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Fasciolopsis PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primers, 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 cumbersome detection steps, high detection cost, easy to cause pollution, etc. Detection efficiency and sensitivity, wide application prospects, labor saving effect

Inactive Publication Date: 2013-08-28
郑秋月
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection steps are cumbersome and the sensitivity of the method is low; the result judgment is mainly based on morphological inspection, which requires high professional ability of inspectors and is easily affected by subjective experience
ELISA is used to detect parasite eggs, which has poor specificity and is prone to false positives
Although the detection cost of PCR-gel electrophoresis is low, the electrophoresis treatment of reaction products is very easy to cause pollution; the 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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  • Fasciolopsis PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primers, kit and detection method
  • Fasciolopsis PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primers, kit and detection method
  • Fasciolopsis PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primers, kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0045] 1. Template Preparation

[0046] The template DNA extraction methods described below are routine operations of those skilled in the art, and all reagents and solutions are prepared by conventional means or obtained from commercial means.

[0047] ① Add 400 μL DNA extraction buffer and 40 μL proteinase K (10 mg / mL) to the 1.5 mL centrifuge tube containing the sample, mix well and digest at 37 °C for 3 h;

[0048] ②Centrifuge at 10000g for 2min, discard the supernatant; add an equal volume of phenol, chloroform, isoamyl alcohol (25:24:1), mix the organic phase and aqueous phase by vigorous shaking, centrifuge at 10000g, 4°C for 10min, transfer the supernatant to another in a centrifuge tube.

[0049] ③ Take the supernatant of equal volume, add chloroform and isoamyl alcohol (24:1) to shake, centrifuge at 12000g for 10min; extract once.

[0050] ④ Take the supernatant and add pre-cooled 2 times the volume of ethanol (or add an equal volume of isopropanol), shake the mixt...

Embodiment 2

[0079] Embodiment 2 specificity test

[0080] Samples of parasites such as Fasciola metacercariae, cysticerci, roundworm eggs, Trichinella spiralis, Toxoplasma gondii, and liver fluke were taken, and genomic DNA was extracted according to the method described in Example 1. Using these genomic DNAs as templates, PCR amplification and DHPLC detection were performed according to the method described in Example 1. The results are attached figure 2 As shown, 1-6 are in order: Fasciola metacercariae, cysticerci, roundworm eggs, trichinella spiralis, toxoplasma gondii, liver fluke; only the samples of Fasciola metacercariae have typical PCR product absorption peaks, and the absorption peak is greater than 3mV. The result of this sample was judged to be positive, and Fasciola fragrans was detected. Other parasite samples such as cysticercus, roundworm eggs, trichinella spiralis, toxoplasma gondii, and liver fluke were all negative, and there were no false positive and false negativ...

Embodiment 3

[0081] Embodiment 3 detection sensitivity test

[0082] A sample of the metacercariae of Fasciola zingiberi was taken, and the count under the microscope was 1.06×10 2 cells / mL, and then diluted successively to 1.06×10 cells / mL, 1.06 cells / mL, 1.06×10 cells / mL -1 individual / mL. 10 μL of each dilution was added to a PCR reaction tube, and DNA was extracted according to the method established in Example 1. Take 2 μL each as a template, and perform PCR-DHPLC detection according to the method established in Example 1. The results are attached image 3 Shown: 1. 1.06×10 metacercariae / mL; 2. 1.06 metacercariae / mL; 3. 1.06×10 metacercariae -1 individual / mL. The results showed that the sensitivity of this method was very high, and about 1 / mL (g) metacercariae of Fasciola zingiberi could be detected.

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Abstract

The invention discloses fasciolopsis PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primers, a kit and a detection method. Primers SEQ ID NO.1-2 are designed according to the gene sequence of fasciolopsis, and a PCR-DHPLC method is utilized to carry out qualitative detection on the fasciolopsis in food or aquatic plants. The kit comprises a 5U / mu L Taq DNA (deoxyribonucleic acid) polymerase, 2.5 mM of PCR reaction solution, 2.5 mM of dNTP (deoxyribonucleotide triphosphate), 10 mu M of SEQ ID NO.1 and 10 mu M of SEQ ID NO.2. The method can detect the fasciolopsis to the precision of 1 fasciolopsis / gram sample. The invention has the advantages of short time consumption and simple operation, can save abundant human resources and material resources, and is suitable for the requirements of quick detection.

Description

technical field [0001] The invention relates to a detection method of fasciola in food and aquatic plants, in particular to a method for detecting fasciola in food and aquatic plants 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] Fasciola brucei is a large trematode parasitic in the small intestines of humans and pigs, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 郑秋月曹际娟徐杨王刚
Owner 郑秋月
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