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Pig viral diarrhea detection primer combination, detection kit and application

A swine viral diarrhea, detection kit technology, applied in the directions of microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of multi-virus mixed infection, unable to meet the needs of epidemic disease detection, and the lack of mature detection methods for viruses, etc. To achieve the effect of convenient detection, high accuracy and good stability

Active Publication Date: 2022-06-28
山东傲农种猪有限公司 +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the detection kits for porcine viral diarrhea are mainly based on the traditional single-plex fluorescent quantitative PCR detection method of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus and porcine rotavirus. However, viral diarrhea often Not only infected with a single virus, but often mixed with multiple viruses, and new viruses of porcine delta coronavirus exist from time to time, and there is no mature detection method for this virus
[0008] Based on this, the existing detection kits for detecting porcine viral diarrhea cannot meet the needs of current disease detection, so there is an urgent need to develop commercial detection kits that meet the current epidemic trend
At present, there is no fluorescent quantitative PCR commercial kit that can detect and identify the above four pathogens at one time

Method used

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  • Pig viral diarrhea detection primer combination, detection kit and application
  • Pig viral diarrhea detection primer combination, detection kit and application
  • Pig viral diarrhea detection primer combination, detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] This embodiment provides a swine viral diarrhea detection kit, which includes a positive control, a negative control, a primer premix, a probe premix, a PCR amplification solution, and an enzyme premix.

[0100] Positive control: recombinant plasmids containing PEDV, TGEV, RV, PDCoV target gene fragments respectively.

[0101] Negative control: double distilled water.

[0102] Primer master mix: including PEDV-F, PEDV-R, TGEV-F, TGEV-R, RV-F, RV-R, PDCoV-F, PDCoV-R at an initial concentration of 10 μM in a molar ratio of 1:1:1: 1:1:1:1:1 premix;

[0103] Probe premix: PEDV-P, TGEV-P, RV-P, PDCoV-P with an initial concentration of 10 μM in a molar ratio of 1:1:1:1;

[0104] PCR amplification solution includes: 2×One Step U+ (Novizan);

[0105] Enzyme premix: One Step U+Enzyme Mix (Novizan).

[0106] Upstream primer PEDV-F (SEQ ID NO.1):

[0107] 5'-CCAACTGGTGTAACGCTAAC-3'

[0108] Downstream primer PEDV-R (SEQ ID NO. 2):

[0109] 5'-GACATAGAAAGCCCAACCAG-3'

[011...

experiment example 1

[0142] In this experiment, the conditions of quadruple fluorescence quantitative PCR were optimized.

[0143] Use Tiangen's plasmid extraction kit to extract the plasmids of the positive strains with correct sequencing in Example 1, use the NanoDrop 2000 nucleic acid concentration analyzer to detect the concentration of the plasmid template, and dilute the four standard plasmids by 10-fold gradient to take the concentration of 10 4copise / μL, 1:1:1:1 was mixed as template. The total system is 30 μL, the upstream and downstream primers and corresponding probes are mixed with different final probe concentrations and primer final concentrations, respectively, and 1.2 μL and 1.2 μL of primers (upstream and downstream) and corresponding probes (the total upstream and downstream primer concentrations) are added respectively. 400nM: probe concentration 400nM); 1.2μL, 0.6μL (upstream and downstream total primer concentration 400nM: probe concentration 200nM), 1.2μL, 0.3μL (upstream and...

experiment example 2

[0152] This experimental example is a repeatable verification experiment.

[0153] The positive control plasmids of 7 concentrations of 10-fold serial dilution were used as templates, and the final concentrations were 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 copise / μL, and perform fluorescence quantitative PCR according to the reaction system and program that provides fluorescence quantification, and set 3 replicates for each gradient to verify the repeatability of the method. The results show that the coefficient of variation (CV value) of the repeated experiments of the present invention is all below 0.5%, indicating that the detection kit provided by the present invention has good repeatability.

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Abstract

The invention discloses a porcine viral diarrhea detection primer combination, a detection kit and application, and relates to the technical field of porcine viral diarrhea detection. The kit comprises a first probe primer pair, a second probe primer pair, a third probe primer pair and a fourth probe primer pair. The detection primer combination and the detection kit can be used for detecting and analyzing porcine epidemic diarrhea, porcine transmissible gastroenteritis, porcine rotavirus and porcine deltacoronavirus in a sample at one time, so that the amplification efficiency of four virus genes is consistent, and the sensitivity of the four virus genes is consistent with that of each single reaction; the method has the characteristics of simplicity, convenience, rapidness, good stability, high detection sensitivity and strong specificity. The detection kit provided by the invention has the advantages of convenience in detection, high accuracy and high detection efficiency, can be applied to daily detection work in pig farms, and provides technical support for clinical diagnosis of four viruses with similar clinical manifestation for porcine viral diarrhea.

Description

technical field [0001] The invention relates to the technical field of swine viral diarrhea detection, in particular to a primer combination, a detection kit and application for swine viral diarrhea detection. Background technique [0002] Porcine viral diarrhea is one of the important infectious diseases that seriously endanger the pig industry. Every year, it causes a large number of piglets to die from diarrhea, rapid dehydration, and is often aggravated by the mixed infection of various diarrhea viruses, resulting in high mortality and serious health problems. Economic losses. The main pathogens of viral diarrhea are porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine rotavirus (RV). In recent years, porcine delta coronavirus (PDCoV) has also been frequently detected as a newly discovered enteric coronavirus. [0003] Among them, Porcine Epidemic Diarrhea (PED) is an acute, highly contagious infectious disease caused ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 张蓉邓红玉陈秀萍凌勇丁能水谢亚磊万文峰吴有林
Owner 山东傲农种猪有限公司
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