Prawn white spot syndrome virus host distinguishing method

A technique for vitiligo syndrome and discrimination method, applied in the field of epidemiological research, can solve the problems of RNA decomposition and long time, and achieve the effects of reducing RNA degradation, fast method and high quality

Inactive Publication Date: 2006-12-13
OCEAN UNIV OF CHINA
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Problems solved by technology

However, the existing RT-PCR reaction method for extracting animal tissue RNA requires operation under low temperature (ice bath) conditions, and the required time is long, requiring more than 5 hours, and it is easy to cause the decomposition of RNA, making it difficult to perform RT-PCR. PCR reaction

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0025] To detect whether Penaeus chinensis is the host of shrimp white spot syndrome virus.

[0026] Select Chinese prawns positive for white spot syndrome virus, take 60 mg of gill tissues of the prawns and put them into 1ml Trizol reagent for homogenization, extract total RNA from animal tissues according to the above steps at room temperature, and detect the UV spectrophotometric absorbance A of the extracted RNA solution 260 / A 280 The ratio reached 1.92. Then use the extracted RNA and M-MLV reverse transcriptase to carry out reverse transcription to synthesize cDNA, then use cDNA and designed specific primers P1 and P2 to carry out PCR reaction, and perform 1% agarose gel electrophoresis on the PCR reaction product, Then, the agarose gel after electrophoresis was observed under ultraviolet light, and a 580bp amplified fragment appeared, which indicated that Penaeus chinensis is the host of white spot syndrome virus.

Embodiment 2

[0028] Artemia at four different developmental stages, namely nauplius, metanauplii, pseudo-adult stage larvae and adult Artemia were tested whether they were hosts of white spot syndrome virus in prawns.

[0029] First select four different developmental stages of Artemia positive for shrimp white spot syndrome virus: nauplius, metanauplius, pseudo-adult stage larvae and adults. Take 80 mg of Artemia at different developmental stages and put them into 1ml Trizol reagent. Slurry, extract animal tissue total RNA at room temperature according to the above steps, and detect the UV spectrophotometric absorbance A of the extracted RNA solution 260 / A 280 The ratios reached 1.91, 1.87, 1.99, 1.96. Then use the extracted RNA and M-MLV reverse transcriptase to carry out reverse transcription to synthesize cDNA, then use cDNA and designed specific primers P1 and P2 to carry out PCR reaction, and perform 1% agarose gel electrophoresis on the PCR reaction product, Then the agarose gel ...

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Abstract

A method for distinguishing the host of prawn-hickie comprehensive viruses includes the following steps: first, under an atmospheric temperature, using Trizol agent to extract the central RNA of the tested animal tissue and making the ratio of ultraviolet spectrophotometer absorbance A260 / A280 of the tested RNA liquor arrive at 1.8-2.0, using available software Primer5.0 to design a pair of primers P1 and P2, and its size of augmentation-aim fragment of is 580bp, then using M-MLV reverse transcriptase to reverse transcription to compound cDNA, the using cDNA and the designed specificity primers P1 and P2 to do PCR reaction and making electrophoresis appraise for the product of PCR reaction. When it comes up a tested animal which has 580bp augmentation fragment, the animal is the host of prawn-hickie comprehensive viruses. The said pair of primers are the upstream primer of P1(5'-GTG GTT TCA CGA GGT TGT-3') and the downstream primer of P2(5'-AAG GAG GAG GTG TTG GAG-3'). This invention is simple and direct, of high sensibility; the method for extracting the central RNA of tissue is rapid and the quality is high, meanwhile, it reduces the degradation of RNA; furthermore, it can distinguish the tested animals earlier than presexisting protein immune crossing technology.

Description

technical field [0001] The invention belongs to the technical field of epidemiological research, and in particular relates to a method for distinguishing the host of prawn white spot syndrome virus (WSSV). Background technique [0002] Since the outbreak of White Spot Syndrome (WSS) nationwide in 1993, the shrimp farming industry in my country has suffered heavy losses. Up to now, White Spot Syndrome is still a fatal threat to Chinese prawn (Fenneropenaeus chinensis) farming. [0003] One of the main reasons why prawn white spot syndrome is difficult to prevent is that its host is not known. According to the detection of polymerase chain reaction (PCR), as many as 40 kinds of arthropods can be infected under cultured, wild and laboratory conditions. Shrimp white spot syndrome virus, showing PCR positive. However, some of these PCR-positive animals were found to be carriers rather than hosts through experiments and production. Because the host and carrier of the virus play ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张家松董双林董云伟刘相义
Owner OCEAN UNIV OF CHINA
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