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PCR (polymerase chain reaction) primer and PCR kit for detecting rhizomyidae circovirus

A kind of circovirus and test kit technology, applied in the field of PCR primers and test kits for detecting bamboo rat circovirus

Active Publication Date: 2017-07-25
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no gene reference sequence for this virus at home and abroad

Method used

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  • PCR (polymerase chain reaction) primer and PCR kit for detecting rhizomyidae circovirus
  • PCR (polymerase chain reaction) primer and PCR kit for detecting rhizomyidae circovirus
  • PCR (polymerase chain reaction) primer and PCR kit for detecting rhizomyidae circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Primer Design

[0023] Primers were designed according to the conserved region of the bamboo rat circovirus gene sequence (see SEQ.ID.No.4 for its full-length sequence) obtained from the previous viral metagenomic sequencing results, and a pair of primers CP1 and CP2 located in the capsid protein gene were obtained (see Table 1), sent to Shenzhen BGI for synthesis, dissolved in DEPC-treated water, and stored at -20°C.

[0024] Table 1 Primer sequences

[0025]

Embodiment 2

[0026] Example 2 Establishment of PCR reaction system and reaction program

[0027] 1. DNA template preparation

[0028] The heart, liver, spleen, lung, kidney and other tissues of the diseased bamboo rat were evenly cut and ground with suction filtration and sterilized DMEM at a volume ratio of 1:5 to prepare a suspension of the diseased material, which was dispensed into a 1.5ml EP tube, and subjected to 3 After the first freezing and thawing, the disease material suspension was centrifuged at 8000 rpm at 4°C for 5 min, and 200 μL of the supernatant was taken, and the positive and negative controls (see Example 6) were simultaneously drawn and numbered in equal amounts. The template DNA was extracted according to the AXYGEN Body Fluid Virus DNA / RNA Mini Kit, and the product was directly used in PCR reaction or stored at -20°C.

[0029] 2. PCR amplification reaction

[0030] Prepare the reaction mixture according to the PCR reaction system (see Table 2) and put it into the ...

Embodiment 3

[0033] Example 3 PCR detection method Determination of annealing temperature

[0034] Use the extracted sample DNA as a template, set different annealing temperatures, and then set other conditions to be the same for PCR. After PCR is completed, take 5 μL of the product and spot it in a 1% agarose gel sample well (containing 0.5 μg / ml). ethidium bromide). The gel after electrophoresis was observed under a UV analyzer to determine its optimal annealing temperature. The results showed that the annealing temperature was from 50°C to 60°C, and the PCR product results were very specific ( figure 1 ), and finally the annealing temperature of the detection method was determined to be 60 °C.

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Abstract

The invention discloses a PCR (polymerase chain reaction) primer for detecting rhizomyidae circovirus. The PCR primer comprises a primer CP1 and a primer CP2, and base sequences of the primer CP1 and the primer CP2 are shown as SEQ.ID.No.1 and SEQ.ID.No.2 respectively. Furthermore, a corresponding PCR kit is prepared, and a corresponding PCR detection method is established. The kit comprises the primer CP1, the primer CP2, an RNA extract agent, a PCR amplification agent, a rhizomyidae circovirus positive control and a rhizomyidae circovirus negative control. According to experiments, the primer and the kit have advantages of specificity, sensitiveness, quickness, simplicity and convenience, fill an existing technical gap in clinical detection of the rhizomyidae circovirus and can be used for quick detection in places such as farms and general laboratories.

Description

technical field [0001] The invention belongs to the technical field of circovirus detection, and in particular relates to PCR primers for detecting bamboo rat circovirus and a kit thereof. Background technique [0002] Circovirus is an animal virus with no envelope and icosahedral symmetry. Its genome is a single-stranded circular DNA, and it is the smallest pathogenic animal virus known so far. Circovirus is often latent infection, which is easy to invade the immune system of animals, resulting in decreased immunity of the body, increasing the susceptibility of animals to various pathogens, and making animals sick. [0003] With the rapid development of my country's special animal breeding industry, the bamboo rat breeding industry has been able to grow continuously. At present, the research and reports in the field of bamboo rat circovirus disease at home and abroad are still in a blank state. And the prevention and treatment of secondary diseases caused by it is also in a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 吴健敏唐海波姜佳佳陈凤莲饶桂波白安斌覃绍敏刘金凤马玲
Owner GUANGXI VETERINARY RES INST
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