Method for extracting scylla paramamosain reovirus RNA (Ribose Nucleic Acid) at room temperature

Abstract

The invention discloses a method for extracting a scylla paramamosain reovirus RNA (Ribose Nucleic Acid) at a room temperature. Quick and accurate detection of reovirus in the body of a blue crab has important practical significance on prediction and forecasting of virus diseases and disease control. The method can be used for quickly detecting the scylla paramamosain reovirus RNA, and comprises the following steps: (1) extracting blue crab blood lymph, and centrifuging to obtain blood cells for extracting the RNA; (2) adding Trizol and chloroform, violently shaking to mix evenly for 20 seconds and then standing for 3 minutes, centrifuging at the room temperature by 12000 rpm for 5 minutes and taking the supernate; (3) adding isopropanol of equal volume, evenly mixing at -20 DEG C and then placing for 20 minutes, centrifuging at the room temperature by 12000 rpm for 5 minutes and abandoning the supernate; and (4) adding 1 mL of 75% ethanol for washing and precipitation, centrifuging at the room temperature by 12000 rpm for 5 minutes and abandoning the supernate, drying in the air for 3-5 minutes at the room temperature, adding Rnase-freedd H2O for dissolving, and then putting the obtained product in a refrigerator for storage for future use. The method disclosed by the invention is simple to operate, less in steps and free of low-temperature condition; the extraction of little blood lymph has no damage on the organism of the blue crab; the method has a great significance in early detection of viruses.

Description

technical field [0001] The invention belongs to the technical field of virus RNA extraction, and in particular relates to a method for extracting virus RNA from the hemolymph of Scylla pseudomaculata, in particular to a method for extracting RNA of Scylla pseudomaculata reovirus at room temperature. Background technique [0002] Pseudomonas blue crab ( Scylla paramamosain ) commonly known as blue crab, is a dominant species of blue crab, widely distributed in the southeast coast of China, is a large edible seafood crab with high economic value, and is a traditional marine treasure in China. Due to the continuous expansion of breeding scale and breeding density, and the deteriorating breeding environment, large-scale epidemics have broken out in the blue crab breeding areas of the southeastern coastal provinces of China. Since 2008, in the process of breeding adult crabs, fattening crabs, and artificial seedlings, the blue crabs of pseudo-caves have died of unknown reasons. ...

AI Technical Summary

Problems solved by technology

The application of this harmful sampling method in the research not only increases the research cost, but also is not conducive to the early virus detection research of the parents in the breeding of mud crabs, and is not conducive to the protection and repeated utilization of wild resources of mud crabs.

Traditionally, the extraction of virus RNA from animal tissues is mostly carried out at low temperature. For example, the patent publication No. CN101220072A discloses a method for extracting viral genome RNA from animal tissues. After freezing and thawing, the diseased tissue is crushed, and the virus is collected by centrifugation at low temperature to separate the RNA. This method requires cumbersome sample collection and processing operations, requires centrifugation at low temperature, and requires removal of host cytoplasm, which is not conducive to easy operation and time-consuming

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