Hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus

A technology of iridescent virus and rolling circle amplification, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the problems of inability to detect diseases in time and accurately, increase the risk of cross-infection, and detection sensitivity Low-level problems, to achieve the effect of increasing the chance of cross-contamination, reducing the risk of reaction contamination, and high sequence specificity

Inactive Publication Date: 2012-01-25
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The current detection methods for Soft-shelled turtle iridovirus (STIV) mainly include conventional diagnostic methods such as serological detection and colloidal gold immunoassay. These methods are complicated in operation, long in detection cycle, and low in detection sensitivity; As a mature molecular detection method, the increased PCR technology has been used for the detection and identification of the virus (Fan Wanhong et al. 2007), but the conventional PCR technology is the amplification of the detection target, which increases the risk of cross-infection and has low sensitivity. The disease cannot be detected timely and accurately; moreover, the above-mentioned detection methods often require specific equipment, laboratories and molecular biology professional experimenters to operate, and there will be great limitations in actual on-site operation

Method used

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  • Hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus
  • Hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus
  • Hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus

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Experimental program
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Effect test

Embodiment 1

[0031] 1. Preparation of DNA sample template and standard sample of SITV genome

[0032]1.1 Extraction of Chinese soft-shelled turtle iridescent virus DNA

[0033] 1.1.1 Purification of virus

[0034] Cut 20 grams of infected tissue into small pieces, put them in 200 ml of TNE buffer, centrifuge at 8000 r / min, 4°C for 20 min, and take the supernatant. Centrifuge at 35000g for 1h at 4°C. Discard the supernatant, except for the precipitated black circle and other precipitates. Resuspend in 5ml TM buffer. Overnight, gently pipette the overnight precipitate evenly, centrifuge at 8000r / min, 4°C for 20min to take the supernatant, and centrifuge at 35000g for 1h at 4°C, the obtained white precipitate is the purified virus.

[0035] 1.1.2 Extraction of purified viral DNA

[0036] Completely transfer the virus to a centrifuge tube with 500 µl of TE buffer. Add 125 microliters of 10% SDS to the tube to make a final concentration of 2%. Add proteinase K to the EP tube to make a fi...

Embodiment 2

[0057] The present invention detects the sensitivity of the STIV method based on HRCA technology

[0058] 1. Preparation of standard substance: measure the concentration of the extracted DNA sample with a spectrophotometer and dilute to 10 10 copies / μL as a standard. Dilute the prepared standard to 10 0 -10 5 copies / μL were used as templates respectively.

[0059] 2. Use the optimal condition method obtained in Example 1 to detect the templates of each concentration, thereby obtaining the sensitivity of the present invention, and perform conventional PCR detection in addition to compare the sensitivity.

[0060] 2.1 Connection of lock probe

[0061] The ligation reaction system is as follows: padlock probe 0.1μM, T4DNA Ligase 1U / μl, 10×T4DNA Ligase Buffer 1μl, sample template DNA 2μl, add water to a total reaction volume of 10μl, and the ligation conditions are: after denaturation at 94°C for 5 minutes, Add 1U / μl T4 DNA Ligase, and react for 10 at 16°C, and the ligation r...

Embodiment 3

[0070] The present invention is based on the specificity determination of the method for detecting STIV by rolling circle amplification technology

[0071] STIV genomic DNA was used as a positive control, and three other viruses with different degrees of correlation were used to verify the specificity of this study, namely white spot syndrome virus (WSSV), Singapore grouper iridovirus (Singapore grouperiridovirus, SGIV) and Portunus reovirus, wherein double distilled water was used as a negative control. Amplified products were detected by agarose gel electrophoresis. The results show that see image 3 , indicating that the HRCA detection method provided by the present invention can ensure the specific detection of STIV and does not cross-react with other related viruses.

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Abstract

The invention discloses a hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus, and the method is characterized by comprising the following steps: (1) designing a padlock probe and a pair of universal primers aiming at the connection sequence of the padlock probe; (2) preparing a padlock probe connection reaction system for performing connection reaction; and (3) preparing an HRCA (hyper-branched rolling cycle amplification) reaction system for performing HRCA amplification reaction, and finally detecting HRCA reaction products. The method has the following advantages: the method has higher sensitivity, specificity and convenience compared with the PCR (polymerase chain reaction) detection method of the soft-shelled turtle iridovirus in the prior art; furthermore, the method is simple and easy to operate, the detection method is fast and efficient, and 2-4 hours can be saved in comparison with the conventional PCR detection.

Description

technical field [0001] The invention relates to a detection method of Chinese soft-shelled turtle iridescent virus, in particular to a hyperbranched rolling circle amplification detection method of Chinese soft-shelled turtle iridescent virus. Background technique [0002] Chinese soft-shelled turtle is a precious aquatic reptile with delicious meat and rich nutrition, so it has important research value. With the improvement of people's living standards, the market demand has increased, and the artificial breeding of Chinese soft-shelled turtles has flourished. Due to the high-density and intensive breeding of Chinese soft-shelled turtles, coupled with the frequent trade of Chinese soft-shelled turtle products and seedlings, and frequent occurrence of various diseases, soft-shelled turtle diseases have seriously threatened my country's soft-shelled turtle industry, with annual economic losses of hundreds of millions of yuan, restricting the cultivation of soft-shelled turtle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 李登峰刘联国陈炯王洪强彭娇周永强李民云王忠平
Owner NINGBO UNIV
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