Primer set for detecting trichina gene by loop-mediated constant temperature amplification, application and method

A loop-mediated isothermal, primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc. problems, to achieve the effect of easy promotion, improved detection sensitivity, and accurate detection

Pending Publication Date: 2020-01-07
ZHEJIANG ACAD OF MEDICAL SCI
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the detection of trichinellosis in animals, microscopic examination or sample digestion method is usually used, but both methods are time-consuming and laborious. so much inconvenience
The use of PCR, Real-time PCR and other methods can improve the detection sensitivity of Trichinella spiralis, but there are limitations such as the need for special equipment and professional technical operators, which is not conducive to the promotion and use at the grassroots level and in the field

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
  • Primer set for detecting trichina gene by loop-mediated constant temperature amplification, application and method
  • Primer set for detecting trichina gene by loop-mediated constant temperature amplification, application and method
  • Primer set for detecting trichina gene by loop-mediated constant temperature amplification, application and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0034] The specific detection of embodiment 2 primers

[0035] Using the genomic DNA of Trichinella spiralis, Angiostrongylus cantonensis, Anisakis elegans, gnathostomum, pinworm, Schistosoma japonicum and Trypanosomiasis as templates, add each template into a reaction tube, and add primer sets F3, B3, FIP, BIP , LB and LF, 8U Bst 2.0 DNA polymerase, 1.5mM dNTP, 7.5mM MgSO4 and ddH2O, in addition, add 1μl of SYTO13 fluorescent dye with a concentration of 25μM, and perform the reaction on Bio-Rad’s fluorescent quantitative PCR instrument. The specific reaction procedure For: react at 65°C for 1.5h and collect fluorescence data, react at 95°C for 5min.

[0036] The result is as figure 1 As shown, the primer set can detect the genomic DNA of Trichinella spiralis, but can not detect the genomic DNA of Angiostrongylus cantonensis, Anisakis elegans, gnathostomum, pinworm, schistosomiasis and Trypanosoma japonicum, indicating that the primers have good specificity .

Embodiment 3

[0037] Sensitivity detection of embodiment 3 primers

[0038] Dilute Trichinella spiralis genomic DNA to 17pg, 1.7pg, 170fg, 17fg, 1.7fg and 0.17fg and add them to reaction tubes respectively. Primers F3, B3, FIP, BIP, LB and LF, 8U Bst are also added to the tubes 2.0 DNA polymerase, 1.5mMdNTP, 7.5mM MgSO4 and ddH2O, in addition, add 1μl of SYTO13 fluorescent dye with a concentration of 25μM, and react on Bio-Rad’s fluorescent quantitative PCR instrument. The specific reaction program is: 65℃ for 1.5h and Fluorescence data were collected and reacted at 95°C for 5 minutes.

[0039] The result is as figure 2 As shown, the primer and reaction system can detect at least 1.7fg of Trichinella spiralis genomic DNA, and have good detection sensitivity.

Embodiment 4

[0040] The detection of embodiment 4 pyrophosphatase detection sensitivity

[0041] Trichinella spiralis genomic DNA was diluted to 17pg, 1.7pg, 170fg, 17fg, 1.7fg and 0.17fg and added to reaction tube 1 respectively. Primers F3, B3, FIP, BIP, LB and LF were also added to the tube, 8U Bst 2.0 DNA polymerase, 1.5mM dNTP, 7.5mM MgSO4 and ddH2O, in addition, add 8U pyrophosphatase, react in a 63°C water bath for 40 minutes, and then mix the reaction product with 4uL molybdic acid-tartaric acid in reaction tube 2 Potassium oxyantimony solution (21mM molybdic acid, 2mM potassium oxyantimony tartrate, 5M sulfuric acid) and 106uL distilled water, and 2uL 10% ascorbic acid and 106uL double distilled water solution in reaction tube 3 are fully mixed, if the solution turns blue, it is positive , which is negative if it remains colorless.

[0042] The result is as image 3 As shown, the pyrophosphatase detection system of the present invention can detect at least 1.7 fg of Trichinella ...

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

No PUM Login to view more

Abstract

The invention discloses a primer set for detecting trichina gene by loop-mediated constant temperature amplification, an application and a method. The primer set comprises a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a pair of loop primers LF / LB, wherein the primer set is used for detecting trichina polypide and ova. The method comprises the steps of extracting sample genomeDNA to be tested, using genome genomic DNA as a formwork, adding the genome DNA to constant temperature amplification reaction liquid, performing a water bath reaction to obtain reaction products, wherein the constant temperature amplification reaction liquid comprises LAMP buffer liquid, a primer set, Bst 2.0 DNA polymerase, pyrophosphatase, dNTP, MgSO4 and ddH2O, and mixing the reaction productswith the detection liquid. If the liquid is changed into blue, results are positive, and if the liquid maintains colorless, the results are negative. The primer set can quickly and accurately detectthe trichina genome DNA, is high in sensibility and high in specificity, has good stability, is simple to operate, can well meet requirements of staff of different levels, and is easy to promote for base.

Description

technical field [0001] The invention belongs to the technical field of parasite detection, and in particular relates to a primer for rapidly detecting Trichinella spiralis by using a loop-mediated isothermal amplification technology combined with a pyrophosphate tracer technology and an application thereof. Background technique [0002] Trichinella is an important zoonotic parasitic disease. Infection can cause severe diseases of humans and other animals (such as pigs, dogs, cats, bears, foxes, and mice) - trichinosis, not only for livestock breeding The industry causes huge economic losses, and also poses a huge threat to human health. [0003] It is of great significance to adopt rapid, sensitive and accurate diagnostic detection methods in the prevention and control of trichinellosis. For the detection of trichinellosis in animals, microscopic examination or sample digestion method is usually used, but both methods are time-consuming and laborious. Come a lot of inconve...

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
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119
Inventor 孔庆明卓洵辉童群波陆绍红丁豪杰郑斌楼涤丁建祖陈睿
Owner ZHEJIANG ACAD OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap