LAMP primer group for detecting porcine circovirus type 3, kit and application thereof

The technology of a porcine circovirus and a kit, applied in the biological field, can solve problems such as long reaction time, low sensitivity, and failure to meet basic needs, and achieve the effects of rapid response, high sensitivity, and easy wide-scale application

Inactive Publication Date: 2018-04-06
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The primer set of the patented loop-mediated isothermal amplification method for detecting porcine circovirus type 3 (application number: 201710757261.1, application date: 2017-08-29; publication number: CN107312875A, publication date: 2017-11-03) combined LAMP The technology is applied to the detection of PCV3, but the method requires a long reaction time (60min) and low sensitivity (76ng / μL), which still cannot meet the basic needs of rapid and sensitive detection methods at entry-exit ports or veterinary grassroots

Method used

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  • LAMP primer group for detecting porcine circovirus type 3, kit and application thereof
  • LAMP primer group for detecting porcine circovirus type 3, kit and application thereof
  • LAMP primer group for detecting porcine circovirus type 3, kit and application thereof

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Experimental program
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Effect test

Embodiment 1

[0046] Design and synthesis of embodiment 1LAMP primer

[0047] According to the principle of primer design, aiming at the conserved region sequence of porcine circovirus type 3 ORF2 gene, the LAMP primer online design software PrimerExplorer V5 was used to design the internal and external primers. According to our experience, the GC content of the primers was guaranteed to be between 40% and 60%. %between. After empirically screening the primer set with the optimal theoretical value, continue to use the software PrimerExplorer V5 to design loop primers. The sequences are shown in Table 1. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The synthesized primers were diluted with sterile triple distilled water to a concentration of 10 μM and stored at -20°C.

[0048] Table 1 LAMP detection primer set for porcine circovirus type 3

[0049]

[0050]

Embodiment 2

[0051] Application of embodiment 2 LAMP primers in detecting the virus to be tested

[0052] 1. Preparation of Nucleic Acid Standards

[0053] After measuring the nucleic acid concentration of the porcine circovirus type 3 ORF2 gene synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., dilute it with ultrapure water to 10 5 The concentration of copies / μL was used as a nucleic acid standard.

[0054] 2. Establishment of LAMP detection method

[0055] With a concentration of 10 5 The copy / μL porcine circovirus type 3 ORF2 gene was used as a template, and a set of primers (as shown in Table 1) with the optimal theoretical value obtained by screening was used for cross-primer amplification, and the obtained product was subjected to agarose gel ( Mass volume ratio 2%) electrophoresis detection.

[0056] 3. Optimization of LAMP reaction conditions

[0057] 1. Optimization of reaction temperature

[0058] In order to obtain the optimal reaction temperature...

Embodiment 3

[0074]Example 3 Suspected PCV3 infected pig disease material tissue detection

[0075] Collect 6 lungs of suspected PCV3-infected pigs from the clinic, take 2-3 mg of clinical disease material, grind them thoroughly and add 1 ml of normal saline. Freezing and thawing were repeated 3 times, and the supernatant was removed by centrifugation. Aspirate 200 μL of the supernatant, and extract the nucleic acid of the virus according to the operating instructions of the viral RNA / DNA extraction kit. Take 1 μL of the extracted nucleic acid for LAMP detection (the reaction system is shown in Table 2, except that the template is changed; the reaction conditions are the optimized conditions determined in step 1) and PCR detection. The result is as Figure 7 and 8 As shown, both the LAMP detection method and the PCR agarose gel electrophoresis detection method can detect 5 positive disease materials, and the coincidence rate of the two is 100%.

[0076] In the present invention, the ke...

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Abstract

The invention discloses an LAMP primer group for detecting porcine circovirus type 3, a kit and application. The primer group contains a primer group with nucleotide sequences shown in SEQ ID NO.1-6.The kit comprises the primer group and a reaction reagent. An application method of the kit comprises the following steps: firstly preparing a cross primer amplification reaction system; and carryingout isothermal reaction, and directly interpreting the obtained products with naked eyes, wherein a positive result is yellow, and a negative result is red. The kit disclosed by the invention is simple to operate, low in cost, rapid in reaction and good in specificity, and results can be easily observed, so that the kit is especially applicable to field detection of export quarantine, food hygieneand livestock farms.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP primer set, kit and application for detecting porcine circovirus type 3. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is an important member of the Circoviridae family. Before 2016, it was generally believed that porcine circovirus included two genotypes, PCV1 and PCV2. PCV1 was found in a porcine kidney epithelial cell line (PK-15) and is not pathogenic to pigs. PCV2 is one of the pathogens that seriously endanger the pig industry. The diseases related to PCV2 include multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, respiratory system diseases, reproductive disorders and intestinal diseases in weaned piglets. [0003] In 2016, Phan T.G. et al. reported the prevalence of a new genotype of circovirus - porcine circovirus type 3 (PCV3) in the United States, and it was also detected in some sows with reproductive d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 勾红潮李春玲蒋智勇蔡汝健宋帅李艳楚品品杨冬霞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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