forensic diatom examination, and particularly to a method for digesting and extracting diatoms in the viscera of a body in water. Background Art

[0002] Diatoms are a type of single-celled organisms that widely exist in various natural water bodies, and also exist in small amounts in soil and air. Currently, about 11,200 species of diatoms on the earth have been recorded, with sizes ranging from 2 to 500 μm. Their growth is affected by factors such as temperature, sunlight, water flow rate, acidity, mineral content in water, and water stratification. Their distribution shows spatio-temporal differences and has prominent regional distribution characteristics, and is known as the "water fingerprint". Diatoms have important applications in the fields of forensic medicine, water environment monitoring, aquaculture, new material development, paleontology, geology, etc.

[0003] In forensic practice, diatom examination is used for the diagnosis of drowning deaths of bodies in water and the inference of drowning locations. The "Technical Specification for Forensic Diatom Examination - Microwave Digestion - Vacuum Filtration - Microscopy Method" (GA / T 1662-2019) is widely used as an industry standard. This method has the advantages of sensitivity and high efficiency. However, because conventional microwave digestion is high-temperature and high-pressure digestion, the microwave digestion instrument and its components are made of special materials, with high costs. Therefore, the digestion tubes need to be reused. To avoid contamination, after use, they need to be successively treated with a series of processes such as alkaline solution soaking, water washing, acid washing, and water washing, and can only be reused after being detected by an electron microscope and found to contain no diatoms. This process is cumbersome and time-consuming. On the other hand, in practice, problems of false positives often occur due to incomplete cleaning of the digestion tubes and residual diatoms. In recent years, some manufacturers have developed a pre-pressurized microwave digestion instrument (also known as a super microwave digestion instrument) using low-cost borosilicate disposable digestion tubes, but because components such as tube caps still need to be reused, the risk of diatom residue still exists. Moreover, the pre-pressurized microwave digestion instrument is expensive (several times the cost of a conventional microwave digestion instrument) and is difficult to be widely applied. Summary of the Invention

[0004] In view of the above deficiencies, the present invention provides a method for digesting and extracting diatoms in viscera based on underwater normal-pressure microwave digestion, using a microwave oven and disposable digestion consumables (including plastic digestion test tubes and tube caps), aiming to solve the problems of high cost of microwave digestion equipment and easy occurrence of false positives due to repeated use of digestion tubes in the existing technology.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for digesting and extracting diatoms in the viscera of a body in water, comprising the following steps:

[0007] (1) Place the organ tissue in a disposable plastic digestion test tube;

[0008] (2) Add 1 - 10 mL of strong acid and 2 - 5 mL of analytical pure hydrogen peroxide solution;

[0009] (3) Use a special test tube cap as the lid of the disposable plastic digestion test tube, and tighten the lid when in use;

[0010] (4) Graphite digestion: Place the test tube from step (3) into the hole slot of a graphite digester, set the digestion temperature at 60 - 110 °C, and the digestion time at 30 - 60 min;

[0011] (5) Underwater microwave - ultraviolet digestion: Place the test tube after graphite digestion into a water tank filled with pure water, then place the test tube on a test tube rack. The test tube cap should be submerged in water and at least 2 cm away from the water surface; then place the water tank into a microwave oven with an ultraviolet lamp inside, and alternately start microwave digestion and ultraviolet digestion:

[0012] a) Microwave digestion: Microwave power is 200 - 600 W, time is 5 - 30 min;

[0013] b) Ultraviolet digestion: Time is 5 - 30 min;

[0014] c) Microwave digestion: Microwave power is 1000 - 2000 W, time is 5 - 30 min;

[0015] d) Ultraviolet digestion: Time is 5 - 30 min;

[0016] If it is found that the grease is not completely digested, then start microwave digestion and ultraviolet digestion again until the grease is completely digested and the digestion solution becomes clear and transparent;

[0017] (6) End the digestion, take out the test tube from the water tank and place it in a fume hood, unscrew the test tube cap, and add pure water to dilute the sample solution.

[0018] The special test tube cap has the following characteristics:

[0019] (1) The central area of the tube cap is a hole with a diameter of 5 - 15 mm;

[0020] (2) The hole is covered with a membrane that is waterproof, breathable, resistant to high pressure and high temperature, and acid - corrosion resistant, and the pore diameter of the membrane is 0.1 - 3 μm;

[0021] (3) Below the membrane and at the inner top surface of the test tube cap, there is a support net for preventing the membrane from collapsing, and the support net, the membrane, and the inner top surface of the test tube cap are firmly adhered. After tightening the cap and submerging the test tube in water, the sealing performance is sufficient to ensure that water cannot enter the test tube.

[0022] After that, vacuum filter, coat the film, and perform scanning electron microscope detection on the sample according to the diatom inspection standard (GA / T 1662 - 2019).

[0023] Preferably, the film that is waterproof, breathable, resistant to high pressure and high temperature, and acid corrosion is a polytetrafluoroethylene film.

[0024] Preferably, the support mesh is made of polytetrafluoroethylene.

[0025] Compared with the prior art, the present invention has the following innovations and beneficial effects:

[0026] 1. For the first time, the atmospheric pressure microwave digestion method is used to digest organ tissues to extract diatoms.

[0027] Different from the closed high-temperature and high-pressure microwave digestion method adopted in the diatom inspection standard (GA / T 1662-2019), the present invention uses the atmospheric pressure microwave digestion method for the first time, and its advantages are as follows:

[0028] (1) Low-cost disposable digestion consumables (including plastic digestion test tubes and test tube caps) can be used, saving the cleaning steps and avoiding the problem of diatom residue.

[0029] (2) Instead of using an expensive microwave digestion instrument, a microwave oven is used, reducing the equipment cost.

[0030] 2. For the first time, the combined technology of graphite digestion - underwater microwave ultraviolet digestion is adopted, which can achieve the effects of complete digestion of organic matter, high diatom extraction rate, anti-pollution, safety and environmental protection. Specifically:

[0031] (1) The combination of graphite digestion and microwave ultraviolet digestion completely digests the organic matter in the organ tissue sample, and the diatom recovery rate is high: first, the graphite digestion method is used to remove the easily digested organic matter in the organ tissue, and then the dual action of high-frequency microwave radiation and ultraviolet radiation is used to catalyze the decomposition of hydrogen peroxide to generate strongly oxidizing hydroxyl radicals, which oxidize and decompose stubborn organic matter (such as grease), thereby improving the diatom recovery rate.

[0032] (2) The advantages of the underwater digestion method include:

[0033] a) The digestion process of the organ tissue sample can proceed stably and continuously. This is because: if the atmospheric pressure digestion is directly carried out in the atmospheric environment, the sample liquid in the disposable digestion tube will quickly dry up, and the plastic tube will also be damaged due to the sudden rise in temperature. After adopting the underwater digestion method, the water in the water tank will timely absorb the heat generated by the digestion reaction, thereby protecting the digestion tube and ensuring the stable progress of the reaction;

[0034] b) Through the isolation of the waterproof and breathable membrane, the diatoms in the tube are prevented from escaping and the diatoms outside the tube from entering; c) The acid gas escaping through the waterproof and breathable membrane during the sample digestion process is absorbed by the water in the water tank, so it will not corrode the microwave oven components and will not pollute the atmosphere.

[0035] Therefore, compared with the existing technical methods, the present invention for the first time adopts the atmospheric pressure microwave digestion method, uses low-cost disposable digestion consumables (including digestion tubes and special tube caps), solves the problem of false positives caused by diatom residues, uses a microwave oven to reduce equipment costs, and for the first time adopts the combined technology of graphite digestion - underwater microwave ultraviolet digestion, which can achieve the effects of complete digestion of organic matter, high diatom extraction rate, anti-pollution, safety and environmental protection. The present invention is economical and practical and has good application prospects in forensic practice. Description of the Drawings

[0036] Figure 1 It is a top view of the special plastic cap structure of the present invention.

[0037] Figure 2 It is a sectional view of the special plastic cap structure of the present invention.

[0038] Figure 3 It is a top view of the test tube rack.

[0039] Figure 4 It is a schematic diagram of the test tube rack placed in the water tank.

[0040] Figure 5 It is a schematic diagram of the microwave oven cavity.

[0041] Figure 6 It is a comparison diagram of the digestion effect of kidney tissue; among them, Figure 6 a is the digestion solution obtained after microwave digestion of the sample according to the method of GA / T1662 - 2019; Figure 6 b is the digestion solution obtained after digesting the sample according to Example 1 of the present invention. Detailed Embodiments

[0042] Example 1: Digestion and extraction of diatoms in the viscera of a corpse in water

[0043] 1. Respectively take 2 g of lung, liver, and kidney tissues and place them in 50 mL disposable plastic test tubes;

[0044] 2. Add 8 mL of analytical pure concentrated nitric acid and 4 mL of analytical pure hydrogen peroxide solution;

[0045] 3. Adopt a special plastic cap and place it on the mouth of the plastic test tube and tighten it. The plastic cap has the following structural features:

[0046] (1) The central area of the tube cap is a hole with a diameter of 10 mm;

[0047] (2) There is a layer of polytetrafluoroethylene film that is resistant to high pressure and high temperature and acid corrosion at the hole in the central area. The film is waterproof and breathable, and the pore diameter is 1 μm;

[0048] (3) There is a support net for preventing the film from collapsing at the inner top surface of the test tube cap below the film. The support net is made of polytetrafluoroethylene that is resistant to high pressure and high temperature and acid corrosion. The support net, the film, and the inner top surface of the test tube cap are firmly bonded together. The schematic diagram of the special test tube cap is as shown in Figure 1 and Figure 2 .

[0049] (4) After tightening the cap, immerse the test tube in water, and water cannot enter the test tube.

[0050] 4. Graphite digestion: Place the test tube containing the organ tissue and concentrated acid into the hole groove of the graphite digestion instrument, set the digestion temperature to 80 °C, and the digestion time to 30 min. After digestion, only a small amount of grease in the test tube is not completely digested.

[0051] 5. Underwater microwave ultraviolet digestion:

[0052] (1) Place the plastic test tube rack into the water tank filled with pure water, and then place the test tube on the test tube rack. The tube cap should be immersed in water and 3 cm away from the water surface. When the plastic test tube rack is placed in water, the test tube is fixed and will not move. The top view structure of the plastic test tube rack is as shown in Figure 3 , and when placed in the water tank, it is as shown in Figure 4 .

[0053] (2) Place the water tank into the microwave oven equipped with an ultraviolet lamp, as shown in Figure 5 , and alternately start the microwave digestion mode and the ultraviolet digestion mode:

[0054] a) Microwave digestion: Microwave power is 500 W, and the time is 10 min;

[0055] b) Ultraviolet digestion: The time is 10 min;

[0056] c) Microwave digestion: Microwave power is 1200 W, and the time is 20 min;

[0057] d) Ultraviolet digestion: The time is 20 min.

[0058] At this time, the grease is completely digested, and the digestion solution is clear and transparent.

[0059] 6. Take out the test tube from the water tank and place it in the fume hood. Unscrew the test tube cap and add pure water to dilute the sample solution.

[0060] After that, vacuum filter, coat the film, and perform scanning electron microscope detection on the sample according to the diatom inspection standard (GA / T 1662-2019).

[0061] Comparison of inspection effects:

[0062] For the same organ tissue sample, analyze it separately according to the existing GA / T 1662-2019 method and Example 1 of the present invention. The results are as follows:

[0063] From the appearance, see Figure 6 Taking kidney tissue digestion as an example, Figure 6 It can be seen that there is no significant difference in the digestion effects of the two methods, and the organ tissues are clear and transparent after digestion.

[0064] Compared with the diatom inspection standard (GA / T1662-2019), although the digestion time required for the present invention is longer, the equipment cost and consumables cost are low. More importantly, in the present invention, the digestion tube is disposable and does not need to be cleaned, which can effectively avoid the problem of false positives caused by contamination (see Table 1).

[0065] It can be seen from Table 2 that for the same organ tissue sample, the diatom extraction rates of the two are basically the same.

[0066] Therefore, the invention is economical, practical and has good application prospects in forensic practice.

[0067] Table 1 Comparison of equipment cost, consumables cost and digestion time

[0068]

[0069]

[0070] Table 2 Comparison of diatom extraction rate

[0071]

Claims

1. A method for digesting and extracting diatoms in cadaver organs in water, characterized in that The steps include: (1) Take the organ tissue and place it in a disposable plastic digestion tube; (2) Add 1-10 mL of strong acid and 2-5 mL of analytical grade hydrogen peroxide solution; (3) Use a special test tube cover as the cover of a disposable plastic digestion test tube and tighten the cover when using it; (4) Graphite digestion: Place the test tube in step (3) into the hole slot of a graphite digestion instrument, set the digestion temperature to 60-110° C., and the digestion time to 30-60 min; (5) Underwater microwave UV digestion: Place the test tube that has been digested with graphite into a sink filled with pure water, and then place the test tube on a test tube rack. The test tube cover should be submerged in water and at least 2 cm above the water surface. Then place the sink into a microwave oven with a built-in UV lamp, and start microwave digestion and UV digestion alternately: a) Microwave digestion: microwave power 200-600W, time 5-30min; b) UV digestion: time 5-30min; c) Microwave digestion: microwave power 1000-2000W, time 5-30min; d) UV digestion: time 5-30min; If it is found that the oil is not completely digested, start the microwave digestion and UV digestion again until the oil is completely digested and the digestion solution becomes clear and transparent; (6) When digestion is complete, remove the test tube from the sink and place it in a fume hood. Unscrew the test tube cap and add pure water to dilute the sample solution.

2. The method for digesting and extracting diatoms in the viscera of a corpse in water as claimed in claim 1, characterized in that The special test tube cover has the following characteristics: The special test tube cover has the following characteristics: (1) The center area of ​​the tube cover is a hole with a diameter of 5-15 mm; (2) The holes are covered with a waterproof, breathable, high pressure and high temperature resistant and acid corrosion resistant membrane, and the pore size of the membrane is 0.1-3 μm; (3) A support net is provided below the membrane and on the inner top surface of the test tube cover to prevent the membrane from collapsing, and the support net, the membrane and the inner top surface of the test tube cover are firmly attached to each other.

3. The method for digesting and extracting diatoms in the viscera of a corpse in water as claimed in claim 2, characterized in that The waterproof, breathable, high pressure and high temperature resistant and acid corrosion resistant membrane is a polytetrafluoroethylene membrane.

4. The method for digesting and extracting diatoms in the viscera of a corpse in water as claimed in claim 2, characterized in that The material of the supporting net is polytetrafluoroethylene.

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