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Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method

A Nodamura virus and fluorescence quantitative technology, which is applied in the field of fluorescence quantitative RT-PCR detection of prawn stealing Nodamura virus. , complex operations, etc.

Inactive Publication Date: 2016-02-17
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first three detection methods are complicated to operate and take a long time. They are generally used in laboratory diagnosis, and their detection sensitivity is low. It is very difficult to detect with these two methods in the very early stage; although the reverse transcription PCR detection method of CMNV overcomes the shortcomings of the previous methods, it cannot quantitatively analyze the viral nucleic acid due to the conventional reverse transcription PCR detection, which is to a certain extent It also limits the popularization and application of reverse transcription PCR detection method in production

Method used

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  • Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method
  • Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method
  • Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method

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Experimental program
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Effect test

Embodiment 1

[0064] Embodiment 1 RNA standard product construction

[0065] 1) Using the CMNV-positive disease RNA as a template, reverse transcription PCR was performed using primers 1 and 2 to obtain a PCR product with a fragment size of 179 bp, and the PCR product was gel-recovered and purified.

[0066] 2) T-VectorpMD TM 20 was used as the transcription vector of the purified PCR product, and then transformed into DH5α competent cells.

[0067] 3) Extract the plasmid using a plasmid extraction kit (Biomed).

[0068] 4) Using EcoRI (TaKaRa Company) for 2 hours at 37° C. to linearize the plasmid. The system is:

[0069]

[0070] 5) Use SP6RNAPolymerase (TaKaRa Company) for in vitro transcription, react at 37°C for 1 hour, the system is:

[0071]

[0072] 6) The synthesized RNA was degraded with DNaseI for plasmid DNA, and reacted at 37°C for 30 minutes. The system was as follows:

[0073]

[0074] 7) Carry out phenol chloroform extraction and purify the extracted RNA

[00...

Embodiment 2

[0085] The making of embodiment 2 standard curve

[0086] Using the concentration of synthetic RNA measured in Example 1, the copy number of synthetic RNA was calculated. Use RNaseFree water to carry out 10-fold gradient dilution of RNA, dilute to 10 7 ~10 2 copies / μL, used as a standard to make a standard curve.

[0087] One-step fluorescent quantitative RT-PCR kit was used, and the reaction solution was prepared according to the instructions of the kit. The total volume of RT-PCR reaction solution was 25 μL: reaction buffer 12.5 μL, TaqHS mixed solution 1 μL, PrimeScript PLUS RTase mixed solution 1 μL, ROX reference The dye is 1ul, the probe is 0.2-0.5μL (10mmol / L), and the two primers and RNA are added at the same time. The final concentration of the primer is 1μL (10mmol / L), and the RNA is 1μL. Use RNase-free water as a supplement 25 μL.

[0088] Use the Roche LightCycler480II real-time fluorescence quantitative PCR system to perform RT-PCR reverse transcription reacti...

Embodiment 3

[0089] Embodiment 3 clinical verification of shrimp samples

[0090] For 15 shrimp samples collected from Shandong, Hebei, Tianjin, Zhejiang and Hainan in 2013-2014, RNA was extracted using a commercial RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The reaction premix was prepared according to the above method, and the RT-PCR reaction was carried out according to the preferred amplification conditions. The results showed that 9 samples were amplified, and the Ct values ​​of the amplification curves were all less than 35 and the Tm values ​​were 81.53±0.14°C, so the positive rate of confirmed samples was 60%. Nested reverse transcription PCR verification was carried out on these 15 samples, and the results showed that 8 of the above 15 samples had positive bands, and the positive rate was 53%. Therefore, the fluorescent quantitative RT-PCR method of the present invention has higher sensitivity than the common nested reverse transcription PCR method. ...

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Abstract

The invention relates to a prawn covert mortality nodavirus (CMNV) fluorescent quantitative PCR (RT-PCR) detection method. The method includes the steps that a primer and a Taqman probe are designed according to a RNA polymerase gene conserved gene segment on which RNA of the CMNV depends, an RNA sample is prepared, and fluorescent quantitative RT-PCR is carried out according to preset reaction conditions. By means of standard curve manufacturing and specificity verification of the detection method, results show that absolute quantification of the CMNV can be achieved through the method. Meanwhile, the CMNV can be accurately identified from the white spot syndrome virus, the baculovirus, the taura syndrome virus, the hepatopancreatic parvovirus, the infectious hypodermal and hematopoietic necrosis virus and the infectious myonecrosis virus through the method, and high specificity is represented. The primer, the probe and the detection method have high popularization value in enhancement of monitoring of the CMNV in water, bait and offspring seeds and prevention of large-scale epidemic outbreak of the CMNV.

Description

technical field [0001] The invention belongs to the technical field of detection of marine biological pathogens, and in particular relates to a fluorescent quantitative RT-PCR detection method for covert mortality nodavirus (CMNV for short). Background technique [0002] Covertmortality disease (CMD) of prawns is a viral disease caused by "Covertmortality nodavirus (CMNV)". CMNV belongs to the genus Nodaviridae, Alphanodavirus of the Nodaviridae family. It is a single-stranded RAN virus with spherical (icosahedral) virions, no envelope, and a diameter of about 32 nanometers. CMD is a new disease that appeared in my country's cultured prawns in recent years. In 2003 or earlier, the disease appeared in high-density cultured Litopenaeus vannamei in Hainan, Guangxi and other places. From 2008 to 2009, a large-scale outbreak occurred in the cultured prawns in various provinces and cities in southern my country. After 2010, it began to spread to my country's Shandong, Hebei and T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/686
Inventor 张庆利黄倢李小平刘爽邱亮
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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