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Specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses and detection kit using the same

A technology for detection kits and amplification primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. problems such as uncertainty and uncertainty, to avoid subjectivity and uncertainty, high sensitivity, and strong specificity

Active Publication Date: 2015-11-11
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes how certain types of DNA can be used to detect harmful bacteria called Chlamydophila pneumoniae (Cp). These microorganisms cause chloroform poisonings or other diseases like swine fever. By designing special probes with unique sequences from this type of organism, these techniques will help diagnose them more accurately than current methods such as PCR analysis.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the ability of studying harmful microorganisms like salmonella, shrimp rotavims, porcine ruminant tick fever vires, yellow sea quaternary tritonfish infectious disease, lymerase serum sickness virus, lycoplasma phosphate coating liquid containing chelated protein called histone proteins, including tentacle cellular membrane receptors involved in DNA damage response mechanisms. Current testing techniques involve invasiveness procedures involving collecting samples from suspected areas around affected individuals, making these processes timeconsuming and expensive. Therefore, new ways to detect and identify harmful organism without causing contamination during sampling process would improve their effectiveness and reduce economic losses associated with damaging livelihood and human resources worldwide.

Method used

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  • Specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses and detection kit using the same
  • Specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses and detection kit using the same
  • Specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses and detection kit using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0037] Operation 1: Acquisition of TSHSV partial nucleic acid sequence

[0038] Step 1: Viral RNA extraction

[0039] The liver, spleen, lung, kidney, and intestinal tissue of the sick soft-shelled turtle (the symptoms of the sick soft-shelled turtle are redness, swelling and erosion of the parotid gland, and obvious bleeding of the liver, intestine, lung, and kidney, etc.), and the sick soft-shelled turtle comes from a local farm in Huzhou) were taken out, Homogenize on ice. The homogenate was centrifuged at 12000g for 20min, the supernatant was taken, filtered through a 0.22μm bacterial filter, the filtrate was centrifuged at 40000g for 3h, and TEN buffer (50mmolTris-HCL, 50mmolNaCl, 5mmolNa 2 EDTA, pH 7.4) to resuspend the pellet as the virus-containing crude extract. Crude virus extracts were treated with RNaseA (Takara Dalian) and DNaseI (Takara Dalian) to remove most of the host's own nucleic acids (background nucleic acids) in the samples. Take 356 μL of virus crude ...

specific Embodiment approach 1

[0056] The operation process of the ordinary RT-PCR virus nucleic acid detection kit is as follows:

[0057] (1) Positive template preparation: use TSHSV-F1 and TSHSV-R1 as specific primers, virus-infected tissue RNA as a template, and use gene cloning technology to construct a 308bp target sequence amplified by ordinary RT-PCR according to the above operation 2. The positive plasmid molecule of the specific sequence, the standard plasmid used is T vector, provided by Dalian Baosheng Biological Takara Company. 0.1-1.0ng is used as a positive template. (2) Extraction of RNA from the sample to be tested: Take 20-30 mg virus-infected soft-shelled turtle lung, spleen, kidney and other tissue homogenate, add 350-500 μL lysate to homogenate, and centrifuge; take the supernatant and mix with an equal volume of 70 % absolute ethanol was mixed evenly, and the mixed solution was combined with the glass cellulose membrane; the glass cellulose membrane was washed with protein-removing so...

specific Embodiment approach 2

[0060] Fluorescence quantitative RT-PCR virus nucleic acid detection kit operation process is as follows:

[0061] (1) Preparation of positive control samples and standard solutions: The preparation of positive control samples is the same as that of the positive template in step (1) of 21 of the specific embodiment. When in use, dilute the positive control sample with ribonuclease-free water, that is, dilute to a concentration of 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 copies / μl of the standard solution, as the reaction template for the positive control sample standard curve. (2) Extraction of RNA from samples to be tested: take 20-30 mg virus-infected soft-shelled turtle lung, spleen, kidney and other tissue homogenate, add 350-500 μL lysate to homogenate, and centrifuge; take the supernatant, and 70% of the same volume After the absolute ethanol is mixed evenly, combine the mixed solution with the glass cellulose membrane; wash the glass cellulose membrane ...

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Abstract

The invention discloses specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses and a detection kit using the same. The amplification primers solve the problem that at present, viral pathogens causing trionyx sinensis explosive death cannot be accurately diagnosed. The specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses can produce a specific detection result, can be determined easily and is conducive to trionyx sinensis virus disease definite diagnosis. The invention also provides a detection kit for detecting trionyx sinensis hemorrhagic disease viruses. The detection kit contains the specific amplification primers. The detection kit is a common RT-PCR kit or a fluorescent quantitative RT-PCR kit. The two types of kits have high specificity and high sensitivity, can be operated simply, and fill a domestic gap in a trionyx sinensis hemorrhagic disease virus nucleic acid molecule detection method, and is suitable for definite diagnosis, screening and prevention of trionyx sinensis hemorrhagic disease viruses.

Description

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Claims

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Application Information

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Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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