Detection primer, detection kit and detection method for clonorchis sinensis and application

The technology of Clonorchis sinensis and detection kit is applied in the field of detection primers of Clonorchis sinensis, and can solve the problems of long time consumption, complicated operation and high cost of detection method

Inactive Publication Date: 2021-02-05
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a low-cost, easy-to-operate, short detection time and high sensitivity Clonorchis sinensis in order to overcome the defects of detection methods such as high cost, cumbersome operation, long time-consuming, missed detection or false detection. (Clonorchis sinensis) detection primers, detection kits and detection methods can complete the detection and interpret the results in 5 minutes at room temperature. The minimum detection concentration of Clonorchis sinensis DNA is 2ng / μL, and the sensitivity is 5 times higher than that of ordinary PCR. , the specificity of detecting Clonorchis sinensis is 100%,

Method used

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  • Detection primer, detection kit and detection method for clonorchis sinensis and application
  • Detection primer, detection kit and detection method for clonorchis sinensis and application
  • Detection primer, detection kit and detection method for clonorchis sinensis and application

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Effect test

Embodiment 1

[0034] One, detect the method for the primer design of Clonorchis sinensis

[0035] (1) Sequence acquisition

[0036] Using DNAman software for sequence comparison and literature search, it was confirmed that COX-1 in Clonorchis sinensis mDNA had a high interspecies difference, and the Clonorchis sinensis COX-1 gene sequence was selected (Genebank: AF096229).

[0037] (2) Three pairs of primers were designed by Primer 5 software, namely COX-1, COX-2, and COX-3.

[0038] (3) Principles of primer design

[0039] The primer design of the recombinase polymerase amplification reaction should pay attention to the length of the primer and the amplicon, the primer sequence and the selectivity of the primer to the homologous sequence. The primer length of the recombinase polymerase amplification reaction is greater than 30bp, generally 30-35bp, because the above-mentioned length is the minimum length for the recombinase-mediated primer to match the homologous double strand, and the l...

Embodiment 2

[0078] 1. Detection method of Clonorchis sinensis

[0079] 1. The method of obtaining Clonorchis sinensis metacercariae

[0080]The metacercariae of Clonorchis sinensis were obtained by artificial digestion method of the ear fish of Songhua River: after removing the head and viscera of the ear fish, they were washed and crushed in a tissue grinder, and then a 10:1 volume of digestive solution ( 7g of pepsin, 10mL of hydrochloric acid, adding normal saline to make the volume to 1000mL) for digestion, each digestion for 3 to 4 hours, carried out in a shaker at 37°C, and then repeatedly washed the precipitate with water until the pure metacercariae were collected.

[0081] 2. Extraction of DNA from test samples

[0082] According to the phenol-chloroform extraction method, the total DNA of the wheat ear fish in the Songhua River Basin was extracted under a microscope to confirm the infection of Clonorchis sinensis metacercariae, and 5 μL of the DNA extraction solution was extrac...

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Abstract

The invention provides a detection primer, a detection kit and a detection method for clonorchis sinensis and an application, belongs to the technical field of biological assays, and aims to overcomethe defects of high cost, complicated operation, large time consumption, missed detection or false detection in existing detection methods. The invention provides a detection primer, a detection kit and a detection method for clonorchis sinensis with low cost, simple operation, short detection time and high sensitivity. According to the invention, detection can be completed within 5 min at the normal temperature, and interpretable results can be obtained. The detection kit of the present invention has 2ng/[mu]L of minimum detection concentration to clonorchis sinensis DNA and 5 times higher sensitivity than common PCR. The detection kit has 100% of specificity to clonorchis sinensis. Results can be judged visually in actual sample detection. clonorchis sinensis metacercariae and eggs can be quickly detected by adding nucleic acid dye Sybr Green I without needing PCR instruments or ultraviolet gel systems. The detection kit is suitable for quick detection in primary care or on site.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to detection primers, detection kits, detection methods and applications of clonorchis sinensis. Background technique [0002] Foodborne parasitic diseases refer to a general term for a class of diseases that are infected by eating raw or not thoroughly heated foods containing parasite eggs or larvae. In recent years, with the improvement of people's living standards and the diversification of dietary sources and methods, outbreaks of foodborne parasitic diseases have occurred from time to time, and food safety problems caused by food parasites have become more prominent. Foodborne parasitic diseases are not only one of the main factors affecting the health of our people, but also one of the important global disease burdens. Clonorchis sinensis is an important food-borne zoonotic parasite. The eggs of Clonorchis sinensis are excreted with the feces o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2521/507
Inventor 刘晓雷刘明远杨勇唐斌丁静张春玲
Owner JILIN UNIV
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