Application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as standard sample in nucleic acid amplification testing

A technology for porcine circovirus and recombinant adenovirus, which is applied in the field of animal disease detection, can solve the problems of complex preparation, inability to control the DNA extraction process, aerosol pollution, etc., and achieves clear adenovirus background, clear adenovirus background, and safe use. handy effect

Inactive Publication Date: 2015-12-09
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The quality control products currently used for PCV2NAT detection are prepared from inactivated virus and plasmid DNA, or the preparation is complicated and requires biosafety facilities, or alth...

Method used

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  • Application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as standard sample in nucleic acid amplification testing
  • Application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as standard sample in nucleic acid amplification testing
  • Application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as standard sample in nucleic acid amplification testing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Acquisition and Identification of Recombinant Adenovirus Containing Porcine Circovirus Type 2 ORF2 Gene

[0040] 1. Materials

[0041] The nucleic acid of porcine circovirus type 2 QD / 2014 strain and HEK293T and HEK293 cell lines are preserved by our laboratory. transfection reagent TransfectionReagent was purchased from Mirus Company; replication defective adenovirus expression system AdenoviralExpressionSystem was purchased from CellBiolabs; pMD19-T and competent cell Top10 were purchased from TaKaRa.

[0042] 2. Method

[0043] 1) Primer design

[0044] According to the PCV2 domestic isolate gene sequence (AY424401.1) published by GenBank, a pair of primers were designed using oligo7 software. It is used to amplify the complete ORF2 gene of PCV2, the fragment size is 702bp, and KpnI and HindIII restriction sites are introduced into the amplification primers. At the same time, primers and probes for PCV2 real-time fluorescent PCR detection were de...

Embodiment 2

[0064] Example 2, Preparation and Inspection of Standard Samples of Recombinant Adenovirus Containing PCV2 ORF2 Gene

[0065] 1. Materials

[0066] The seventh generation pacAd5CMV-ORF2 was prepared by our laboratory. Trehalose was purchased from Beijing Zhongke Sanhuan Company.

[0067] 2. Method

[0068] 1) Aliquoting and freeze-drying of standard samples of recombinant adenovirus containing PCV2ORF2 gene

[0069] After repeated freeze-thawing of the 7th generation pacAd5CMV-ORF2 inoculated cells cultured in large quantities for 3 times, centrifuge at 1000g / min for 10min to remove the precipitate, add trehalose at 1% as a freeze-drying protective agent, and distribute at 0.5mL / bottle under sterile conditions. Packed in a vial and freeze-dried for 24 hours under vacuum.

[0070] 2) Uniformity inspection of standard samples

[0071] Randomly select 10 tubes of prepared standard products, extract DNA, and make a 103-fold dilution of each DNA. The established PCV2 fluo...

Embodiment 3

[0081] Embodiment 3, definite value and application of standard sample

[0082] 1. Materials

[0083] The freeze-dried product of the standard sample containing PCV2ORF2 gene for NAT detection of recombinant adenovirus was prepared by our laboratory; the primer probes PCV2F, PCV2R and PCV2P were synthesized by TAKARA Company. Viral DNA extraction kit was purchased from Tiangen Biological Company; dNTPs (2.5mmol / L) was purchased from TAKARA Company; TaqDNA polymerase was purchased from Promega Company.

[0084] 2. Method

[0085] 1) Determination of recombinant adenovirus standard samples containing PCV2ORF2 gene NAT (GEq / mL determination)

[0086] The value determination method is to calculate the copy number of the purified plasmid pMD19-T-ORF2 by measuring its absorbance value at 260nm and 280nm, and further serially dilute it to prepare a series of external standard products. The established PCV2 fluorescent PCR method was used to indirectly determine the copy number...

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Abstract

The invention discloses application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as a standard sample in nucleic acid amplification testing. The adenovirus recombination technology is used to prepare the recombinant adenovirus containing the porcine circovirus type 2 ORF2 genes, the recombinant adenovirus is used as the standard sample of the nucleic acid amplification testing (NAT), the standard sample is stable, simple to prepare and free of biosafety risks, virus particular structure of the recombinant adenovirus is generally similar to that of pig prokaryotic virus, whole-process quality control of virus DNA extraction and amplification detection can be achieved, features such as certain quantity value are achieved, and the recombinant adenovirus is suitable for the quality control of a porcine circovirus type 2 nucleic acid amplification detection method and evaluation of detection reagents and related abilities of laboratories.

Description

technical field [0001] The invention belongs to the technical field of animal disease detection and relates to the application of a recombinant adenovirus containing porcine circovirus type 2 ORF2 gene as a standard sample in nucleic acid amplification detection. Background technique [0002] Porcine circovirus type 2 (porcine circovirus type 2, PCV2) has a worldwide distribution and is found in domestic pigs and wild boars. At present, PCV2 is believed to be an important pathogen that endangers the pig industry worldwide, and can cause various diseases of pigs, which are called PCV2-associated diseases (PCV2-associated diseases, PCVAD). PCVAD includes weaning piglet multisystem wasting syndrome (post-weaningmultisystemic wasting syndrome, PMWS), porcine dermatitis and nephrotic syndrome (porcinedermatitisandnephropathyysyndrome, PDNS) and reproductive disorders and other diseases. PCV2 infection can cause the decline of herd immunity, which can lead to the failure of vario...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/63C12R1/93
CPCC12Q1/701C12N15/63C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 张鹤晓张伟谷强刘环林祥超高志强乔彩霞蒲静张利峰
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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