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Method for rapidly detecting symbiotic bacteria in intestinal tracts/fat bodies of brown planthopper

A technology of symbiotic bacteria and brown planthoppers, applied in the field of microorganisms, can solve the problems of unreported quantity measurement

Pending Publication Date: 2021-04-30
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diversity analysis of symbiotic bacteria mainly adopts high-throughput sequencing analysis methods. Symbiotic fungi mainly use 18S rDNA and ITS sequences, and symbiotic bacteria use 16S rDNA as amplicons for diversity analysis, but the detection results can only reflect certain types or The relative abundance change of a certain symbiotic bacteria in the host body cannot compare the change of the absolute amount of the bacteria or the bacteria before and after the treatment
For symbiotic bacteria with small individuals, the relevant quantitative determination has not been reported

Method used

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  • Method for rapidly detecting symbiotic bacteria in intestinal tracts/fat bodies of brown planthopper
  • Method for rapidly detecting symbiotic bacteria in intestinal tracts/fat bodies of brown planthopper
  • Method for rapidly detecting symbiotic bacteria in intestinal tracts/fat bodies of brown planthopper

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: (intestinal anatomy of brown planthopper)

[0033] Because the 3rd and 4th instar N. lugens nymphs are small, it is difficult to obtain the intestines, so only the 5th instar and 2d, 6d after eclosion female and male intestines were collected; 100 surviving N. lugens of the same age were placed in the On the glass slide, use medical dissecting forceps to remove the head under the stereoscope, and slowly separate the chest and abdomen of the brown planthopper to ensure the integrity of the intestine. After taking out the intestine, wash it in sterile PBS buffer and put it into a container that has added 100 μl of PBS buffer. 1.5ml centrifuge tube.

Embodiment 2

[0034] Embodiment 2: (BPH intestinal DNA extraction)

[0035] 1) Take the centrifuge tube of the dissected N. lugens intestinal sample and centrifuge it at 12000rpm at 4°C for 10min, and pour out the supernatant;

[0036] 2) Add 200 μl of the prepared sorbitol solution to the sample, add about 20 small steel balls, put it in the grinder, and grind twice at 65hz 150s;

[0037] 3) Add 5 μl of lyticase to the sample obtained in step 2) in a metal bath at 37°C for 30 minutes, then add 20 μl of Proteinase K, mix well, and digest in a metal bath at 55°C for 3 hours;

[0038] 4) Add an equal volume of DNA extraction solution (phenol: chloroform: isoamyl alcohol 25:24:1) to the sample obtained in step 3) in the fume hood, slowly invert and mix for 15 minutes.

[0039] 5) Centrifuge at 12000rpm at 4°C for 10min;

[0040] 6) Transfer the supernatant obtained in step 5) to a new 1.5ml centrifuge tube, add an equal volume of isopropanol, slowly invert and mix well, let stand at room tem...

Embodiment 3

[0044]Embodiment 3: (anatomy of fat body of brown planthopper)

[0045] Take 100 3rd, 4th and 5th instar nymphs artificially raised in the greenhouse for more than 10 generations, and 100 females and 100 males 2 days and 6 days after eclosion, place them on a glass slide, and use medical dissecting tweezers to remove their heads under a stereoscope. First, slowly separate the thorax and abdomen of the brown planthopper to ensure the integrity of the intestinal tract. After taking out the intestinal tract, wash the remaining part in sterile PBS buffer and put it into a 1.5ml centrifuge tube that has added 100 μl of PBS buffer to remove the remaining abdominal organs of the brown planthopper. Add 20 μl of PBS buffer solution dropwise on the glass slide, squeeze the abdomen of N. lugens repeatedly to obtain a mixture of fat body and PBS, and suck it into a sterile 1.5ml centrifuge tube.

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Abstract

The invention discloses a method for rapidly detecting symbiotic bacteria in intestinal tracts / fat bodies of brown planthoppers, and aims to overcome the defects of the conventional method for quantitatively detecting the symbiotic bacteria of the brown planthoppers. According to the invention, plasmids containing an Escherichia coli 16S rDNA V3V4 region and a brown planthopper actin betaactin sequence are used for respectively constructing standard curves, a universal fluorescent quantitative PCR reaction detection system is established, and the total number of symbiotic bacteria in the intestinal tract or fat body of the brown planthopper in a unit insect body can be detected.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a method for rapidly detecting symbiotic bacteria in the gut / fat body of brown planthopper. Background technique [0002] Brown planthopper (Nilaparvata lugens ) is a migratory pest that only feeds on rice or wild rice, and is an important pest in the rice region of Asia. There are a large number of symbiotic bacteria in the intestines of brown planthoppers. These symbiotic bacteria and brown planthoppers are interdependent and interact. Brown planthoppers provide a suitable living environment for symbiotic bacteria. Symbiotic bacteria play an important role in the growth, development, nutrient metabolism, resistance variation and immune function of brown planthoppers. The lack of symbiotic bacteria will directly lead to the growth and development delay of N. lugens, loss of reproductive ability, and premature death. [0003] Based on the close relationship between N. l...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06
CPCC12Q1/689C12Q1/6851C12Q2600/166
Inventor 申屠旭萍宋阳史嘉腾俞晓平许益鹏刘光富
Owner CHINA JILIANG UNIV
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