Kit for detecting porcine pseudorabies virus (PRV) by combining centrifugal micro-fluidic chip with loop-mediated isothermal amplification (LAMP) technology

A technology of porcine pseudorabies virus and microfluidic chip, which is applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the unfavorable rapid detection of porcine pseudorabies virus and the lack of POCT detection products of porcine pseudorabies virus and other issues, to achieve efficient real-time detection, good practical significance, and broad market prospects

Inactive Publication Date: 2021-01-05
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented describes an improved way to diagnose Porcoidum parvovirus (PPR) disease caused by bacteria called Pseudoraimus or Pasteurium spp., through testing different sequence types from genome DNA found within these organisms. By doing that we have developed a simpler but more reliable process than previous methods such as PCR. Overall, our new approach provides faster and better ways to identify diseases associated with Psoriasis without requiring expensive specialized tests like ELISA.

Problems solved by technology

This patented describes different techniques for identifying and measuring PPRV antibodies in order to prevent veterinary issues such as severe respiratory syndrome associated with swines due to this type of virus. However current technologies require expensive instruments like electronics and advanced analyzer systems, making their practicality limited at home settings where they may lack accessibility. There exists a new method for quick identification of PPRvims while reducing costs compared to existing methods.

Method used

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  • Kit for detecting porcine pseudorabies virus (PRV) by combining centrifugal micro-fluidic chip with loop-mediated isothermal amplification (LAMP) technology
  • Kit for detecting porcine pseudorabies virus (PRV) by combining centrifugal micro-fluidic chip with loop-mediated isothermal amplification (LAMP) technology
  • Kit for detecting porcine pseudorabies virus (PRV) by combining centrifugal micro-fluidic chip with loop-mediated isothermal amplification (LAMP) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This example describes the microfluidic chip used in the present invention.

[0050] Such as 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 are injection holes and exhaust holes; each reaction detection area is equipped with 4 amplification pools.

[0051] 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 realizes the LAMP reaction; the capillary microvalve 4 mainly utilizes the resistance of the capillary...

Embodiment 2

[0053] This example is the preparation of the LAMP primer composition and positive standard used in the present invention for detecting porcine pseudorabies virus.

[0054] The steps of its design and synthesis are as follows:

[0055] Firstly, the full-length genome sequences of all PRVs 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 PRV genome. According to the obtained target sequence, use PrimerExplorer V5 to design the above LAMP primers and synthesize them;

[0056] 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 as SEQ ID NO: 1-5.

[0057] SEQ ID NO: 1

[0058] F3: CCGTGCTC...

Embodiment 3

[0069] This embodiment is the primer composition and kit used in the present invention for detecting porcine pseudorabies virus.

[0070] The kit involved in the present invention includes the microfluidic chip described in Example 1, the porcine pseudorabies virus primer set described in Example 2 and the positive standard for porcine pseudorabies virus LAMP method, and a constant temperature amplification premix .

[0071] For the primer set of porcine pseudorabies 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.

[0072] 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.).

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Abstract

The invention belongs to the technical field of molecular detection, discloses a kit for detecting porcine pseudorabies virus (PRV) by combining a centrifugal micro-fluidic chip with a loop-mediated isothermal amplification (LAMP) technology, and establishes a PRV nucleic acid detection method which is rapid, sensitive, high in accuracy and high in repeatability by combining the micro-fluidic chiptechnology. The micro-fluidic chip detection method is high in specificity, and the lowest detection limit can reach 10<2>copies/[mu]L. According to the micro-fluidic chip kit for detecting porcine pseudorabies virus (PRV), a chip analysis device is small and portable, and the defects of time consumption, labor consumption and high cost of PRV detection can be overcome, so that the detection sensitivity and specificity are improved, the labor and equipment cost is reduced, the detection period is shortened, and the requirements of field detection are met. The rapid detection technology can bepopularized and applied to epidemiological investigation and epidemic situation monitoring of the porcine pseudorabies virus (PRV), and has good practical significance and wide market prospects.

Description

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Claims

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Application Information

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Owner SOUTH CHINA AGRI UNIV
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