METHOD OF ELUTING NUCLEIC ACID

Abstract

The invention relates to a method of eluting nucleic acid from a swab. The invention also relates to a kit for eluting nucleic acid from a swab and an elution solution.

Claims

1. A method of eluting nucleic acid from a swab comprising cellulose, the method comprising incubating the swab in an elution solution, the elution solution comprising: a) a polymer comprising pyrrolidone side chains; b) a non-ionic or zwitterionic detergent; c) a protease; and d) a buffer at pH 5-8.5.

2. The method of claim 1, wherein the polymer is polyvinylpyrrolidone (PVP).

3. The method of claim 1, wherein the detergent is a polysorbate.

4. The method of claim 1, wherein the incubation is carried out at 50-60 C. for at least 20 minutes.

5. The method of claim 1, wherein after incubating the swab in the elution solution, the swab is centrifuged to enable extraction of the elution solution from the swab.

6. The method of claim 1, wherein the protease is proteinase K.

7. The method of claim 1, wherein the method additionally comprises heating the collected elution solution to at least 90 C. to destroy the protease activity.

8. The method of claim 1, wherein PCR is performed on the elution solution.

9. The method of claim 1, wherein before incubating the swab in the elution solution, the swab is washed with ethanol.

10. A kit for eluting nucleic acid from a swab comprising cellulose, the kit comprising: a) a polymer comprising pyrrolidone side chains; and any one or more of the following: b) a non-ionic or zwitterionic detergent, preferably polysorbate 20; c) a protease, preferably proteinase K; d) a buffer at pH 5-8.5; and e) one or more spin baskets.

11. An elution solution comprising: a) a polymer comprising pyrrolidone side chains; and b) a non-ionic or zwitterionic detergent.

12. The method of claim 1, wherein the detergent is polysorbate 20.

13. The kit of claim 10, wherein the polymer comprises PVP.

14. The elution solution of claim 11, further comprising: c) a buffer at pH 5-8.5.

Description

DESCRIPTION OF THE FIGURES

[0110] FIG. 1: Comparison of 1-2% PVP in extraction of free DNA dried onto cotton swabs.

[0111] FIG. 2: Proteinase K treatment releases the cellular DNA from swabs. 1e6 cells or 93 ng free DNA was dried onto swabs and then extracted in the presence or absence of proteinase K followed by heat killing the proteinase K.

[0112] FIG. 3: Agarose gels of PCR products from cellular material treated as indicated in Example 3.

[0113] FIG. 4: Diagram of different ways of collecting DNA on swab, see Example 4.

[0114] FIG. 5: 10 ul samples were taken before heat treatment to kill the proteinase K and DNA concentration estimated by Qubit dye binding assay. Panel A (top): shows estimated DNA in ng and Panel B (bottom) shows percentage DNA recovered relative to the direct to buffer controls (DB).

[0115] FIG. 6: This figure shows the recovery and % recovery (relative to the direct to buffer controls) of free DNA deposited directly into extraction buffer, onto a cotton swab or recovered by swabbing a glass slide using a cotton swab. DNA content was estimated by qPCR (sybre Green) using trout primer pair 2 (free DNA).

[0116] FIG. 7: This figure shows the recovery and % recovery (relative to the direct to buffer controls) of cellular DNA deposited directly into extraction buffer, onto a cotton swab or recovered by swabbing a glass slide using a cotton swab. DNA content was estimated by qPCR (sybre Green) using mouse primer pair 1 (cellular DNA).

[0117] FIGS. 8-9: FIGS. 8 and 9 shows that PVP outperforms other polar polymers such as dextran (D). However, other detergents work in the elution solution as well as Tween, here we show Zwittergent 3-08 (Z).

[0118] FIGS. 10-11: These figures compare the recovery of DNA using a commercially available Qiagen kit and the claimed method.

EXAMPLES

Example 1: Comparison of Different Amounts of PVP for Elution from Swabs

[0119] Method: [0120] 1. 10 ng/ul Trout DNA stock. Spotted 5 ul=50 ng onto swabs and dried. [0121] 2. Elution solution: 1% to 2% PVP40, 1% tween 20 in 20 mM TrisHCl.Math.DNA quantified by Qbit. [0122] 3. Extracted 30 min on shaker at 56 degrees in 200 ul of buffer. [0123] 4. DNA quantified by Qbit.

[0124] FIG. 1 shows that 1 and 2% PVP have high rates of recovery of DNA from the swab.

Example 2: The Addition of Proteinase K Enhances Recovery of DNA from Swabs

[0125] Method: [0126] 1. Swabs were seeded with Stock DNA 10 ul, Qbit verified at 9.3 ng/ul total of 93 ng. [0127] 2. Swabs also seeded with PBS washed cells 10 ul of 110.sup.6 cells/ml suspension approximately 60 ng DNA. [0128] 3. Controls with DNA or cells as above added directly to 200 ul conditions 1 to 2 as below. [0129] A: EBP (20 mM TrisHCl, pH 7.5; 1% Tween20 and 1% PVP) [0130] B: EBP plus Proteinase K (PK) at 100 g/ml. [0131] 4. Swabs were incubated in elution solution at 56 C. for 1 hour. [0132] 5. The swabs were then added to a spin basket inserted into an Eppendorf tube. The spin baskets and Eppendorfs were then spun for 3 minutes at full speed. The spin basket was thrown away. [0133] 6. The solution collected in the Eppendorf was heated to 95 C. for 10 minutes to destroy Proteinase K activity. [0134] 7. A sample of 10 l was taken from the Eppendorf (into which the elution solution and eluted DNA were collected) for Qbit analyses.

[0135] FIG. 2 shows that the proteinase K has a marked effect on the release of cellular DNA. In addition, the presence or absence of calcium or EGTA has no effect

Example 3: Test of DNA Extraction from Cells in PVP Buffers and Suitability for PCR

[0136] Method: [0137] 1. Approximately 10{right arrow over ()}6 cells were resuspended in PBS and 5 ul added to 200 ul of the PVP buffers. [0138] 2. Samples and buffer blanks were incubated at room temperature for 30 min and EGTA then added where indicated without heat kill at 95 degrees. [0139] 3. Samples were also incubated at 56 degrees for 30 min and then EGTA added where indicated. Samples were also incubated at 56 degrees for 30 min and then EGTA added where indicated and heated to 95 degrees for 10 min. [0140] 4. 10 ul samples were then taken for PCR annealing temp 68 degrees.

[0141] Results are shown in FIG. 3. Incubation at 56 degrees enhances the digestion with EGTA reducing the yield. Including the 95 degree step enhances yield across the board probably due to the proteinase K (PK) being completely destroyed and removing any inhibitory effect on the PCR reaction. There is still PCR in the presence of active Proteinase K. This may be due to the 2 min 95 degree activation step in the PCR cycles which may be enough to destroy the PK activity.

[0142] The results of these experiments indicate that the addition of calcium or EGTA is not required for the inactivation of the proteinase K and will be omitted from all future experiments as simple heat treatment seems to be sufficient to inactivate the proteinase K and allow PCR.

Example 4: Comparing Free and Cellular DNA Recovery from Swab

[0143] Trial run of a comparison of extraction/recovery efficiency using the elution solution using free trout DNA and cellular mouse DNA. The protocol is show diagrammatically in FIG. 4.

[0144] All samples were extracted at 56 degrees for 60 min and then spin basket collected (in the case of swabs)

[0145] 10 ul samples were taken for Qbit before heating at 95 degrees for 10 min.

[0146] Free trout DNA was 14.5 ng/ul and diluted 1:3=4.83 ng/ul seeded at 10 and 5 ul ie: 48.3 ng and 24.1 ng As estimated by Qubit Mouse cells were resuspended in PBS at approx. 0.72e6/ml=4.32 ng/ul and seeded at 10 and 5 ul approximating to 43.2 and 21.6 ng DNA.

[0147] Samples with a 1:1 mix of trout cells and trout DNA were also used. Sample ID key is given below: [0148] DB=direct to buffer; Material added directly to 250 ul PVP PK buffer [0149] DS=direct onto swab; Material added directly to a swab and dried over night [0150] SS=slide followed by swab; Material dried onto a glass slide swabbed with cotton swab wetted with 100 ul 250 mM NaCl and dried overnight. [0151] T1 and T2 high and low trout free DNA [0152] M1 and M2 high and low cells. [0153] MT1 and MT2 high and low mixture of free and cellular DNA.

[0154] The results of the Qubit assay are shown in FIG. 5.

[0155] The Qubit data corresponds well with the estimated free DNA but suggests that the input from cellular material is underestimated. As a result, qPCR was ran and the results are shown in FIGS. 6 and 7.

[0156] The results relative to the direct to buffer control samples as shown in the data expressed as percent recovery clearly shows 80% to 90% recovery of free DNA from material applied directly to swabs and 70% to 80% of cellular DNA applied directly to swabs. For free DNA the recovery from the swabbed glass slide is still high at 70% to 80% of the applied material and for cellular DNA this is only slightly reduced at around 60% to 70% of the applied material.

Example 5: Method with Zwittergent 3-08

[0157] The effect of substitution with another high molecular weight polymer Dextran Sulphate and substitution of the detergent Tween 20 with Zwittergent 3-08 was investigated.

[0158] Swabs were spotted with either 55 or 27.5 ng (10 or 5 ul 5.5 ng/ul) Trout DNA and dried overnight. DNA extraction was carried out with 250 ul of extraction buffers, as detailed below, with shaking at 56 C. for 1 hour. DNA extracted was quantified by Qbit analysis of a 10 ul sample and qPCR of a 5 ul sample.

[0159] The results of the extraction and Qbit quantification are shown in FIG. 8.

[0160] From the data there appears to be a decrease of around 30-40% in the effectiveness of the extraction buffer when PVP is replaced by Dextran sulphate (DT1) and a small decrease in the effectiveness when Tween 20 is replaced by Zwittergent 3-08 (PZ1).

[0161] Subsequent quantitation of the DNA by qPCR resulted in the data shown in FIG. 9.

TABLE-US-00001 Op quant ng/5 ul Total DNA recovered ng in PT1 0.430 15 0.445414 0.595155 0.431461 41.9 1 43.36102 57.93834 42.00276 DT1 ND ND ND ND ND ND ND ND PZ1 0.518175 0.633273 0.682663 0.811742 32.69685 39.9595 43.07603 51.2209 DZ1 ND ND ND ND ND ND ND ND 27.5 ng in PT2 0.220086 0.25 151 0.2 51 0.18 107 21.42533 25.1 096 24.60288 18.31218 DT2 ND ND ND ND ND ND ND ND PZ2 0.2338 0.3104 9 0.314482 0.30 786 14.75708 19.58 7 19.84383 19.2 DZ2 ND ND ND ND ND ND ND ND % Recovered ave sd ng in PT1 76.25419 78.83822 105.3524 76.3 5 84.20087 13.18181 DT1 ND ND ND ND ND ND PZ1 59.44882 72.65363 78.32005 3.12893 75.88786 9.05667 DZ1 ND ND ND ND ND ND 27.5 ng in PT2 77. 10 1.38529 89.79228 66.58975 81.41941 11.34702 DT2 ND ND ND ND ND ND PZ2 53.6621 71.23627 72.15 7 70.16395 66. 0542 2.337387 DZ2 ND ND ND ND ND ND indicates data missing or illegible when filed

[0162] The data clearly shows that Dextran Sulphate is a potent inhibitor of the PCR reaction since no product was detectable in any samples in which it was present. This makes Dextran sulphate unsuitable as a substitute for PVP (as well as that Dextran does not remove the nucleic acid from the cellulose as effectively as PVP). The substitution of Zwittergent 3-08 for Tween 20 is however acceptable.

Example 6: Comparison of Method with Qiagen Kit

[0163] As in previous experiments either free DNA (trout) or cellular (mouse) was extracted and quantified by qPCR using the appropriate trout primers for detection of free DNA and mouse primers for the detection of cellular DNA.

[0164] The results are shown in FIGS. 10-11.

[0165] The data shows the percentage recovery of two different amounts of the input DNA where samples denoted 2 have 50% the amount of samples denoted 1. The DNA was extracted from buffer alone (DB), swabs with the DNA directly applied to the swab (DS) and where the DNA was first applied to glass slides then swabbed (SS). Panel A shows the % recovery of free DNA and Cellular DNA applied to buffer or swabs as described above as recovered using either the Qiagen kit or extraction buffer. Panel B shows the % recovery of free DNA and Cellular DNA applied to buffer or swabs as for panel A with the exception that the cellular and free DNA was applied as a combined mixture. The data clearly shows that the extraction buffer consistently outperforms the Qiagen kit in terms of % DNA recovered in all conditions.