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Trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as kit and detection method

A PCR-DHPLC, detection kit technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of short storage time, easy pollution and high detection cost of fluorescent probes, Achieve the effect of improving detection efficiency and sensitivity, short detection time, and good repeatability

Inactive Publication Date: 2013-08-21
郑秋月 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method makes up for the shortcomings of conventional methods such as long detection cycle, tediousness, and easy to be affected by human factors.
Although the detection cost of PCR-gel electrophoresis is low, the electrophoresis treatment of reaction products is very easy to cause pollution; the real-time fluorescent PCR method with strong specificity and high sensitivity also has problems such as high detection cost and short storage time of fluorescent probes

Method used

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  • Trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as kit and detection method
  • Trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as kit and detection method
  • Trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Design and synthesis of primers and assembly of kits:

[0046] The present embodiment determines that the primer sequences and amplified fragment lengths used for detection are as follows:

[0047] Upstream primer: 5′-gcg aat tct tgg atc gga gac ggc ctg-3′

[0048] Downstream primer: 5′–gct cta gac gag atg tcg tgc ttt caa cg-3′

[0049] A 740bp specific target gene fragment was amplified from Trichinella spiralis.

[0050] On this basis, a kit for PCR-DHPLC detection was designed. The kit includes Taq DNA polymerase and PCR reaction solution at a concentration of 5U / μL; the PCR reaction solution contains 10mM Tris HCl, 50mM KCl, 25mM MgCl 2 , dNTP (dATP, dGTP, dCTP and dTTP) 2.5mM each and trichinella detection primer pair 10μM.

[0051] (2) Establishment of PCR-DHPLC detection method:

[0052] This detection method uses the detection kit established in this embodiment, comprising the following steps:

[0053] 1) Sample preparation

[0054] ① For morphologica...

Embodiment 2

[0083] Embodiment 2 specificity test

[0084] Samples of parasites such as Trichinella spiralis, cysticercus, roundworm eggs, whipworms, hookworms, liver flukes, Fasciola zingiberi, Trichostrongylus and other parasite samples were taken, and genomic DNA was extracted according to the method described in Example 1. Using these genomic DNAs as templates, carry out PCR amplification and DHPLC detection according to the method described in Example 1, the results are shown in the attached figure 2 1-8 shown in order are: Trichinella spiralis, cysticercosis, roundworm eggs, whipworm, hookworm, liver fluke, fascia zingiberi, Trichostrongylus; only Trichinella samples have typical PCR product absorption peaks, the absorption peak is greater than 3mV, The result of this sample was judged to be positive, and Trichinella spiralis was detected. Other parasite samples such as cysticercus, roundworm eggs, whipworms, hookworms, liver flukes, fasciola, Trichostrongylus and other parasites w...

Embodiment 3

[0085] Embodiment 3 detection sensitivity test

[0086] Count the Trichinella spiralis bodies under the microscope, extract 5 Trichinella spiralis bodies and 1 Trichinella spiralis body DNA respectively according to the method established in Example 1, take 2 μ L each as a template, and perform PCR according to the method established in Example 1- DHPLC detection, the results are attached image 3 Shown: After extracting DNA from 1 Trichinella spiralis body, adding 2 μL of template DNA can still detect a typical positive absorption peak by PCR-DHPLC. The results showed that the sensitivity of this method was very high, and DNA from a single tissue of Trichinella spiralis could be detected.

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Abstract

The invention discloses a trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as a kit and a detection method. In allusion to a gene sequence of the trichina, detection primers SEQ ID NO.1-2 are designed and a PCR-DHPLC method is used for the qualitative detection of the trichina in detected animal derived food. The kit comprises Taq DNA polymerase with concentration of 5U / microlitre, 2.5mM of PCR reaction liquid, 2.5mM of dNTP (Diethyl-Nitrophenyl Thiophosphate), 10 microns of SEQ ID NO.1 and 10 microns of SEQ ID NO.2. The method disclosed by the invention can be used for precisely detecting the polypide sample of one trichina, and has the advantages of being short in detection time and simple to operate, saving lots of manpower and material resources and being suitable for rapid detection.

Description

technical field [0001] The invention relates to a detection method for Trichinella spiralis in animal-derived foods, in particular to a method for detecting Trichinella spiralis in animal-derived foods by using PCR and denatured high-performance liquid chromatography (DHPLC) techniques. It also relates to the composition used for the detection, ie the kit. Background technique [0002] Harmful organisms in food and agricultural by-products mainly include bacteria, viruses and parasites. According to the zoonotic diseases announced by the World Health Organization in 1999, there are 58 zoonotic diseases. According to the Fourth Military Medical University of the PLA in 2004 From April 2005 to February 2005, 91 species of zoonotic parasitosis in my country were investigated. Zoonotic parasitic diseases have brought serious threats and harm to public health, people's health, and social stability. [0003] Trichinella nematodes, referred to as Trichinella spiralis, belong to Tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 郑秋月曹际娟赵昕徐静
Owner 郑秋月
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