Real-time fluorescent LAMP detection primer group, kit and detection method of African swine fever virus non-structural gene

An African swine fever virus and non-structural gene technology, applied in the field of real-time fluorescent LAMP detection of African swine fever virus non-structural genes, can solve problems such as economic loss, interference with normal production and living order, and ASF cannot be vaccinated, and achieve specific Good reproducibility and reproducibility, good effect, excellent sensitivity

Inactive Publication Date: 2017-07-14
TECH CENT OF GUANGZHOU CUSTOMS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since ASF cannot be vaccinated and effectively treated, culling and purification measures must be taken in the prevention and control work, which seriously interferes with normal production and living order and causes major economic losses. It has become a huge threat to my country's pig industry, and its research results have important political Economic significance

Method used

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  • Real-time fluorescent LAMP detection primer group, kit and detection method of African swine fever virus non-structural gene
  • Real-time fluorescent LAMP detection primer group, kit and detection method of African swine fever virus non-structural gene
  • Real-time fluorescent LAMP detection primer group, kit and detection method of African swine fever virus non-structural gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The establishment of embodiment 1 reaction system

[0056] 1. Design LAMP primers

[0057] The online software BLAST was used to compare the ASFV gene sequences, the online software PrimerExplorer was used to design the primer set of ASFV fluorescent quantitative LAMP, and 8 sets of G1211R gene LAMP primers were designed online, and a set of primers was optimized by using sequence conservation, structural characteristics and Ct value, as shown in Table 1 shown.

[0058] Table 1

[0059]

[0060] 2. Design fluorescent PCR primers

[0061] Fluorescent PCR primers and probes are in accordance with the OIE method, and their sequences are: the upstream primer is SEQ ID No.6: 5'-CTGCTCATGGTATCAATCTTATCGA-3'; the downstream primer is SEQ ID No.7: 5'-GATACCACAAGATCRGCCGT-3'; the probe It is SEQ ID No.8: 5'CCACGGGAGGAATACCAACCCAGTG-3', with a FAM group attached to its 5' end and a TAMRA group attached to its 3' end. The probe is a TaqMan fluorescent probe, the reporter fl...

Embodiment 2

[0067] 1. LAMP reaction system

[0068] Preparation of lyophilized reaction tube: 0.04nmols of FIP primer, 0.04nmols of BIP primer, 0.005nmols of F3 primer, 0.005nmols of B3 primer, 8U of BstDNA polymerase large fragment, 35nmols of dNTPs mixture and 0.01nmols of SYTO-9 fluorescent dye were mixed and then freeze-dried. become.

[0069] The LAMP reaction solution consists of:

[0070] One freeze-dried reaction tube, 15 μL of reconstitution solution, 2 μL of template to be tested (ASFV nucleic acid, concentration 1050 ng / μL), 8 μL of DEPC distilled water. Different temperatures from 60°C to 65°C, each temperature was reacted for 10sec to 30sec, and the fluorescence value of FAM was collected at 65°C, 60 cycles.

[0071] The conditions were further optimized at 60°C for 10 sec, 61°C for 10 sec, 62°C for 10 sec, 63°C for 10 sec, 64°C for 10 sec and 65°C for 10 sec.

[0072] 2. Fluorescence PCR reaction system

[0073] The fluorescent PCR reaction was carried out according to t...

Embodiment 3

[0093] 30 imported and exported pig serum samples, 15 pig nose swab samples, 2 pig urine samples, 3 pig farm insects, and 2 pig feed samples were collected from the laboratory, a total of 52 samples. The ASFV Arm 07 strain was used to simulate the strong and weak positive samples, and the MagNA Pure96 high-throughput nucleic acid separation and purification system and supporting nucleic acid extraction reagents were used to extract nucleic acid, and the established LAMP method and fluorescent PCR were used for detection, and the nucleic acid of the ASFV E70 strain was used as the positive As a control, DEPC distilled water was used as a negative control, and the detection practicability of the two methods was compared, and the results are shown in Table 3.

[0094] LAMP method reaction system (for each sample): 1 lyophilized reaction tube, 15 μL of reconstitution solution, 2 μL of template to be tested or positive control or negative control, 8 μL of DEPC distilled water. Diff...

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Abstract

The invention provides a real-time fluorescent LAMP detection primer group, a kit and a detection method of an African swine fever virus (ASFV) non-structural gene. The primer group is designed based on a non-structural DNA polymerase G1211R gene and comprises an FIP primer, a BIP primer, an F3 primer and a B3 primer. A detection result shows that a typical S-shaped nucleic acid amplification curve, and an amplification product has a specific melting curve. An ASFV70 strain virus nucleic acid is taken as a template, and LAMP detection is better than a fluorescent PCR (Polymerase Chain Reaction) method in sensitivity. Intra-assay and inter-assay variable factors of repetitive testing LAMP detection are both smaller than 5 percent. An ASFVArm07 stain is used for preparing various clinic simulated samples, and detected positive rate is up to 17.31 percent. The detection method provided by the invention can provide a new technological means for preventing and controlling African swine fever virus, and detection on different genetic stains and quick screening for exit and entry are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set, a kit and a detection method for real-time fluorescent LAMP detection of African swine fever virus nonstructural genes. Background technique [0002] African Swine Fever (ASF) is a severe infectious disease of pigs caused by the African Swine Fever virus (ASF), which has an important impact on the international trade of live pigs and meat products. The disease is a first-class animal disease in my country, and it is also one of the animal diseases legally reported by OIE (World Organization for Animal Health). Currently, the ASF epidemic is spreading from west to east in Europe, affecting Russia, Ukraine and several EU member states. According to the OIE report, in the first quarter of this year, domestic pigs and wild boars continued to be infected in countries such as Russia, Poland, Lithuania, Ukraine and South Africa. Among them, since 2014 in Russia, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 林志雄田纯见杨舒展段喻燕刘志玲罗琼陈茹
Owner TECH CENT OF GUANGZHOU CUSTOMS
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