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Method for isolating nucleic acids from a veterinary whole blood sample

A whole blood sample, veterinary technology, applied in the field of nucleic acid isolation, can solve problems such as inability to perform standard detection tests, inability to isolate target pathogen nucleic acid, low purification efficiency, etc.

Inactive Publication Date: 2014-05-28
QIAGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The observed low purification efficiencies and limitations in input volumes result in conventional nucleic acid isolation methods often not isolating target pathogen nucleic acids in sufficient quantities for follow-up to standard detection assays such as polymerase chain reaction

Method used

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  • Method for isolating nucleic acids from a veterinary whole blood sample
  • Method for isolating nucleic acids from a veterinary whole blood sample
  • Method for isolating nucleic acids from a veterinary whole blood sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Different veterinary samples (and PBS buffer as a positive control) were spiked with BVDV virions, and different protocols under different binding conditions were used to isolate nucleic acids. In all protocols, Proteolytic Enzyme K (20 μl) was added for lysis.

[0086] In Protocol 1, lysis was performed by adding a lysis buffer containing polyoxyethylene (20) cetyl ether and GTC. Then, binding buffer containing GTC, isopropanol and polyoxyethylene (20) cetyl ether was added to the lysed samples. The resulting bonded mixture contained 2.31M GTC, 13% isopropanol, and 6.5% polyoxyethylene (20) cetyl ether.

[0087] In protocol 2, lysis was performed by adding lysis buffer containing guanidine hydrochloride and a mixture of non-ionic detergents (Tween 20 and Triton X-100). Then, binding buffer containing GTC, isopropanol and polyoxyethylene (20) cetyl ether was added to the lysed samples. The resulting binding mixture contained the chaotropic salts GTC and GuHCL (total ...

Embodiment 2

[0091] 200 μl whole blood samples from different species (and 0.9% sodium chloride solution as a positive control) were spiked with BVDV particles. The samples were processed using a standard method using a binding mixture comprising guanidine hydrochloride (GuHCL) and Tween 20, or a different variation of the teaching of the present invention using different concentrations in the binding mixture of guanidinium thiocyanate (GTC) and polyoxyethylene (20) cetyl ether. Add proteolytic enzyme K to aid in cleavage:

[0092] Method A: 1.81M GuHCl, 32.2% EtOH, 6.5% Tween 20 in binding mixture

[0093] Method B: 1.9M GTC, 24% Isopropanol, 8.5% Polyoxyethylene (20) Cetyl Ether in a combined mixture

[0094] Method C: 1.5M GTC, 17% Isopropanol, 7.6% Polyoxyethylene (20) Cetyl Ether, 0.3% SDS in the binding mixture

[0095] Method D: 1.5M GTC, 19% isopropanol, 6.8% polyoxyethylene (20) cetyl ether, 0.3% SDS in binding mixture

[0096] Processing was done on QIAamp96 plates using vacu...

Embodiment 3

[0098] Bovine and sheep blood aspirate with BHV1 virus particles and processed using different variations of the present invention. Samples were lysed using proteolytic enzyme K and a lysis solution containing a mixture of non-ionic detergents (Tween 20 and Triton X-100) and a chaotropic salt (GuHCL). For binding, different volumes of binding solution containing chaotropic salts (GTC), isopropanol and polyoxyethylene (20) cetyl ether were added. Thus, different combined mixtures with different concentrations of chaotropic salts (2.14M-2.36M), alcohols (12.8%-23.4%) and polyoxyethylene (20) cetyl ethers (3.8%-7%) were obtained. The use of different concentrations of polyoxyethylene (20) cetyl ether can effectively prevent clogging. All of the different variations of the invention are capable of isolating nucleic acid from a whole blood sample to be tested, e.g. Image 6 shown in the results. A difference in ct between 2.5 μl to 10 μl template volume of approximately 2 indica...

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Abstract

The present invention provides a method for isolating nucleic acids from a veterinary whole blood sample, said method comprising at least the following steps a) preparing a binding mixture comprising - the lysed sample - at least one chaotropic agent - at least one alcohol - at least one polyoxyethylene fatty alcohol ether; b) passing the binding mixture through a column comprising a nucleic acid binding solid phase thereby binding the nucleic acids to the nucleic acid binding solid phase; c) optionally washing the nucleic acids while being bound to the solid phase; d) optionally eluting the nucleic acids from the solid phase. It was found that the addition of the specific non-ionic detergent overcomes the problems of the prior art methods, wherein the column clogs what prevents the efficient nucleic acid isolation from this difficult sample. When the specific non-ionic detergent is included into the binding mixture, no clogging occurs thereby allowing the efficient isolation of nucleic acids from veterinary whole blood samples.

Description

field of invention [0001] The present invention relates to methods for isolating nucleic acids from veterinary whole blood samples. In particular, the present invention provides methods for isolating pathogenic nucleic acids from veterinary whole blood samples. Background of the invention [0002] Several methods for isolating nucleic acids from different samples are known in the prior art. However, each method worked differently for all samples. For example, nucleic acids can be efficiently isolated from certain samples (eg, tissue, plasma, serum, or urine) using one method. However, the method may not be effective for isolating nucleic acids from other samples, such as whole blood samples. This is especially challenging with biological samples containing high cell numbers, high protein and / or lipid content, such as veterinary whole blood samples obtained from large livestock such as horses or cattle. Methods known in the prior art are often unsatisfactory in efficientl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q1/6806C12Q1/70
Inventor D·弗卢杰L·克鲁格M·谢雷尔
Owner QIAGEN GMBH