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Real-time fluorescence PCR primer for perkinsosis detection and probe

A real-time fluorescence, piquing worm technology, applied in the field of epidemiology and food hygiene detection, can solve the problems of low sensitivity, low detection sensitivity, long time consumption, etc., and achieve the effect of strong specificity

Inactive Publication Date: 2012-04-11
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique is time-consuming, requires highly skilled technicians, and has low detection sensitivity
Ray (1952) reported that adopting FTM to cultivate the method for the dormant spores of Piedia is to cultivate the trophozoites of Piedia, and observe after the size of the insect body increases, which is a traditional and effective method for quantitative detection of Piedia, but this The culture method can only be detected by culturing Pijenia to the stage of dormant spores, and the whole process takes up to a week, so the sensitivity is low, and it can only be detected when the number of protozoa in a gram of tissue is higher than 1000

Method used

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  • Real-time fluorescence PCR primer for perkinsosis detection and probe
  • Real-time fluorescence PCR primer for perkinsosis detection and probe
  • Real-time fluorescence PCR primer for perkinsosis detection and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Piedia FTM culture method

[0027] 1. Sample pretreatment

[0028] Take shellfish samples that reach a certain statistical significance, place them in a sterilized mortar, measure the length, width, and thickness, discard the shells, weigh the net weight of the tissue with a balance, and place it in a sterilized research mortar. Bowl, grind to 0.1mm 3 about.

[0029] 2. Incubation of samples

[0030] Place the samples in sterilized centrifuge tubes containing 5mL of FTM medium, add 250μL of 2.5% chloramphenicol and shake well, cover the liquid with 1mL of 1% nystatin, close the lid, and place in a dark room at 22-24°C Culture 5-7d.

[0031] 3. Purification of samples

[0032] Centrifuge the cultured sample at 4000r / min for 10min, discard the supernatant, add 6mL of 2mol / L NaOH, vortex and mix well, and place it in an oven at 55°C for overnight digestion. Centrifuge at 4000r / min for 10min, discard the supernatant, add 2mL ddH 2 O wash, centrifuge at 4...

Embodiment 2

[0037] Preparation of embodiment 2 primers and probes

[0038] 1 Design and synthesis of primers and probes

[0039] Select the relatively conserved ITS-2 region in the ribosomal DNA sequence of Pietia worm, and design it with the software Beacon Designer7.0. The primer pair was determined to be the specific primer pair of Pijenia by online sequence BLAST analysis. And set a specific fluorescent TaqMan probe in the amplification region of the primer pair.

[0040] The primer sequences used in this example are:

[0041] Upstream primer: (forward): 5'-GAAGAATGGCGTGATCAAGGAAC-3';

[0042] Downstream primer: (reverse): 5'-TGCAAGGCTATAATCTCGTATTGTAG-3', the length of the amplified product is 126bp;

[0043] The sequence of the probe is: 5'FAM-CAACACAGTCGGACTTGCGAGCATCCAAFAMRA-3', the 5' end of the probe is marked with a fluorescent reporter group 6-carboxyfluorescein (6-carboxyfluorescein, FAM), and the 3' end is marked with a fluorescence quencher Killing group 6-carboxytetra...

Embodiment 3

[0046] Embodiment 3 Establishment of the detection method of Piedia worm fluorescent PCR

[0047] 1 Genomic DNA extraction from diseased tissue

[0048] Take 25 mg of the gill tissue of the Philippine clam that was infected with Piechinia by FTM culture, extract the genomic DNA according to the instructions of the kit (QIAGEN, 69506), and store it at -20°C for future use.

[0049] 2PCR amplification of the target gene

[0050] Using primers to use the genomic DNA of the diseased tissue as a template, the above-mentioned extracted DNA was amplified by PCR using the Piedia specific primers AF and AR published by Liang Yubo (2005). The primer sequences are: AF: 5'-CCGCTTTGTTTGGMTCCC-3'; AR: 5'-ACATCAGGCCTTCTAATGATG-3. PCR reaction system reference: 0.5 μL of primer AF (10 μmol / L), 0.5 μL of primer AR (10 μmol / L), 0.5 μL of fluorescent probe (10 μmol / L), 10×buffer (containing Mg 2+ ) 2.5 μL, dNTP (2.5mmol / L) 2 μL, rTaq DNA polymerase (5U / μL) 0.5 μL, sterilized ddH 2 O suppleme...

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Abstract

The invention provides a real-time fluorescence PCR primer for perkinsosis detection and a probe; the specificity of the primer aims at a guard area of ITS2 sequence in perkinsosis genome DNA, the specificity of the probe aims at a CC high content area of the primer in an amplification area, the 5'end of the probe has a fluorescence dye reporting mark and the 3'end thereof has a quenching fluorescence dye mark; the real-time fluorescence PCR primer fully utilizes the high-efficiency amplification of the fluorescence PCR technology, good nucleotide hybridization specificity and rapid sensitivity of the fluorescence detection technology; when the reaction is finished, whether a tissue sample to be detected contains the perkinsosis can be judged according to an amplification curve; in the invention, the genome area for PCR amplification is the perkinsosis ITS2 guard area, the specificity is strong, and the primer has no cross reaction with other parasitism protozoa such as bonamiasis protozoa, cryptosporidiuin and the like and can not be disturbed by shellfish tissue DNA.

Description

technical field [0001] The invention belongs to the field of epidemiology and food sanitation detection. Specifically, the present invention relates to real-time fluorescent PCR primer pairs and probes for the detection of Piedia worms, which are especially useful for the detection of Piedias worms carried in shellfish products. The detection of pijon worms provides an effective technical means. Background technique [0002] Perkinsosis is caused by marine Perkinsus marinus, Perkinsus olseni, etc. in the blood cells of shellfish hosts or free in connective tissue, gills, viscera or mantle epithelium A serious parasitic disease caused by aquatic animals. It is the main pathogen affecting the development of shellfish farming in the world, and it is one of the must-report aquatic animal diseases stipulated by OIE. So far, the infected hosts of pike worms have been found to include oysters, abalones, clams, scallops, pearl oysters, and cockles. and mussels etc. [0003] It is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64C12N15/11
CPCY02A50/30
Inventor 吴绍强林祥梅刘建贾广乐
Owner CHINESE ACAD OF INSPECTION & QUARANTINE