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Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof

A technology of Macrobrachium rosenbergii and bicistronic, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as lack of detection methods, incalculable economic losses in the Macrobrachium rosenbergii nursery industry, and obstacles to disease diagnosis and prevention.

Active Publication Date: 2012-02-15
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Macrobrachium rosenbergii larval syndrome (MRLS) broke out in 2009 and 2010, causing a large number of deaths of Macrobrachium rosenbergii larval syndrome in the larval stage, especially The death of larvae at the age of 6-9 days is particularly serious. The mortality rate is generally 80-90%, and the severe case reaches 100%. It involves the main Macrobrachium rosenbergii nursery areas in my country, and the economic losses caused to the Macrobrachium rosenbergii nursery industry are immeasurable.
After research, it was found that its main pathogen was a new dicistronic virus, tentatively named Macrobrachium rosenbergii dicistrovirus (MRDV), MRDV is a single positive-strand RNA virus, classified Virus family, since it was discovered, due to the lack of information about the virus genome and the lack of detection methods, it has seriously hindered the diagnosis and prevention of diseases caused by the virus. Therefore, the analysis of the full sequence of the virus genome and its related applications are particularly important

Method used

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  • Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof
  • Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof
  • Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Extraction of viral RNA

[0082] Macrobrachium rosenbergii larvae infected with bicistronic virus strain CN-NTH were taken for tissue grinding, and PBS buffer was added at a volume ratio of 1:10, and the cells were disrupted by ultrasonication for 3-5 seconds. After centrifugation at 6000 rpm at 4°C for 30 min, the supernatant was taken, sterilized by filtration with a 0.22um filter membrane, and then subjected to discontinuous density gradient centrifugation of sucrose / glycerol to obtain high-purity MRDV virus, and the viral RNA was extracted with a viral RNA extraction kit.

[0083] Reverse transcription PCR (RT-PCR)

[0084] Design and synthesize primers: 5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3' (SEQ ID NO: 4). Take 5uL of the above-prepared RNA virus and place it in a 200uL small tube dedicated to PCR amplification, add 100pmol of primer 1, place it at 65°C for 10min to denature, put it on ice immediately, add 2uL of 10×reverse transcription buffer, l mmol / L 4×dNTP, 20U R...

Embodiment 2

[0098] Verification of Genome Sequence of Macrobrachium rosenbergii Bicistronic Virus

[0099] According to the nucleotide sequence shown in SEQ ID NO: 1, primers were designed according to the length of each 800bp or so, PCR reaction was carried out, and the PCR product was sequenced and verified. The result showed that the nucleotide sequence of SEQ ID NO: 1 was correct.

Embodiment 3

[0101] Kit for detection of Macrobrachium rosenbergii bicistronic virus strain

[0102] Based on the genomic sequence shown in SEQ ID NO: 1, the following PCR primers and probes were synthesized:

[0103] Sense primer 1: the sequence is 9625-9644 in SEQ ID NO:1.

[0104] Antisense primer 2: the sequence is the complementary sequence of 9755-9774 in SEQ ID NO:1.

[0105] Probe 3: the sequence is the complementary sequence of positions 9646-9667 in SEQ ID NO:1.

[0106] Prepare a test kit (detect 100 times), it contains:

[0107] name concentration sense primer 1 100pmol antisense primer 2 100pmol Probe 3 100pmol PCR reaction solution Containing Taq Enzyme dNTP Magnesium Ion PCR Reaction Buffer

[0108] Macrobrachium rosenbergii bicistronic virus strain CN-NTH was used to infect Macrobrachium rosenbergii larvae, and the Macrobrachium rosenbergii bicistronic virus strain was obtained as in Example 1, and a cDNA sample was obtained by...

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Abstract

The invention relates to a macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and an application thereof, which belong to the technical field of virus genomics. The invention provides the macrobrachium rosenbergii bicistronic mRNA viral genome complete sequence shown as SEQIDNO: 1. In addition, the invention provides a macrobrachium rosenbergii bicistronic mRNA viral specific primer, a probe or a reagent kit. The macrobrachium rosenbergii bicistronic mRNA viral genome complete sequence and the application lay the reliable foundation to the purposes of macrobrachium rosenbergii dicistrovirus (MRDV) detection, MRDV infection path study and the like.

Description

technical field [0001] The invention belongs to the technical field of viral genomics, and in particular relates to the genome sequence of Macrobrachium rosenbergii bicistronic virus and its application. Background technique [0002] Macrobrachium rosenbergii (Macrobrachium rosenbergii DeMan, 1879), also known as freshwater long-armed prawn, belongs to the genus Macrobrachium (Macrobrachium), and is the main cultured species of freshwater crustaceans in my country. According to the statistics of the Food and Agriculture Organization of the United Nations (FAO) in 2009, the total global aquaculture output of Macrobrachium rosenbergii reached 207,749 tons in 2008, of which the output in Asia was 127,627 tons, accounting for 98% of the global output. China is the largest aquaculture industry in the world. country. [0003] Macrobrachium rosenbergii larval syndrome (MRLS) broke out in 2009 and 2010, causing a large number of deaths of Macrobrachium rosenbergii larval syndrome i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/11C12Q1/70C12Q1/68
Inventor 潘晓艺沈锦玉石正丽曹铮郝贵杰姚嘉赟徐洋尹文林
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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