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RAA constant temperature fluorescence detection method and reagent for salmonid alphavirus (SAV)

A technology for detecting kits and alphaviruses, applied in the field of molecular biology, can solve the problems of high false positives, limited application, and few, and achieve the effects of high sensitivity, simple identification, and simple operation

Pending Publication Date: 2020-03-20
HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, real-time fluorescent PCR is time-consuming and expensive, and it is not widely used in the routine detection of pathogens in aquaculture animals.
LAMP isothermal amplification technology also has high false positives and low accuracy, so its application in the detection of aquatic pathogens is still relatively limited

Method used

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  • RAA constant temperature fluorescence detection method and reagent for salmonid alphavirus (SAV)
  • RAA constant temperature fluorescence detection method and reagent for salmonid alphavirus (SAV)
  • RAA constant temperature fluorescence detection method and reagent for salmonid alphavirus (SAV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The present invention searches the salmon alpha virus strain gene sequence in the Genebank database for salmon alpha virus, and uses DNAMAN 6.0 software to compare multiple sequences to find out conserved segments. Four sets of primers and probes were designed in the conserved region, and BLAST alignment was performed in the NCBI database. The sequences of the primers and probes are shown in Table 1. The positive sample amplification curve is as figure 1 Shown.

[0032]

[0033] Table 1 Primer and probe sequence:

[0034]

[0035] by figure 1 The results show that the amplification curve of the fourth set of primers and probes is the most typical, with obvious exponential phase and plateau phase, high fluorescence intensity (ordinate value), and small CT value (the intersection of the curve and the threshold line) The corresponding abscissa) result analysis is shown in Table 2. Other primer probe curves have a low rise height, a large CT value, and the plateau period is no...

example 3

[0048] Example 3: The kit of the present invention is effective against salmon alpha virus

[0049] 1. Extraction of nucleic acid from positive samples

[0050] 1.1. Nucleic acid extraction: Take about 100mg of the muscle tissue of the fish to be tested, grind it thoroughly with a grinding rod, and put it into a 1.5mL centrifuge tube, then add 1mL Trizol to the centrifuge tube, shake and mix, and ice bath for 10 minutes. Add 350μL of chloroform, shake well, and then stratify after a little static, then centrifuge at 4℃, 12000r / min for 15min. Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol (pre-cooled at 4°C) and mix well. Centrifuge at 12000r / min for 15min at 4℃. Discard the supernatant, centrifuge again at 12000r / min for 30s, add 200μL of 75% ethanol, shake and wash once, and carefully discard the ethanol. Dry at room temperature. Then add 20 μL DEPC water to dissolve the precipitate (the precipitate is the total RNA requir...

Embodiment 4

[0057] Example 4: Evaluation of the RAA detection kit of the present invention in clinical practical applications

[0058] The test kit of the present invention is used to conduct a clinical blind sample experiment, and 50 fish samples are detected; the experimental results show that the fourth primer pair of the present invention can distinguish salmon alpha virus, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 samples, 28 of the reverse transcription PCR results were positive, 22 were negative, 29 were positive by the RAA method, and 21 were also negative. There was one positive result with different results This sample is amplified by reverse transcription PCR and sequenced, and the sequencing result shows that the sample is positive, indicating that the RAA detection reagent of the present invention has a higher accuracy rate.

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Abstract

The invention discloses a RAA constant temperature fluorescence detection method and a detection kit for a salmonid alphavirus (SAV). The detection kit comprises a forward primer SEQ ID NO.1, a reverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, a reaction liquid, reverse transcriptase, recombinant polymerase and a reference substance. The kit disclosed by the invention is high in specificity, high in detection sensitivity which can reach 1.93 fg / [mu]L, high in accuracy, reliable, simple and rapid to operate and suitable for field detection, and has wide application scenes.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, relates to a detection method in the marine aquaculture industry, and specifically relates to a RAA constant temperature fluorescent detection method and a kit for salmon alpha virus. Background technique [0002] Salmonid alphavirus ((Salmonid alphavirus, SAV) has been popular in Ireland, Norway, the United Kingdom and other countries since the late 1990s. It mainly infects salmonids such as Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), causing pancreas, Symptoms such as myocardial inflammation and lethargy have a fatality rate of 1% to 48%. They have outbreaks at all growth stages of fish farming. It is also called salmon pancreatic disease or sleeping sickness. In 2013, salmon alphavirus infection was included in OIE aquatic animals. In the list of epidemic diseases, salmon alphavirus belongs to the Togaviridae family (Togaviridae), an RNA-type virus of the alphav...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 郑晓聪钱冬程奇史秀杰刘荭张建勋肖文余国君贾鹏王津津于力何俊强刘莹温智清
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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