METHOD AND APPARATUS FOR ENRICHING PATHOGEN DNA

Abstract

A method and an apparatus are provided for enriching pathogen DNA. The method includes adding a selective lysis buffer to a sample, incubating the mixture formed thereby, and filtering the mixture. The apparatus for enriching pathogen DNA includes: a lysis chamber; a reservoir containing selective lysis buffer, connected to the lysis chamber; and a filter connected to the lysis chamber; that achieves a limit of detection of pathogens of 500 cfu/ml or less.

Claims

1. A method of enriching pathogen DNA from a sample suspected to contain pathogen cells, the method comprising: adding a selective lysis buffer to the sample to provide a mixture; wetting a filter with a universal lysis buffer to lyse filtered intact pathogen cells; passing through the filter a lysis buffer containing guanidine thiocyanate; contacting the mixture with a matrix and incubating the mixture to thereby form a complex with the pathogen DNA; and separating the complex from the mixture.

2. The method of claim 1, further comprising: quantifying bound pathogen DNA from the complex.

3. The method of claim 1, wherein the filter has a pore size in the range of 0.2 microns to 0.8 microns.

4. The method of claim 1, wherein the filter is a stack filter having one or more layers of filters of pore sizes in a range of 10 μm to 0.2 μm.

5. The method of claim 1, wherein the selective lysis buffer is a composition of a non-ionic detergent and a buffer, and wherein the selective lysis buffer has a pH in a range of 9.5 to 11.

6. The method of claim 1, wherein the universal lysis buffer is a composition of lithium acetate and sodium dodecyl sulfate (SDS).

7. The method of claim 6, wherein concentration of the lithium acetate in the universal lysis buffer is in a range of 0.01M to 0.5 M.

8. The method of claim 6, wherein concentration of the SDS in the universal buffer is in a range of 0.1% to 0.5% v/v or w/v.

9. The method of claim 1, wherein the lysis buffer has a pH in a range of 8 to 9.5.

10. The method of claim 1, wherein the matrix is a silica coated magnetic bead.

11. The method of claim 1, wherein the matrix is a silica coated column.

12. The method of claim 1, wherein separating the complex from the mixture using a magnet.

13. An apparatus for enrichment of pathogen DNA, the apparatus comprising: a lysis chamber; a reservoir containing a selective lysis buffer, connected to the lysis chamber; a filter connected to the lysis chamber; a reservoir containing a universal lysis buffer, connected to the filter; an incubation chamber; a reservoir containing a lysis buffer having guanidine thiocyanate, and a matrix, connected to the incubation chamber; and an analysis chamber.

14. The apparatus of claim 13, wherein the lysis chamber is configured to receive a sample suspected to contain pathogen cells.

15. The apparatus of claim 13, wherein the incubation chamber is configured to receive lysate from the filter after treatment with universal lysis buffer.

16. The apparatus of claim 13, wherein the analysis chamber is configured to receive one or more complexes from the incubation chamber.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present disclosure is further described hereinafter with reference to illustrated embodiments shown in the accompanying drawings, in which:

[0031] FIG. 1 illustrates a schematic diagram of a flow chart of an embodiment of a method.

[0032] FIG. 2 illustrates a graph of the effect of selective lysis and filtration based enrichment on the detection of 1 cfu/ml Candida tropicalis.

DETAILED DESCRIPTION

[0033] Hereinafter, embodiments 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.

[0034] FIG. 1 illustrates a schematic diagram of a flowchart of an embodiment of the method 1. The method 1 enriches pathogen DNA from a sample suspected to contain pathogen cells. In act 10 of the method 1, a selective lysis buffer is added to the given sample. In the embodiment described herein, the given sample is whole blood. The selective lysis buffer is a composition of a non-ionic detergent, e.g., Triton X-100 in sodium carbonate buffer. The concentration of Triton X-100 is ranging from 2% to 7%, or from 4% to 6%, in 200 mM sodium carbonate buffer. The selective lysis buffer has a pH in the range of 9.5 to 11. Around 10 ml of selective lysis buffer is added to the given sample and the mixture is vortexed and incubated at room temperature for 5 minutes. The selective lysis buffer selectively lyses the eukaryotic cells present in the given sample. Post lysis, the mixture is neutralized by addition of neutralizing buffer. In the present embodiment, the neutralizing buffer is a Tris buffer having a concentration in the range of 0.5M and 1.5M and a pH of 7.0.

[0035] In act 11, the mixture is passed through a filter. In the present embodiment, the filter used is a 25 mm GDx PVDF syringe filter having a pore size in the range of 0.2 microns to 0.8 microns, or 0.4 to 0.6 microns. Alternatively, a stack filter may also be used to filter the lysed contents of the mixture. Alternatively, the lysed mixture may also be centrifuged to isolate pathogen cells. The filter was washed with neutralizing buffer, following which the filter is wet with a universal lysis buffer in act 12. The universal lysis buffer lyses the pathogen cells concentrated on the filter to release pathogen DNA. In the present embodiment, the universal lysis buffer is a composition of lithium acetate (LiOAc) and sodium dodecyl sulfate (SDS). The concentration of lithium acetate varies from 0.01M to 0.5M or from 0.1M to 0.3M. The concentration of SDS varies from 0.1% to 5% v/v or w/v or from 0.5% to 2% v/v or w/v. The filter was incubated in the universal lysis buffer for a period varying from 10 seconds to 60 minutes or 3 to 5 minutes. The incubation was done at a temperature ranging from 50° C. to 90° C., or 70° C. to 90° C.

[0036] In act 13, a preheated lysis buffer containing guanidine thiocyanate is added to the filter. In the present embodiment, the lysis buffer used is VERSANT® lysis buffer. To the mixture formed, matrix is added in act 14. The matrix is a silica coated magnetic bead. The isolated pathogen DNA binds to the silica coated magnetic beads in the presence of guanidine thiocyanate and forms a complex. The mixture is vigorously vortexed to dislodge pathogen lysate from the beads. In act 15, the complexes are separated from the mixture using a magnet. The beads bound to pathogen DNA may be directly subjected to quantitative polymerase chain reaction (qPCR) for quantification.

[0037] FIG. 2 illustrates a graph on the effect of selective lysis and filtration based enrichment on the detection of 1 cfu/ml Candida tropicalis. The X-axis represents the number of amplification cycles and the Y-axis represents the intensity of normalized fluorescence. The threshold for normalized fluorescence is set at 0.032058. For the experiment, 5.0 ml fresh blood, collected in EDTA vacutainers is spiked with 5 cfu of Candida tropicalis to obtain blood sample with final concentration of 1 cfu/ml. Following lysis and formation of complex, the pathogen DNA was subjected to quantitative polymerase chain reaction (qPCR). The qPCR curves depicted on the graph indicate the effect of selective lysis buffer treatment and filtration on the detection of 1 cfu/ml Candida tropicalis. The samples that were subjected to qPCR include a positive reference that has a no blood background; a negative template control; and one repeat of the pathogen DNA obtained from the blood sample.

[0038] The Ct.sub.18s rDNA average, an average of 3 PCR replicates for positive reference is 32.99 cycles; selective lysis buffer treated blood is 38.83 cycles; blood only treated with selective lysis buffer is undetermined; and negative template control is undetermined. Therefore, the method provides for detection of pathogen cells in the blood to a level as low as 1 cfu/ml.

[0039] It is to be understood that the elements and features recited in the appended claims may be combined in different ways to produce new claims that likewise fall within the scope of the present disclosure. Thus, whereas the dependent claims appended below depend from only a single independent or dependent claim, it is to be understood that these dependent claims may, alternatively, be made to depend in the alternative from any preceding or following claim, whether independent or dependent, and that such new combinations are to be understood as forming a part of the present specification.

[0040] While the present disclosure has been described above by reference to various embodiments, it may be understood that many changes and modifications may be made to the described embodiments. It is therefore intended that the foregoing description be regarded as illustrative rather than limiting, and that it be understood that all equivalents and/or combinations of embodiments are intended to be included in this description.