Multi-fluorescence immunity analysis method for quickly distinguishing PEDV, TGEV and PoRV

A multiple fluorescence and immunoassay technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve difficult problems and achieve lower detection costs, less sample consumption, and strong specificity Effect

Active Publication Date: 2015-12-16
GUANGDONG LAB ANIMALS MONITORING INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. Big

Method used

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  • Multi-fluorescence immunity analysis method for quickly distinguishing PEDV, TGEV and PoRV
  • Multi-fluorescence immunity analysis method for quickly distinguishing PEDV, TGEV and PoRV
  • Multi-fluorescence immunity analysis method for quickly distinguishing PEDV, TGEV and PoRV

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1: Construction of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine group A rotavirus plasmid

[0096] Use the kit MiniBESTViralRNA / DNAExtractionKitVer.4.0 to extract the RNA of PEDV, TGEV, and PoRV viruses, reverse transcribe into cDNA, and perform PCR amplification with the corresponding primers above. The amplification system is as follows:

[0097]

[0098] The reaction procedure for amplification is:

[0099] Pre-denaturation at 94°C for 5 minutes;

[0100] Denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s; cycle 35 times;

[0101] Final extension at 72°C for 10 min.

[0102] The amplified products were detected by agarose gel electrophoresis and purified by gel cutting.

[0103] The purified cDNA was ligated into the pMD-19T vector with a kit from TaKaRa Company. The vector ligation reaction system was as follows (10 μl):

[0104]

[0105] Reaction conditions: 16°C, over 4...

Embodiment 2

[0107] Embodiment 2: Establishment of multiple fluorescent immunoassay methods for porcine epidemic diarrhea, porcine transmissible gastroenteritis, and porcine group A rotavirus:

[0108] 1. Plasmid PCR amplification

[0109] Single-fold, double-fold and triple-fold PCR amplification was performed with specific amplification primers for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine group A rotavirus.

[0110] Preparation of upstream primer mixture: mix P1, T1 and R1 at a ratio of 1:1:1; preparation of downstream primer mixture: mix P2, T2 and R2 at a ratio of 1:1:1. The specific regions of the above three viruses are amplified by using three specific templates, double templates, and triple templates. The preparation of double templates: mix two or two plasmids at a ratio of 1:1, and the preparation of triple templates: mix three plasmids at a ratio of 1:1:1.

[0111] The PCR amplification reaction system is as follows:

[0112]

[0113...

Embodiment 3

[0128] Embodiment 3: Multiple fluorescence immunoassay detection sensitivity experiment of PEDV, TGEV, PoRV

[0129] The prepared plasmid was quantified, diluted to 0.01 fg / ul by 10-fold dilution method, and detected by the multiple fluorescence immunoassay method established above. The sensitivity test results of multiple fluorescence immunoassays for PEDV, TGEV, and PoRV are as follows: image 3 As shown, the experimental results show that the sensitivity of PEDV, TGEV, and PoRV is high, and the detection limit is 1fg / PCR.

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Abstract

The invention discloses a multi-fluorescence immunity analysis method for quickly distinguishing PEDV, TGEV and PoRV. The method is easy to operate, a target amplified fragment is obtained through PCR, then hybridization is conducted on an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin, the MFi value is read through a detection instrument, and different types of viruses are distinguished. By means of the method, porcine epizootic diarrhea, swine transmissible gastroenteritis and pig group A rotavirus can be accurately detected at the same time, the specificity is high, the sensitivity is high, and the repeatability is good. Compared with a traditional detection method, various molecules of different purposes in the same sample are detected at the same time, the sample consumption is little, operation is simple and fast, and the detection cost can be greatly lowered.

Description

technical field [0001] The invention belongs to the field of virus detection in aquaculture, and in particular relates to a method for quickly distinguishing PEDV, TGEV and PoRV [0002] Multiplex fluorescence immunoassay method. Background technique [0003] In recent years, the prevalence of porcine diarrhea disease has been on the rise, and the epidemic law has also changed greatly. The disease usually occurs frequently in winter and spring, but it has also become popular in summer in recent years, and pigs of all ages and physiological conditions have occurred. Among them, piglets have the highest morbidity and mortality. Porcine diarrhea is one of the most serious piglet diseases and an important cause of piglet death. According to the survey, the death of piglets due to diarrhea accounted for 39.8% of the total number of piglet deaths. In recent years, diarrhea in piglets in my country has been very common. According to reports, piglets under 30 kg have an annual av...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/131C12Q2563/149
Inventor 朱余军郭鹏举黄韧
Owner GUANGDONG LAB ANIMALS MONITORING INST
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