LAMP primer combination, detection method and kit capable of distinguishing porcine circovirus type 2 and type 3 typing detection

A technology of porcine circovirus and primer combination, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve problems such as laboratory pollution, aerosol pollution, false positive results, etc., and achieve specificity Good, avoid error, high sensitivity effect

Inactive Publication Date: 2019-11-08
CAPITALBIO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, most of the currently established LAMP reaction methods require agarose gel electrophoresis or need to add a chromogenic reagent after opening the cover, which is likely to cause laboratory contamination or lead to false positive results.
In addition, most of the chromogenic methods used by the LAMP method for direct observation are to open the cover after the reaction and add fluorescent dyes for color reaction, and observe whether there is color development to interpret the test results, which will cause aerosol pollution and cannot distinguish specific amplification. and non-specific amplification, thus increasing the probability of false positive diagnostic results; and for weak positive reactions, it is likely to be misjudged as negative by human naked eyes

Method used

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  • LAMP primer combination, detection method and kit capable of distinguishing porcine circovirus type 2 and type 3 typing detection
  • LAMP primer combination, detection method and kit capable of distinguishing porcine circovirus type 2 and type 3 typing detection
  • LAMP primer combination, detection method and kit capable of distinguishing porcine circovirus type 2 and type 3 typing detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the screening preparation of primer combination

[0058] 1. Screening of primer combinations

[0059] 1. The primer sequences used were synthesized by Sangon Company. Multiple sets of primer combinations were designed for the porcine circovirus type 2 Rep gene, and multiple sets of primer combinations were designed for the porcine circovirus type 3 cap gene. The sequences of each set of primer sets are shown in Table 1. Show.

[0060] Table 1 Primer Set Sequence

[0061]

[0062]

[0063]

[0064] In the above primer combinations, each single-stranded DNA is packaged independently.

[0065] In the above primer combination, the molar ratios of primer F3, primer B3, primer FIP, primer BIP, primer LF and primer LB are all 0.3:0.3:2.4:2.4:1:1. If the primer LF is missing in the primer set, replace it with pure water.

[0066] 2. Using the porcine circovirus type 2 and type 3 detection gene plasmid DNAs as templates, the primer sets prepared in ste...

Embodiment 2

[0076] Embodiment 2, specificity experiment

[0077] The samples used were as follows: porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine Influenza virus (SIV), highly pathogenic porcine blue ear virus (HP-PRRSV), porcine parvovirus (PPV1)

[0078] Each sample to be tested carries out the following steps respectively:

[0079] 1. Extract the genomic DNA or RNA of the sample to be tested.

[0080] 2. Using the genomic nucleic acid extracted in step 1 as a template, each primer set prepared in Example 1 was used to perform loop-mediated isothermal amplification.

[0081] Reaction system (20 μL): 10 μL reaction solution (product of Boao Biological Group Co., Ltd., catalog number: CP.440020), 2.96 μL primer mixture, 2 μL genome template (5pg-50pg), 0.5 μL AMV reverse transcriptase (template is Add when adding RNA), RNase-free water rehydration to 20μL. The pri...

Embodiment 3

[0087] Embodiment 3, preliminary screening of sensitivity

[0088] Sample to be tested: the plasmids of porcine circovirus type 2 and type 3 prepared in Example 1.

[0089] 1. Extract the plasmid DNA of the sample to be tested, and perform gradient dilution with sterile water to obtain each dilution.

[0090] 2. Using the dilution obtained in step 1 as a template, the primer combination screened in Examples 1 and 2 was used to perform loop-mediated isothermal amplification.

[0091] Reaction system (20 μL): 10 μL reaction solution (product of Boao Biological Group Co., Ltd., catalog number: CP.440020), 2.96 μL primer mixture, 2 μL template diluent (1 μL of the genome copy number contained in the diluent is 10 4 and 10 3 ), replenish water to 20 μL. The primer mixture is the mixture of each primer in the primer combination. In the reaction system, 0.12 μL each of 0.3 mM outer primers F3 and B3; 0.96 μL each of 2.4 mM inner primers FIP and BIP; 0.4 μL each of 1 mM loop prime...

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Abstract

The present invention belongs to the technical field of biology and discloses a LAMP primer combination, detection method and kit capable of distinguishing porcine circovirus type 2 and type 3 typingdetection. The primer combination can respectively detect the porcine circovirus type 2 and the porcine circovirus type 3, and also has no cross reactions with other porcine viruses (such as circovirus type 1, porcine parvovirus, pseudorabies virus, porcine cholera virus, swine influenza virus, highly pathogenic porcine reproductive and respiratory syndrome virus, etc.). Fluorescent dye is added into a LAMP reaction system, so that detection of the porcine circovirus type 2 and / or type 3 can be realized by a one-step method, whole-process real-time monitoring is realized by combining fluorescence quantification, results can be obtained within 1 hour, and detection sensitivity can reach 100 copies / [mu]L. The detection method has advantages of simplicity, convenience, rapidness, sensitivityand specificity, and also avoids errors caused by artificial judgment and environmental pollution caused by uncovering.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer combination, a detection method and a kit of LAMP capable of distinguishing porcine circovirus types 2 and 3 by typing detection, which are used for detecting and distinguishing porcine circovirus types 2 and 3 type. Background technique [0002] Porcine circovirus (PCV, Porcine circovirus) is a member of the genus Circovirus of the family Circoviridae, a single-stranded circular DNA virus, and is one of the smallest animal viruses discovered so far. According to the pathogenicity, nucleic acid sequence and antigenicity of PCV, PCV can be divided into two serotypes, namely PCV1 and PCV2. The two have about 80% homology in nucleotide sequence, and PCV1 has no pathogenicity. PCV2 is the main pathogen causing multisystem wasting syndrome (PMWS) and dermatitis and nephrotic syndrome (PNDS) in weaned piglets. The PCV3 virus was first discovered in the United States ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844
Inventor 张岩马银平陈燕旌邢婉丽程京冯小宇梅力韦海涛王英超高晓龙
Owner CAPITALBIO CORP
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