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Chemical derivation-based detection method for hemolymph metabolin of migratory locust

A detection method, hemolymph technology, applied in the field of animal ecology and analytical chemistry, can solve the problems of hemolymph metabolic fingerprint detection of migratory locusts, high cost and operation cost, narrow coverage of biological information, etc. The method is convenient, efficient, The effect of low instrument requirements and complete and comprehensive biological information

Inactive Publication Date: 2014-07-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, there are no other reports on the metabolic fingerprint analysis of small molecule metabolites in migratory locust hemolymph
The technology invented by Lenz et al. can detect multiple types of metabolites including amino acids, carbohydrates and organic acids, but this invention also has obvious defects: 1) the technical core of this invention is a 500MHz nuclear magnetic resonance instrument, and the cost and high operating costs, which are far beyond what ordinary laboratories can possess, and cannot meet the research needs of most biological laboratories; 2) Due to the complex composition and diverse chemical structures of metabolites in locust hemolymph, the metabolites collected in this invention There is a serious peak overlap phenomenon in the fingerprint, which not only poses a severe challenge to the analysis of the metabolite structure, but also interferes with the integration result of the peak area, thereby affecting the accuracy of the determination of the relative content of the metabolite; 3) more critical Unfortunately, the metabolic information obtained by this technology is limited, and only more than 20 metabolites have been analyzed from the hemolymph of desert locusts. The coverage of biological information is narrow, which is far from meeting the needs of understanding the physiological and metabolic functions of migratory locusts from the perspective of systems biology.
Through further literature search, so far, there has been no relevant report on the detection of hemolymph metabolic fingerprints of migratory locusts based on chemical derivatization and gas chromatography-mass spectrometry

Method used

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  • Chemical derivation-based detection method for hemolymph metabolin of migratory locust
  • Chemical derivation-based detection method for hemolymph metabolin of migratory locust
  • Chemical derivation-based detection method for hemolymph metabolin of migratory locust

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Experimental program
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Embodiment 1

[0020] Take 4-year-old gregarious migratory locusts, use a micro-syringe to puncture the epidermis at the junction of the pronotum and abdomen to aspirate body fluid, centrifuge at 13,000 g for 10 min at 4°C to obtain hemolymph, and store at -80°C before analysis. After thawing at room temperature, put 50 μL of hemolymph into a 1.5 mL Eppendorf tube, add 150 μL of ethanol-acetonitrile mixed solvent (9 / 1, v / v) to precipitate protein, vortex vigorously for 1 min, ultrasonically extract for 2 min, and centrifuge at 15,000 g for 15 min at 4°C , Pipet 120 μL of the supernatant extract into another 1.5 mL Eppendorf tube, and dry under reduced pressure in a lyophilizer for 5 h. Add 50 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the extract, dissolve it by ultrasonication for 1 min, vortex mix, and oximate in a constant temperature water bath at 40°C for 90 min; then, add 50 μL of N-methyl-N-trimethyl Silane-trifluoroacetamide reagent, vortex mixed, derivatized by...

Embodiment 2

[0034] Take the gregarious East Asian migratory locust with knockout carnitine-acetyltransferase, use a micro-syringe to puncture the epidermis at the junction of the pronotum and abdomen to aspirate the body fluid, centrifuge at 13000g for 10 minutes at 4°C to obtain hemolymph, and use dry ice to cool down during transportation . After thawing at room temperature, take 50 μL of hemolymph and place it in a 1.5 mL Eppendorf tube, add 150 μL of ethanol-acetonitrile mixed solvent (9 / 1, v / v) to precipitate protein, vortex vigorously for 1 min, and ultrasonically extract metabolites for 2 min, 15000 g at 4 °C Centrifuge for 15 min, pipette 120 μL supernatant extract into another 1.5 mL Eppendorf tube, and dry under reduced pressure in a lyophilizer for 5 h. Add 50 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the extract residue, dissolve it by ultrasonication for 1 min, vortex and mix well, and oximate in a constant temperature water bath at 40°C for 90 min; the...

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Abstract

The invention discloses a chemical derivation-based detection method for hemolymph metabolin of migratory locust, and relates to the field of animal ecology and analytical chemistry. The detection method specifically comprises the following steps of: taking hemolymph, deproteinizing by adding ethanol-acetonitrile mixed solvent, supersonically extracting, and centrifuging at a high speed to obtain metabolin extracting solution; drying the extracting solution by means of pressure reduction, adding methoxamine hydrochloride-pyridine solution, supersonically dissolving, blending by means of vortex, and deriving in an oximation way in a constant-temperature water bath; adding N-methyl-N-trimethylsilane-trifluoroacetamide reagent, blending by means of vortex, and deriving in an alkylation way in the constant-temperature water bath; and adding methyl oleate-normal heptane solution to compensate the volume, blending by means of vortex, and analyzing by a spare gas chromatography-mass spectra coupling technique. The detection method provided by the invention is convenient, high-efficiency, mild in reaction conditions, good in repeatability, wide in metabolin coverage, suitable for developing the multi-center and large-sample research, and capable of providing help for knowing the biological law of various migratory locust variations and the occurrence mechanism of the locust plague.

Description

technical field [0001] The invention relates to the fields of animal ecology and analytical chemistry, and is a method for detecting hemolymph metabolites of migratory locusts based on two-step chemical derivation. Background technique [0002] Locusta migratoria is a major agricultural pest widely distributed in the eastern hemisphere. It is known that there are 9 subspecies in the world. Among them, there are East Asian migratoria, Asian migratoria and Tibetan migratoria in China. The three subspecies are the main culprits of locust outbreaks in my country. Migratory locusts have polymorphism, and have different ecotypes in nature, namely: gregarious and scattered. The two types can be transformed into each other, which is called the metamorphosis of migratory locusts. The main features of locust plague outbreaks are the transformation of scattered migratory locusts into gregarious locusts, and the large-scale relocation of gregarious migratory locust clusters. Studies h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88G01N30/06
Inventor 许国旺吴泽明赵春霞周佳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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