RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for EHP (enterocytozoon hepatopenaei)

A technology of hepatic Enterocystis and detection kit, which is applied in the field of molecular biology, can solve the problems of application limitations, high false positives, and low accuracy, and achieve the effect of simple identification and simple operation

Active Publication Date: 2018-05-04
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 (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for EHP (enterocytozoon hepatopenaei)
  • RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for EHP (enterocytozoon hepatopenaei)
  • RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for EHP (enterocytozoon hepatopenaei)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] In the present invention, the SSU gene sequence of Enterocystis prawnii strain is searched in the Genebank database, and DNAMAN 6.0 software is used to compare multiple sequences to find 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.

[0030] Table 1 Primer and probe sequences:

[0031]

[0032] 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 ...

example 3

[0044] Example 3: Kit of the present invention Enterococcus hepatica

[0045] 1. Extraction of positive sample nucleic acid

[0046] 1.1. Nucleic acid extraction: DNA extraction was performed using a marine animal tissue DNA extraction kit.

[0047] 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.

[0048] table 3:

[0049] 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

wxya 2 o

28.4μL

total capacity

50μL

[0050] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0051] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and perform RAA amplification according to the following pr...

Embodiment 4

[0053] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0054] The kit of the present invention is used to carry out a clinical blind sample experiment, and 500 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 nested PCR is very high. Among the 500 copies, nested PCR, 318 were positive results, 182 were negative results, 319 were positive by the RAA method, 181 were also negative results, and one positive result was different. The sample DNA was amplified by 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 dislcoses an RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and a detection kit for EHP (enterocytozoon hepatopenaei). The detection kit comprisesa forward primer SEQ ID NO. 1, a reverse primer SEQ ID NO. 2, a specific fluorescence probe SEQ ID NO. 3, reaction liquid, recombinant polymerase and a reference substance. The kit has the advantagesthat the specificity is strong; the detection sensitivity is high, and can reach 100fg/mu L; high accuracy and reliability are achieved; the operation is simple and rapid, and the kit 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 of the marine aquaculture industry, in particular to a RAA constant-temperature fluorescence detection method and a kit of the prawn enterocystoma hepatica. Background technique [0002] Enterocytozoon hepatopenaei (EHP), also known as Enterocytozoa, is a microsporidia found in prawns in recent years. It mainly infects important cultured shrimps such as Litopenaeus vannamei and Penaeus monodon. species, was found in 2009 in stunted P. monodon in aquaculture ponds in Thailand. It has been reported that EHP has a high detection rate in Penaeus monodon and Litopenaeus vannamei with white faeces syndrome (WFS) in Thailand and Vietnam, and the shrimp is seriously infected with Enterocystoma hepatica. Shrimp infected with Enterociasis can be found to grow slowly, which seriously affects the production cycle of shrimp culture. Once the microsporidia in the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6893
CPCC12Q1/6844C12Q1/6893C12Q2521/507C12Q2522/101C12Q2563/107
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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