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Reaction system and kit for detecting African swine fever virus nucleic acid and application thereof

A technology of African swine fever virus and reaction system, applied in the direction of microbe-based methods, microbiological measurement/inspection, biochemical equipment and methods, etc., can solve the problems of low sensitivity and sensitivity, long reaction time, etc., and achieve the goal of equipment The effect of low requirements and fast detection speed

Pending Publication Date: 2020-06-09
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the low sensitivity and sensitivity to African swine fever virus (ASFV) nucleic acid detection in the prior art, and the defects such as long reaction time, the invention provides a kind of detection method for African swine fever virus (ASFV) nucleic acid Reaction system, kit and its application, using SHERLOCK technology to establish a rapid detection method for ASFV nucleic acid, which has high specificity and sensitivity, and provides a fast, simple and accurate detection method for the diagnosis of ASFV infection

Method used

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  • Reaction system and kit for detecting African swine fever virus nucleic acid and application thereof
  • Reaction system and kit for detecting African swine fever virus nucleic acid and application thereof
  • Reaction system and kit for detecting African swine fever virus nucleic acid and application thereof

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Experimental program
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Effect test

Embodiment 1

[0061] The design of embodiment 1-RPA primer and crDNA and the synthesis of crRNA

[0062] (1) Design of RPA primers and crDNA

[0063]Vector NTI Suite software was used to analyze the conserved sequence of the P72 protein gene of ASFV isolated from different countries and regions, and Primer Express software was used to design primers and crDNA. Primers and crDNA were synthesized by Shanghai Sangon Biotechnology Co., Ltd. Primer with DEPC-H 2 O dilute the primers to 10 μM and store at -20°C for later use.

[0064] Table 1 Primer pairs and crDNA combinations

[0065]

[0066] (2) Using crDNA to synthesize crRNA

[0067] Use the HiScribe T7 Rapid RNA Synthesis Kit and use the crDNA provided in Table 1 as a template to prepare a 20 μL reaction system:

[0068]

[0069] React at 37°C for 4 hours to obtain crRNA, freeze-dry and store at -20°C, and add RNase-free water until the crRNA concentration is 4 μM.

Embodiment 2

[0070] The establishment and optimization of embodiment 2-reaction system

[0071] 1. Nucleic acid extraction and cDNA template preparation

[0072] For DNA virus samples, take 100 μL of tissue supernatant or body fluid samples, carry out virus DNA extraction according to the instructions of DNA extraction kits (commercially available, such as kits purchased from TIANGEN), and finally dissolve the DNA with 50 μL of water, The amplified template is obtained; for RNA virus samples, 100 μL of supernatant or body fluid is taken, RNA is extracted by TRIzol method, and then reverse-transcribed to obtain cDNA template.

[0073] The specific steps of DNA extraction are as follows:

[0074] Add 700 μL of 65°C preheated buffer 1 centrifuge tube to 100 μL of tissue supernatant or body fluid sample, mix well and place the centrifuge tube in a 65°C water bath for 20 minutes. During the water bath, invert the centrifuge tube to mix the sample several times;

[0075] Add 700 μL of chlorofo...

Embodiment 3

[0102] Embodiment 3-based on the sensitivity comparison of Cas12a and Cas13a detection method

[0103] To test the sensitivity of the established detection method based on Cas12a and Cas13a, the ASFV positive plasmid with a concentration of 1pg / μL was used as a template, and the negative control was set as ddH 2 O, and then use the Cas12a and Cas13a detection methods to detect. See the test results figure 2 , the results showed that the fluorescent signal intensity of Cas13a-based detection of ASFV plasmid was higher than that of Cas12a-based detection method, and had higher sensitivity.

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Abstract

The invention relates to a reaction system for detecting African swine fever virus nucleic acid. The reaction system is an SHERLOCK reaction system. The system comprises an RPA primer pair for amplifying a target nucleic acid fragment and / or crRNA, the crRNA is used for combining ssRNA transcribed by the amplified target nucleic acid fragment, the primer pair comprises primers with sequences shownas SEQ ID NO.1 and SEQ ID NO.2, the crRNA is synthesized from crDNA, and the sequence of the crDNA is shown as SEQ ID NO.3. The system further comprises Cas13a, a fluorescence labeling probe and thelike, the Cas13a is combined with the crRNA combined with the ssRNA so as to have RNA enzyme activity, the Cas13a with the RNA enzyme activity cuts the fluorescence labeling probe to generate fluorescence, and the generated fluorescence can be detected in real time. The invention also relates to a kit comprising the reaction system and related application of the RPA primer pair and / or the crRNA. The kit and a detection method are used for detecting the African swine fever virus nucleic acid, can realize constant-temperature detection at 37 DEG C, have short reaction time, high detection speed,and high sensitivity and the specificity.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering, in particular to a reaction system for detecting African swine fever virus (ASFV) nucleic acid, a kit and an application thereof. Background technique [0002] African swine fever is a highly contagious, explosive and highly fatal infectious disease causing hemorrhagic symptoms in domestic and wild boars. African swine fever spreads rapidly, causing serious socio-economic crisis, and is an important surveillance disease in the international trade of animals and animal products. The pathogen of African swine fever is African swine fever virus (ASFV), which is a member of the African swine fever virus family of African swine fever virus. ASFV is an enveloped double-stranded DNA virus. In the wild, it is mainly transmitted by soft ticks, but in domestic environments it can also be transmitted independently of soft ticks. More than 20 ASFV genotypes have been identified by se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/327C12Q2521/507C12Q2522/101C12Q2531/119C12Q2563/107
Inventor 薛俊欣李健熊炜张强林颖峥蒋原
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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