Multi-RT-PCR method for monitoring pollution of six viruses in pig farm environment through one system and application

A technology of RT-PCR and swine fever virus, applied in the field of multiple RT-PCR technology

Inactive Publication Date: 2015-10-28
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing technology is mainly used for the diagnosis of pigs after the disease, but lacks the monitoring technology for virus pollution in the environment, let alone establish an early warning mechanism, prevent and control virus infection in advance, and cannot monitor the purification of pathogens in pig farms

Method used

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  • Multi-RT-PCR method for monitoring pollution of six viruses in pig farm environment through one system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the preparation of special Marker

[0041] The present invention utilizes the reference sequences of Genbank to perform sequence comparison, select conserved sequences, use biological information technology to design primers for each sequence, extract virus nucleic acid, perform PCR or RT-PCR, and recover the PCR product by glue, and connect it to pMD- 18T vector, transform it into DH 5α Store in competent cells, carry out PCR amplification with the recombinant plasmid as a template, recover each fragment by gel, measure its concentration, dilute it to the corresponding concentration and add protective agent (36% glycerol, 0.05% bromophenol blue, 0.05% xylene Nitrile Blue FF, 30 mM EDTA). And according to the protective agent and DNA ratio of 1:5 mixed evenly, that is to make a special Marker. The dedicated marker is composed of DNA fragments 1045bp, 829bp, 636bp, 382bp, 275bp and 142bp, and there are six bands in total. The DNA amount of the 636bp ba...

experiment example 2、6

[0042] Experimental example 2, 6 kinds of virus multiplex RT-PCR method

[0043]Mix the extracted DNA of PCV-2, PPV, and PRV with the cDNA of JEV, CSFV, and PRRSV as templates for multiplex RT-PCR detection. Multiplex PCR uses a 50 μL reaction system: 2×Taq MasterMix is ​​25 μL, and 6 kinds of virus mixed primers (20 μM / L) 3 μL in total, 3 μL of mixed nucleic acids of 6 viruses in total, and 19 μL of sterilized distilled water. Briefly centrifuge to mix well. The reaction conditions are: 94°C for 5 minutes; 94°C for 45s; 56°C for 1m30s; 72°C for 50s, the number of cycles is 40 times, and finally 72°C for 10 minutes, and stored at 4°C. The reaction products were identified by 1.5% agarose gel electrophoresis.

experiment example 3

[0044] Experimental example 3, monitoring of pig farm environment

[0045] Connect the portable gas sampling pump to the bubble collection bottle, add 20 mL of PBS buffer solution (containing 5% glycerol) with pH=7.2 into the collection bottle, the working flow rate is 6 L / min, the collection time is 30 minutes, and the samples are processed within 24 hours complete.

[0046] Concentrate the air sample by ultrafiltration. Add the above collected solution into an ultrafiltration tube with a molecular weight cut-off (10kD), centrifuge at 7000rpm for 30min at 4°C, then centrifuge the concentrated solution at 10000rpm for 10min, and extract the supernatant Nucleic acid, multiplex RT-PCR detection.

[0047] Feces and feed samples Weigh 5g of the sample, add 15mL of 0.1M PBS solution, vortex to make a suspension, centrifuge at 3000r / min at 4°C for 10min, take the supernatant, and centrifuge the supernatant at 10000r / min for 30min, take the supernatant; Take swab eluate from uten...

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Abstract

The invention provides a multi-RT-PCR method for monitoring pollution of six main viruses in a pig farm environment through one system and application. According to the method, primer design is conducted for CSFV, JEV, PRRSV, PCV-2, PRV and PPV, a multi-RT-PCR reaction system for the six viruses is established, air samples are collected through liquid collision, the viruses are subjected to separation concentration through ultrafiltration centrifugation, and the method for detecting the pollution of the main viruses in the pig farm environment is established. The method can be used for monitoring the pollution of the six viruses in air, fodder, drinking water, appliances, excrement and pig groups in the pig house and pig farm environment, and technical support is provided for establishing a main virus pollution early-warning mechanism and a main virus disease preventing, controlling and purifying system in the pig farm environment.

Description

technical field [0001] The invention relates to a multiple RT-PCR technology for monitoring main virus pollution in pig farm environment, and belongs to the technical field of bioengineering. Background technique [0002] The pig farming industry is gradually developing towards intensification and scale. Intensive feeding is the main breeding mode at present. Pig infectious diseases are mainly transmitted through air, feed, drinking water, utensils, and feces in the environment. Classical swine fever virus (CSFV), Japanese encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2), porcine pseudorabies virus (PRV) and porcine parvovirus Viruses (PPV) etc. are the main viruses that contaminate large-scale pig farms. The existing technology is mainly used for the diagnosis of pigs after the onset of the disease, but there is a lack of monitoring technology for virus pollution in the environment, and it is impossib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 刘兴友魏小兵张秀林欧长波刘明成王秋霞贺会利刘长忠王丽荣
Owner HENAN INST OF SCI & TECH
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