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

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...

AI Technical Summary

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 ORF3 has been established, but the real-time PCR method is expensive to quantify the virus, and the detection sensitivity can only be increased by dozens of times compared with ordinary RT-PCR. Therefore, it is limited by economic cost in production practice

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