Method for directly amplifying single ovum DNA (Deoxyribonucleic Acid) of human ascarid without extraction

A human roundworm, extraction-free technology, applied in the biological field, can solve the problems of difficulty in extracting DNA, low content of human roundworm egg genome DNA, and the release of egg genome DNA, so as to reduce the loss and reduce the loss of DNA. Effect

Inactive Publication Date: 2017-01-25
NANCHANG UNIV
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Problems solved by technology

[0003] For the direct amplification of any kind of trace DNA, the release of genomic DNA is one of the key factors to determine whether the DNA amplification is successful. The special feature of human roundworm eggs is that the roundworm eggs have a layer of hard, dense protein The shell and the impermeable lipid layer make the eggs highly resistant to various physical and chemical substances and adverse external environments, but have a certain impact on the release of genomic DNA from the eggs
At the same time, the genome DNA content of human roundworm eggs is very small, so it is very difficult to extract DNA

Method used

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  • Method for directly amplifying single ovum DNA (Deoxyribonucleic Acid) of human ascarid without extraction

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Embodiment 1

[0014] Example 1. A method for directly amplifying the DNA of a single human roundworm egg without extraction is as follows.

[0015] 1. Wash the eggs with distilled water.

[0016] Use a 1.5ml centrifuge tube, add distilled water and eggs, vortex at 3200rpm for 1min, then centrifuge at 10000r / min for 2min, discard the supernatant, repeat this process 3 times. Pick an egg under a microscope and put it in a 0.2ml PCR tube with 2 PowerBeads and 5μl high-purity water.

[0017] 2. Broken eggs.

[0018] The PCR tube equipped with PowerBead was alternately placed in 100°C boiling water and -80°C for 1 min. After 6 times of repeated freezing and thawing, it was placed horizontally on a vortex shaker at 3200 rpm for 10 min.

[0019] 3. PCR amplification.

[0020] Pipette 15 μl of the PCR reaction solution into a PCR tube for amplification. The formula of PCR reaction solution is: Premix Taq Version2.0 (Takara) 10μl, upstream and downstream primers 0.2 μM each (1.5μl each), high...

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Abstract

A method for directly amplifying the DNA of a single human roundworm egg without extraction, comprising four steps of washing the egg with distilled water, breaking the egg, PCR amplification and result detection. The invention can obtain PCR products with good stability and high amplification efficiency by optimizing the fragmentation method of eggs and the PCR amplification system, and can avoid the amplification failure caused by the loss of DNA in the extraction process. The amplified product obtained by the present invention can effectively perform downstream gene sequencing and STR scanning, and provide important technical methods for molecular epidemiology, genetic diversity, genetic structure, phylogenetics, phylogenetic geography and paleontological research of human roundworms , the invention is also of great significance to the molecular diagnosis, prevention and control of human ascariasis.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a method for amplifying the DNA of a human ascaris single egg. Background technique [0002] human roundworm ( Ascaris lumbricoides Linnaeus, 1758), belonging to the phylum Nematodes, Class Caudosensilla, Order Ascariae, and the genus Ascaris. It is one of the most common intestinal parasites in the human body, especially children. It parasitizes in the human small intestine and causes ascariasis. It is a large human parasitic nematode. Mature females and males mate and lay eggs in the small intestine. The fertilized egg is excreted with the patient's feces and develops under suitable conditions. It can make people infected with ascariasis, which is called infectious egg. If this infectious egg enters the human body along with food or other ways, the larvae hatch in the duodenum, and the larvae drill into the blood vessels of the intestinal wall, pass through the liver, heart, lungs...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q1/686
Inventor 周春花石琴华吴萍萍
Owner NANCHANG UNIV
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