RAA constant temperature fluorescence detection method and kit for yellow head virus (YHV) of shrimps

A yellow head virus and detection kit technology, applied in the field of molecular biology, can solve the problems of limited application, high false positives, low accuracy, etc., and achieve the effect of simple identification and simple operation

Inactive Publication Date: 2018-03-23
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 kit for yellow head virus (YHV) of shrimps
  • RAA constant temperature fluorescence detection method and kit for yellow head virus (YHV) of shrimps
  • RAA constant temperature fluorescence detection method and kit for yellow head virus (YHV) of shrimps

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] In the present invention, the shrimp yellow head virus strain gene sequence is searched in the Genebank database, and DNAMAN 6.0 software is used to compare multiple sequences to find 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.

[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 is lower, the C...

example 3

[0047] Example 3: Kit prawn yellow head virus of the present invention

[0048] 1. Extraction of positive sample nucleic acid

[0049] 1.1. Nucleic acid extraction: Take about 100 mg of shrimp muscle tissue to be tested, grind it thoroughly with a grinding rod, put it into a 1.5 mL centrifuge tube, then add 1 mL Trizol to the centrifuge tube, shake and mix, and place in an ice bath for 10 min. Add 350 μL of chloroform, oscillate fully, after a little rest, layers appear, and then centrifuge at 4°C, 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°C. Pour off the supernatant, centrifuge again at 12000r / min for 30s, add 200μL of 75% ethanol, shake and wash once and pour off the ethanol carefully. Dry at room temperature. Then add 20 μL of DEPC water to dissolve the precipitate (the precipitate is the desired total RNA). The ext...

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 is used to carry out a clinical blind sample experiment, and 50 prawns are detected; the experimental results show that the fourth primer pair of the present invention can distinguish Hepatocystis prawns, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 samples, reverse transcription PCR, 28 samples were positive results, 22 samples were negative results, 29 samples were positive results by RAA method, 21 samples were also negative results, and one positive result was different. This sample was amplified by reverse transcription PCR and sequenced, and the sequencing result showed that the sample was positive, 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 fluorescence detection method and a detection kit for yellow head virus (YHV) of shrimps. 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 solution, reverse transcriptase, recombinant polymerase and a reference substance. The kit disclosed by the inventionhas the advantages of high specificity, high detection sensitivity as high as 0.10fg / mu L, high accuracy, reliability, simplicity and rapidness in operation, suitability for field detection and broadapplication prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method of marine aquaculture industry, in particular to a RAA constant temperature fluorescence detection method and a kit of shrimp yellow head virus. Background technique [0002] Shrimp yellow head virus (YHV) is a single-stranded RNA virus with a genome length of about 26,000 nucleotides. The virion is rod-shaped, has a capsule, and has a capsule protuberance. Virions are composed of nucleoprotein p24 and envelope proteins gp64 and gp116. Yellow head virus genotype I is the pathogen that causes yellow head disease. [0003] The virus was first discovered in Thailand's Black tiger shrimp in 1990. Shrimp infected with yellow head virus had symptoms of yellowing of the head and chest and albinism of the muscles of the whole body. Within 2-4 days of the initial stage of infection, the shrimp would only Abnormal large-scale feeding occurs, and then almost co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/701C12Q2563/107C12Q2521/507C12Q2522/101
Inventor 程奇钱冬黄震巨张建勋肖文余国君陶智勇徐锦余霍胜楠沈弘郑晓叶郑天伦沈伟良吕文浩
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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