Unlock instant, AI-driven research and patent intelligence for your innovation.
Real-time fluorescent quantitative RT-PCR detection method for bovine norovirus
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 real-time fluorescence quantitative and detection method technology, applied in the field of molecular biology, to achieve the effect of high sensitivity, strong specificity and high amplification efficiency
Pending Publication Date: 2020-03-17
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
View PDF5 Cites 0 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
Fluorescent quantitative PCR detection method is an internationally recognized gold standard for detecting viruses. However, there is currently no rapid diagnostic method specifically for BNoV in China, and an early and rapid diagnostic method for BNoV is urgently needed
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
[0039] 1. Primer design and synthesis
[0040] Using MEGA 7.0 software to analyze and compare the nucleotide sequences of BNoV RdRp genes published in GeneBank, and combined with the existing BNoV RdRp gene sequences in the laboratory, a pair of specific primers were designed using Oligo 6.0 software. The primer sequences are shown in Table 1. The primers were Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0041] The primer sequence of table 1 RdRp gene of the present invention
[0042]
[0043] 2. Extract the total nucleic acid of the sample to be tested
[0044] Suspend 221 stool samples in PBS at 1:10 (w:v), freeze and thaw three times, vortex, centrifuge at 8000r / min at 4°C for 5min, harvest supernatant, and store at -80°C for subsequent experiments ; Refer to the extraction kit to extract viral RNA, and store the viral RNA at -80°C; refer to the reverse transcription kit for cDNA, and store it at -20°C.
[0045] 3. Preparation of recombinant plasmid sta...
Embodiment 2
[0052] Example 2: Establishment of bovine norovirus real-time fluorescent quantitative RT-PCR detection method
[0053] 1. Optimization of reaction conditions for real-time fluorescent quantitative RT-PCR
[0054] (1) Determination of annealing temperature
[0055] Using the recombinant plasmid pMD18T-BNoV-RdRp as a template, using a 20 μL reaction system, the annealing temperature was set to 6 temperature gradients of 57, 58, 59, 60, 61, and 62°C, and the Ct values at different annealing temperatures were compared. According to the reaction system provided by the qPCR kit, that is, SuperMix (2×) 10 μL, ROX Reference Dye (50×) 0.4 μL, upstream and downstream primers 0.4 μL (10 μmol / L), template 1 μL, ddH 2 O supplemented to 20 μL, optimized annealing temperature. The results showed that when the annealing temperature was 60°C, the Ct value was the smallest, indicating that the amplification efficiency was the highest when the annealing temperature was 60°C.
[0056] (2) D...
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
Property
Measurement
Unit
correlation coefficient
aaaaa
aaaaa
PCR efficiency
aaaaa
aaaaa
Login to View More
Abstract
The invention relates to the technical field of molecular biology, in particular to a real-time fluorescent quantitative RT-PCR detection method for bovine norovirus (BNoV). According to the method, apair of specific primers is designed based on a targeted RdRp gene of the BNoV and a reaction system and reaction conditions of real-time fluorescent quantitative PCR are optimized, so that a fluorescent quantitative PCR method for detecting the BNoV is established. The method is good in linear relation and good in amplification efficiency; the sensitivity of the disclosed method is 1000 times ofthat of the conventional PCR method and is relatively high. The method only has a good amplification curve for BNoV, so that the specificity is high; and the difference in the groups and the difference between the groups are small and the high repeatability is realized.
Description
technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a real-time fluorescent quantitative RT-PCR detection method for bovine norovirus. Background technique [0002] Calf diarrhea is a common clinical syndrome, which affects the health of calves, and then affects the production of high-quality livestock products, causing serious harm to the cattle industry. Its etiology is complex, and virus infection is an important cause of diarrhea. According to reports, in addition to common viruses such as bovine coronavirus (BCoV), bovine rotavirus (Bovine rotavirus, BRoV) and bovine viral diarrheavirus (Bovine viral diarrheavirus, BVDV) that can cause diarrhea in calves, bovine Emerging viruses such as Bovine norovirus (BNoV), bovine torovirus (BToV) and bovine nebovirus (BNeV) have also attracted more and more attention in the pathogenic spectrum of calf diarrhea. [0003] Norovirus (NoV) is a single-stranded positi...
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