Extracting method of viral nucleic acid capable of being used for high flux full automation extraction by magnetic bead method

A virus nucleic acid, fully automated technology, applied in the biological field, can solve the problems of lack of versatility, failure to adopt magnetic separation method, and inability to realize high-throughput fully automated operation, so as to achieve simple and fast operation and realize high-throughput automatic operation Effect

Active Publication Date: 2010-10-06
宁波市博坤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether it is precipitation centrifugation or gel adsorption, it involves complex equipment (centrifuge or chromatography equipment). This process is difficult to integrate with the automatic extraction operation based on magnetic separation in the magnetic bead virus nucleic acid extraction technology, and manual extraction is still required. operate
At present, there is also a technology that uses antibody-coated magnetic beads to separate viruses, but the subsequent nucleic acid extraction does not use magnetic separation.
At the same time, different antibodies need to be coated for different types of viruses, and this technology is not universal
Therefore, none of the currently reported magnetic bead virus nucleic acid extraction technologies can achieve a universal high-throughput fully automated operation from virus extraction to viral nucleic acid extraction.

Method used

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  • Extracting method of viral nucleic acid capable of being used for high flux full automation extraction by magnetic bead method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Step 1: Magnetic Beads I——1.2μm Fe 3 o 4 - Preparation of Hydroxyapatite Magnetic Composite Particles

[0033] Synthesis of 12nm Fe by co-precipitation method reported in literature 3 o 4 magnetic nanoparticles. After mixing 20ml of glacial acetic acid and 780ml of water, add 5g of Fe 3 o 4 Magnetic nanoparticles, ultrasonically dispersed. Under the condition that the stirring speed is 100rpm, add 200ml containing 14.11g Ca(NO 3 ) 2 , 7.02g KH 2 PO 4and 4ml of glacial acetic acid, and stirred for 15 minutes. Slowly add 2mol / L NaOH solution dropwise to the mixed solution to keep the pH of the solution greater than 13. The reaction was carried out for 12 hours under the condition that the stirring speed was 150 rpm. Use a permanent magnet to separate the solid from the reaction solution, and wash the resulting solid 3 times with high-purity water to obtain Fe 3 o 4 - hydroxyapatite magnetic composite particles, the average size of which is 1.2 μm;

[0034] ...

Embodiment 2

[0047] Step 1: Magnetic beads I——Preparation of 0.6 μm Ni-hydroxyapatite magnetic composite particles

[0048] After mixing 25ml of glacial acetic acid and 775ml of water, add 5g of 8nm commercial Ni magnetic nanoparticles and ultrasonically disperse them. Under the condition that the stirring speed is 150rpm, add 200ml containing 8.5g Ca(NO 3 ) 2 , 4.21g KH 2 PO 4 and 7.5ml of glacial acetic acid, and stirred for 30 minutes. Slowly add 2mol / L NaOH solution dropwise to the mixed solution to keep the pH of the solution greater than 13. The reaction was carried out for 6 hours under the condition that the stirring speed was 200 rpm. Use a permanent magnet to separate the solid from the reaction solution, and wash the obtained solid with high-purity water for 3 times to obtain Ni-hydroxyapatite magnetic composite particles with an average size of 0.6 μm;

[0049] Step 2: Extract viral nucleic acid from various biological samples

[0050] The magnetic beads I used in this e...

Embodiment 3

[0055] Step 1: Magnetic Beads I——2μm Ni-Fe 3 o 4 - Preparation of Hydroxyapatite Magnetic Composite Particles

[0056] After getting 40ml of glacial acetic acid and 760ml of water to mix, add 5g of commercialized average size of 8nm Ni magnetic nanoparticles and 5g of commercialized average size of 10nm Fe 3 o 4 Magnetic nanoparticles, ultrasonically dispersed. Under the condition that the stirring speed is 150rpm, add 200ml containing 28.22g Ca(NO 3 ) 2 , 12.64g KH 2 PO 4 and 8ml of glacial acetic acid, stirred for 30 minutes. Slowly add 4mol / L NaOH solution dropwise to the mixed solution to keep the pH value of the solution greater than 13. The reaction was carried out for 6 hours under the condition that the stirring speed was 200 rpm. Use a permanent magnet to separate the solid from the reaction solution, and wash the resulting solid with high-purity water for 3 times to obtain Ni-Fe 3 o 4 - hydroxyapatite magnetic composite particles, the average size of which...

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Abstract

The invention provides an extracting method of viral nucleic acid capable of being used for high flux full automation extraction by a magnetic bead method, which is characterized by comprising the steps of separating viruses via a magnetic bead I: absorbing the viruses to the magnetic bead I from a biological sample to obtain the magnetic bead I with the viruses which is called as a virus-magnetic bead I composite for short, additionally adding a magnetic field to separate the virus-magnetic bead I composite from the sample, suspending the virus-magnetic bead I composite in a lysis buffer I, dissociating the virus from the magnetic bead I in the lysis buffer, additionally adding a magnetic field to remove the magnetic bead I, and heating to crack the virus; and separating a viral nucleic acid via a magnetic bead II: absorbing the viral nucleic acid in a viral lysate via the magnetic bead II, additionally adding a magnetic field to separate the magnetic bead II with the viral nucleic acid from the viral lysate, washing the magnetic bead II with the viral nucleic acid by using a washing solution, and eluting the viral nucleic acid from the magnetic bead II by using a solution to obtain a purified viral nucleic acid. In the invention, cracking cells and separating viruses are completed at one step in one tube by using the magnetic beads; and extraction of the viruses and the viral nucleic acid can be achieved by using two magnetic beads so that the extraction process is simple and rapid without a complicated device, and high pass automatic operation is easy to be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting viral nucleic acid by a magnetic bead method that can be used for high-throughput fully automatic extraction. Background technique [0002] With the rapid development of molecular biology, PCR and RT-PCR have become powerful tools for highly sensitive detection of DNA viruses and RNA viruses. Compared with other current detection techniques, PCR and RT-PCR methods have made great progress in terms of detection sensitivity, specificity and time-consuming. Moreover, since a large number of viral nucleic acid sequences are included in the Genbank database, this is very important for viruses. Specific PCR primer design provides unique advantages. With the increasing application of PCR and RT-PCR in virus detection, the workload of virus nucleic acid extraction has increased rapidly, and traditional manual operations can no longer meet the requirements of high-thr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王刚王延旭戴洁董涛王凌云吴晓明
Owner 宁波市博坤生物科技有限公司
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