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This extended run time prevented the customer from processing a full tray of 88 samples plus calibrators and controls, including hc assays, in one shift (8 hours)
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[0181] According to one embodiment, the method of the present invention comprises the following steps d) and e):
[0182] Step d):
[0183] - contacting the sample containing the released and denatured nucleic acid with one or more target nucleic acid-specific probes under conditions that allow the probes to hybridize to single-stranded target nucleic acid molecules to form double-stranded nucleic acid hybrids;
[0184] Step e):
[0185] - detecting the presence or absence of a double-stranded nucleic acid hybrid.
[0186] Preferably, the method of the present invention comprises the following steps d) and e):
[0187] Step d):
[0188] - subjecting a sample containing released and denatured nucleic acid and magnetic particles for binding cells to one or more target nucleic acids under conditions that allow hybridization of the probes to single-stranded target nucleic acid molecules to form double-stranded nucleic acid hybrids sex probe contact;
[0189] Step e):
[0190...
Embodiment 1
[0386] Example 1: Comparison of manual AXpH-direct and manual conversion
[0388] In MC, which is a standard prior art method, sample preparation is as follows: Samples of HPV-negative clinical pools and HPV-positive clinical pools formulated in culture medium (see below). Obtained by density gradientcentrifugation Post-gradient samples, corresponding obtained samples were further processed as described below.
[0389] First, vortex the sample thoroughly. 2.8ml of each sample was transferred to a 15ml centrifuge tube and centrifuged at 800g for 10 minutes. After centrifugation, the supernatant was decanted and the centrifuge tube (opening facing down) was tapped 3 times on a cloth. 200 μl of sample transport medium (Specimen Transport Medium-STM, Digene - containing chaotropic agent) was added to each pellet, ie sample, and vortexed at maximum speed for 15 seconds until all pellets were resuspended...
Embodiment 2
[0407] Example 2: AXpH-direct method for the applicability of automation
[0408] In Example 2, the following samples were processed:
[0409] 1) HPV-negative clinical library in medium (RLU / CO about 0.2),
[0410] 2) SiHa cells in medium (HPV positive; 1 × 10 5 cells / ml),
[0411] 3) HPV-positive clinical pool 1 in medium (RLU / CO approximately 186.4), and
[0412] 4) HPV positive clinical pool 2 in medium (RLU / CO ~530).
[0413] 1.1) Reference: Manual Conversion (MC)
[0414] See Example 1 for the manual conversion (MC) protocol.
[0415] 1.2) Manual AXpH-direct method
[0416] First, vortex the sample thoroughly. Combine 2.8ml of each sample, i.e., with 1.4ml 1.4ml of the original sample mixed with medium, transferred to a 5ml PP-tube. 50 [mu]l of Bead Suspension I (see Example 1) was added. Secure the tube with the cap, carefully invert 10 times, and vortex for 30 seconds. Samples were then incubated at room temperature for 5 minutes. After incubati...
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Abstract
The present invention provides a method of determining the presence or absence of a target nucleic acid in a cell sample. The method includes the steps of: a) contacting a surface comprising anion exchange moieties with the sample under conditions suitable to induce binding between the cells and the surface; b) separating the surface with the bound cells from the remaining sample to collect the cells; c) releasing nucleic acids from the cells; d) generating a hybrid between the target nucleic acid and a probe specific for the target nucleic acid; and e) detecting the presence or absence of thehybrid. A rapid and automatable method is provided, wherein cells, such as epithelial cells originating from cervical swab samples, are collected from the surrounding liquid medium, such as a liquidbased cytology medium, by binding them to an anion exchange surface. The cells bind directly with high affinity and quick kinetics to the anion exchange surface which preferably is provided by magnetic particles carrying anion exchange moieties. The cells that are bound to the anion exchange surface can be easily separated from the surrounding medium and can be directly resuspended in a composition that is suitable for a subsequent hybrid capturing assay which is performed in step d) to detect e. g. pathogen nucleic acids such as HPV nucleic acids.
Description
[0001] This application is a divisional application of the Chinese invention patent application with the application number 2013800456094 filed on August 16, 2013. [0002] field of invention [0003] The present invention relates to a method for detecting the presence or absence of a target nucleic acid in a liquid sample containing cells. [0004] Background of the invention [0005] To detect the presence or absence of a target nucleic acid, several methods are known in the prior art based on capturing hybrids comprising the target nucleic acid to be detected, referred to herein as hybrid capturing assays. The basis of each technique is described, for example, in WO 93 / 10263 and further developments are described in WO 2010 / 127223. Techniques for the detection and characterization of specific nucleic acid sequences and sequence alterations, such as detecting the presence or absence of viral or bacterial nucleic acid sequences indicative of infection, the presence or absence...
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