Roundworm egg detecting real-time fluorescence PCR primer and probe

A real-time fluorescence and probe technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., which can solve the problems of high initial volume requirements, time-consuming and labor-intensive, and inoperability.

Inactive Publication Date: 2007-03-21
中国检验检疫科学研究院动植物检疫研究所
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AI Technical Summary

Problems solved by technology

The above studies are all carried out using the PCR-RFLP method, that is, according to the difference in the ITS nucleic acid sequence, the method of enzyme digestion after the PCR reaction and then electrophoresis is carried out, which is very time-consuming and laborious, and requires a high initial amount of DNA. Operate at the individual egg level

Method used

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  • Roundworm egg detecting real-time fluorescence PCR primer and probe

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Embodiment 1

[0043] The preparation of embodiment 1 primer and probe

[0044] 1.1 Design and synthesis of primers and probes

[0045] According to the published sequences of Ascaris suum and human Ascaris 18S-28S rDNA genes (GenBank NO.AB110029 and AB110030), the gene sequences were analyzed by MegAlign software, and according to the design principles of primers and probes, BeaconDesigner 5.0 was used to screen the conserved regions of these sequences to a pair of universal primers with the same base sequence. And set a universal fluorescent TaqMan probe in the amplification region of the primer pair.

[0046] The primer sequences are:

[0047] P1 (Forward): 5'-TGCGATAAATAGTGCGAATTGC-3'

[0048] P2 (reverse): 5'-TCTTTCAATAATTCAACCCTCAGC-3', the length of the amplified product is 87bp;

[0049] The sequence of the probe is: 5'-ACGTGCCAACGGGAATGAACCC-3'

[0050] The 5' end of the probe was labeled with the reporter fluorescent dye FAM, and the 3' end was labeled wi...

Embodiment 2

[0054] The establishment of embodiment 2 fluorescent PCR detection method

[0055] 2.1 Isolation of suspected roundworm eggs in pickles

[0056] I. Elution: get 200 grams of sauerkraut (containing soup part) sample and put into large beaker, fully clean each blade with 1.0L 0.5% SDS solution 3 times, combine eluent for subsequent use.

[0057] II. Filtration: Filter the eluate with a 40-mesh sieve (above 0.2 mm in sieve diameter), wash the residue on the sieve with deionized water, and keep the filtrate for later use; if testing fluid products such as chili sauce, you can directly sieve , and rinse the residue with 0.5% SDS solution.

[0058] III. Centrifugation: centrifuge the filtrate at 1500g for 10 minutes, pour off the supernatant carefully, add deionized water to wash, centrifuge twice, and take the precipitate for later use.

[0059] IV. Floating: Add 5 times the volume of saturated salt solution to the sediment, vortex and mix with an oscillator, ...

Embodiment 3

[0068] Example 3. Sensitivity test of Ascaris ovum fluorescent PCR detection method

[0069] Add 1-5 eggs of Ascaris suum to the PCR reaction tube, use the extracted Ascaris suum DNA as a positive control, and sterilize ddH 2 O was used as a negative control to determine the sensitivity of the above roundworm egg fluorescent PCR detection method to the eggs.

[0070] 3.1 Collection of Ascaris suum eggs

[0071] Carried out by flotation in saturated saline. The specific operation is: take 10 grams of feces collected from a pig farm, add 100 ml of saturated saline, mix them, pass through a 250 μm (60 mesh) copper sieve, filter them into a beaker, let stand for half an hour or centrifuge at a low speed for 10 minutes, and the eggs will float up. Use a wire ring with a diameter of 5-10mm to touch the liquid surface in parallel to pick up the surface liquid film, shake it off on a glass slide pre-added with a small amount of sterilized water, and wait for inspection...

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Abstract

The present invention provides roundworm egg detecting real-time fluorescence PCR primer and probe and the detection method therewith. Roundworm egg causing contamination on kraut and other botanical product is hard to detected morphologically with microscope. Through designing superfamily roundworm primer and probe to separate out suspected parasitic egg from kraut, repeated freezing and thawing with liquid nitrogen and fluorescent PCR operation, the suspected parasitic egg from kraut may be identified in high accuracy. The present invention has sensitivity enough high for detecting single roundworm egg, high specificity, and no cross reaction with other parasite egg and plant nematode egg.

Description

technical field [0001] The invention belongs to the field of epidemiology and food sanitation detection, in particular, the invention relates to real-time fluorescent PCR primers and probes for detecting parasite eggs in plant-derived products, and methods for detection thereof, more specifically Said to be real-time fluorescent PCR primers and probes for the detection of roundworm eggs carried in plant-derived products, and a method for using them for detection. Background technique [0002] Human parasites number in the hundreds and pose potential threats to human health. Many animal parasites can also endanger human health. After humans eat vegetables contaminated by parasite eggs, it can cause larval migratory disease, and Japan has reported many cases of human infection with Ascaris suum. A domestic survey of nematode-contaminated vegetables found that a variety of so-called "clean vegetables" sold in the domestic market also carry parasites. Therefore, parasite eggs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 吴绍强林祥梅陈洪俊韩雪清葛建军朱水芳梅琳刘建贾广乐孙晓智石鑫
Owner 中国检验检疫科学研究院动植物检疫研究所
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