Method Of Isolating Nucleic Acid From Specimens In Liquid-Based Cytology Preservatives Containing Formaldehyde

A technology of cytology and preservatives, applied in the direction of biochemical equipment and methods, organic chemistry, microbiological determination/inspection, etc., can solve problems such as unresolved and insufficiently given

Active Publication Date: 2016-11-16
GEN PROBE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, Khripin et al. do not address how nucleic acids can be made suitable for use as templates in nucleic acid amplification reactions, nor do they give sufficient disclosure to enable detection of RNA targets from formaldehyde-fixed samples.

Method used

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  • Method Of Isolating Nucleic Acid From Specimens In Liquid-Based Cytology Preservatives Containing Formaldehyde
  • Method Of Isolating Nucleic Acid From Specimens In Liquid-Based Cytology Preservatives Containing Formaldehyde
  • Method Of Isolating Nucleic Acid From Specimens In Liquid-Based Cytology Preservatives Containing Formaldehyde

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0080] Example 1 describes the method used to assess the analytical sensitivity of the experimental system by testing an experimental panel comprising in vitro transcripts for each of the 14 high-risk HPV genotypes. The success of nucleic acid processing techniques involving treatment with 2-imidazolidinone and proteinase K at elevated temperature conditions was measured by detection of HPV RNA using a commercially available assay. As described below, the results show that treatment conditions do not affect the amplification and detection of HPV RNA.

[0081] Example 1

[0082] Establishing Analytical Sensitivity of HPV Assays Using Synthetic Transcripts

[0083] Transcripts synthesized in vitro serve as templates for amplification in conventional TMA reactions using HPV gene probe assays were performed. Transcript copy numbers for each of the different HPV types correspond to the limit of detection (LOD) of the APTIMA HPV Gene Probe assay, which has been preserved in ...

example 2

[0086] Example 2 describes a method for evaluating the analytical sensitivity of an HPV assay by testing a panel of HPV-containing human cells. The method was generally as described in Example 1 except: (1) an HPV expressing cell line was used instead of in vitro transcripts; and (2) samples were incubated for extended periods of time in the presence of formaldehyde-containing preservatives.

[0087] Example 2

[0088] Establishing Analytical Sensitivity of HPV Assays Using HPV-Containing Human Cell Lines

[0089] HPV-containing human cell lines were spiked into sample pools of SUREPATH liquid-based cytology preservative, stored at 25°C for 7 days, and then treated with a combination of recovery agent solution and proteinase K at 90°C for 15 minutes, or at 65°C °C were tested in half-log dilutions (3 to 30 cells / reaction) after proteinase K treatment alone for 2 hours. As in Example 1, the combination of recovery agent solution and proteinase K is conveniently provided a...

example 3

[0091] The method described in Example 3 demonstrates how the combined use of 2-imidazolidinone and proteinase K under elevated temperature conditions improves the recovery of amplifiable nucleic acids from samples stored for long periods of time in liquid-based cytological preservatives containing formaldehyde Rate. As discussed below, the difference in RNA recovery was most pronounced over extended time periods compared to experiments treated with proteinase K alone.

[0092] Example 3

[0093] Enhanced recovery of amplifiable mRNA from cell samples stored in formaldehyde-containing liquid-based cytology preservatives

[0094] To mimic clinical samples, ten pools of residual samples in SUREPATH Liquid-Based Cytology Preservative previously confirmed as HPV-negative using the APTIMA HPV Gene Probe assay were split in half and spiked into either SiHa or HeLa cells. All tubes were stored neat at 25°C for up to 42 days. On each test day, aliquots of each pool were diluted...

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Abstract

Method, composition, kit and system for isolating amplifiable nucleic acid from specimens preserved in a liquid-based cytology preservative that contains formaldehyde. The technique relies on the use of 2-imidazolidone and a protease enzyme, such as proteinase K, at elevated temperatures. Advantageously, RNA can be isolated and used as a template in nucleic acid amplification reactions.

Description

[0001] Cross references to related patent applications [0002] This patent application is formal application 61 / 946,637 filed February 28, 2014, which is incorporated by reference in its entirety for all purposes. technical field [0003] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for isolating nucleic acid from a sample fixed in a liquid-based cytological preservative containing formaldehyde, wherein the isolated RNA is suitable for use as a template in nucleic acid amplification procedures. Background technique [0004] Laboratory workers engaged in the molecular analysis of nucleic acids isolated from formaldehyde-fixed samples recognize that there are certain limitations to the usefulness of this sample type. This is because formaldehyde and certain other chemical fixatives chemically modify proteins and nucleic acids. These modifications are known to impair the utility of the nucleic acid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C07H21/04
CPCC12N15/1003C12Q1/6806C12Q2521/537C12Q2527/101C12Q2527/125
Inventor D·C·杰森B·W·柯康奈尔T·J·威尔逊
Owner GEN PROBE INC
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