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RAA constant-temperature fluorescent detection method and reagent for viral haemorrhagic septicemia (VHSV)

A technology of viral hemorrhage and detection kit, which is applied in the field of molecular biology, can solve the problems of few, high cost, and long time-consuming real-time fluorescent PCR, and achieve the effect of simple identification and simple operation

Inactive Publication Date: 2019-12-20
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.

Method used

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  • RAA constant-temperature fluorescent detection method and reagent for viral haemorrhagic septicemia (VHSV)
  • RAA constant-temperature fluorescent detection method and reagent for viral haemorrhagic septicemia (VHSV)
  • RAA constant-temperature fluorescent detection method and reagent for viral haemorrhagic septicemia (VHSV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The present invention searches the gene sequence of viral hemorrhagic sepsis strains in the Genebank database, uses DNAMAN 6.0 software to compare multiple sequences, and finds out the conserved segments. Four sets of primers and probes were designed in the conserved regions, and BLAST comparison 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 follows figure 1 shown.

[0033]

[0034] Table 1 primers and probe sequences:

[0035] Depend on figure 1The results show that the amplification curves of the fourth group of primers and probes are the most typical, with obvious exponential phase and plateau phase, higher fluorescence intensity (ordinate value), and smaller CT value (the intersection of the curve and the threshold line Corresponding abscissa) result analysis is shown in Table 2. For other primers and probes, the rising height of the curve is lower, the CT value ...

example 3

[0048] Example 3: kit viral hemorrhagic sepsis according to the present invention

[0049] 1. Extraction of positive sample nucleic acid

[0050] 1.1. Nucleic acid extraction: use traditional Trizol-RNA reagent or an equivalent RNA extraction kit.

[0051] 2. The configuration of the RAA reaction system: each test sample corresponds to a RAA reaction dry powder tube, and the reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 3.

[0052] table 3:

[0053] RAA reaction system components Volume (μL) A Buffer 12.5μL B Buffer 2.5μL primer mix 4μL specific fluorescent probe 0.6μL DNA template 2μL DEPC treated water 28.4μL total capacity 50μL

[0054] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0055] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and perform RAA amplification according to the following procedure: 37°C, ...

Embodiment 4

[0058] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0059] The kit of the present invention is used to carry out a clinical blind sample experiment, and 60 shrimps are detected; the experimental results show that the fourth primer pair of the present invention can distinguish viral hemorrhagic sepsis, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 copies, 33 were positive results and 27 were negative results by reverse transcription PCR. The results detected by the RAA method were 33 positive and 27 were also negative results. The results were completely consistent with the actual samples. Consistent, indicating that the RAA detection reagent of the present invention has a higher accuracy rate.

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Abstract

The invention discloses an RAA constant-temperature fluorescent detection method and detection kit for viral haemorrhagic septicemia (VHSV). The detection kit comprises a forward primer SEQ ID NO.1, areverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, a reaction solution, reverse transcriptase, recombinant polymerase and a reference substance. The kit has high specificity and high detection sensitivity reaching 10fg / [mu]L, is high and reliable in accuracy, is simple and quick to operate, is suitable for field detection, and has an extensive application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method for marine aquaculture industry, in particular to a RAA constant temperature fluorescence detection method and a kit for viral hemorrhagic sepsis. Background technique [0002] Viral hemorrhagic septicemia virus (VHSV), is a disease that infects salmon of all ages and a variety of free-living marine fish such as cod, herring, sole, catfish, northern pike, and turbot. , flounder, etc.) fatal, systemic infectious disease. It is usually popular at the end of winter and early spring and when the water temperature is 8-10°C. When the water temperature rises above 15°C, the incidence rate decreases. The disease is endemic throughout continental Europe, North America, and Japan, and is highly contagious, with 1,000 viruses per liter of water in affected fish farms. The main feature of viral hemorrhagic sepsis is bleeding, and the incubation period of the dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107C12Q2521/107C12Q2521/319
Inventor 刘荭钱冬程奇刘莹黄震巨张建勋肖文余国君史秀杰郑晓聪贾鹏王津津于力何俊强温智清
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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