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A specific primer, method and kit for detecting and identifying wolbachia infection in insects

A specific, insect-based technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of non-specificity and false negatives, and achieve the goal of improving the detection rate and broad application prospects Effect

Active Publication Date: 2022-02-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of false negatives and non-specificity in existing Wolbachia detection primers and methods, utilize the existing Wolbachia16S gene sequence on the NCBI network to redesign and pass through a large number of screenings, and obtain more sensitive and more efficient Precise 16S-specific primers to enable identification of Wolbachia

Method used

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  • A specific primer, method and kit for detecting and identifying wolbachia infection in insects
  • A specific primer, method and kit for detecting and identifying wolbachia infection in insects
  • A specific primer, method and kit for detecting and identifying wolbachia infection in insects

Examples

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Embodiment 1

[0038] Example 1 Wolbachia Detection Primer Design and Screening

[0039] 1. Extraction of total DNA from Trichogramma population

[0040] According to the preparation method of insect trace DNA template, it is obtained after being improved. The operation steps are as follows:

[0041] (1) 10 bees were randomly selected from each Trichogramma population, first soaked in absolute ethanol for 24 hours, then washed 5 times with sterile water, and finally washed once with 75% ethanol, blotted dry with filter paper, and placed Put in 200μL sterilized PCR tubes and number them well.

[0042] (2) Mix proteinase K working solution (20mg / mL) with STE buffer solution (100mmol / L NaCl, 10mmol / L Tris-HCl, 1mmol / L EDTA, pH 8.0) in a ratio of 1:9 to obtain the STE buffer containing PK (hereinafter referred to as STE) for extracting DNA.

[0043] (3) Add 1 μL of STE-containing solution to the PCR tube.

[0044] (4) Grind the worm bodies with a melted 200 μL sterilized pipette tip until a...

Embodiment 2

[0071] Example 2 A kit for detecting Wolbachia infection in insects

[0072] 1. A kit for detecting Wolbachia infection in insects, comprising a specific amplification primer Tw16s1-F / R, whose sequence is as follows:

[0073] Tw16s1-F: 5'-ATACCCTGGTAGTCCACGCT-3' (SEQ ID NO: 1);

[0074] Tw16s1-R: 5'-GCAGTGTGTACAAGACCCGA-3' (SEQ ID NO: 2);

[0075] The kit also contains reagents required for PCR amplification reaction: 2x TSINGKE Master Mix, 5x GCbuffer, sterilized water.

[0076] The kit also includes insect total DNA extraction reagents: 1.5mL / 2.0mL centrifuge tube, PCR tube, absolute ethanol, 75% ethanol, sterile water, proteinase K, STE buffer.

[0077] 2. How to use the kit

[0078] The method for using the kit to detect Wolbachia infection in insects comprises the following steps:

[0079] S1. extracting the total DNA of the insect to be tested;

[0080]S2. Using the DNA of S1 as a template, performing a PCR amplification reaction using the above primers;

[0081] S...

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Abstract

The present invention disclosed a test and identified insect body Wolbachia The specific primers, methods and kits of infection.TW16S1, which is special amplifier, includes positive primer F and reverse primer R, and its sequences are shown in sequel: 1-2.The detection method is: S1. Extract the total DNA of insects to be tested; S2. With S1 DNA as a template, using the primer to perform the PCR amplification reaction; S3.EssenceThe primers described by the present invention are strong, the amplification products are a single destination fragment, and the sensitivity is high. It can screen a false negative and non -specific sample, thereby increasing the detection rate.It only takes about 4 hours to obtain the results from mentioning DNA.You can accurately and quickly detect whether to detect whether the sample is subject to Wolbachia Infection provides a basis for further use of disease prevention and reproductive regulation of insects.

Description

technical field [0001] The invention relates to the technical field of molecular detection of symbiotic bacteria in insects, and more specifically, to a primer set, method and kit for detecting and identifying Wolbachia infection in insects. Background technique [0002] Wolbachia is a kind of commensal bacteria widely distributed in reproductive tissues of arthropods, and about 75% of species infect Wolbachia. Wolbachia has a symbiotic relationship with the host. For example, the filarial worm that causes lymphatic filariasis has a mutualistic symbiotic relationship with Wolbachia in its body. Wolbachia controls the growth, development and survival of the filarial worm in mosquitoes. The detection of Wolbachia is the first condition to prevent the transmission of lymphatic filariasis by vector insects. [0003] In addition, Wolbachia also has a reproductive regulation effect on the host. The role of Wolbachia on the host is mainly reflected in its regulation of reproducti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689
Inventor 何晓芳林良冠张美娇
Owner SOUTH CHINA AGRI UNIV
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