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RPA-LED visualized reagent kit for detecting porcine epidemic diarrhea viruses

A porcine epidemic diarrhea and kit technology, applied in DNA/RNA fragments, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of limiting the popularization and application of grassroots and clinical testing, complex primer design, etc. Easy-to-use effects

Inactive Publication Date: 2019-04-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RPA technology has great advantages in gene amplification, but primer design is more complicated
[0008] The deficiencies of the above methods limit the popularization and application in primary and clinical testing.

Method used

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  • RPA-LED visualized reagent kit for detecting porcine epidemic diarrhea viruses
  • RPA-LED visualized reagent kit for detecting porcine epidemic diarrhea viruses
  • RPA-LED visualized reagent kit for detecting porcine epidemic diarrhea viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Preparation of Recombinase Polymerase Isothermal Amplification Primer Set for Detection of Porcine Epidemic Diarrhea Virus

[0076] This embodiment discloses the design and screening process of primers and probes.

[0077] The M gene is a relatively conserved gene in the PEDV genome. By comparing and analyzing the homologous sequences of M gene of 18 strains published in Genbank, the conserved region of M gene of porcine epidemic diarrhea virus was determined and used as a template for RPA primer design. The primer length requirement of RPA is 30-35bp, the amplification product is within 500bp, and the amplification efficiency is high. To establish a rapid and sensitive detection method, it is necessary to screen multiple primers, and the individual base differences of primers will affect the amplification effect. Therefore, in the conserved gene region, a series of gradient candidates are designed, and pairwise combinations of them are used. Choose the best...

Embodiment 2

[0084] Example 2 Optimization of Reverse Transcription Recombinase Polymerase Isothermal Amplification Reagent for Detection of Porcine Epidemic Diarrhea Virus and Assembly of Kit

[0085] The reagents included recombinase polymerase, dNTPs, and the primer set described in Example 1.

[0086] A reverse transcriptase recombinase polymerase isothermal amplification kit for detecting porcine epidemic diarrhea virus, the kit contains the reagents described in Example 1 or as described above.

[0087] The kit also includes hydrolysis buffer and magnesium acetate solution. The magnesium acetate solution is a 280 mM magnesium acetate solution.

[0088] The nucleic acid detection test strip is a universal nucleic acid detection test strip.

[0089] The kit includes a fully enclosed target nucleic acid rapid detection device. The fully enclosed target nucleic acid rapid detection device is obtained by placing nucleic acid detection test strips into a palm-top plastic detection devic...

Embodiment 3

[0124] Example 3 Optimization of reaction conditions of PEDV RPA-LFD detection method

[0125] (1) First, we determined the optimal reaction temperature of the RPA-LFD detection method. We tested different temperatures at 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, and 50°C, respectively. temperature, and the reaction time was set to 20 min. The test results are as Figure 4 As shown, under the condition of 37°C, the detection line developed the brightest color, that is, the amount of the reaction product was the largest. It shows that the optimal reaction temperature of this test is 37°C, therefore, we choose 37°C as the reaction temperature of this detection method.

[0126] (2) Secondly, we determined the effect of different incubation times on the amplification results at 37°C. We tested the experimental results of 5min, 10min, 15min, 20min, 25min, 30min, and 35min. The test results are as follows Figure 5 As shown, a weak band appeared on the test strip when the a...

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Abstract

The invention relates to the field of virus detection, in particular to a RPA-LED visualized reagent kit for detecting porcine epidemic diarrhea viruses. The invention provides a recombinase and polymerase isothermal amplification primer set for detecting the porcine epidemic diarrhea viruses. The reagent kit comprises an upstream primer, a downstream primer and a probe, of which the sequences areshown as SEQ ID NO: 1-3. The invention further provides a reverse transcription recombinase polymerase isothermal amplification reagent for detecting the porcine epidemic diarrhea viruses. The reverse transcription recombinase polymerase isothermal amplification reagent comprises recombinase polymerase, dNTPs and the primer set. In addition, the invention provides a reverse transcription recombinase polymerase isothermal amplification reagent kit for detecting the porcine epidemic diarrhea viruses, and the reagent kit comprises the primer set or reagent. The detection reagent kit is high in specificity and high in sensitivity, results are easy to observe, complex equipment is not needed, and portable on-the-site quick nucleic acid detection of the porcine epidemic diarrhea viruses can bereally realized.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to an RPA-LED visualization kit for detecting porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (PED) is an acute intestinal infectious disease caused by Porcine epidemic diarrhea virus (PEDV). The main clinical features are diarrhea, vomiting and dehydration. Its clinical symptoms, pathological changes It is very similar to porcine transmissible gastroenteritis virus (TGEV) and difficult to distinguish. PEDV infection can occur in pigs of different ages, and the most serious infection is piglets, with morbidity and mortality often reaching 100%. PEDV has been widely spread in pig herds in my country. In October 2010, the mutation of PEDV caused a large-scale outbreak of PED in my country, resulting in the death of a large number of piglets and causing huge economic losses to my country's pig industry. [0003] PEDV is an enveloped single-stran...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2565/625
Inventor 赵明秋李晓明张静远张媛媛易琳陈金顶丁红星
Owner SOUTH CHINA AGRI UNIV
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