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RAA constant-temperature fluorescence detection method and reagent for epidemic hematopoietic organ necrosis virus (EHNV)

A hematopoietic organ necrosis and detection kit technology, applied in the field of molecular biology, can solve the problems of expensive equipment, high technical requirements, unsuitable on-site detection and popular use, etc., and achieve the effect of 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

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Problems solved by technology

The PCR molecular detection method is the most commonly used detection method. It is sensitive, accurate and fast, and is widely used. However, it is not suitable for On-site testing and popularization

Method used

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  • RAA constant-temperature fluorescence detection method and reagent for epidemic hematopoietic organ necrosis virus (EHNV)
  • RAA constant-temperature fluorescence detection method and reagent for epidemic hematopoietic organ necrosis virus (EHNV)
  • RAA constant-temperature fluorescence detection method and reagent for epidemic hematopoietic organ necrosis virus (EHNV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The present invention searches the gene sequence of the epidemic hematopoietic organ necrosis virus strain in the Genebank database, uses DNAMAN 6.0 software to compare multiple sequences, and finds out the conserved segment. 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.

[0032] Table 1 primers and probe sequences:

[0033]

[0034] Depend on figure 1 The 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 i...

example 3

[0047] Example 3: Kit of the present invention epidemic hematopoietic necrosis virus

[0048] 1. Extraction of positive sample nucleic acid

[0049] 1.1. Nucleic acid extraction: use traditional Trizol-RNA reagent or an equivalent RNA extraction kit.

[0050] 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.

[0051] table 3:

[0052] RAA reaction system components Volume (μL) A Buffer 12.5μL B Buffer 2.5μL primer mix 4μL specific fluorescent probe 0.6μL RNA template 2μL DEPC treated water 28.4μL total capacity 50μL

[0053] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0054] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and carry out RAA amplification according to the following procedure: 3...

Embodiment 4

[0057] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0058] The kit of the present invention was used to carry out a clinical blind experiment, and 20 large yellow croakers were detected; the experimental results showed that the fourth primer pair of the present invention can distinguish epidemic hematopoietic organ necrosis virus, and the positive coincidence rate with reverse transcription PCR is very high. Among the 20 samples, reverse transcription PCR, 16 samples were positive results, 4 samples were negative results, 4 samples were positive results detected by the RAA method, and 4 samples were also negative results. The results of the two groups of experiments were consistent. It shows that the RAA detection of the present invention has the same reliability as the PCR detection.

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Abstract

The invention discloses an RAA constant-temperature fluorescence detection method and detection kit for epidemic hematopoietic organ necrosis virus (EHNV). The detection kit comprises a forward primerSEQ ID NO. 1, a reverse 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 of the present invention has high specificity; the detection sensitivity is high, and can reach 6 fg / [mu]L; the kit has high accuracy and reliability; and the kit has simple and fast operation, is suitable forfield detection, and has wide application scenarios.

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 epidemic hematopoietic organ necrosis virus. Background technique [0002] In the world's aquaculture industry, the harm caused by viruses has always been huge. Among them, the harm of iridescent virus is more serious, and the distribution range of its harm is wider, and once fish are infected with iridescent virus and parasite at the same time, the fatality rate can reach more than 90%. Epidemic hematopoietic necrosis virus (EHNV) is a member of the genus Ranavirus in the family Iridoviridae, which can cause visceral necrosis and death in juvenile and adult fish such as perch and rainbow trout. It was first discovered in Australia in 1986. It can infect the kidney hematopoietic tissue, liver and spleen of necrotic fish, and the fata...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 贾鹏钱冬程奇温智清刘荭张建勋肖文余国君史秀杰郑晓聪王津津于力何俊强刘莹
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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