Nested primer for early warning of cultured and wild Portunus trituberculatus microsporidium infection, and application thereof

Abstract

The invention relates to a nested primer for early warning of cultured and wild Portunus trituberculatus microsporidium infection, and an application thereof. The nested primer comprises a pair of outer primers MI1F / MI545R and a pair of inner primers MI230F / MI426R, the sequence of the MI1F is represented by TGGCGACCAGTTCTAAG, the sequence of the MI545R is represented by AAGCCGAACAATCCACA, the sequence of the MI230F is represented by CARATAGATAGGGGCAGTAGC, and the sequence of the MI426R is represented by ATCGGTCACTGCTAGAACTAC. The primer has good detection sensitivity and high specificity, is greatly better than universal primers, is suitable for early detection of Portunus trituberculatus microsporidium in muscle tissues and other samples of microsporidium slightly or latently infected crabs, can be applied in molecular detection and disease early warning of the Portunus trituberculatus microsporidium, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of prevention and treatment of Portunus trituberculatus diseases, and in particular relates to a nested primer for early warning of Microsporidia infection in cultured and wild Portunus trituberculatus in my country and an application thereof. Background technique [0002] Portunus trituberculatus is an important fishery production object in my country, and it is also an important aquaculture species with high economic value. However, the prevalence of "toothpaste disease" in recent years has caused a huge threat to Portunus trituberculatus aquaculture. Our laboratory has proved that the pathogen is a microsporidia through pathology and molecular biology methods. Although the microsporidiosis of portunus trituberculatus has a slow onset, it is highly contagious. The initial infection characteristics of the disease are relatively hidden, and the typical symptoms appear only in the middle and late stages of cultivation...

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Problems solved by technology

Although the onset of microsporidiosis in Portunus trituberculatus is relatively slow, it is highly contagious

However, there have been no reports on the identification and sequence analysis of the endemic species of Microsporidia in wild Portunus trituberculatus in my country, and there is no specific molecular detection technology for the endemic species of Microsporidia in cultured and wild species in China.

[0004] Because microsporidia are intracellular parasites, they also have a hard chitin shell, which not only affects the extraction efficiency of DNA, but also tends to produce shorter fragments. Therefore, microsporidia based on general primers (amplification of long fragments) The detection rate of insects is much lower than that of bacteria, viruses and other pathogens. Although the sensitivity of quantitative PCR technology has been improved to a certain extent, the detection cost has been greatly increased, and the requirements for instruments are higher.

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