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A method and kit for rapidly quantifying nematode survival rate

A technology of survival rate and kit, which is applied in the field of rapid quantitative nematode survival rate, can solve the problems of high cost, time-consuming, and laborious, and achieve the effects of low cost requirements, fast operation, and saving manpower, material resources and time

Active Publication Date: 2015-12-02
李红玉
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can color the dead nematodes, but not the live nematodes. However, after dyeing, the nematodes need to be cracked to release the dye and then the absorbance value of the dye should be measured. The steps are cumbersome and time-consuming.
[0005] As can be seen from the analysis of the prior art related to the above-mentioned nematode death or survival judgment, the prior art is time-consuming, laborious, and costly or requires expensive instruments and equipment. Expensive instruments and equipment can quickly quantify the new method of nematode survival rate, which can be widely used in the fields of new drug screening, pesticide screening, environmental evaluation and water quality evaluation

Method used

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  • A method and kit for rapidly quantifying nematode survival rate

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment one, dyeing result under different temperature

[0036] Staining agent: 5% red tetrazolium (TTC), prepared with phosphate buffer (pH7.0).

[0037] Test nematodes: 1500 Caenorhabditis elegans synchronized adults.

[0038] Dyeing time: 30min.

[0039] C. elegans were cultured according to the method described by Brenner (Brenner. Genetics, 1974, 77:71-94). Wash the nematodes in the mixed stage after 4-5 days of culture with M9 buffer solution (Brenner, Genetics, 1974, 77:71-94) into the eppendorf tube, centrifuge at 4000rpm for 4min to settle the nematodes, add 1ml alkaline lysate (0.4-0.5M NaOH , 2-4% bleaching powder), after fully lysing, centrifuge at 4000rpm for 4min; after discarding the supernatant, wash twice with M9 buffer, resuspend in 1ml M9 buffer, and incubate at 18°C ​​for 8h, the eggs basically hatch into L1 stage Nematodes: Discard the supernatant, spread the nematodes evenly on the surface of the medium, and culture them at 18°C. After 2.5 da...

Embodiment 2

[0043] Embodiment two, different dyeing time dyeing results

[0044] Staining agent: 5% red tetrazolium (TTC), prepared with phosphate buffer (pH7.0).

[0045] Test nematodes: 300 Caenorhabditis elegans synchronized adults.

[0046] Dyeing temperature: 32°C.

[0047] Synchronized adult worms were washed with M9 buffer into eppendorf tubes, and the supernatant was discarded by centrifugation, then washed twice with M9 buffer. Add 500 microliters of staining solution, and the staining time is 5-45 minutes. At different times, nematodes can be colored to different degrees. After staining, the absorbance was measured at 485 nm with a microplate reader, and the results are shown in Table 2.

[0048] Table 2. Absorbance values ​​measured by staining at different staining times

[0049] Dyeing time (min) Absorbance 5 0.159 15 0.207 25 0.178 35 0.164 45 0.166

Embodiment 3

[0050] Embodiment three, the dyeing result of different concentration dyeing agent

[0051] Staining agent: different concentrations of red tetrazolium (TTC), prepared with phosphate buffer (pH7.0).

[0052] Test nematodes: 1000 Caenorhabditis elegans synchronized adults.

[0053] Dyeing temperature: 32°C.

[0054] Dyeing time: 30min.

[0055] Synchronized adult worms were washed with M9 buffer into eppendorf tubes, and the supernatant was discarded by centrifugation, then washed twice with M9 buffer. Add 500 microliters of staining solution, the concentration of the staining solution is 0.1-20%, and the nematodes can be colored to varying degrees. After staining, the absorbance was measured at 485 nm with a microplate reader, and the results are shown in Table 3.

[0056] Table 3. Absorbance values ​​measured by dyeing with different dye concentrations

[0057] TTC concentration (%) Absorbance 0.1 0.063 1 0.093 4 0.165 8 0.121 20 0.16...

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Abstract

The invention relates to a method and a kit for setting the survival rate of nematode quickly. In the method, the nematode is dyed by using a dye which is 2,3,5-triphenyl tetrazolium chloride (TTC); live nematode can be dyed, dead nematode cannot be dyed, and a light absorption value is in proportion to the number of the live nematode, so the survival rate of the nematode can be reflected directly, and the kit is prepared according to the survival rate. The method and the kit can be applied in aspects of medicinal screening, pesticide screening, environmental evaluation, water quality evaluation which take the survival rate of the nematode as indexes. The method and the kit are quick, simple and easy in operation, low in requirement on equipment and low in cost, and have a high application value.

Description

technical field [0001] The invention relates to a method and a kit for quickly quantifying the survival rate of nematodes. The method and the kit can be applied in drug screening, pesticide screening, environmental evaluation, water quality evaluation, etc., and belong to the field of biotechnology. Background technique [0002] Caenorhabditis elegans ( C. elegans ) is a soil-growing nematode that feeds on bacteria and is easily grown in the laboratory. Adults are 1 mm long and transparent, making them easy to observe under a microscope. L3-L4 stage is sensitive to the outside world. It only takes 3 days for a fertilized egg to develop into an adult worm that can lay eggs. In the natural state, most individuals of Caenorhabditis elegans are hermaphrodites, and they can produce about 300 fertilized eggs in their lifetime. If a few nematodes are placed on a petri dish, a large number of offspring can be obtained in a few days (Pang Linhai et al. , 2007). These characteris...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31G01N1/30
Inventor 李红玉周婷支德娟
Owner 李红玉
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