METHODS AND PRODUCTS FOR ISOLATING NUCLEIC ACIDS

Abstract

The present invention relates to methods and products for isolating nucleic acids from samples containing biological material. In particular, the present invention relates to silica-coated magnetic particles, processes for their preparation and their use in methods of isolating nucleic acids samples containing biological material.

Claims

1. A process for the preparation of silica coated magnetic particles comprising (I) combining magnetic particles and an alkoxysilane, such as tetraorthoethylsilicate, in water and a C1-4-alcohol at a temperature in the range of 15 to 90° C. in the presence of a hydroxide in order to form silica coated magnetic particles; (II) washing the silica coated magnetic particles of step (I) with water and/or alcohol until the pH of the silica coated magnetic particles when suspended in water is between 8 and 11, preferably 9 to 10.

2. A process for the preparation of a silica coated magnetic particles as claimed in claim 1 comprising (I) combining magnetic particles and an alkoxysilane in a C1-4-alcohol to form a mixture; (II) adding water and a hydroxide to the mixture of step (I) and heating the resulting mixture to a temperature in the range of 15 to 90° C. in order to form silica coated magnetic particles; (III) washing the silica coated magnetic particles of step (II) with water and/or alcohol until the pH of the silica coated magnetic particles, when suspended, in water is between 8 and 11.

3. A process for the preparation of silica coated magnetic particles comprising (I) combining magnetic particles and an alkoxysilane, such as tetraorthoethylsilicate, in water and a C1-4-alcohol such that the weight ratio of C1-4-alcohol to water in the mixture is 1:1 to 15:1 at a temperature in the range of 15 to 90° C. in the presence of a hydroxide in order to form silica coated magnetic particles; (II) washing the silica coated magnetic particles of step (I) with water and/or alcohol until the pH of the silica coated magnetic particles when suspended in water is between 8 and 11, preferably 9 to 10.

4. A process for the preparation of a silica coated magnetic particles as claimed in claim 3 comprising (I) combining magnetic particles and an alkoxysilane in a C1-4-alcohol to form a mixture; (II) adding water and a hydroxide to the mixture of step (I) such that the weight ratio of C1-4-alcohol to water in the mixture is 1:1 to 15:1 and heating the resulting mixture to a temperature in the range of 15 to 90° C. in order to form silica coated magnetic particles; (III) washing the silica coated magnetic particles of step (I) with water and/or alcohol solvent, preferably until the pH of the silica coated magnetic particles when suspended in water is between 8 and 11.

5. A process as claimed in any preceding claim wherein the C1-4-alcohol is ethanol or isopropanol.

6. A process as claimed in any preceding claim wherein the weight ratio of C1-4-alcohol to water in the mixture is 3:1 to 12:1 or 3:2 to 5:1.

7. A process as claimed in any preceding claim wherein the particles are iron oxide nanoparticles.

8. A process as claimed in any preceding claim wherein the temperature is 75 to 85° C. or 20 to 30° C.

9. A process as claimed in any preceding claim wherein the pH of the silica coated magnetic particles when suspended in water is 9 to 10.

10. A process as claimed in any preceding claim wherein the alkoxysilane is TEOS.

11. A process as claimed in any preceding claim further comprising suspending the silica coated magnetic particles in water wherein the concentration of said particles in water is 5 to 35 mg/ml, preferably 5 to 30 mg/ml, such as 5 to 12 mg/ml.

12. A process as claimed in any preceding claim further comprising suspending the silica coated magnetic particles wherein the zeta potential of the suspension is −20 to −90 mV.

13. A process as claimed in any preceding claim wherein the magnetic particles that are combined with the alkoxysilane in step (I) are coated with an organic polyacid or organic polyacid salt coating, such as citrate.

14. An aqueous suspension of silica coated magnetic particles wherein said suspension has a pH of 8 to 11, preferably 9 to 10 and the concentration of silica coated magnetic particles in water is 5 to 30 mg/ml such as 5 to 12 mg/ml.

15. An aqueous suspension of silica coated magnetic particles wherein said suspension has a pH of 8 to 11, preferably 9 to 10, and has a zeta potential of −20 to −90 mV, preferably −40 to −70 mV.

16. An aqueous suspension of silica coated magnetic particles wherein the concentration of silica coated magnetic particles in water is 5 to 30 mg/ml such as 5 to 12 mg/ml and the suspension has a zeta potential of −20 to −90 mV.

17. An aqueous suspension of silica coated magnetic particles as claimed in claims 14 to 16 wherein the particles are monodisperse, e.g. having a PDI of less than 0.005 or a CV of 5% or less.

18. A lysis solution comprising: (a) a buffer; (b) a chelating agent; (c) a chaotropic agent; and (d) a detergent; and optionally (e) a reducing agent; and/or (f) a nucleic acid carrier.

19. The lysis solution of claim 18, comprising: (a) a buffer; (b) a chelating agent; (c) a chaotropic agent; (d) a detergent; and (e) a nucleic acid carrier.

20. The lysis solution of claim 18 or 19, comprising: (a) a buffer; (b) a chelating agent; (c) a chaotropic agent; (d) a detergent; (e) a reducing agent; and (f) a nucleic acid carrier.

21. The lysis solution of any one of claims 18 to 20, wherein: (i) the buffer is Tris (tris(hydroxymethyl) aminomethane); (ii) the chelating agent is ethylenediaminetetraacetic acid (EDTA); (iii) the chaotropic agent is a guanidinium salt, preferably guanidinium isothiocyanate (iv) the detergent is sodium lauroyl sarcosinate (sarkosyl) or 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100); (v) the reducing agent is tris(2-carboxyethyl)phosphine (TCEP); and/or (vi) the nucleic acid carrier is glycogen.

22. The lysis solution of any one of claims 18 to 21, comprising: (a) a buffer solution at a concentration of about 10-200 mM, preferably 40-60 mM, with a pH of about 6.0-9.0, preferably about 7.4-8.2; (b) a chelating agent at a concentration of about 5-50 mM, preferably about 15-30 mM; (c) a chaotropic agent at a concentration of at least about 3M, preferably about 3-6M; and (d) a detergent at a concentration of about 0.5-5.0% w/v, preferably about 0.75-3.0% w/v, such as about 1.0-1.5% w/v or about 1.75-2.25% w/v.

23. The lysis solution of claim 22 further comprising a nucleic acid carrier at a concentration of about 0.1-5 mg/ml, preferably about 0.5-3.0 mg/ml.

24. The lysis solution of claim 22 or 23 further comprising a reducing agent at a concentration of about 1-20 mM, preferably about 5-16 mM.

25. The lysis solution of any one of claims 18 to 24 comprising: (a) Tris-HCl at a concentration of about 40-60 mM, with a pH of about 7.4-8.2; (b) EDTA at a concentration of about 15-30 mM; (c) a guanidinium salt at a concentration of about 3-6M; (d) sodium lauroyl sarcosinate at a concentration of about 1.75-2.25% w/v or 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100) at a concentration of about 1.0-1.5% w/v; and optionally (e) TCEP at a concentration of about 5-16 mM; and/or (f) glycogen at a concentration of about 0.5-3.0 mg/ml.

26. The lysis solution of any one of claims 18 to 25, comprising: (a) Tris-HCl at a concentration of about 50 mM, with a pH of about 7.6-8.0, preferably about 7.8; (b) EDTA at a concentration of about 20 mM; (c) guanidinium isothiocyanate at a concentration of about 4M; (d) 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100)) at a concentration of about 1.2% w/v or sodium lauroyl sarcosinate at a concentration of about 2.0% w/v; and (e) glycogen at a concentration of about 1.0 mg/ml.

27. The lysis solution of any one of claims 18 to 26, comprising: (a) Tris-HCl at a concentration of about 50 mM, with a pH of about 7.6-8.0, preferably about 7.8; (b) EDTA at a concentration of about 20 mM; (c) guanidinium isothiocyanate at a concentration of about 4M; (d) sodium lauroyl sarcosinate at a concentration of about 2.0% w/v or 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100)) at a concentration of about 1.2% w/v; (e) TCEP at a concentration of about 10 mM; and (f) glycogen at a concentration of about 1.0 mg/ml.

28. Use of a lysis solution of any one of claims 18 to 27 to release nucleic acids from biological material for adsorption onto the surface of silica-coated magnetic particles.

29. The use of claim 28, wherein the lysis solution is used in a method of isolating nucleic acids from a sample containing biological material using silica-coated magnetic particles.

30. A method for isolating nucleic acids from a sample containing biological material comprising: (a) contacting the sample containing biological material with a lysis solution of any one of claims 18 to 27; (b) contacting the sample from (a) with silica-coated magnetic particles under conditions suitable to adsorb nucleic acids in the sample to the silica-coated magnetic particles; (c) washing the silica-coated magnetic particles from (b); and (d) desorbing the nucleic acids from the silica-coated magnetic particles.

31. The method of claim 30, wherein step (b) comprises contacting the sample from (a) with at least an equal volume of a suspension of silica-coated magnetic particles.

32. The method of claim 30 or 31, wherein the biological sample is an oral or nasal sample.

33. The method of claim 31 or 32, wherein the suspension of silica-coated magnetic particles comprises silica-coated magnetic particles suspended in a C.sub.2-C.sub.4 alcohol, preferably isopropanol.

34. The method of any one of claims 31 to 33, wherein the suspension of magnetic particles contains proteinase K or wherein proteinase K is added to the sample from (a) prior to contact with the silica-coated magnetic particles.

35. The method of any one of claims 31 to 34, wherein the concentration of silica-coated magnetic particles in the suspension is at least about 0.4 mg/ml, preferably about 0.4-0.6 mg/ml.

36. The method of any one of claims 30 to 35, wherein step (c) comprises washing the silica-coated magnetic particles with a washing liquid comprising a C.sub.2-C.sub.4 alcohol.

37. The method of any one of claims 30 to 35, wherein step (c) comprises washing the silica-coated magnetic particles with isopropanol followed by 80% ethanol.

38. The method any one of claims 30 to 37, wherein step (d) comprises contacting the silica-coated magnetic particles with an aqueous solution comprising a blocking reagent, preferably wherein the blocking reagent is polyoxyethylene (20) sorbitan monolaurate.

39. An in vitro method of detecting a nucleic acid from an infectious agent in a biological sample comprising: (a) isolating nucleic acids from a biological sample suspected of containing an infectious agent using the method of any one of claims 30 to 38; (b) analysing the nucleic acids from (a) for the presence of a nucleic acid from the infectious agent.

40. The in vitro method of claim 39 being a method of determining whether a subject is infected with an infectious agent comprising: (a) isolating nucleic acids from a biological sample from a subject suspected of having an infection using the method of any one of claims 18 to 26; (b) analysing the nucleic acids from (a) for the presence of nucleic acids from the infectious agent, wherein detection of nucleic acids from the infectious agent indicates that the subject has an infection.

41. The in vitro method of claim 39 or 40, wherein the infectious agent is a virus, preferably a virus with an RNA genome.

42. The in vitro method of claim 41, wherein the virus is a coronavirus, preferably covid-19 (SARS-CoV2).

43. A kit comprising: (a) a lysis solution of any one of claims 18 to 27; and (b) silica-coated magnetic particles.

44. The use, method or kit of any one of claims 28 to 43, wherein the silica-coated magnetic particles: (i) are obtained or obtainable from the process of any one of claims 1 to 13; (ii) are as defined in any one of claims 14 to 17; and/or (iii) have the properties of the silica-coated magnetic particles in the aqueous suspension of silica-coated magnetic particles of any one of claims 14 to 17.

Description

[0253] The invention will be further described with reference to the following non-limiting Examples with reference to the following drawings in which:

[0254] FIG. 1 shows the silica coated magnetic particles of MB1.

EXAMPLES

Example 1—Synthesis of Iron Oxide Nanoparticles (IONP)

[0255] 8.0 g of FeCl.sub.2.Math.4H.sub.2O and 21.6 μm of FeCl.sub.3.Math.6H.sub.2O were weighed into separate 100 ml volumetric flasks and each flask filled to 100 ml with MQ-water. 84.6 g MQ water was placed in a beaker and 15.4 ml of 25 wt % NH.sub.4OH was added. 10 ml of iron chloride stock solution was added to the NH.sub.4OH/water dropwise using a burette under vigorous stirring (4-500 rpm) to create a suspension.

[0256] 40 mL of the suspension was transferred to a 40 mL centrifuge tube, and the magnetically formed particles were separated and the supernatant discarded. The rest of the suspension was added to the particles and the separation process repeated until all the slurry is separated.

[0257] The separated particles are washed thrice with MQ-water and suspended in 15 mL MQ-water resulting in the final volume of 20 mL. The particle weight is adjusted to 50 mg/ml.

Example 2—Iron Oxide Magnetic Nanoparticles—Small Scale

[0258] 2 ml of TEOS was mixed with 20 ml of ethanol in a vial and kept stirring at 500 rpm for 15 minutes. 1 ml of IONP (˜50 mg) aq. dispersion from example 1 was first magnetically separated from the water and cleaned thrice with ethanol. The washed IONPs were added into the reaction mixture and stirred for 30 minutes.

[0259] 4 ml of MQ water followed by 5 ml of 25 wt % NH.sub.4OH was added to the reaction mixture and stirred for 30 minutes. The reaction mixture was heated to 82° C. over 15 minutes and the reaction left running overnight (at least 12 hours). The ratio of ethanol to added water is approx 5:1 (and to total water is 2.6:1)

[0260] The reaction mixed was cooled to room temperature and the formed silica coated IONPs magnetically separated. These were washed two times with ethanol and seven times with MQ water. The washed silica coated IONPs were finally re-dispersed in 25 ml MQ water. This sample is called MB1. The concentration of silica coated IONPs in water for the small scale batch is 6-7 mg/ml.

Example 3—Scaled Up Synthesis of Silica Coated IONPS (Magnetic Beads)

[0261] 20 ml of TEOS was mixed with 380 ml of ethanol in a water jacketed reactor and kept stirring at 500 rpm for 15 minutes. 10 ml of IONP (˜500 mg) dispersion from example 1 was first magnetically separated from the water and cleaned thrice with Ethanol. The particles are redispersed in 20 ml of ethanol and the dispersion added into the reaction mixture and stirred for 30 minutes.

[0262] 40 ml of MQ water followed by 5 ml of 25 wt % NH.sub.4OH was added to the reaction mixture and stirred for 30 minutes. The reaction mixture was heated to 82° C. over 15 minutes and the reaction left running overnight (at least 12 hours). The ratio of ethanol to added water is approx. 10:1 (and to total water 9.1:1)

[0263] The reaction mixed was cooled to room temperature and the formed silica coated IONPs magnetically separated. These were washed two times with ethanol and seven times with MQ water. The washed silica coated IONPs were finally re-dispersed in 250 ml MQ water. This sample is called MB2. The concentration for the scaled up batch is between 9-10 mg/ml.

Properties of the Silica Coated IONPs

[0264] The pH of MB1 and MB2 was measured. The pH was 9.3 for MB1 and 9.1 for MB2. PDI was measured as 0.0014 and 0.0013 for MB1 and MB2 respectively.

[0265] The zeta potential of MB1 and MB2 was measured using a Malvern NanoSizer. The zeta potential was −52 mV for MB1 and −62 mV for MB2.

SEM Analysis

Sample Preparation

[0266] Bead suspension is prepared at a concentration of approximately 0.1 to 0.01 percent by volume, and dispersion is ensured by vortexing or ultrasonicating the diluted sample. A flat, conductive substrate is prepared. Clean Si wafer pieces or mica with a conductive coating—e.g. thick gold coating—are ideal. The conductive sample is plasma cleaned using O.sub.2 plasma for a time of 1 minute, increasing hydrophilicity.

[0267] Apply 50 μL of dilute bead suspension to the freshly plasma treated substrate, and dry at room temperature or elevated temperature until bone dry. Samples are affixed to a SEM stub using conductive adhesive, and electric contact from sample to stub is confirmed.

Imaging

[0268] A SEM capable of high resolution needs to be utilized, e.g. a Field-Effect gun SEM (FE-SEM/FEG-SEM). Samples are introduced to the microscope, and imaged at an adequate resolution, e.g. 5-20k. Several images must be taken of different areas on each sample, totalling a number of identifiable beads, approaching 100, at different positions on the sample surface. These micrographs are ideally, for automation purposes, recorded at the same resolution. Where possible, a detector or detector combination with low topographic contrast is desirable.

Image Analysis

[0269] Visual inspection is the initial inspection of the samples, samples exhibiting obvious deviation from the desired particle shape, size and uniformity of either can be rejected at this point. After an initial visual inspection, images can be analyzed and beads measured manually. More optimally they are run through an algorithm which thresholds the image, producing a binary mask separating the particles and the background by contrast. After thresholding the image is analyzed counting the particles per unit area, measuring their size and shape descriptors, e.g. circularity, aspect ratio, Feret's diameter, etcetera are measured and exported for statistical analysis. Each sample is then analyzed using appropriate computer software for this purpose, returning size and shape descriptor distributions, and relevant statistics to the operator.

[0270] For both the batches, MB1 and MB2, mean particle diameters are found to be 0.41 and 0.45 μm respectively. Beads in both batches were very monodisperse (CV=4%). The particles of MB1 are shown in FIG. 1.

Example 4—Large Scale Synthesis of Silica Coated IONPS (Magnetic Beads)

[0271] 48 ml of TEOS was added to 700 ml of ethanol in a vial and stirred at 500 rpm for 15 minutes. ˜20 ml of IONP (1200 mg) dispersion from example 1 was first magnetically separated from the water and cleaned thrice with Ethanol. The washed IONPs were redispersed in 260 mL ethanol and added into the reaction mixture. The reaction mixture was stirred for 30 minutes and 200 ml of MQ water was then added followed by 50 ml of 25 wt % NH.sub.4OH and stirred for 30 minutes. The mixture was heated to 82° C. over 15 minutes and left overnight (at least 12 hours). The ratio of ethanol to added water is approx. 4.8:1 (and to total water 4:1).

[0272] The reaction mixed was cooled to room temperature and the formed silica coated IONPs magnetically separated. These were washed two times with ethanol and seven times with MQ water. The washed silica coated IONPs were finally re-dispersed in 200 ml MQ water. This sample is called MBL (Magnetic Beads Large Scale).

[0273] The concentration for the scaled up batch is between 26.3 mg/ml.

[0274] The pH of Magnetic Beads Large Scale was measured. The pH was 9.5.

Example 5—Large Scale Synthesis of Silica Coated IONPS (Magnetic Beads) at Room Temperature

[0275] 48 ml of TEOS was added to 700 ml of isopropanol in a vial and stirred at 500 rpm for 15 minutes. ˜20 ml of IONP (1200 mg) dispersion from example 1 was first magnetically separated from the water and cleaned thrice with isopropanol. The washed IONPs were redispersed in 260 mL isopropanol and added into the reaction mixture. The reaction mixture was stirred for 30 minutes and 200 ml of MQ water was then added followed by 50 ml of 25 wt % NH.sub.4OH and stirred for 30 minutes. The mixture was left overnight (at least 12 hours). The ratio of ethanol to added water is approx. 4.8:1 (and to total water 4:1).

[0276] The formed silica coated IONPs are magnetically separated. These were washed two times with isopropanol and seven times with MQ water. The washed silica coated IONPs were finally re-dispersed in 200 ml MQ water. This sample is called MBL (Magnetic Beads Large Scale).

[0277] The concentration for the scaled up batch is between 29.3 mg/ml.

[0278] The pH of Magnetic Beads Large Scale was measured. The pH was 10.2.

Example 6—Method for Nucleic Acid Extraction from a Biological Sample Using Silica-Coated Magnetic Particles

[0279] A lysis solution was prepared according to Table 1. A suspension of silica-coated magnetic particles (prepared using the method described in Example 3) was prepared according to Table 2 (magnetic particle mix). An elution buffer was prepared according to Table 3.

TABLE-US-00001 TABLE 1 to 100 ml (for Final concentration 5*96 Wplates) Guanidine thiocyanate 4M 47.3 g Tri-HCl pH-7.8 50 mM 5 ml of 1M stock N-lauroyl Sarcosine 2%   2 g EDTA 20 mM 4 ml of 0.5M stock Glycogen 1 mg/ml 500 ul of 200 mg/ml stock TCEP 10 mM

TABLE-US-00002 TABLE 2 Per sample For 100 samples Isopropanol 400 μl 40 ml Proteinase K 100 μg/ml 700 μl of 10 mg/ml stock Magnetic Particles 20 μl of a suspension  2 ml comprising about 10 mg/ml

TABLE-US-00003 TABLE 3 Per sample For 100 samples Nuclease free water 50 μl 5 ml Tween 20 1% 250 ul of 20% stock

[0280] Protocol

[0281] 1. Pipette out 200 μl Lysis solution (per sample tube or per well if using plate, e.g. 96 well plate)

[0282] 2. Add 100 μl sample (i.e. comprising biological material), mix (pipetting/vortexing)

[0283] 3. Resuspend magnetic particle-mix. Vortex thoroughly to resuspend all particles

[0284] 4. Add 400 μl particle-mix to each sample/lysis tube, mix (pipetting/vortexing)

[0285] 5. Keep the particles in solution for 10 minutes by mixing/shaking

[0286] 6. Remove supernatant using a magnet

[0287] 7. Wash the particles in 400 μl isopropanol, mix for 2 minutes

[0288] 8. Remove supernatant using a magnet

[0289] 9. Wash the particles in 400 μl 80% EtOH, mix for 2 minutes

[0290] 10. Remove supernatant using a magnet

[0291] 11. Wash the particles in 400 μl 80% EtOH, mix for 2 minutes

[0292] 12. Remove supernatant using a magnet

[0293] 13. Dry the particles for 10 minutes at room temperature

[0294] 14. Resuspend the particles in 50 μl elution buffer, mix for 5 minutes

[0295] 15. Collect the supernatant for qPCR.

Set-Up for KingFisher Robot:

[0296]

TABLE-US-00004 Position Plate type Content Volume 1 KF 96 deep-well Lysate (Lysis buffer, 700 ul sample, bead mix) 2 KF 96 deep-well Isopropanol wash 400 ul 3 KF 96 deep-well 80% Ethanol wash 400 ul 4 KF 96 deep-well 80% Ethanol wash 400 ul 5 KF 96 standard Elution buffer  50 ul 6 KF 96 standard 96 tip comb for deep- well maqnets

Protocol

[0297] 1. Transfer 200 μl Lysis buffer to each well

[0298] 2. Add 100 ul Sample to Lysis Buffer, leave at RT for minimum 5 minutes

[0299] 3. Add 400 ul Bead-mix

[0300] 4. Prepare reagents in plates according to table.

[0301] 5. Start program on instrument

[0302] 6. After run, collect eluted sample in Plate 6 to PCR.

Example 7—Comparison of Methods for Detection of Covid-19 Virus from Viscous Expectorate Samples

[0303] Nucleic acid was isolated from viscous expectorate samples from patients suspected of being infected with the covid-19 virus (a coronavirus) using the protocol described in Example 6 and a commercial nucleic acid extraction kit, NucliSENS® EASYMAG® (bioMerieux), which also uses silica-coated magnetic particles. The protocol described in Example 6 used 100 μl of patient sample, whereas 200 μl was used as the input for the commercial kit. Nucleic acid obtained from each sample was used in a standard reverse transcription qPCR (RT-qPCR) to detect covid-19 target nucleic acids.

[0304] Table 4 below shows the cycle threshold (Ct) values from the RT-qPCR and the results demonstrate that the Ct values are lower for almost all patient samples, indicating that the nucleic acid isolated using the protocol in Example 6 results in a more sensitive assay. It was determined that the isolation protocol of Example 6 results in the isolation of about 3 times more RNA compared to the commercial kit. This facilitates the detection of nucleic acids that are present in a biological sample at very low levels.

[0305] Samples from subjects not infected with the covid-19 virus yielded negative results, as expected.

TABLE-US-00005 TABLE 4 Parallel Ct-values Ct-values sample from NucliSENS ® Example 3 Example 3 Samples kit protocol protocol 2 18.89 18.34 3 19.24 20.58 4 18.12 18.81 5 14.45 13.13 6 29.35 26.89 7 33.07 32.64 31.21 8 23.13 22.56 9 35.11 36.73 10 21.7 19.28 11 28.46 28.06 12 24.51 24.93 13 31.65 31.77 32.78 14 25.39 24.97 15 16.51 15.25 16 34.71 34.41 33.89 17 30.37 29.27 30.31 18 26.02 24.71 19 31.27 30.14 29.32 20 18.99 17.8 21 25.85 24.62 22 17.15 16.82 23 29.16 27.22 24 31.98 29.12 30.26 25 32.77 32.03 30.89

Example 8—Effects of Various Components in the Isolation Protocol

[0306] The effects of various components used in the nucleic acid protocol described in Example 6 were assessed using a viscous expectorate sample from a Covid-19 positive patient. The nucleic acids obtained from each variant isolation method from the sample were used in a standard qPCR to detect covid-19 target nucleic acids.

[0307] Variations of the protocol described in Example 6 as outlined in Table 5. For instance, the elution step was performed with and without Tween. The reducing agent in the lysis buffer was changed, i.e. from TCEP to DTT. Also, in some cases, proteinase K was added to the lysis solution after contact with the expectorate sample but before the addition of the particle mix (without proteinase K)—see “Proteinase K in Lysis buffer”.

TABLE-US-00006 TABLE 5 Well Parameters Cq A01 Standard setup Undetermind B01 Standard setup Undertermind C01 Standard setup 1% Tween in elution buffer 35.74 D01 Standard-setup 1% Tween in elution buffer 35.05 A02 10 mM TCEP in Lysis Buffer 28.33 B02 10 mM TCEP in Lysis Buffer 28.35 C02 10 mM TCEP in Lysis Buffer 1% Tween in 28.22 elution buffer D02 10 mM TCEP in Lysis Buffer 1% Tween in 27.68 elution buffer E02 20 ug Proteinase K in Lysis Buffer 34.46 F02 20 ug Proteinase K in Lysis Buffer 32.45 G02 20 ug Proteinase K in Lysis Buffer 1% 34.48 Tween in elution buffer H02 20 ug Proteinase K in Lysis Buffer 1% 30.14 Tween in elution buffer A03 200 ug Proteinase K in Lysis Buffer 29.06 B03 200 ug Proteinase K in Lysis Buffer 28.10 C03 200 ug Proteinase K in Lysis Buffer 1% 28.15 Tween in elution buffer D03 200 ug Proteinase K in Lysis Buffer 1% 28.10 Tween in elution buffer E01 80 mM DTT in Lysis Buffer 29.89 F01 80 mM DTT in Lysis Buffer 29.54 G01 80 mM DTT in Lysis Buffer 1% Tween in 29.40 elution buffer H01 80 mM DTT in Lysis Buffer 1% Tween in 29.49 elution buffer E03 Pos CTRL 31.53 F03 Neg CTRL

[0308] The results demonstrate that the use of TCEP significantly enhances the detection of covid-19 nucleic acid. Similarly, the use of high amounts of proteinase K during the lysis step also improves detection of covid-19 nucleic acid.

Example 9—Formulation for Alternative Lysis Solutions

[0309] An alternative lysis solution for use in the protocol described in Example 6 may be prepared according to the formulation in Table 6.

TABLE-US-00007 TABLE 6 Final concentration to 1000 ml Guanidine thiocyanate 4M 473 g Tri-HCI pH-7.8 50 mM 50 ml of 1M stock Triton X-100 1.20% 12 g EDTA (pH 7.8) 20 mM 40 ml of 0.5M stock Glycogen 1 mg/ml 1.0 g

[0310] A further alternative lysis solution can be prepared by omitting the glycogen component from the formulation in Table 6.