Probe and kit for in situ hybridization detection of spiroplasma pathogen in shrimp and crab

Abstract

The invention relates to an in-situ hybridization detection probe of shrimp and crab spiroplasma pathogens, and a kit thereof. The sequences of the probe are represented by SEQIDNO.1 and SEQIDNO.2 respectively. The above two oligonucleotide probes are labeled with digoxin, the labeled probes are hybridized with spiroplasma 16SrDNA in shrimp and crab tissue slices, signal enlargement is carried out through an antigen-antibody reaction, and then enzyme reaction color development is carried out; the pathogen detection and pathological change can be visually combined; the infection rate and the infection intensity are analyzed on sample slices while spiroplasma is efficiently and accurately detected; the hybridization of the probes and the 16SrDNA is specific, and the signal is amplified step by step, so the probes are more sensitive and accurate than routine dyeing observation; and the slices obtained after the detection result hybridization color development can be preserved for a long term.

Description

technical field [0001] The invention relates to an in situ hybridization detection probe and a detection kit for the pathogen of shrimp and crab spiroplasma. 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. Among the many diseases of river crabs, "shivering disease" is the most common and the most harmful. Since the disease was first discovered in Jiangsu in 1994, it has successively broken out in Shanghai, Zhejiang, Anhui, Jiangxi and other places, and it has been increasing year by year. Later, It has spread to almost all river crab breeding areas in the country. In 2008, the Ministry of Agriculture has listed the disease as the newly revised "List of Types 1, 2, and 3 Animal Diseases". Procambarus clarkii (Procambarus ...

AI Technical Summary

Problems solved by technology

At present, ultra-thin section electron microscope observation is the most widely used detection method, but this method requires high operating technology and equipment, and the experimental period is long, so it is difficult to popularize; PCR is also another commonly used detection method[3]. Although this method is relatively simple and highly sensitive, it cannot visually observe and prove the inclusion body of spiroplasma, and it is prone to false positives due to template contamination during operation.

The false positive rate of in situ hybridization is low, and it is a detection method that cannot be replaced by any other method in terms of intuition, accuracy and sensitivity. However, there is no in situ hybridization detection method for aquatic spiroplasma pathogens.

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