Method for quickly extracting plasmodium DNA in high through-put mode and application of method

A malaria parasite, high-throughput technology, applied in the field of nucleic acid extraction and separation of parasites, can solve the problems of low throughput, time-consuming, and complicated DNA preparation, and achieve the effects of low extraction throughput, shortened time, and high purity

Active Publication Date: 2017-11-07
辽宁省农业科学院大连生物技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problems of complicated, time-consuming and low-throughput preparation of target DNA in the current Plasmodium detection technology, the present invention provides a high-throughput and rapid extraction method of Plasmodium DNA

Method used

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  • Method for quickly extracting plasmodium DNA in high through-put mode and application of method
  • Method for quickly extracting plasmodium DNA in high through-put mode and application of method
  • Method for quickly extracting plasmodium DNA in high through-put mode and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Select a case of filter paper blood from a patient infected with Plasmodium vivax, and a case of filter paper blood from an artificially bred Plasmodium falciparum 3D7 strain. Use a puncher to take disc blood samples with a diameter of 3 mm and place them in two 1.5 mL EP tubes, numbered Pv, respectively. Pos. Add 200 μL of lysate S (0.2M NaOH, 1% SDS) to the Pv tube, vortex and mix well, let it stand at room temperature for 5 minutes, add 10 μL of magnetic bead Buffer (C@SiO 2 @Fe 3 o 4 ) and 150mL of absolute ethanol, shake and mix gently, place the EP tube on a magnetic stand for separation, and when the magnetic beads are completely separated from the liquid phase, pour off the liquid phase, remove the magnetic stand, add 150 μL of washing buffer, shake and mix After uniformity, put it on the magnetic stand for separation, discard the liquid, remove the magnetic stand, add 150 μL of washing buffer again, oscillate, mix and place on the magnetic stand for separatio...

Embodiment 2

[0060] Take filter paper blood samples of artificially bred Plasmodium falciparum strain 3D7, use a puncher to take disc blood samples with a diameter of 3mm and place them in two 1.5mL EP tubes, numbered 1# and 3# respectively. Add 200 μL of 0.2M NaOH to the tube, vortex and mix well, place 1# at room temperature for 5 minutes, and 3# at room temperature for 40 minutes, then transfer the blood slices into new EP tubes, numbered 2# and 4# respectively. Add 10 μL of magnetic bead Buffer (SiO 2 @Fe 3 o 4 ) and 150 mL of absolute ethanol, shake and mix gently, place the EP tube on a magnetic stand for separation, and when the magnetic beads are completely separated from the liquid phase, pour off the liquid phase, remove the magnetic stand, add 150 μL of cleaning buffer, shake and mix After uniformity, put it on the magnetic stand for separation, discard the liquid, remove the magnetic stand, add 150 μL of cleaning buffer again, oscillate, mix and place on the magnetic stand fo...

Embodiment 3

[0067]Take the filter paper blood of patients infected with Plasmodium vivax, and use a puncher to take a disc blood sample with a diameter of 3mm and place it in a 1.5mL EP tube. Mix well, let stand at room temperature for 5 minutes, add 10 μL of magnetic bead buffer (Sigma) and 150 mL of absolute ethanol, shake gently to mix, place the EP tube on a magnetic stand for separation, and pour off the liquid phase after the magnetic beads are completely separated from the liquid phase , remove the magnetic stand, add 150 μL of cleaning buffer, oscillate and mix well, place on the magnetic stand for separation, discard the liquid, remove the magnetic stand, add 150 μL of cleaning buffer again, oscillate and mix, place on the magnetic stand for separation, transfer Aspirate as much liquid as possible with the liquid gun, remove the magnetic stand, place the EP tube in a 37°C incubator to dry for 30 minutes, and then elute with 20 μL of elution buffer. The DNA was frozen at -20°C unt...

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Abstract

The invention discloses a method for quickly extracting plasmodium DNA in a high through-put mode and application of the method. Particularly, the template extracting method comprises the steps that release, enrichment, separation and purification of DNA are conducted through alkaline lysis and magnetic beads. The method is simple and fast in operation, low in cost, high in through-put, low in contamination risk, high in quality of the extracted DNA, and capable of achieving automation and being widely used in molecular detection, the detection through-put and the operation convenience can be remarkably improved, the false positive rate of detection is obviously reduced, and the detection sensitivity and accuracy and the detection efficiency are improved. The visible and automatic sample diagnosis technology that extraction and detection of a mass of DNA on a perforated plate are integrated can be achieved through series connection of an automatic nucleic acid extractor and a PCR instrument and the PCR technology which is visible in optimizing result. The problems that in an existing plasmodium detection technology, the preparation of the target DNA is complex, wastes time, is low in through-put and the like are solved, and the method has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of nucleic acid extraction and separation of parasites, and in particular relates to a method for rapidly extracting Plasmodium DNA with high throughput and an application thereof. Background technique [0002] Malaria is a parasitic disease transmitted through the bite of Anopheles mosquito. It is a major infectious disease that seriously endangers human health and life safety. It is listed by the World Health Organization (WHO) as one of the three major global public health problems. Malaria control relies on three main approaches: interrupting the source of transmission, chemoprevention, and case management. Case management includes malaria diagnosis in malaria-endemic areas and treatment of positive patients. WHO calls for testing all people in malaria-endemic areas for Plasmodium infection in order to block transmission, control epidemics, and ultimately achieve the goal of eliminating malaria. In the face of a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C12Q1/04C12R1/90
CPCC12N15/1013C12Q1/6806C12Q2523/308Y02A50/30
Inventor 李佩佩范琦赵振军叶博曹雅明
Owner 辽宁省农业科学院大连生物技术研究所
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