Nematode larva activity detection method and application

A detection method and technology for viability detection, applied in pharmaceutical formulations, preparations for in vivo tests, luminescence/biological dyeing preparations, etc., can solve problems such as difficulty in consistency and standardization of detection results, differences in subjective judgments, and complex judgment processes. , to achieve the effect of being conducive to separation and culture, low cost, and expanded practicability

Active Publication Date: 2020-08-07
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] Most of the in vitro detection methods of parasite drug resistance currently used are aimed at parasite larvae, and the effects of drugs on parasite larval vigor or lethality are used to judge the parasite's resistance to parasites. Sensitivity of anthelmintics, and the existing detection metho

Method used

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  • Nematode larva activity detection method and application
  • Nematode larva activity detection method and application
  • Nematode larva activity detection method and application

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preparation example Construction

[0053] 4. Preparation of the third-stage nematode larvae suspension

[0054] Weigh an appropriate amount of fresh sheep feces, add an appropriate amount of water to mash, filter through double-layer gauze until the filtrate is clear, collect the feces and sterilize them under high pressure to prepare sterilized feces for later use. Pipette 500 μL of the egg suspension prepared above into a petri dish, add an appropriate amount of fecal residue (0.2-0.5 mm thick) and mix with the egg suspension, add 5 mL of 1640 culture solution dropwise, and culture in a 27°C incubator During the 7th day, 1640 culture solution was added appropriately to prevent the sample from drying and affecting the development of the worm body. After 7 days, the nematode larvae were separated by the Bellman method, the larvae suspension was collected, centrifuged at 1500r / min for 5min, the supernatant was removed, and PBS buffer solution was added, which contained 250mg / L amphotericin B and 10mL / L blue chai...

Embodiment 1

[0057] This embodiment provides a method for detecting the vigor of nematode larvae.

[0058] Preparation of nematode larvae with different vigor:

[0059] 700 μL (containing about 350 larvae) of the above nematode larvae suspension and 200 μL of 1640 culture solution were respectively added to 5 wells of a 24-well culture plate.

[0060] Set these 5 wells as 1 blank control well and 3 drug gradient wells, and mark them as 1-1, 1-2, 1-3, 1-4 respectively. Add 100 μL DMSO to the 1-1 well as the blank control, Add 100 μL (1 μg / mL) albendazole stock solution to wells 1-2 (final concentration of albendazole is 0.1 μg / mL), add 100 μL (2 μg / mL) albendazole stock solution (final The concentration is 0.2 μg / mL), and 100 μL (3 μg / mL) albendazole stock solution is added to wells 1-4 (the final concentration of albendazole is 0.3 μg / mL). Place in a 27°C incubator to continue culturing for 12 hours.

[0061] Vitality detection:

[0062] Drop 10 μL of the above MB stock solution into e...

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Abstract

The invention relates to the field of larva activity detection, and particularly discloses a nematode larva activity detection method and application. The nematode larva activity detection method comprises the following steps: (1) dyeing nematode larvae by using methylene blue as a coloring agent; and (2) 5-7 minutes later, determining the activity of the nematode larvae according to the colors ofthe nematode larvae: when the nematode larvae are blue or light blue, it is determined that the nematode larvae are dead or low-activity larvae, and when the nematode larvae are in an undyed state, it is determined that the nematode larvae are high-activity larvae. According to the method, MB is used as a coloring agent to visually identify the activity condition of the digestive tract nematode larvae according to the dyeing depth effect, convenience and rapidness are achieved, the cost is low, subjective judgment difference can be effectively avoided, help is provided for researching the pesticide effect, drug resistance and other aspects of anthelmintics, and the method has great significance in selecting sensitive drugs to guide clinical medication and the like.

Description

technical field [0001] The invention relates to the technical field of larval vitality detection, in particular to a nematode larval vitality detection method and application. Background technique [0002] Nematodes are a common type of parasite, which mostly parasitize in the digestive tract of animals and have adverse effects on the health of the host. Therefore, a lot of research has been carried out in the prior art on the way of eliminating nematodes, but the current common anthelmintics generally have the problem of drug resistance, which plagues fields such as livestock breeding. [0003] Sheep gastrointestinal nematode disease is one of the important diseases that endanger the healthy development of sheep farming. The infection is very common and can easily cause large economic losses. It is the focus of prevention and control during breeding. In production practice, due to the long-term unscientific application of anthelmintics, many digestive tract nematodes have ...

Claims

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Application Information

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IPC IPC(8): A61K49/00
CPCA61K49/006Y02A50/30
Inventor 王文龙陆静吕旭翟帅刘春霞刘晓磊苏倩王腾宇林洋
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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