Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms

Abstract

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.

What is claimed is: 
     
         1 . A method for producing a lysate from cells contained in a liquid sample, comprising agitating the liquid sample within a bead beating system in the absence of lysis buffer and in the presence of one or more blocking agents under conditions sufficient to lyse the cells, said bead beating system comprising a bead beating tube and beads. 
     
     
         2 . The method of  claim 1 , wherein the liquid sample contains an endogenous blocking agent. 
     
     
         3 . The method of  claim 2 , wherein the agitating is carried out in the absence additives. 
     
     
         4 . The method of  claim 1 , wherein the one or more blocking agents are exogenous to the liquid sample. 
     
     
         5 . The method of  claim 2 , wherein the beads:
 a) are adapted to lyse bacteria, yeast, filamentous fungi, spores, plant cells, or animal cells;   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).   
     
     
         6 . The method of  claim 1 , further comprising a step of placing the liquid sample within the sample tube prior to agitating the liquid sample. 
     
     
         7 . The method of  claim 1 , wherein the agitating comprises subjecting the bead beating system to an oscillating motion. 
     
     
         8 . The method of  claim 1 , wherein the sample is a biological sample, an environmental sample, or a food product. 
     
     
         9 . The method of  claim 8 , wherein the sample is a biological sample selected from blood, serum, saliva, urine, gastric fluid, digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluid, fluid derived from tumorous tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid, hair, fingernails, skin cells, plasma, fluid obtained from a nasal swab, fluid obtained from a nasopharyngeal wash, cerebrospinal fluid, a tissue sample, fluid or tissue obtained from a throat swab, fluid or tissue obtained from a wound swab, biopsy tissue, placental fluid, amniotic fluid, peritoneal dialysis fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, or a sample processed, extracted or fractionated from any of the foregoing. 
     
     
         10 . The method of  claim 9 , wherein the biological sample is:
 a) urine, sputum or a sample processed, extracted or fractionated from urine;   b) sputum or a sample processed, extracted or fractionated from sputum;   c) a wound swab or a sample processed, extracted or fractionated from a wound swab;   d) blood or a sample processed, extracted or fractionated from blood; or   e) peritoneal dialysis fluid or a sample processed, extracted or fractionated from peritoneal dialysis fluid.   
     
     
         11 . The method of  claim 8 , wherein the sample is an environmental sample selected from soil, groundwater, surface water, wastewater, or a sample processed, extracted or fractionated from any of the foregoing. 
     
     
         12 . The method of  claim 1 , wherein the cells comprise one or more pathogens. 
     
     
         13 . The method of  claim 12 , wherein the one or more pathogens comprise:
 a) one or more bacterial pathogens, viral pathogens, fungal pathogens, or a combination thereof; or   b) one or more of  Mycobacterium tuberculosis, Mycobacterium avium  subsp  paratuberculosis, Staphylococcus aureus , methicillin resistant  Staphylococcus aureus  (MRSA),  Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfuluezae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter  sp.,  Bordetella pertussis, Neisseria meningitidis, Bacillus anthracis, Nocardia  sp.,  Actinomyces  sp.,  Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella  species,  Pneumocystis jiroveci , influenza A virus, cytomegalovirus, rhinovirus,  Enterococcus faecium, Acinetobacter baumannii, Corynebacterium amycolatum, Enterobacter aerogenes, Enterococcus faecalis  CI 4413,  Serratia marcescens, Streptococcus equi , and  Candida albicans.      
     
     
         14 . The method of  claim 12 , in which the sample is sputum or a sample processed, extracted or fractionated from sputum and the one or more pathogens comprise one or more of  Mycobacterium tuberculosis, Staphylococcus aureus , methicillin resistant  Staphylococcus aureus  (MRSA),  Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfuluezae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter  sp.,  Bordetella pertussis, Neisseria meningitidis, Bacillus anthracis, Nocardia  sp.,  Actinomyces  sp.,  Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella  species,  Pneumocystis jiroveci , influenza A virus, cytomegalovirus, and rhinovirus. 
     
     
         15 . The method of  claim 14 , in which the one or more pathogens comprise  Mycobacterium tuberculosis.    
     
     
         16 . The method of  claim 12 , in which the sample is milk or semen, or a sample processed, extracted or fractionated from milk, soil, feces, or semen, and the one or more pathogens comprises  Mycobacterium avium  subsp  paratuberculosis.    
     
     
         17 . The method of  claim 12 , in which the sample is a wound swab or a sample processed, extracted or fractionated from a wound swab and the one or more pathogens comprise one or more of  E. coli, P. aeruginosa, E. faecium, S. aureus, K. pneumoniae, A. baumannii, C. amycolatum, E. aerogenes, E. faecalis  CI 4413,  S. marcescens, S. equi  and  C. albicans.    
     
     
         18 . The method of  claim 12 , in which the sample is peritoneal dialysis fluid or a sample processed, extracted or fractionated from peritoneal dialysis fluid and the one or more pathogens comprise  S. aureus  and/or  P. aeruginosa.    
     
     
         19 . The method of  claim 1 , further comprising recovering the lysate from the sample tube following cell lysis, and optionally further comprising purifying the nucleic acids from the lysate. 
     
     
         20 . The method of  claim 1 , further comprising analyzing one or more of the nucleic acids, optionally wherein:
 a) the analysis is performed using a microarray or by sequencing the one or more nucleic acids;   b) a target sequence is amplified prior to performing the analysis, optionally by PCR.   
     
     
         21 . A bead beating system comprising (i) a sample tube having an inner cavity that is accessible by an aperture, (ii) beads, and (iii) a dry blocking agent, wherein the beads and dry blocking agent are located within the inner cavity of the sample tube. 
     
     
         22 . The bead beating system of  claim 21 , wherein the blocking agent comprises:
 a) one or more chaotropic agents, creatinine, one or more nucleotides, one or more oligonucleotides, or a combination thereof;   b) one or more chaotropic agents comprising urea, one or more guanidine salts, one or more lithium salts, one or more magnesium salts, one or more detergents, or a combination thereof;   c) one or more guanidine salts comprising guanidine isocyanate, guanidine chloride, or a combination thereof;   d) one or more lithium salts comprising lithium perchlorate, lithium acetate, or a combination thereof;   e) comprises magnesium chloride;   f) one or more detergents comprising sodium dodecyl sulfate, sodium lauroylsulfate sarcosinate, polyoxyethylene (20) sorbitan monolaurate, or a combination thereof;   g) one or more nucleotides comprising naturally occurring nucleotides, non-naturally occurring nucleotides, or a combination thereof;   h) one or more naturally occurring deoxyribonucleotides, one or more naturally occurring ribonucleotides, or a combination thereof;   i) one or more deoxyribonucleotides comprising deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytosine monophosphate, deoxythymidine monophosphate, or a combination thereof;   j) one or more ribonucleotides comprising adenosine monophosphate, guanosine monophosphate, cytosine monophosphate, thymidine monophosphate, or a combination thereof;   k) one or more oligonucleotides comprising ribonucleotides, deoxyribonucleotides, nucleotide analogs, or a combination thereof; or   l) one or more oligonucleotides each of which is independently 2 to 120 nucleotides long, 2 to 100 nucleotides long, 2 to 50 nucleotides long, 2 to 25 nucleotides long, 5 to 40 nucleotides, 2 to 15 nucleotides long, 8 to 120 nucleotides long, 8 to 80 nucleotides long, 10 to 100 nucleotides long, 15 to 50 nucleotides long, 50 to 100 nucleotides long, or 100 to 120 nucleotides long, optionally wherein the oligonucleotides comprise DNA and/or RNA.   
     
     
         23 . The bead beating system of  claim 21 , in which the inner cavity of the sample tube is at least partially coated with a layer or film of the blocking agent. 
     
     
         24 . The bead beating system of  claim 21 , in which some or all of the beads are partially or fully coated with a layer or film of the blocking agent. 
     
     
         25 . The bead beating system of  claim 21 , comprising a powder containing the blocking agent, which is optionally a lyophilized powder containing the blocking agent. 
     
     
         26 . The bead beating system of  claim 21 , wherein the beads:
 a) are adapted to lyse bacteria, yeast, filamentous fungi, spores, plant cells, or animal cells;   b) comprise:
 i. mineral beads, ceramic beads, glass beads, metal beads, or a combination thereof; or 
 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).   
     
     
         27 . The bead beating system of  claim 21 , further comprising:
 a) ethylenediaminetetraacetic acid (EDTA) and/or a sodium salt thereof located within the inner cavity of the sample tube; and/or   b) creatinine located within the inner cavity of the sample tube.   
     
     
         28 . A method for lysing cells contained in a liquid sample, comprising agitating the liquid sample within the sample tube of the bead beating system according to  claim 21  under conditions sufficient to lyse the cells. 
     
     
         29 . A kit for lysing cells contained in a liquid sample, comprising (i) the bead beating system of  claim 21  and (ii) one or more components for preparing the liquid sample, one or more oligonucleotides for amplifying one or more of the nucleic acids, one or more probes for detecting one or more of the nucleic acids, or a combination thereof, optionally wherein the one or more components for preparing the liquid sample comprise water, saline, a buffer, a filter, or a combination thereof. 
     
     
         30 . A kit for obtaining the bead beating system of  claim 21 , comprising a sample tube, beads, and a dry blocking reagent, optionally (i) in which the beads and/or dry blocking reagent are included in the kit separately from the sample tube, or (ii) in which the beads and/or dry blocking reagent are included in the kit within the sample tube.