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Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof

A technology of RT-LAMP and Chinese mitten crab, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of complex instruments, cumbersome operation process, and inability to detect latent virus samples, etc. Reach the effect of low equipment requirements and easy result identification

Inactive Publication Date: 2016-04-20
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Studies have shown that Chinese mitten crab reovirus is a double-stranded RNA virus with 9-12 genome fragments, its sequence has been determined and published on GenBank (accession number: KP638402-KP638413), and the current detection method of the virus is electron microscopy method, RT-PCR detection method, etc., but these methods have many problems in actual operation: first, the operation process is cumbersome, and the required instruments are more complicated, which is not conducive to large-scale census and on-site detection; second, the sensitivity is not high enough to prevent Latent virus samples detected

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  • Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof
  • Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof
  • Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof

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Embodiment 1

[0039] The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples. In the present invention, unless otherwise specified, the raw materials and equipment used are more commonly used in this field. Example 1: The establishment of a RT-LAMP detection kit for Chinese mitten crab reovirus

[0040] RT-LAMP detection kit for Chinese mitten crab reovirus, including RT-LAMP primer set, RT-LAMP amplification reaction solution, virus RNA extraction reagent, RT-LAMP chromogenic solution, BstDNA polymerase, AMV reverse transcription Enzymes, positive and negative controls.

[0041] (1) RT-LAMP primer set: primers including the following sequences:

[0042] Primer EsRV-FIP:

[0043] CAAGAACCCACTTCGGAGCGT-GCTGCACCTGTAGTACTTGG.

[0044] (SEQ ID NO.1)

[0045] Primer EsRV-BIP:

[0046] TGCGACTATCGAAGGCGTGC-GATAGCCCTGGATAAGCCAC.

[0047] (SEQ ID NO.2)

[0048] Primer EsRV-F3:

[0049] TG...

Embodiment 2

[0059] Embodiment 2: The specific detection method of the RT-LAMP detection kit of Chinese mitten crab reovirus is as follows:

[0060] (1) RNA extraction of samples to be tested:

[0061] 1. Take 30-50mg Chinese mitten crab muscle tissue, add 1ml Trizol (TaKaRaBio, Dalian, China) to homogenate, then add 200μl chloroform, and centrifuge at 12000rpm for 15min;

[0062] 2. Transfer the supernatant obtained by centrifugation in step 1 into a new centrifuge tube, add 500 μl of isopropanol to mix, and centrifuge at 12,000 rpm for 10 minutes;

[0063] 3. Pour out the centrifuged liquid in step 2, add 75% absolute ethanol to wash the precipitate, and centrifuge at 7500rpm for 5min;

[0064] 4. Pour out the centrifuged liquid in step 3, add 10-12 μl DEPC water to dissolve the precipitate at room temperature; store at -20°C for later use.

[0065] (2) RT-LAMP amplification reaction:

[0066] Constant temperature amplification reaction: 25 μl reaction system includes: 1.6 μmol / LEsRV-...

Embodiment 3

[0072] Embodiment 3: RT-LAMP optimization test

[0073] In order to screen the optimum temperature for this system, under the same conditions as other experiments, several different temperature gradients such as 55°C-65°C were set up for RT-LAMP amplification reaction. After the reaction was completed, agarose gel electrophoresis showed that 65°C When the amplification results are best (see figure 2 ).

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Abstract

The invention discloses an eriocheir sinensis reovirus RT-LAMP detection reagent kit and a detection method thereof. According to the detection method, primers needed for an EsRV RT-LAMP reaction system are designed according to a conservative region of the sequence of an eriocheir sinensis reovirus(EsRV) disclosed in a GenBank, and the detection primers include a pair of outer primers and a pair of inner primers. The detection reagent kit comprises the detection primers, RT-LAMP amplified reaction liquid, DNA polymerase, AMV reverse transcriptase and a reference substance. The reagent kit further comprises a color developing agent. Compared with a common detection technology of the virus, the reagent kit is easy, convenient and fast to operate, high in specificity, high in detection sensitivity, suitable for on-site detection of front-line production technicians and farmers and favorable for diagnosis and prevention of eriocheir sinensis tremor diseases and has wide application prospects.

Description

technical field [0001] The invention relates to the field of aquatic animal detection, in particular to a Chinese mitten crab reovirus RT-LAMP detection kit and a detection method thereof. Background technique [0002] Chinese mitten crab tremor disease (tremordisease, TD), also known as tremor disease, Chinese mitten crab reovirus (Eriocheirsinensisreovirus, EsRV) is the main pathogen of Chinese mitten crab tremor disease, since 1994 in Jiangsu Province for the first time was detected Since the report, it has subsequently spread to major Chinese mitten crab breeding areas such as Anhui, Zhejiang, and Hubei. The main symptoms of diseased crabs are obvious tremors in the feet, red knuckles accompanied by liver erosion, slightly black gill filaments, triangular membrane edema, etc., and the mortality rate is as high as 70%. cause great harm to the industry. The disease is mainly based on prevention, early detection of the disease and effective preventive measures to prevent ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/107C12Q2537/1376C12Q2531/119
Inventor 沈怀舜马源潮周鑫
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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