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Freeze-drying PCR reagent for detecting shrimp seed enterocytozoon hepatopenaei

A technology of Enterocytoplasma hepatis and a freeze-dried protective agent is applied in the field of freeze-dried PCR reagents and their recombination solutions for detecting Enteroplasma hepatis in shrimp fry, and can solve the problems of DNA degradation detection failure, inconvenient transportation, reagent deterioration and failure, and the like. Achieve the effect of ensuring sequence specificity, good application prospects, and reduced storage costs

Inactive Publication Date: 2019-03-12
GUANGZHOU HUAFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the time-consuming and high cost limit the wide application of real-time fluorescent PCR in the routine detection of pathogens in aquaculture animals.
[0004] In addition, shrimp farms are generally located in remote rural areas, and the transportation is very inconvenient. It often takes a lot of time to transport the samples to the city center, and the degradation of the DNA during the long-distance transportation of the samples may cause the detection to fail.
Conventional liquid PCR reagents contain temperature-sensitive DNA polymerases, primers, probes and other components, which require cold-chain transportation. While increasing the cost of reagents, it is very likely that the cold chain will fail during transportation, resulting in reagent deterioration and failure.

Method used

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  • Freeze-drying PCR reagent for detecting shrimp seed enterocytozoon hepatopenaei
  • Freeze-drying PCR reagent for detecting shrimp seed enterocytozoon hepatopenaei
  • Freeze-drying PCR reagent for detecting shrimp seed enterocytozoon hepatopenaei

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Preparation of freeze-dried PCR reagents for detecting Enterocystis hepatica in shrimp larvae

[0024] 1. Primer Design

[0025] The applicant used the conserved sequence of the gene sequence of Enteroplasma hepatica SWP1 (spore wall protein 1) as a target, and used the primer design software "Beacon designer 8" to design primers and oligonucleotide fluorescent probes: oligonucleotide upstream primer sequence is "5'-CACTGTAAACCTTAAAGCA-3'", the oligonucleotide downstream primer sequence is "5'-TCTCCAACTGTATTTGAAAG-3'", and the oligonucleotide fluorescent probe sequence is "5'-AGAGACGATATTTACACAGACACAGCA-3'", The 5' end of the oligonucleotide fluorescent probe is labeled with FAM, and the 3' end is labeled with BHQ1. Wherein, FAM represents carboxyfluorescein (6-carboxy-fluorescein), as the fluorescent group of the oligonucleotide fluorescent probe of the present embodiment, BHQ1 represents Black Hole Quencher 1, as the oligonucleotide of the present embodiment Quenche...

Embodiment 2

[0032] Detection of Hepatic Enterocystis in Shrimp Larvae

[0033] 1. DNA extraction

[0034] Prepare three samples of shrimp seedlings infected with Enterocystis hepatica and one sample of healthy shrimp seedlings, take 30 mg of shrimp seedlings into sterile centrifuge tubes, and use the Aquatic Animal Virus DNA / RNA Extraction Kit (Spin Column Method) (Guangzhou Hua Peak Biotechnology Co., Ltd.) to extract DNA, and the extracted DNA was labeled as EHP sample 1, EHP sample 2 and EHP sample 3 in sequence.

[0035] 2. Reconstitution of freeze-dried PCR reagents

[0036] The complex solution was prepared according to the following formula: 0.05 μmol magnesium ion, 1.25 μmol Tris, 2.25 μmol potassium ion, 0.1% (w / v) Tween 20.

[0037] The freeze-dried PCR reagent used in this example is the freeze-dried PCR reagent prepared in Example 1 for detecting Enterococcus hepatica of shrimp larvae. Add reconstitution solution to the lyophilized PCR reagent to restore the lyophilized rea...

Embodiment 3

[0047] Long-term Stability Test of Freeze-dried PCR Reagents

[0048] The freeze-dried PCR reagents used in this example are all freeze-dried PCR reagents prepared according to Example 1. Add 22.5 μl of the complex solution prepared according to Example 2 to the freeze-dried PCR reagents stored at 45°C for one, two, four weeks and at 25°C for three, six, and twelve months and the freshly prepared freeze-dried PCR reagents respectively, and then respectively Add 2.5 μl of Enterocystis hepatica DNA and carry out PCR amplification. The PCR amplification program is: react at 95° C. for 1 minute; react at 95° C. for 5 seconds; react at 60° C. for 20 seconds, a total of 40 cycles.

[0049] PCR amplification test results such as figure 2 As shown, the amplification curves obtained by using freshly prepared freeze-dried PCR reagents were used as controls, and the amplification curves of freeze-dried PCR reagents stored at 25°C for three, six, and twelve months and 45°C for one or tw...

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Abstract

The invention discloses a freeze-drying PCR reagent for detecting shrimp seed enterocytozoon hepatopenaei. The freeze-drying PCR reagent comprises TaqDNA polymerase, a freeze-drying protective agent,an oligonucleotide upstream primer, an oligonucleotide downstream primer and an oligonucleotide fluorescence probe. A sequence of the oligonucleotide upstream primer is 5'-CACTGTAAACCTTAAAGCA-3', a sequence of the oligonucleotide downstream primer is 5'-TCTCCAACTGTATTTGAAAG-3', and a sequence of the oligonucleotide fluorescence probe is 5'-AGAGACGATATTTACACAGACACAGCA-3'. The freeze-drying PCR reagent for detecting the shrimp seed enterocytozoon hepatopenaei can tolerate a higher temperature, is convenient in transportation and storage, and capable of realizing sensitive, specific, rapid and convenient detection on the shrimp seed enterocytozoon hepatopenaei, and has a good application prospect.

Description

technical field [0001] The invention relates to the field of detection of marine biological pathogens, in particular to a freeze-dried PCR reagent for detecting enterococcus hepatica of shrimp larvae, its reconstituted solution and its application. Background technique [0002] Enterocytozoon hepatopenaei (EHP), a microsporidia found in prawns in recent years, mainly infects important cultured shrimps such as Litopenaeus vannamei and Penaeus monodon. E. hepatica was first isolated and named in 2009 from slow-growing P. monodon cultured in Thailand. Infection with Enterocystis hepatica causes slow growth of prawns, because the sick prawns are still eating feed, so farmers will waste time and feed on these small prawns, making it difficult to obtain effective economic benefits. An effective method for preventing and resisting Enteroplasma hepatica is to strictly screen and detect the seedlings to avoid mixing the shrimp seedlings carrying pathogens into the breeding ponds. T...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686
CPCC12Q1/686C12Q1/6895
Inventor 熊槐黄昱阳肖周婷李会琴马春平刘阳
Owner GUANGZHOU HUAFENG BIOTECH
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