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Eriocheir sinensis Microsporidia in-situ hybridization detection probe and kit

A technology of Chinese mitten crab and detection kit, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc., can solve the problem of low false positive rate of in situ hybridization, and achieve sensitive dyeing detection Effect

Inactive Publication Date: 2015-12-23
FRESHWATER FISHERIES RES INSITUTE OF JIANGSUPROVINCE
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Problems solved by technology

The false positive rate of in situ hybridization is low, and it is a detection method that cannot be replaced by any other method considering the comprehensive consideration of intuition, accuracy and sensitivity. to report

Method used

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  • Eriocheir sinensis Microsporidia in-situ hybridization detection probe and kit
  • Eriocheir sinensis Microsporidia in-situ hybridization detection probe and kit
  • Eriocheir sinensis Microsporidia in-situ hybridization detection probe and kit

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Embodiment 1

[0021] Chinese mitten crab sample:

[0022] The diseased Chinese mitten crab was taken from a farm in Jiangsu Province where severe mortality occurred in August 2014, with an average weight of 91.2g (sample size n=30). Healthy Chinese mitten crabs were taken from Nanjing Chinese mitten crab farm in September 2014, with an average weight of 94.3g (sample size n=24). Healthy Chinese mitten crabs were determined to have no microsporidia infection through conventional light microscope, electron microscope observation and PCR detection, and were used as a negative control; as a positive control.

[0023] Sample fixation and slice preparation:

[0024] (1) Fix: Take the hepatopancreas, a susceptible tissue of the Chinese mitten crab, and place it in 4% paraformaldehyde containing 0.1% DEPC (add Na to 1000ml distilled water). 2 HPO 4 .12H 2 O36g, paraformaldehyde dry powder 40g, add NaH after heating and stirring to dissolve 2 PO 4 .2H 2 (03 g, pH 7.0; DEPC was added while ho...

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Abstract

The invention discloses an Eriocheir sinensis Microsporidia in-situ hybridization detection probe and kit. The sequence of the probe is disclosed as SEQ ID NO.1. After the probe is labeled by digoxin and combined with Microsporidia 18S subunit ribosome DNA (18S SSU rDNA) by hybridization, the Microsporidia infection can be judged under a microscope after alkaline phosphatase detection system color development. The invention also discloses a kit containing the probe, which has favorable specific, is simple to operate and can intuitively combine the pathogen detection and pathological changes. On the premise of efficiently and accurately detecting the Spiroplasma, the kit can analyze the infection rate and infection intensity of the sample section. The hybridization between the probe and 18S SSU rDNA is specific, the signal is progressively amplified, and thus, the kit disclosed by the invention is more sensitive and accurate than the conventional dyeing observation detection. The detection result proves that the section subjected to hybridization color development can be stored for a long time.

Description

technical field [0001] The invention relates to an in situ hybridization detection probe and a detection kit for Microsporidia sinensis. Background technique [0002] Chinese mitten crab (Eriocheirsinensis), commonly known as river crab, is a unique aquaculture treasure in my country. However, with the intensive and large-scale development of aquaculture, various diseases have become an important problem that endangers the crab industry. In recent years, microsporidiosis has been widely prevalent in the main breeding areas of river crabs in Xinghua City, Yandu District of Yancheng City, and Sihong County in Jiangsu Province. The epidemic has caused huge losses to the local river crab breeding industry. The main symptom is long-term empty appendages of river crabs. Although they can continue to shed their shells, they grow slowly. The color of the hepatopancreas gradually becomes lighter from golden yellow to grayish white when dissected. Some of them also have the symptoms o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6804C12Q2543/10C12Q2563/131Y02A50/30
Inventor 丁正峰薛晖刘洪岩孙梦玲赵彦华
Owner FRESHWATER FISHERIES RES INSITUTE OF JIANGSUPROVINCE
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