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Kit used for detecting porcine Japanese encephalitis virus by combining centrifugal micro-fluidic chip technology with loop-mediated isothermal amplification technology

A microfluidic chip and porcine Japanese encephalitis technology, applied in recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., can solve unfavorable rapid detection of porcine Japanese encephalitis, lack of POCT detection products, etc. problems, to achieve efficient and real-time detection, reduce labor and equipment costs, and shorten the detection cycle

Inactive Publication Date: 2021-01-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The existing technology lacks POCT detection products related to porcine Japanese encephalitis virus, which is not conducive to the rapid detection of porcine Japanese encephalitis virus

Method used

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  • Kit used for detecting porcine Japanese encephalitis virus by combining centrifugal micro-fluidic chip technology with loop-mediated isothermal amplification technology
  • Kit used for detecting porcine Japanese encephalitis virus by combining centrifugal micro-fluidic chip technology with loop-mediated isothermal amplification technology
  • Kit used for detecting porcine Japanese encephalitis virus by combining centrifugal micro-fluidic chip technology with loop-mediated isothermal amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] This example describes the microfluidic chip used in the present invention.

[0052] like figure 1 As shown, the microfluidic chip used in the present invention is a disc-shaped microfluidic chip, which is produced by Shanghai Suchuang Diagnostic Products Co., Ltd., and its model is 8×4, which includes 8 reaction detection areas 1, Each reaction detection zone 1 includes a liquid storage pool 2, a distribution pool 5, a capillary microvalve 4, and an amplification pool 3 that are connected in sequence. There is a sample injection hole, and the distribution pool 5 is connected with the waste liquid pool and the exhaust hole through an arc channel; each reaction detection area 1 is equipped with 4 amplification pools.

[0053] Wherein the main function of the liquid storage pool 2 is to load the reaction solution; the main function of the distribution pool 5 is to evenly distribute the reaction solution to the amplification pool 3; the amplification pool 3 specifically r...

Embodiment 2

[0055] This example is the preparation of the LAMP primer composition and positive standard used in the present invention for detecting porcine Japanese encephalitis virus.

[0056] The steps of its design and synthesis are as follows:

[0057] Firstly, the full-length genome sequences of all JEVs were retrieved from the Genbank database, and the homology analysis was performed by BLAST software to find the relatively conserved target gene sequences of the JEV genome. According to the obtained target sequence, use PrimerExplorer V5 to design the above LAMP primers and synthesize them;

[0058] The third step is to screen the primer combination: after dissolving the synthesized primers, perform primer screening, and finally obtain the specific and sensitive primer combination required for the microfluidic chip. The primers are respectively outer primer F3 / B3, inner primer FIP / BIP, and loop primer LF, and the sequences are shown in SEQ ID NO: 1-5.

[0059] The sequence of the ...

Embodiment 3

[0072] This example is the primer composition and kit used in the present invention for detecting porcine Japanese encephalitis virus.

[0073] The kit involved in the present invention includes the microfluidic chip described in Example 1, the porcine Japanese encephalitis virus primer set and porcine Japanese encephalitis virus LAMP method positive standard described in Example 2, and also includes a constant temperature Amplification master mix, reverse transcriptase solution, RNase inhibitor.

[0074] For the primer set of porcine Japanese encephalitis virus, the preferred molar ratio of the five primers is outer primer F3 / B3: inner primer FIP / BIP: loop primer LF is 1:8:4.

[0075] For the constant temperature amplification master mix, it is a reaction solution containing 800 U / mL of Bst DNA polymerase and 50 μM fluorescent dye SYBRGreen (provided by Shanghai Suchuang Diagnostic Products Co., Ltd.).

[0076] For the reverse transcriptase solution, the solvent is water, th...

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Abstract

The invention belongs to the technical field of molecular detection, discloses a kit used for detecting a porcine Japanese encephalitis virus by combining a centrifugal microfluidic chip technology with a loop-mediated isothermal amplification technology. A Japanese encephalitis virus (JEV) nucleic acid detection method rapid, sensitive, high in accuracy and strong in repeatability is establishedby designing and screening specific primers, and combining with the microfluidic chip technology. The micro-fluidic chip detection method provided by the invention is high in specificity, and the lowest detection limit can reach 10<1> copies / [mu]L. According to the micro-fluidic chip kit for detecting the porcine Japanese encephalitis virus provided by the invention, a chip analysis device is small and portable, the defects that JEV detection is time-consuming, labor-consuming and high in cost can be remedied, the detection sensitivity and specificity are improved, the labor and equipment costis reduced, the detection period is shortened, and the requirements of field detection are met; and the method can be popularized and applied to epidemiological investigation and epidemic situation monitoring of the porcine Japanese encephalitis virus, and has good practical significance and broad market prospects.

Description

technical field [0001] The present invention relates to the technical field of molecular detection, more specifically, to a kit for the detection of porcine Japanese encephalitis virus by combining a centrifugal microfluidic chip combined with a loop-mediated isothermal amplification technology, in particular to a microfluidic chip, A constant temperature microfluidic chip analysis device and a microfluidic detection method combined with a LAMP technology kit for detecting porcine Japanese encephalitis virus. Background technique [0002] Japanese encephalitis (JE), also known as Japanese encephalitis, is an acute zoonotic infectious disease caused by Japanese encephalitis virus (JEV). Mosquitoes are used as the medium of transmission, and pigs are the main storage host of JEV, which can amplify JEV. When the disease occurs, it can cause abortion, stillbirth or mummified fetuses in sows, and it can also cause orchitis or infertility in boars. cause huge economic losses. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2565/629C12Q2521/107C12Q2527/127C12Q2563/107C12Q2545/113
Inventor 孙媛陈咏辉周玲李倩妞蓝天马静云卢先东刘艳红
Owner SOUTH CHINA AGRI UNIV
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