Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
The present disclosure provides improved methods for bead beating and a bead beating system useful therefor. The bead beating system comprises a sample tube, beads, and a dry blocking agent, and methods for using the bead beating system to extract nucleic acids from cells containing the nucleic acids.
1. A method for producing a lysate from cells contained in a blood sample, comprising agitating the blood sample within a bead beating system in the absence of a buffer, in the absence of a detergent, and in the presence of one or more endogenous blocking agents comprising urea under conditions sufficient to (i) lyse both a fungal pathogen and a bacterial pathogen and (ii) provide a lysate suitable for PCR amplification of DNA from the fungal pathogen, when present, and the bacterial pathogen, when present, said bead beating system comprising a bead beating tube and beads.
2. The method of claim 1 , wherein the agitating is carried out in the absence of any additives.
3. The method of claim 1 , wherein the beads:
a) are adapted to lyse yeast or filamentous fungi;
b) comprise:
i. mineral beads, ceramic beads, glass beads, metal beads, or a combination thereof;
ii. zirconium beads, zircon beads, zirconia beads, quartz beads, aluminum oxide beads, silicon carbide beads, ceramic beads, silicon dioxide glass beads, stainless steel beads, chrome steel beads, or a combination thereof;
c) have a diameter ranging from 50 μm to 3 mm; or
d) any combination of a)-c).
4. The method of claim 1 , further comprising a step of placing the blood within the bead beating tube prior to agitating the blood.
5. The method of claim 1 , which comprises agitating the blood under conditions sufficient to (i) lyse Candida albicans and (ii) provide a lysate suitable for PCR amplification of DNA from Candida albicans , when the Candida albicans is present.
6. The method of claim 1 , which comprises agitating the blood under conditions sufficient to (i) lyse Staphylococcus aureus and (ii) provide a lysate suitable for PCR amplification of DNA from Staphylococcus aureus , when the Staphylococcus aureus is present.
7. The method of claim 5 , which comprises agitating the blood under conditions sufficient to (i) lyse Staphylococcus aureus and (ii) provide a lysate suitable for PCR amplification of DNA from Staphylococcus aureus , when the Staphylococcus aureus is present.
8. The method of claim 1 , which comprises agitating the blood under conditions sufficient to (i) lyse Escherichia coli and (ii) provide a lysate suitable for PCR amplification of DNA from Escherichia coli , when the Escherichia coli is present.
9. The method of claim 5 , which comprises agitating the blood under conditions sufficient to (i) lyse Escherichia coli and (ii) provide a lysate suitable for PCR amplification of DNA from Escherichia coli , when the Escherichia coli is present.
10. The method of claim 6 , which comprises agitating the blood under conditions sufficient to (i) lyse Escherichia coli and (ii) provide a lysate suitable for PCR amplification of DNA from Escherichia coli , when the Escherichia coli is present.
11. The method of claim 7 , which comprises agitating the blood under conditions sufficient to (i) lyse Escherichia coli and (ii) provide a lysate suitable for PCR amplification of DNA from Escherichia coli , when the Escherichia coli is present.
12. The method of claim 1 , wherein the agitating comprises subjecting the bead beating system to an oscillating motion.
13. The method of claim 12 , wherein the agitating comprises subjecting the bead beating system to 2000 or more oscillations per minute.
14. The method of claim 1 , wherein the agitating is performed with a vortexer or a homogenizer.
15. The method of claim 14 , wherein the agitating is performed with a homogenizer.
16. The method of claim 15 , wherein the agitating is performed for 25-60 seconds.
17. The method of claim 15 , wherein the agitating is performed for 1-2 minutes.
18. The method of claim 1 , wherein the blood comprises one or more pathogens.
19. The method of claim 18 , wherein the one or more pathogens comprise one or more bacterial pathogens, viral pathogens, fungal pathogens, or a combination thereof.
20. The method of claim 19 , wherein the one or more pathogens comprise Candida albicans.
21. The method of claim 19 , wherein the one or more pathogens comprise Staphylococcus aureus.
22. The method of claim 19 , wherein the one or more pathogens comprise Escherichia coli.
23. The method of claim 1 , wherein the blood comprises cells from multiple species.
24. The method of claim 1 , further comprising recovering the lysate from the bead beating tube following cell lysis.
25. The method of claim 24 , further comprising purifying nucleic acids from the lysate.
26. The method of claim 1 , further comprising analyzing one or more nucleic acids from the lysate.
27. The method of claim 26 , wherein the analyzing comprises PCR to amplify DNA from the fungal pathogen, when present, and DNA from the bacterial pathogen, when present.
28. The method of claim 27 , further comprising using a microarray to analyze the one or more nucleic acids or sequencing the one or more nucleic acids following PCR.