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PPV (porcine parvovirus) LAMP (loop-mediated isothermal amplification) kit and application thereof

A loop-mediated isothermal, parvovirus technology, applied in the field of microbial detection, can solve the problems of false positive results of laboratory contamination, high cost, long time required, etc., and achieve the effect of simple interpretation, rapid acquisition, and easy operation.

Pending Publication Date: 2016-02-24
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional method is the isolation and identification of viruses, and the results are accurate and reliable, but the conventional identification method has the disadvantages of complex operation, complex equipment and long time, which is not conducive to the rapid diagnosis of porcine parvovirus disease
The method of molecular biology is to detect the specific gene of porcine parvovirus virus by polymerase chain reaction (PCR). Although the PCR method is faster and more accurate than the conventional method, it needs to design specific primers and expensive equipment. The cost is high, and agarose gel electrophoresis is required to determine the results, which is likely to cause laboratory pollution and lead to false positive results, and is not suitable for grassroots and on-site testing

Method used

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  • PPV (porcine parvovirus) LAMP (loop-mediated isothermal amplification) kit and application thereof
  • PPV (porcine parvovirus) LAMP (loop-mediated isothermal amplification) kit and application thereof
  • PPV (porcine parvovirus) LAMP (loop-mediated isothermal amplification) kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0082] The specificity result of embodiment 1LAMP detection method

[0083] LAMP amplification was performed on 1 strain of porcine parvovirus, 7 strains of control virus and water control, and the results were as follows figure 1 As shown, the porcine parvovirus reaction tube showed a rising curve of turbidity in about 13 minutes, which was a positive result, and the curves of the 7-strain control virus reaction tube and the water control reaction tube showed no amplification, which was a negative result.

Embodiment 2

[0084] The sensitivity result of embodiment 2LAMP detection method

[0085] The initial concentration of porcine parvovirus original DNA was 2.16×10ng / μL. After 10-fold serial dilution, LAMP and PCR amplification were performed, and the results were as follows: figure 2 and image 3 As shown, the results show that the detection limit of the LAMP method of the present invention is about 2.16×10-10 ng / μL, while the detection limit of the conventional PCR method is 2.16×10-8 ng / μL.

Embodiment 3

[0086] Fluorescence visualization detection result of embodiment 3 LAMP detection method

[0087] According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added to the reactor, and after 60 minutes of reaction at 63°C, observed under ultraviolet light, Figure 4 To observe the results, the left tube is the reaction with the porcine parvovirus genome DNA as the template, which is a positive result, and the right tube is a negative control, which is a negative result. The test results show that the established LAMP method can be used conveniently at the grassroots level. You only need to use the kit with the LAMP primer designed by this method, add the sample, and use an inexpensive water bath to keep it at 63°C for 60 minutes to quickly observe the results without the need for Open the lid to avoid contamination.

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Abstract

The invention discloses a PPV (porcine parvovirus) loop-mediated isothermal amplification kit and application thereof. The kit comprises LAMP primers, a 2*reaction buffer solution, Bst DNA polymerase, a fluorescent visual detection reagent, ultrapure water and a PPV DNA template, wherein the LAMP primers comprise outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The application lies in that the kit is used for detecting PPV lesion tissue samples and carrying out qualitative research on PPV pathogens. Detection and sensitivity detection results prove that the LAMP detection method provided by the invention can monitor the reaction in real time, and quantitatively detect the copy number of PPV, quickly and accurately obtains the detection result, and brings convenience to easy, convenient, quick and reliable PPV detection.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a fast, visualized and real-time quantitative detection of porcine parvovirus loop-mediated isothermal amplification kit and its application. Background technique [0002] Porcine parvovirus (Porcine Parvovirus, PPV) is one of the main pathogens that cause reproductive disorders in sows. It is characterized in that when pregnant pigs are infected in the early stages of pregnancy, the embryos or fetuses are invaded through the placenta, causing sows to abort, embryos to die, and fetuses to die. Deformation, fetal mummification and infertility, etc., can also cause dermatitis and diarrhea in piglets, and a large number of newborn piglets die. [0003] PPV was first discovered as a small DNA-contaminated virus from the cells of porcine parvovirus. In 1967, Carwtright et al. isolated PPV from infertility, abortion and stillbirth pigs, and later found that the virus was ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q2531/119C12Q2563/107
Inventor 陈忠伟何颖赵武卢冰霞梁家幸秦毅斌段群棚李斌周英宁蒋冬福杨思仪苏乾莲
Owner GUANGXI VETERINARY RES INST
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