WO2017080714A1

WO2017080714A1 - Method of processing nucleic acids

Info

Publication number: WO2017080714A1
Application number: PCT/EP2016/073317
Authority: WO
Inventors: Divya KHANDIGE SHARMA; Ragavendar MS; Nivedita MITRA; Ramya VUTUKURU
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2015-11-11
Filing date: 2016-09-29
Publication date: 2017-05-18

Abstract

A method of processing nucleic acids is disclosed. In one embodiment, the method includes obtaining a sample containing one or more pathogen cells and one or more free floating nucleic acids. The method further includes degrading the one or more free floating nucleic acids in the sample.

Description

METHOD OF PROCESSING NUCLEIC ACIDS

FIELD OF TECHNOLOGY

[0001] The present disclosure generally relates to the field of processing of nucleic acids. In particular, the disclosure relates to a method of removal of free floating nucleic acids from a given sample.

BACKGROUND

[0002] Application of polymerase chain reaction (PCR) techniques in detecting and identi- fying pathogens directly from the sample, instead of the traditional sample culturing methods has potential to revolutionize the diagnosis and management of sepsis. Polymerase chain reaction is one of the essential methods through which presence of pathogen in a sample can be detected. An important step in such an assay is the removal of free floating nucleic acids that may be present in the sample. The free floating nucleic acids may be pathogen deoxyribose nucleic acids (DNA) that may remain in the blood from previous infections. The free floating pathogen nucleic acids may also be sourced from pathogens that have been apoptized due to immune response or cleaved pathogens in response to antibiotics. The presence of free floating nucleic acids may provide false positive results when detection of pathogen nucleic acids may be performed for the infected blood sample. Known polymerase chain reaction techniques fail to distinguish between nucleic acids of viable pathogen and the free floating pathogen nucleic acid, once the sample is lysed to extract for the nucleic acids. The free floating pathogen nucleic acid can be found even in the blood of a healthy individual, which can also contribute to the false positive PCR results. The analytic test, therefore, fails to provide the correct estimate of the actual pathogen load, which can lead to incorrect clinical diagnosis. [0003] In order to separate intact pathogens and thereby the intact pathogenic DNA from circulating free floating pathogen DNA, a pre-analytic enrichment step may be performed. The pre-analytic enrichment step may help in removal of free floating pathogen DNA that result in non-specific polymerase chain reaction signals which is important for making right clinical decisions for sepsis management. Currently, separation of the free floating pathogen nucleic acids may be carried out using selective lysis buffer and a filter. At first, the selective lysis buffer may be used to selectively lyse the mammalian blood cells. Then, the intact pathogen cells are concentrated on a filter such that the free floating pathogen nucleic acids are washed away through the filter pores. However, the above method of separation is not accurate as there are chances of some free floating pathogen nucleic acids not being completely washed through the filter pores. Filter based methods are also prone to pathogen losses which makes it not suitable to detect pathogens at low concentrations. This may lead to incorrect results when the diagnostic test is performed. Therefore, there exists a need to provide a method that effectively removes the free floating pathogen nucleic acids from the sample so as to increase the accuracy of detection of actual patho- gen load present in the sample.

[0004] The object of the invention is to provide a method that processes nucleic acids in a sample to increase the accuracy of detection of actual pathogen load present in the sample. SUMMARY OF THE INVENTION

[0005] A method of processing nucleic acids is disclosed. In one aspect, a method includes obtaining a sample containing one or more pathogen cells and one or more free floating nucleic acids. In another aspect, the method includes degrading the one or more free floating nucleic acids present in the sample.

[0006] Therein, degrading one or more free floating nucleic acids includes contacting the sample containing the free floating nucleic acids with a nuclease enzyme.

[0007] Therein, degrading one or more free floating nucleic acids includes treating the sample containing free floating nucleic acids with chemical reagents that degrade the free floating nucleic acids. [0008] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the following description. It is not intended to identify features or essential features of the claimed subject matter. Furthermore, the claimed subject matter is not limited to implementations that solve any or all disadvantages noted in any part of this disclosure.

BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 illustrates a flowchart of an exemplary method of processing nucleic acids in a sample, according to an embodiment. DETAILED DESCRIPTION

[0010] Hereinafter, embodiments for carrying out the present invention are described in detail. The various embodiments are described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purpose of explanation, numerous specific details are set forth in order to provide a thorough understanding of one or more embodiments. It may be evident that such embodiments may be practiced without these specific details. In other instances, well known materials or methods have not been described in detail in order to avoid unnecessarily obscuring embodiments of the present disclosure. While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0011] Figure 1 provides an illustration of a flowchart of an exemplary method 10 of processing nucleic acids present in a sample. At step 11, the sample comprising pathogen cells is obtained from an individual, for example, a patient. The obtained sample may contain free floating nucleic acids along with pathogen cells. In an embodiment, the sample may be an infected blood sample. Alternatively, the sample may also be saliva, urine, soft tissue, cerebrospinal fluid or nasopharyngeal fluid. The blood sample may be drawn from the patient and may be collected in heparin tubes. At step 12, a nuclease enzyme may be added to the blood sample such that the nuclease enzyme is brought in contact with the free floating nucleic acids that may be present in the blood sample. In an embodiment, the nuclease enzyme may be a deoxyribonuclease (DNase) enzyme. DNase enzyme breaks down deoxyribose nucleic acid (DNA) by hydrolysing the phosphodiester bonds present in the DNA backbone. The sample containing free floating nucleic acids is incubated with DNase enzyme for a period ranging between 1 minute to 30 minutes and preferably between 5 minutes to 15 minutes. The incubation may be performed at a temperature ranging between 20°C to 40°C and preferably between 30°C to 40°C. The DNase enzyme hydrolyzes the free floating nucleic acids that may be present in the sample, thereby degrading them.

[0012] In an alternate embodiment, the free floating nucleic acids in the sample can be degraded by treating the sample with one or more chemical reagents. The chemical reagents may be chosen from a group comprising of ethidium monoazide and propidium monoazide. The chemical reagents that bind irreversibly bind to nucleic acids may be chosen for treating the free floating nucleic acids present in the sample. The irreversible binding of chemical agents to the free floating nucleic acids prevent amplification of the free floating nucleic acids when subjected to polymerase chain reaction. For example, ethidium monoazide covalently binds to nucleic acids present in the sample. Ethidium monoazide is impermeable to living cells and therefore binds specifically to free floating nucleic acids that may be present in the sample. Therefore, on covalent binding with chemical reagents such as ethidium monoazide, the free floating nucleic acids are degraded and eliminated from further downstream processes that may be performed on the sample. [0013] The degraded free floating nucleic acids may be filtered from the sample at step 13 and the sample may be subjected to further downstream processing for analysis of the pathogen cells. For example, the sample can be enriched to remove mammalian genomic DNA background, such that the pathogen cells are isolated. In an alternative embodiment, the sample may be enriched before the free floating nucleic acids that are present in the sample are degraded. The sample may be further processed to extract pathogen DNA and amplify the pathogen DNA using polymerase chain reaction to identify the pathogen present in the sample.

Claims

CLAIMS What is claimed is:

1. A method of processing nucleic acids, the method comprising:

obtaining a sample containing one or more pathogen cells and one or more free floating nucleic acids; and

degrading the one or more free floating nucleic acids in the sample.

2. The method according to claim 1, wherein degrading the one or more free floating nucleic acids comprises contacting the sample containing free floating nucleic acids with a nuclease enzyme.

3. The method according to claims 1 or 2, wherein degrading the one or more free floating nucleic acids comprises treating the sample containing the free floating nucleic acids with chemical reagents that degrade the free floating nucleic acids.

4. The method according to claim 2, wherein the nuclease enzyme is a DNase enzyme.

5. The method according to claim 2 or 4, further comprising incubating the sample containing the one or more free floating nucleic acids with DNase for a time period ranging between 1 to 30 minutes and at a temperature ranging between 20°C to 40° C.

6. The method according to claim 3, wherein the chemical reagent is chosen from a group comprising ethidium monoazide and propidium monoazide.

7. The method according to claim 3, wherein the chemical reagent binds with the one or more free floating nucleic acids in the sample to prevent amplification of the free floating nucleic acids, thereby degrading the free floating nucleic acids.

8. The method according to any of the claims 1 to 7, wherein the sample is chosen from a group comprising blood, saliva, urine, soft tissue, cerebrospinal fluid and nasopharyngeal fluid.

9. The method according to any of the claims 1 to 8, further comprising:

enriching the sample by removing mammalian cells;

extracting nucleic acids from pathogen cells in the sample;

amplifying the extracted nucleic acids from the pathogens; and

identifying the pathogens in the sample from the amplified nucleic acids.