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Method for extraction and identification of nucleic acids

A nucleic acid and nucleic acid extraction technology, which is used in the field of nucleic acid extraction and identification, and can solve problems such as inability to detect infection, reduction of test sensitivity, and delay in diagnosis.

Inactive Publication Date: 2008-09-17
NEW YORK BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The drawback of serological screening tests is that they cannot detect infection if the body has not yet mounted an antibody response
Unfortunately, pooled samples reduce the sensitivity of the test and, if pooled samples test positive, delay the final diagnosis

Method used

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  • Method for extraction and identification of nucleic acids
  • Method for extraction and identification of nucleic acids
  • Method for extraction and identification of nucleic acids

Examples

Experimental program
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preparation example Construction

[0038] Another suitable preparation technique involves removing cells from the sample. For example, to prepare a serum sample, cells are typically, but not necessarily, removed from the sample, such as from a whole blood sample. Cells can be removed from a sample by any method known to those skilled in the art. For example, centrifugation or filtration can be used to prepare cell-free samples. For example, serum is typically obtained from clotted blood by centrifugation to remove cellular components. Plasma is usually obtained in a similar manner to serum, except that an anticoagulant is added to the blood.

[0039] In another specific embodiment, optionally, the method further includes the step of concentrating cells or viruses in the sample. Any concentration method known to those skilled in the art can be used. Suitable methods of concentration include, but are not limited to, the use of polyethylene glycol.

[0040] In general, the proper fraction of concentration to ...

Embodiment 1

[0100] Example 1 :Material

[0101] West Nile virus was obtained from the New York State Department of Health and cultured in Vero cells. The number of infectious virus particles was determined by conventional plaque assays (Beaty et al, Arboviruses, p. 797-856. In N.J. Schmidt, and R.W. Emmons (ed.), Diagnostic procedures for viral, rickettsial and chalmydial infections. American Public Health Association, Washington. D.C. 1989). Viral genome equivalent values ​​were determined by quantitative RT-PCR using a WNV RNA quantifier QWN702 (BBI Diagnostics, West Bridgewater, MA) as a quantitative standard.

[0102] The New York Blood Center provided HBV-infected plasma as well as plasma from chronic HCV-infected blood donors. HIV stocks (stocks, stocks) are cell-free supernatants from cultures of HIV-infected human peripheral blood lymphocytes.

[0103] Normal human plasma, pre-assayed for blood-borne pathogens, was used for serial dilution of positive or spiked samples.

Embodiment 2

[0104] Example 2 : Extraction of multiple virus genomes in plasma

[0105] Aliquots of HBV, HCV, HIV and WNV samples were pipetted in different volumes into 96 well 2.2ml storage plates (ABgene, Surrey, KT, UK). Plasma volumes for direct extraction varied from 150 to 450 μl.

[0106] Optimized amounts of Proteinase K (Qiagen, Chatsworth, CA) and AL Lysis Buffer (Qiagen, Chatsworth, CA) were added and mixed briefly. AL Lysis Buffer contains, among other components, detergents and guanidinium salts. After incubating the storage plate at 58 °C for 25 min in a shaking water bath, a predetermined amount of absolute ethanol (Table 1) was gently mixed with the lysate. The above preparations were transferred into 0.7 μm glass fiber filter plates (GF / F, Whatman, Clifton, NJ) and filtered at -450 mmHg vacuum. Transfers were accomplished in 1 to 3 pipetting steps, depending on the total volume of the lysate preparation. Subsequently, the loaded filter plates were washed with AW2 wa...

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Abstract

The invention provides a method for extracting nucleic acids from a sample. The sample contains cells, viruses, or both cells and viruses. The method include adding a lysing solution containing a detergent to the sample to lyse the cells or viruses to form a lysate, adding alcohol to the lysate to aggregate or precipitate the nucleic acids, and purifying the nucleic acids from the lysate-alcohol mixture by filtering the mixture through a glass-fiber filter.

Description

[0001] This application claims priority to US Provisional Application Serial No. 60 / 645,905, filed January 21, 2005, the entire contents of which specification are hereby incorporated by reference. Background technique [0002] The public health sector increasingly requires highly sensitive genetic assays for viruses, bacteria, fungi, parasites or cells. For screening (e.g. blood supply, arboviruses in mosquitoes), surveillance (e.g. West Nile virus in flocks), water analysis, diagnosis of infection, gene-based diagnosis (e.g. hemophilia, High-throughput sample processing of breast cancer susceptibility, cancer cells, etc. would be very beneficial. [0003] Blood supply contamination with pathogenic viruses such as human immunodeficiency virus (HIV), hepatitis A, B or C, parvovirus, cytomegalovirus, and Epstein-Barr virus (African lymphoma virus), as well as bacterial infections such as Lyme disease, has become an increasingly increasingly serious problem. The prevailing opi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C07H21/04
CPCC07H21/02C07H21/04C12N15/1017C12Q1/6806C12Q1/6888
Inventor 沃尔弗拉姆·H.E.·普尔法艾尔弗雷德·M·普林斯李敦勋琳达·安德勒斯
Owner NEW YORK BLOOD CENT