Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology

A nano-gold and nano-gold particle technology, which is applied in the field of rapid detection of PEDV early infection and low-dose porcine epidemic diarrhea virus infection based on nano-gold label amplification technology, can solve the problems of low sensitivity, cumbersome operation, and poor specificity

Inactive Publication Date: 2016-08-17
NORTHWEST A & F UNIV
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Problems solved by technology

These methods have various degrees of disadvantages such as cumbersome operation, time-consuming and laborious, poor specificity, low sensitivity, or limited detection range.
At present, the real-time PCR method for detecting PEDV subgenome S, M, N, and ORF

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  • Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology
  • Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology
  • Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology

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Embodiment Construction

[0080] The invention designs a specific probe according to the ORF1a gene encoding replicase located in the whole genome of PEDV, which can reflect the level of infectious virus particles more accurately. In addition, although a method for detecting TGEV early blood samples based on DNA tags has been established, this method can detect PEDV in pig feces. At the same time, this method designed 16 specific probes based on the conserved region of the ORF1a gene, and screened them through a series of tests. And optimize the 2-segment nucleic acid probe with strong specificity, virus enrichment and high amplification ability to further improve the specificity, sensitivity and repeatability of this method. At the same time, the technical method is further improved in the detection method. After the sample is lysed, it can be directly hybridized with the functionalized magnetic beads without going through the nucleic acid precipitation and purification steps, which greatly shortens th...

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Abstract

The invention provides a method for enriching viruses from faeces rapidly and efficiently, amplifying signals through gold nanoparticles crosslinked through specific DNA labels and then rapidly detecting early-state PEDV infection with a PCR. According to the method, an PRF1a gene located in a PEDV whole genome is adopted as a target sequence, specific nucleic acid probes are designed, second-segment probes which are high in specificity, great in virus enriching capability and easy to amplify and detect are screened out of the specific nucleic acid probes and optimized, and by means of functionalized magnetic particles and nanogold particles of the second-segment probes, a PCR detection technology based on nanoparticle PEDV specificity is established; meanwhile, by means of the technical method, the technical breakthrough of conducting a direct hybridization reaction between faeces sample lysate and the functionalized magnetic particles is achieved, the step of purifying a sample nucleic acid is completely omitted, specificity and sensitiveness are improved, meanwhile, the detection step is simplified, and detection time is shortened. By means of the method, time and labor are saved, cost is low, and in addition, the animal virus infection level can be prompted accurately.

Description

technical field [0001] The invention relates to a method for rapidly detecting porcine epidemic diarrhea virus (PEDV) low-dose infection from early-stage infected pig feces, in particular to a method for rapidly detecting early-stage PEDV infection based on nano-gold label amplification technology, which belongs to veterinary detection technology field. Background technique [0002] With the development of the large-scale pig industry, the impact of porcine viral diarrhea on the pig industry is becoming more and more serious, which has caused huge economic losses to the pig industry in my country. Come to a new challenge. [0003] The pathogens of porcine viral diarrhea mainly include: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV). The main pathogen for the healthy development of pig industry. Porcine epidemic diarrhea is a highly contagious porcine intestinal infectious disease caused by porcine ep...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2563/137
Inventor 童德文黄勇邢娜赵晓民
Owner NORTHWEST A & F UNIV
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