Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A fluorescence quantification and detection method technology, applied in microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as S gene and dye method not involved, and achieve shortened detection time and specificity. High, not easy to contaminate effect
Inactive Publication Date: 2011-08-17
SHANGHAI JIAO TONG UNIV
View PDF5 Cites 17 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Problems solved by technology
But the S gene and dye method are not involved in this method
Method used
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0035] (1) Design and synthesis of primers and probes
[0036] According to the S gene sequence of the TGEV strain preserved in our laboratory, primers were designed with primer5.0 and other software. The target fragment size was 111bp, which was synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0037] The upstream primer is sequence listing 1; the downstream primer is sequence listing 2.
[0038] (2) Cloning of S gene
[0039] Inoculate 10% of the TGEV strain into a cell bottle that has grown to 80% of the cells, adsorb at 37°C for 1 hour, change 2% of the maintenance solution, and CO 2 Cultivate in an incubator at 37°C for 3-6 days, collect the virus after 80% of the lesions appear, freeze and thaw the virus cell liquid three times repeatedly, collect the virus, and store it at -80°C for later use. Viral RNA was extracted according to Reagent kit instructions for use.
[0040] TGEV DNA was synthesized according to the instructions of the one-step reverse transcrip...
Embodiment 2
[0054] (1) Collection of clinical stool samples, adding 2 times the volume of PBS to the clinical stool samples, freezing and thawing 3 times, taking the supernatant as the extraction stock solution and storing it at -80°C for later use. Viral RNA was extracted according to Reagent kit instructions for use.
[0055] (2) PCR system optimization
[0056] Element
[0057] (3) Optimal reaction conditions
[0058]
[0059] (4) Calculate the virus content in the sample according to the fluorescent quantitative PCR result and the standard curve.
[0060] (5) Fluorescent quantitative PCR results, the fluorescent quantitative PCR instrument will automatically display the concentration of the sample, the unit is Copies / 2μL.
[0061]
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
Abstract
The invention discloses a fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for a porcine transmissible gastroenteritis virus gene S and a primer thereof in the technical field of biotechnology. The method comprises the following steps of: cloning a PCR amplification target segment identified as a positive PCR product to a vector pMD18-T, transforming to a competent cell DH5alpha, selecting positive clone by screening blue and white spots and identifying sequencing; extracting a positive recombinant plasmid, quantifying by using an ultraviolet spectrophotometer, diluting a standard product series by 10 times of gradient until the final concentration is 1.0*10<3>-1.0*10<11> copies/mL, undergoing a fluorescence quantitative PCR by taking the standard product series as a template, and establishing a fluorescence quantitative PCR standard curve; and extracting virus RNA (Ribonucleic Acid) of a clinical excrement sample, undergoing a fluorescence quantitative PCR, and calculating the content of viruses in the sample according to a result and the standard curve, wherein the sequences of the primer are sequence 1 and sequence 2. The method and the primer have theadvantages that: a fluorescent probe does not need to be designed additionally, the cost is lowered, operation is easy and convenient, and detection can be completed within 2 hours. The detection method and the primer are suitable for any fluorescence quantitative PCR instrument, and can be applied to the detection of large-scale and high-flux samples.
Description
technical field [0001] The invention relates to a detection method and gene primers in the field of biotechnology, in particular to a fluorescent quantitative PCR detection method and primers for porcine transmissible gastroenteritis virus S gene. Background technique [0002] Porcine transmissible gastroenteritis (TGE) is a highly contagious infection caused by porcine transmissible gastroenteritis virus (TGEV), characterized by vomiting, watery diarrhea, and dehydration in sick pigs. The disease is highly lethal to piglets under 1 week of age. After recovery, the piglets were stunted and stunted. The disease can impair the disease resistance and immune function of recovered adult pigs, lose fat, reduce feed utilization, waste medicine and manpower, etc., resulting in serious economic losses. Therefore, it is necessary to establish a fast, sensitive, low-cost and easy-to-operate detection method. [0003] TGEV encodes four structural proteins, namely the filament (S) pro...
Claims
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more