Chlamys Farreri pathogeny rickettsia in situ hybridization detection method and its reagent kit

Abstract

This invention relates to the original position cross checking method of a Hence Kong waters disease type Pamela Ci's body and its reagent box. This invention includes the following steps: composing the three oligoribonucleotides probes of the High Sim markings according to the special DNA list design of the Pamela Ci's body's 16S rDNA list in the bacteria taxonogy, this unusual probe crosses and combines with the Pamela Ci's body 16s rRNA of the Hence Kong waters slice up, and then enlarges signal through the antigen antibody reaction, and displays colour through enzyme reaction. This checking method can combine the pathogeny checking with its pathology together; it can analyze the infection rate and infection intension on the stylebook slice when effiently and correctly checking the Pamela Ci's body; and the checking result is sensitive and precisious; and the slice after being checked can be preserved perennially.

Description

technical field [0001] The invention relates to an in situ hybridization detection method for the pathogenic rickettsiae of Chlamys farreri and a kit used for the method. Background technique [0002] Scallop (scallop) farming is an important pillar industry that dominates shellfish farming or even the entire mariculture in my country. Chlamys farrei is an important marine cultured scallop species in China. However, since 1995, large-scale scallops have continuously appeared Death, especially during 1997-1999, the mortality rate of scallops reached more than 90%, and in some sea areas 100% died. In 1997, the death area of ​​Chlamys farreri in northern culture reached 330,000 mu, causing economic losses of 1.5 billion yuan. It has been found that rickettsia-like prokaryote (or rickettsia-like organism) is the pathogen that causes its onset. At present, the most conventional detection method is to use tissue section optical microscope observation and ultra-thin section electron...

AI Technical Summary

Problems solved by technology

[0002] Scallop (scallop) farming is an important pillar industry that dominates shellfish farming or even the entire mariculture in my country. Chlamys farrei is an important marine cultured scallop species in China. However, since 1995, large-scale scallops have continuously appeared Death, especially during the period from 1997 to 1999, the mortality rate of scallops reached more than 90%, and 100% died in some sea areas. In 1997, the dead area of ​​Chlamys farreri in northern culture reached 330,000 mu, causing economic losses of 1.5 billion yuan

It has been found that rickettsia-like prokaryote (or rickettsia-like organism) is the pathogen that causes its onset. At present, the most conventional detection method is to use tissue section optical microscope observation and ultra-thin section electron microscope observation. , but these traditional methods are not only time-consuming and labor-intensive, but also the biggest defect is that the Rickettsia-like inclusion bodies observed by the optical microscope in the tissue section cannot be compared with the Rickettsia-like inclusion bodies observed by the electron microscope in the ultrathin section. Intuitively corresponding to the body, thus lacking sensitivity and reliability

Serological detection methods require a large amount of pure pathogens to prepare enough serum, which is cumbersome and not sensitive enough for rickettsiae that cannot be artificially cultured

Recently, there have been a small number of international literature reports on the use of PCR methods to detect Rickettsia-like infections in aquaculture animals. Although the PCR method is fast and simple to operate, has high sensitivity, low cost, and strong specificity, the PCR method still cannot visually observe and prove the inclusion bodies of Rickettsia-like species, and it is easy to be contaminated by templates in the operation. False positives occur (see references: 1. Kellner-Cousin K, Le Gall G, DespresB, et al. Genomic DNA cloning of rickettsia-like organisms (RLO) of Saint-Jacquesscallop Pecten maximus: Evaluation of prokaryote diagnosis by hybridization with anon -isotopically labeled probe and by polymerase chain reaction.Diseases of Aquatic Organisms, 1993, (15): 145-152; 2.Andree K B, Friedman C S, Moore J D, et al.A polymerase chain reaction assay for the detection of genomic DNA of arickettsiales-like prokaryote associated with withering syndrome in California abalone. Journal of Shellfish Research, 2000, 19: 213-218.)

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

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