WO2023023051A2

WO2023023051A2 - Rapid, sensitive detection of nucleic acid sequences in environmental samples

Info

Publication number: WO2023023051A2
Application number: PCT/US2022/040458
Authority: WO
Inventors: Christopher T. FRENCH; Wayne Wong; Jeffrey F. Miller
Original assignee: The Regents Of The University Of California
Priority date: 2021-08-16
Filing date: 2022-08-16
Publication date: 2023-02-23
Also published as: WO2023023051A3

Abstract

Rapid polymerase chain reaction (PCR)-based detection of nucleic acid sequences in bulk soil is dependent on the removal of PCR inhibitors, such as metals and humic acids, and the protection of genetic material from destructive endo- and exonuclease enzymes. We have designed and tested a unique methodology and reagent combination that facilitates the PCR-based detection of nucleic acid sequences in soil without the need to isolate DNA. Illustrative embodiments of our system were developed for detection of Burkholderia pseudomallei, a pathogenic bacterium found in high-humic soil.

Description

RAPID, SENSITIVE DETECTION OF NUCLEIC ACID SEQUENCES IN ENVIRONMENTAL SAMPLES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application Serial No 63/233,442, filed on August 16, 2021, and entitled “RAPID, SENSITIVE DETECTION OF NUCLEIC ACID SEQUENCES IN ENVIRONMENTAL SAMPLES” which application is incorporated by reference herein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant Number HDTRA1-17-1-0015, awarded by the U.S. Department of Defense, Defense Threat Reduction Agency. The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure relates to methods and materials useful for detecting nucleic acid sequences in environmental samples.

BACKGROUND OF THE INVENTION

Nucleic acid sequences have a wide variety of applications in the field of molecular biology. They are a valuable tool in many analytical and application techniques used in the field of molecular biology, health and medicine (gene therapy, diagnostics, recombinant protein expression), bioterrorism (agent detection and analysis), forensics, space science, and food science. Some examples of these techniques include genotyping microorganisms, DNA fingerprinting plants and animals, detecting pathogens and beneficial microorganisms in soils, water, plants and animals, forensic identification of biological samples and environmental samples contaminated with different biological entities. All these techniques are based on identifying a specific sequence of nucleic acid in either a biological sample, such as a microorganism, plant tissues or animal tissues, or in any environment capable of supporting life. Identifying target nucleic acid sequences directly in biological samples and in environmental samples has the advantages of speed, accuracy, high- throughput and a low limit of detection to picogram or femtogram quantities of nucleic acids. The target nucleic acid sequence, in order to be used as a diagnostic tool in such applications, should be free of contaminants that inhibit polymerase chain reaction (PCR) polynucleotide amplification and other downstream applications. These contaminants are often from the groups that include polyphenols, polysaccharides and humic substances.

The field of nucleic acid extraction and subsequent amplification of this DNA by polymerase chain reaction has revolutionized the rapid analysis of genetic composition of several ecosystems. Methods and kits are available for isolating genomic DNA from a wide range of biological entities, and from the environment in which these living entities dwell. The polymerase chain reaction is a very powerful and sensitive analytical technique with applications in many diverse fields, including molecular biology, clinical diagnosis, forensic analysis, and population genetics. However, the success rate in soil and plant genomic analysis has been relatively slow due to the poor quality of DNA isolated. In plant genomic DNA analysis, the DNA is invariably co-extracted with other plant components such as polyphenols and polysaccharides which inhibit PCR applications. In the field of soil ecosystems, nucleic acid extraction methods suffer from compounded inefficiencies of DNA sorption to soil surfaces and co-extraction of enzymatic inhibitors from soils. Both the clay and organic fractions of soil affect DNA isolation and purification. Clay has a tendency to bind DNA adsorptively, whereas humic polymers found in the organic fraction tend to co-purify with DNA during the extraction procedure. The higher the montmorillonite clay and organic matter content, the higher the buffering capacity of the soil system and also greater the amount of DNA adsorbed to the soil particles. Thus methods developed for a particular soil type with a clay: organic ratio may not work for any other soil type with different clay: organic ratio. An additional concern when isolating microbial DNA from compost is that plant material in various stages of decomposition may be present in significant concentrations in compost.

Direct extraction of total nucleic acid from soils or sediments usually results in co-extraction of other soil components, mainly humic acids or other humic substances, which negatively interfere with DNA transforming and detecting processes. It has been reported that these substances inhibit restriction endonucleases and Taq polymerase, the key enzyme of PCR, and decrease efficiencies in DNA-DNA hybridizations. Separation of humic substances from DNA usually involves timeconsuming and tedious steps. For these reasons, there is a need in the art for improved methods and materials useful for detecting nucleic acid sequences in environmental samples, and in particular, those methods and materials that can be used in the PCR-based detection of nucleic acid sequences in soil without the need to isolate DNA.

SUMMARY OF THE INVENTION

Rapid polymerase chain reaction (PCR)-based detection of nucleic acid sequences in bulk soil is dependent on the removal of PCR inhibitors, such as metals and humic acids, and the protection of genetic material from destructive endo- and exonuclease enzymes. As disclosed herein we have designed and implemented a unique methodology and reagent combination that facilitates the PCR-based detection of nucleic acid sequences in environmental samples without the need to isolate nucleic acids as required by conventional methodologies. Embodiments of the methods and materials disclosed can be used in efficient and inexpensive assays for detecting nucleic acid sequences in soil and water, for example the nucleic acid genomes of bacterial pathogens that may be used in bioterrorism and biowarfare.

As discussed below, illustrative working embodiments of the invention are used in the detection of nucleic acids from Burkholderia pseudomallei, a pathogenic bacterium found in high-humic soil. Burkholderia pseudomallei can cause a potentially deadly illness known as melioidosis — a disease that mostly occurs in tropical climates, especially in Southeast Asia and northern Australia. In July of 2022, the United States Centers for Disease Control and Prevention issued a health advisory after Burkholderia pseudomallei was found in the Gulf Coast region of southern Mississippi.

The invention disclosed herein has a number of embodiments. Embodiments of the invention include compositions of matter that are useful in the “Direct-Detect” methodologies disclosed herein for detecting nucleic acid sequences in environmental samples. Such embodiments of the invention include compositions of matter comprising a methylmethacrylate polymer having hydrophobic organic groups with an affinity for humic and fulvic acids; a styrene-divinylbenzene co-polymer comprising iminodiacetic acid groups; an aminopolycarboxylic acid; a nonionic detergent; and an emulsifier. Typically such compositions comprise a DAX-8 resin; a Chelex resin; a buffering agent such as tris(hydroxymethyl)aminomethane; an ethylenediaminetetraacetic acid; nonionic surfactant that has a hydrophilic polyethylene oxide chain such as Triton X-100; and glycerol. In one illustrative working embodiment, this composition comprises 25% v/v DAX-8 resin; 10% v/v Chelex resin; 15 mM Tris, pH 8.0; 1 mM EDTA; 0.1% Triton x 100; and 5% glycerol. Optionally the composition is disposed within a vessel or matrix, for example a cylindrical column that facilitates the processing of environmental samples.

While Burkholderia pseudomallei is the focus of the illustrative working embodiments disclosed herein, those of skill in the art understand that the nature of this invention allows it to be adapted to a wide variety other organisms such as Bacillus anthracis, Bacillus Cereus, Campylobacter jejuni, Psuedomonas synringae, pathovars, Ralstona solanacearum, Agrobacterium tumefaciens, Xanthomonas oryzae, Xanthomonas Campestris pathovars, Listeria monocytogenes, Clostridium tetani, Clostridium botulinum, Histoplasma capsulatum, Enterohemorrhagic E. coli, Enterotoxigenic E. coli, Salmonella spp., Acinetobacter spp., Rhizobium spp., and Fusarium oxysporum, as well as Cryptosporidium parvum, Coccidioides spp,, Aspergillus spp. and the like.

In typical embodiments of the invention, the composition is cooled to a temperature of less than 5°C and/or heated to a temperature of at least 90°C. In certain embodiments, these compositions are combined with a soil sample (e.g., from 0.1 to 20 grams of soil) containing an organism such as a bacteria or a fungi, for example a soil sample selected from an environment observed to inhabited by a specific pathogenic organism such as Burkholderia pseudomallei. Typically this soil sample has been pretreated with a surfactant buffer solution comprising phosphate salts and a nonionic detergent (e.g. phosphate buffered saline and Triton X-100).

Another embodiment of the invention is a method of preparing a soil sample for a polymerase chain reaction process comprising combining the soil sample with a Direct-Detect compositions disclosed herein, and homogenizing the combination. Typically the soil sample has been pretreated with a surfactant buffer solution comprising phosphate salts and a nonionic detergent. In certain embodiments of the invention, pretreating the soil sample includes at least one of: vortexing the soil sample following combination with the surfactant buffer solution; incubating the soil sample following combination with the surfactant buffer solution for at least 30 minutes (and typically not more than 3 hours); and/or centrifuging the soil sample following combination with the surfactant buffer solution. In typical embodiments of the invention, centrifuging the soil sample comprises a low-speed centrifugation that sediments solids but does not sediment bacterial cells followed by a high-speed centrifugation that sediments bacterial cells present in a supernatant. Typically in these methods, the temperature of the buffer and soil mixture is precisely controlled, for example so that at least one methodological step is performed at 4°C, and/or at least one methodological step is performed at least 90°C etc.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic of a “Direct-Detect” embodiment of the invention.

Figure 2. Direct-Detect provides high sensitivity comparable to methods based on soil DNA isolation using commercial kits. Detection of B. pseudomallei in soil using the species specific ylf PCR-based assay (Tuanyok et al. (2007) J Bacteriol 189, 9044- 9049). A. Results of detection for two Bp-positive (“Bp (+))” soil samples each prepared by three methods: Method “E”, broth culture containing endemic soil followed by Direct- Detect and PCR; Method “X”, Water extraction of soil according to the method in this invention report followed by Direct-Detect and PCR; Method “D” commercial-kit-based DNA extraction of soil and PCR. B. Results of detection using Bp-negative (“Bp(-)) soil sample from the same geographical location as for (A). C. Control samples: B. thailandensis (Bt) (negative control), blank, and B. pseudomallei (positive control). M; molecular weight marker.

DETAILED DESCRIPTION OF THE INVENTION

In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

Many of the aspects of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. For example, a wide variety of publications describing PCR reagents and processes that can be adapted for use with embodiments of the invention are described in U.S. Patent Application Publication Numbers 20200123528, 20180195135, 20150368624, 20120129706, 20120028259, 20110027832, 20110020813, 20100255573,

20090325157, 20090111159, 20080293931, 20080099395, 20070065841,

20070059749, 20060230468, 20050282202, 20050136410, 20040229344,

20040161767 and 20030104383, the contents of which are incorporated herein by reference.

As noted above, we have designed and implemented a unique methodology and reagent combination that facilitates the PCR-based detection of nucleic acid sequences in environmental samples without the need to isolate DNA. Embodiments of the invention include compositions of matter comprising a methylmethacrylate polymer having hydrophobic organic groups with an affinity for humic and fulvic acids; a styrene-divinylbenzene co-polymer comprising iminodiacetic acid groups; an aminopolycarboxylic acid; a nonionic detergent; and an emulsifier. Optionally such compositions comprise a DAX-8 resin; a Chelex resin; a buffering agent such as tris(hydroxymethyl)aminomethane; an ethylenediaminetetraacetic acid; nonionic surfactant that has a hydrophilic polyethylene oxide chain such as Triton X-100; and glycerol. In this context, specific illustrative reagents useful in embodiments of the invention such as a DAX-8 resin, a Chelex resin and Triton X-100 are well known in the art. DAX-8 is a resin with strong hydrophobic organic matter endorsed to humic and fulvic acids. It is also referred as polymethylmethacrylate resin. See, e.g., Limura et al., CHARACTERIZATION OF DAX-8 ADSORBED SOIL FULVIC ACID FRACTIONS BY VARIOUS TYPES OF ANALYSES: 2012 Soil Science and Plant Nutrition 58(4):404-415; D01:10.1080/00380768.2012.708318; and Watanabe, A. 2007. “Isolation and purification of soil humic substances by IHSS method”. In Handbook of Analytical Methods for Humic substances, Edited by: Watanabe, A, Fujitake, N and Nagao, S. Nagoya: Sankeisya Co. Chelex resin is a styrene divinylbenzene copolymer containing paired iminodiacetate ions, which act as chelating groups in binding polyvalent metal ions. Triton X-100 is a poly(ethylene glycol) derivative that is poly(ethylene glycol) in which one of the terminal hydroxy groups has been converted into the corresponding p-(2,4,4-trimethylpentan-3- yl)phenyl ether. In one illustrative embodiment of the invention, the composition comprises from 20% to 30% (e.g., about 25%) v/v DAX-8 resin; from 5% to 15% (e.g., about 10%) v/v Chelex resin; from 10 mM to 20 mM (e.g., about 15 mM) Tris, pH 8.0; from 0.5 mM to 1.5 mM (e.g., about 1 mM EDTA); from 0.05% to 0.2% (e.g., about 0.1%) Triton X-100; and from 2.5% to 7.5% (e.g., about 5%) glycerol. Optionally the composition is disposed within a vessel or matrix, for example a cylindrical column that facilitates the processing of environmental samples.

In typical embodiments of the invention, the composition is cooled to a temperature of less than 5°C and/or heated to a temperature of at least 90°C. In certain embodiments, these compositions are combined with a soil sample (e.g. from 0.1 to 20 grams of soil) containing an organism such as a bacteria or a fungi, for example a soil sample selected from an environment observed to inhabited by a specific pathogenic organism such as Burkholderia pseudomallei. Typically this soil sample has been pretreated with a surfactant buffer solution comprising phosphate salts and a nonionic detergent (e.g. phosphate buffered saline and Triton X-100).

Another embodiment of the invention is a method of preparing a soil sample for a polymerase chain reaction process comprising combining the soil sample with a Direct-Detect compositions disclosed herein, and homogenizing the combination. Typically the soil sample has been pretreated with a surfactant buffer solution comprising phosphate salts and a nonionic detergent. In certain embodiments of the invention, pretreating the soil sample includes at least one of: vortexing the soil sample following combination with the surfactant buffer solution; incubating the soil sample following combination with the surfactant buffer solution for at least 30 minutes (and typically not more than 3 hours); and/or centrifuging the soil sample following combination with the surfactant buffer solution. In typical embodiments of the invention, centrifuging the soil sample comprises a low speed centrifugation that sediments solids but does not sediment bacterial cells followed by a high-speed centrifugation that sediments bacterial cells present in a supernatant. Typically in these methods, the temperature of the buffer and soil mixture is precisely controlled, for example so that at least one methodological step is performed at 4°C, and/or at least one methodological step is performed at least 90°C etc. Embodiments of the invention also include kits having a constellation of reagents selected to facilitate the PCR-based detection of nucleic acid sequences in environmental samples without the need to isolate nucleic acids as required by conventional methodologies. In certain embodiments of the invention, the kit comprises a surfactant buffer disclosed herein, a chelating buffer disclosed herein, and optionally one or more primers adapted for use in a polymerase chain reaction process. Such one or more primers can be those adapted to amplify polynucleotides present in at least one of: Burkholderia pseudomallei, Bacillus anthracis, Bacillus Cereus, Campylobacter jejuni, Psuedomonas synringae, pathovars, Ralstona solanacearum, Agrobacterium tumefaciens, Xanthomonas oryzae, Xanthomonas Campestris pathovars, Listeria monocytogenes, Clostridium tetani, Clostridium botulinum, Histoplasma capsulatum, Enter ohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Salmonella species, Acinetobacter species, Rhizobium species, Fusarium oxysporum, Cryptosporidium parvum, Coccidioides species or Aspergillus species.

The following disclosure provides a number of illustrative embodiments of the invention.

Illustrative Working Embodiments of Compositions of the Invention

The following disclosure describes materials used in an illustrative and nonlimiting working embodiment of the invention. Such illustrative embodiments include the use of reagents that can be mixed with soil including a surfactant buffer and a chelating buffer. Illustrative recipes for such buffers are as follows:

Surfactant Buffer (SB)

• lx Phosphate Buffered Saline (PBS)

• 0.1 % non-ionic detergent such as Triton X-100 The incubation of bulk soil with Surfactant Buffer (SB) serves two purposes. First, phosphate salts reduce clumping of charged inorganic and organic particles. This allows bacteria to be dislodged from soil particles and facilitates better sedimentation of inorganic solids. The release of bacterial cells from soil particles is further promoted by the non-ionic detergent Triton X-100 (0.1% final concentration). More importantly, Triton X-100 will not damage bacteria at this concentration, but is effective at promoting lysis of unicellular eukaryotes (e.g. protozoa) that may mask detection by sheltering bacteria intracellularly.

Chelating Buffer Mix (CBM)

• 25% v/v DAX-8 resin

• 10% v/v Chelex resin

• 15 mM Tris, pH 8.0

• 1 mM EDTA

• 0.1% Triton x 100

• 15% glycerol

A central component of CBM is the DAX-8 polymeric adsorbent resin (Sigma- Aldrich) (see, e.g. Schriewer et al., (2011) Microbiol Methods 85, 16-21). DAX-8 binds to PCR-inhibiting humic acids found in the soil, and facilitates their removal from the final preparation by filtration or centrifugation. Another component, Chelex Resin (Sigma Aldrich) is a styrene-divinylbenzene copolymer containing paired iminodiacetate ions which act as chelating groups for polyvalent metal ions such as Ca2+ and Mg2+, thus depriving DNAses of a required cofactor and inhibiting exo- and endonuclease activity. The inclusion of ethylene diamine tetraacetic acid (EDTA) provides additional metal chelating and nuclease inhibiting capacity. Glycerol helps to keep both DAX-8 and Chelex slurries in suspension for quantitative transfers of buffer volumes from one container to another. Typically, CBM is assembled with all components at a 2X concentration, and mixed 1:1 (vol:vol) with soil, water or other environmental sample. Tris-hydroxymethyl-aminomethane (Tris) serves as a general, inert organic pH buffer to optimize molecular amplification of target DNA.

Illustrative Working Embodiments of “Direct-Detect” Methodology of the Invention

Embodiments of the invention have advantages over conventional methods in this technology in that, for example, there is no recovery step, and no overnight incubation step needed.

ASSAY

• Preheat heating block or water bath to 95°C.

• Prepare Surfactant and Chelating Buffers beforehand. Cap and shake CBM tube immediately before use to prevent resin slurries from settling.

1. In 50 ml conical tubes, incubate 10 g of soil or 10 ml water sample with 10 ml Surfactant Buffer (SB). Vortex well, and shake for 1 h at 100 RPM, 25°C.

The incubation in SB suspends bacteria in liquid suspension and loosens clumps of soil.

Typically perform steps 2.-7 below on ice or with refrigeration.

2. Centrifuge samples at 200 x g for 5 min, 4°C in a swinging-bucket rotor.

Low-speed centrifugation sediments solids but does not sediment bacterial cells, which remain in the supernatant. The resultant pellet contains small soil particles, microbes, and organic PCR inhibitors. 3. Carefully transfer supernatants to chilled 15 ml conical tubes or to several microcentrifuge tubes

4. Spin samples: i. 15 ml conical tubes: 4,200 x g, 25 min, 4°C li. Microcentrifuge tubes: 10,000 x g, 2 min, 4°C

High-speed spin to pellet the bacterial fraction of the supernatant

5. Decant supernatant.

6. Using a 1 mL wide bore pipet tip, add 200 μl of CBM into each sample tube.

7. Homogenize the sample by brief, vigorous vortexing.

8. Transfer samples to a heating block or water bath at 95°C for 10 min. Mix well by vortexing, and centrifuge: i. 15 ml Falcon tubes: 4,200xg, 25 minutes ii. 1.5ml microcentrifuge tubes: 10,000xg, 2 minutes

Heating at 95°C for 10 minutes will lyse bacteria and sterilize each sample, and deactivate DNAse enzymes.

9. Proceed with PCR assay on supernatant.

1-2 μl of supernatant is adequate as template material for PCR reaction.

10. Store samples frozen at -70°C.

The development of the Direct-Detect methodology disclosed herein was facilitated by our study of environmental Burkholderia pseudomallei, which is the causative agent of melioidosis, a serious and often fatal human infection. In Southeast Asia and Australia, where the endemicity of the soil-dwelling organism is high, infections are nearly always acquired from the environment.

Soils that host B. pseudomallei are often clay-rich and high in humic acids, and the same is true for many microbes that are important from an agricultural or health context. Current PCR-based methods rely on the isolation and cleanup of nucleic acids to remove humic PCR inhibitor substances from environmental samples using expensive commercial kits. In contrast, the Direct-Detect method can be performed directly on environmental specimens - it does not require propagation of the organism(s) of interest or DNA isolation. As shown in Fig. 2, Direct-Detect provides detection sensitivity comparable to commercially-available DNA isolation kits at a fraction of the cost and time.

Reliance on conventional assays is cost prohibitive and time intensive, highlighting a key advantage of Direct-Detect methodologies disclosed herein. In addition to its use in detecting pathogenic organisms, there is great potential for the application of Direct-Detect for clinical science, public health and agriculture. For example, agriculturists can use embodiments of the invention to confirm the presence of economically important nitrogen fixing bacteria among relevant crops. Also, physicians and public health experts studying outbreaks may use embodiments of the invention to obtain information about a patient’s exposure to contaminated soil. Direct-Detect methodologies can also be utilized in veterinary and clinical diagnostics with heavily soiled tissue samples.

CONCLUSION

This concludes the description of the illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

All publications mentioned herein are incorporated herein by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications.

Claims

CLAIMS:

1. A composition of matter comprising: a methylmethacrylate polymer comprising hydrophobic organic groups having affinity for humic and fulvic acids; a styrene-divinylbenzene co-polymer comprising iminodiacetic acid groups; an aminopolycarboxylic acid; a nonionic detergent; and an emulsifier.

2. The composition of claim 1, wherein the composition comprises:

DAX-8 resin;

Chelex resin; a buffering agent;

Ethylenediaminetetraacetic acid (EDTA);

Triton X-100; and glycerol.

3. The composition of claim 2, wherein the composition comprises:

25% v/v DAX-8 resin;

10% v/v Chelex resin;

15 mM Tris, pH 8.0;

1 mM EDTA;

0.1% Triton x 100; and

5% glycerol.

4. The composition of claim 1, wherein the composition has a temperature of less than 5°C.

5. The composition of claim 1, wherein the composition has a temperature of at least 90°C.

6. The composition of claim 1, further comprising a soil sample containing a pathogenic microorganism.

7. The composition of claim 6, wherein the soil sample is selected from an environment observed to inhabited by Burkholderia pseudomallei.

8. The composition of claim 6, wherein the soil sample has been pretreated with a surfactant buffer solution comprising phosphate salts and a nonionic detergent.

9. The composition of claim 8, wherein the surfactant buffer solution comprises: Phosphate buffered saline; and

Triton X-100.

10. A method of preparing a soil sample for a polymerase chain reaction process comprising combining the soil sample with a composition of claim 1; and homogenizing the combination.

11. The method of claim 10, wherein the soil sample has been pretreated with a surfactant buffer solution comprising phosphate salts and a nonionic detergent.

12. The method of claim 11, wherein pretreating the soil sample includes at least one of: vortexing the soil sample following combination with the surfactant buffer solution; incubating the soil sample following combination with the surfactant buffer solution for at least 30 minutes; and/or centrifuging the soil sample following combination with the surfactant buffer solution.

13. The method of claim 12, wherein centrifuging the soil sample comprises: a low-speed centrifugation step selected to sediment solids but not sediment bacterial cells present in a supernatant followed by a high-speed centrifugation step that sediments bacterial cells present in a supernatant.

14. The method of claim 12, wherein at least one step is performed at 4°C.

15. The method of claim 10, wherein the soil sample comprises from 0.1 to 20 grams of soil.

16. The method of claim 10, further comprising performing a polymerase chain reaction process on a sample obtained from the homogenized combination.

17. The method of claim 11, wherein the polymerase chain reaction process comprises the use of one or more primers adapted to amplify polynucleotides present in at least one of: Burkholderia pseudomallei, Bacillus anthracis, Bacillus Cereus, Campylobacter jejuni, Psuedomonas synringae, pathovars, Ralstona solanacearum, Agrobacterium tumefaciens, Xanthomonas oryzae, Xanthomonas Campestris pathovars, Listeria monocytogenes, Clostridium tetani, Clostridium botulinum, Histoplasma capsulatum, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Salmonella species, Acinetobacter species, Rhizobium species, Fusarium oxysporum, Cryptosporidium parvum, Coccidioides species or Aspergillus species.

18. The method of claim 11, wherein the polymerase chain reaction process comprises the use of one or more primers adapted to amplify polynucleotides present in Burkholderia pseudomallei.

19. A kit compri sing : a composition of claim 1; a composition of claim 8; and one or more primers adapted for use in a polymerase chain reaction process.

20. The kit of claim 20, wherein the one or more primers are adapted to amplify polynucleotides present in at least one of: Burkholderia pseudomallei, Bacillus anthracis, Bacillus Cereus, Campylobacter jejuni, Psuedomonas synringae, pathovars, Ralstona solanacearum, Agrobacterium tumefaciens, Xanthomonas oryzae, Xanthomonas Campestris pathovars, Listeria monocytogenes, Clostridium tetani, Clostridium botulinum, Histoplasma capsulatum, Enter ohemorrhagic Escherichia coli,

Enterotoxigenic Escherichia coli, Salmonella species, Acinetobacter species, Rhizobium species, Fusarium oxysporum, Cryptosporidium parvum, Coccidioides species or Aspergillus species.