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A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses

A recombinase polymerase, African swine fever virus technology, applied in recombinant DNA technology, microbial assay/inspection, microorganism-based methods, etc. Cold chain transportation, easy to carry, small size

Active Publication Date: 2017-03-22
HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report of ASF in my country, but there is a border line of more than 4,300 kilometers between my country and Russia. At the same time, China-Africa trade and ties have been further strengthened, and the risk of African swine fever in my country has further increased.

Method used

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  • A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses
  • A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses
  • A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Specificity and sensitivity test of ASFV real-time RPA method

[0028] 1.1 Main reagents

[0029] TwistAmp TM DNA amplification kit was purchased from TwistDx Company in the UK; viral DNA extraction kit and DNA purification and recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; PrimeScript Ⅱ 1st strand cDNA Synthesis Kit, Premix Ex Taq, competent cell DH5α were purchased from From Takara Company; other required reagents are provided by our laboratory.

[0030]1.2 Main instruments

[0031] Isothermal amplification fluorescence detector (Genie III), British Optigene Company; real-time fluorescent quantitative PCR instrument (ABI7500), American ABI Company; nucleic acid protein analyzer (BioPhotometer Plus), Germany Eppendorf Company; small desktop centrifuge (SIGMA 1-14 ), Sigma Company of Germany; electric heating constant temperature water tank (DK-8D), Shanghai Yiheng Technology Co., Ltd.

[0032] 1.3 P72 gene recombinan...

Embodiment 2

[0048] Embodiment 2 is to the detection of clinical simulation sample

[0049] The 35 clinical whole blood samples were all from pig farms in different regions of Hebei, and were confirmed negative for ASFV by the real-time fluorescent PCR method recommended by OIE. will be 10 7 copies~10 2 Copies P72 gene recombinant plasmid was added to 35 samples of 200 μL pig whole blood respectively, 5 samples of each concentration were paralleled, mixed thoroughly, and the remaining 5 samples were not treated as a control. Viral DNA was extracted using Tiangen Viral DNA Extraction Kit, using 30 μL ddH 2 O was dissolved, and 3 μL was taken as a template for real-time fluorescent PCR and real-time RPA detection. The experimental results are shown in Table':

[0050] Table 2 Comparison of ASFV real-time RPA and real-time fluorescent PCR methods for the detection of simulated samples

[0051]

[0052]

[0053] TT: threshold time; Ct: threshold period

[0054] ND: No amplification...

Embodiment 3

[0057] The composition of embodiment 3 detection kits

[0058] Products in the kit (50 times):

[0059] Forward primer, concentration 10μM 110μL

[0060] Reverse primer, concentration 10μM 110μL

[0061] Probe, concentration 10μM 35μL

[0062] 50 0.2ml tube tubes containing polymerase, recombinant enzyme and single-chain binding protein lyophilized enzyme preparation

[0063] MgAc, concentration 280mM 140μL

[0064] Buffer 2ml

[0065] Distilled water 5ml.

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Abstract

A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses is disclosed. A forward primer sequence for detecting the African swine fever viruses through a method provided by the kit is shown as SEQ ID NO:1, a reverse primer sequence is shown as SEQ ID NO:2 and a probe sequence is shown as SEQ ID NO:3. A real-time isothermal recombinase-polymerase amplification method provided by the invention for ASFV detection is simple and convenient in operation, rapid in reaction and low in detection cost, can be used for ASFV detection in a laboratory and on site, particularly ASFV detection in quarantine ports, airports and epidemic disease outbreak sites, and provides a novel and reliable technique support for ASF control in China.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for rapidly detecting African swine fever virus by using real-time isothermal recombinase polymerase amplification and a corresponding detection kit. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious infectious disease of pigs caused by African swine fever virus (ASFV), which can infect domestic pigs of all breeds and ages and wild boar. The high morbidity and mortality of ASF, the huge economic loss caused by restrictions on international trade, and the current lack of effective vaccines make ASF one of the most serious diseases that endanger the swine industry. ASFV is currently the only member of the African swine fever virus family (Asfarviridae), the African swine fever virus genus (Asfivirus), and the only known DNA arbovirus. The ASFV genome is a linear dsDNA molecule with a size of 170-193 kb...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2561/113C12Q2521/507C12Q2563/107
Inventor 王建昌王金凤刘立兵孙晓霞马付坤闫慧张静依
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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