Environmental RNA assays

Abstract

Provided are methods of detecting a living target organism, for example, living Bursaphelenchus xylophilus, from an environmental nucleic acid sample by detecting a rapid kill RNA marker of the target organism. Also provided are primers and probes specific to rapid kill RNA markers, and kits including primers and probes that are useful for detecting rapid kill RNA markers.

Description

[0001] ENVIRONMENTAL RNA ASSAYS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 715,407, filed November 1, 2024, which is incorporated by reference in its entirety.

[0004] FIELD

[0005] This relates to assays and kits for detecting environmental RNA.

[0006] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0007] The Sequence Listing is submitted as an XML filed named “Sequence.xml,” created on October 29, 2025, 53,419 bytes, which is incorporated by reference herein.

[0008] BACKGROUND

[0009] Pests and invasive species can cause costly economic and ecological damage, such as natural forest decimation, crop decimation, clogging of water facilities and waterways, disease transmission, increased fire vulnerability, etc. Specific, sensitive detection increases the likelihood that an invasive species population is found, contained, and eradicated before it becomes widely established. In addition, determining the viability of pests or invasive species in the context of environmental samples has important regulatory implications because live organisms are often the focus of attention for environmental protection and biosurveillance. Thus, improved methods of detecting invasive species, particularly living invasive species, are needed.

[0010] SUMMARY

[0011] Disclosed are methods of detecting a living target organism from an environmental nucleic acid sample. The methods include detecting a rapid kill RNA marker of the target organism in the environmental nucleic acid sample, thereby detecting the living target organism. The rapid kill RNA marker is low or not detectable when the target organism is deceased. The methods can further include detecting a genomic DNA molecule of the target organism. Detecting the rapid kill RNA marker or genomic DNA molecule can include: i) contacting the environmental nucleic acid sample with forward and reverse primers specific to the rapid kill RNA marker and / or genomic DNA molecule; ii) amplifying the rapid kill RNA marker and / or genomic DNA molecule, thereby producing amplicons, and iii) detecting the amplicons, thereby detecting the rapid kill RNA marker or genomic DNA molecule. In some aspects, the amplicons are detected using a probe that binds the amplicon of interest. In some aspects, the environmental nucleic acid sample is obtained from a sample of wood. The target organism can be a pest or invasive species, for example, Bursaphelenchus xylophilus (pinewood nematode). In some aspects, the environmental nucleic acid sample is obtained from a sample of wood that is infested with, or suspected of being infested with, Bursaphelenchus xylophilus. In some aspects, the rapid kill marker is a sequence specific to mRNA of contig 280 or contig 3172 of Bursaphelenchus xylophilus. Exemplary primers and probes for detecting Bursaphelenchus xylophilus are provided herein.

[0012] Also provided are kits including a forward and reverse primer specific to a rapid kill RNA marker. In some aspects, the kits further include a forward and reverse primer specific to a genomic DNA molecule. The kits can further include probes, for example, to detect amplicons of the rapid kill RNA marker or genomic DNA molecule. In some aspects, the kits include a primer and / or probe disclosed herein. The kits can include further, for example, a container for holding or collecting a sample, one or more DNA isolation reagents, one or more qPCR reagents, one or more filters, or combinations thereof.

[0013] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects, which proceeds with reference to the accompanying figures.

[0014] BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Schematic of an exemplary BxCheck test. In this example, the BxCheck test is comprised of four components: the IntegritE-DNA® and IntegritE-RNA™ assays that check for sample inhibition and degradation of gDNA and RNA, respectively, plus specific assays for the detection of Bursaphelenchus xylophilus gDNA (eBUXY) and cDNA derived from RNA (erBUXY). Typically, a sample is run using qPCR in eight technical replicates (shown in Sample columns) with assay performance controls: (“+” includes gBlocks™ synthetic DNA template; “-“is a no template control to detect contamination). Each column represents eight technical replicates. The erBUXY detection kit can identify live organisms in an environmental sample such as pinewood nematode in wood product. The eBUXY and erBUXY results are used to generate a relative viability index.

[0015] FIG. 2. Glass Plate with 5 chambers.

[0016] FIG. 3. Glass plate set up in incubator.

[0017] FIG. 4. Burette setup.

[0018] FIG. 5. Baermann funnel setup.

[0019] FIG. 6. Overview of multiple glass plate set up in incubator.

[0020] FIGS.7A-7D. Rationale and depiction of the use of sample integrity evaluation in the context of the BxCheck assay. FIG. 7A: Samples with good integrity containing either DNA or RNA converted to cDNA can either contain the target nucleic acid resulting in a detection (check mark) or not contain the target nucleic acid resulting in no detection (“X”). However, false negative results or underestimation of target nucleic acid amounts can occur if a sample has poor integrity due to degradation, the presence of inhibitors, or a combination of both (“X”). FIG. 7B: The IntegritE assays (IntegritE-DNA® and IntegritE-RNA™ for DNA and RNA sample integrity, respectively) are run on each sample; if the sample passes the quality criteria, then the sample is deemed as being of good quality. If the sample fails, then it is subjected to a clean-up step and retested. If the sample then passes, then it is deemed as being of good quality. If it fails, it is deemed poor quality, and the user should proceed with caution in interpreting targeted assay results. FIG. 7C: Overview of the required results from a sample for a valid outcome confirming the presence of dead nematodes e.g., phytosanitary measures were successful. FIG. 7D: Overview of the required results from a sample for a valid outcome confirming the presence of live nematodes e.g., phytosanitary measures failed.

[0021] FIGS. 8A-8C. RNA expression profiles of BUXY-280 in adult, JIII juvenile, and egg stages. FIGS. 9A-9C. RNA expression profiles of BUXY-3172 in adult, JIII juvenile, and egg stages. FIG. 10. Example results from specificity testing of eBUXY5-contig280 (left panel) and erBUXY1-contig280 (right pandel).

[0022] FIG. 11. Protein translation and Alphafold2 results for BUXY280. SEQ ID NO. 31 shown is the translated protein.

[0023] FIG. 12. Protein translation and Alphafold2 results for BUXY3172. SEQ ID NO. 32 shown is translated protein.

[0024] FIGS. 13A-13C. Exemplary plate set ups to run a full BxCheck assay. The example shows eight biological replicates that are each run as four technical replicates for the IntegritE-DNA® and IntegritE-RNA™ assays. Controls with no reverse transcriptase (Minus RT) are run in duplicate for each sample to detect any target DNA contamination. One or more of the eBUXY assays to detect DNA and one or more of the erBUXY assays to detect cDNA converted from RNA samples are run on separate 96-well plates. Each plate, irrespective of what assay is run on it, contains eight technical replicates of no template controls (“-”) and duplicate reactions of an appropriate double stranded DNA positive control at 20 copies per reaction (“+”).

[0025] FIG. 14. Binary heat map showing the results using wood block samples inoculated with the Nel2 / 02 strain of Bursaphelenchus xylophilus and then heated to a kill temperature of 56°C for 30 minutes in a Humble water bath (Heat-treated) or kept at 25°C as a control (Control). There were three biological replicates. Nucleic acids were extracted from the infected wood, then assessed using assays that target DNA (IntegritE-DNA®, eBUXY5, and eBUXY9) and assays that target RNA via cDNA (IntegritE-RNA™, erBUXY7, erBUXY4). Four technical replicates per sample were run for the IntegritE tests while eight technical replicates were run per sample for the other assays. Additionally, eight technical replicates of no template control (“- Plate Control”) and duplicate reactions of an appropriate double stranded DNA positive control at 20 copies per reaction (“+”) were run on each plate and returned the expected results. A further control for DNA contamination of RNA samples where no reverse transcriptase was added to the cDNA synthesis reactions (“-RT Control”) were run in duplicate for all samples. No contamination was detected in any of these reactions.

[0026] FIGS. 15A-15E. DNA (FIGS. 15A, 15C, 15E) and RNA (FIGS. 15B, 15D) integrity and targeted assay amplification of Bursaphelenchus xylophilus (Q 14-26 strain) following kiln heat treatment of infected lumber (Heat-treated; circles). The lumber was treated to meet the following Canadian Food Inspection Agency (CFIA) schedule: Heat Treatment Schedule for Option A for a Lumber Thickness of up to 60 mm. The included the following parameters: Minimum Heat Treatment Run Time - 6 hours and 26 minutes, Wet Bulb Temperature Continuous Run Time > 60°C (140°F) - 2 hours and 3 minutes and Minimum Final Wet Bulb Temperature - 63°C (145°F). This treatment kills all nematodes at all life stages in the lumber.

[0027] Similarly infected lumber was not heat-treated but maintained at 25 °C as a control (Control; triangles) and processed at the indicated time points with heat-treated samples. Nucleic acids were extracted from wood shavings from lumber, then assessed using assays which target DNA (IntegritE-DNA®, eBUXY5, and eBUXY9) and assays that target RNA via cDNA (IntegritE-RNA™, erBUXY7). Four technical replicates per sample were run for the IntegritE tests while eight technical replicates were run per sample for the other assays. Each timepoint is a mean of 5 samples; error bars are standard error of the mean. There are no error bars associated with heat-treated erBUXY7 results as all samples returned no detections.

[0028] FIGS. 16A-16F. DNA (FIGS. 16A, 16C, 16E) and RNA (FIGS. 16B, 16D, 16F) integrity and targeted assay amplification of Bursaphelenchus xylophilus (Nel2 / 02 strain) following kiln heat treatment of infected lumber (Heat-treated; orange circles). The lumber was treated to meet the following Canadian Food Inspection Agency (CFIA) schedule: Heat Treatment Schedule for Option A for a Lumber Thickness of up to 60 mm. The included the following parameters: Minimum Heat Treatment Run Time - 6 hours and 26 minutes, Wet Bulb Temperature Continuous Run Time > 60°C (140°F) - 2 hours and 3 minutes and Minimum Final Wet Bulb Temperature - 63°C (145°F). This treatment kills all nematodes at all life stages in the lumber. Similarly infected lumber was not heat-treated but maintained at 25°C as a control (Control; triangles) and processed at the indicated time points with heat-treated samples. Nucleic acids were extracted from wood shavings from lumber, then assessed using assays which target DNA (IntegritE-DNA®, eBUXY5, and eBUXY9) and assays that target RNA via cDNA (IntegritE-RNA™, erBUXY7). Four technical replicates per sample were run for the IntegritE tests while eight technical replicates were run per sample for the other assays. Each timepoint is a mean of 5 samples; error bars are standard error of the mean. There are no error bars associated with heat-treated erBUXY7 results as all samples returned no detections.

[0029] SEQUENCES

[0030] Any nucleic acid and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C. F. R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0031] SEQ ID NOs: 1-28 are exemplary nucleic acid sequences of primers, probes, and amplicons provided in Table 3.

[0032] SEQ ID NO: 29 is the genomic nucleic acid sequence for the BUXY 280 gene located on chromosome 1 [(+ / - strands) 1,725,043-1,716,636]. The sequence includes 12 exons (introns are shown in lowercase).

[0033] ATGTCTCAACAGCGGAACACTGTCGCAATAAATCGGAGCTTATGggtgagttggaatttcgggcaaaaaatg atttttacttttccaaaaattgcaaaattagtcctccaatttccaGCTCCTCCTCTTCTCCGCAATTTTGGT GCACGCCGACTCCCGCAACAGCAATGTCCCTCAGGCAAAGTCGCCGCCGTTGCCGGCGCCCAAAATCCAAAC CATCGATGGGCACAGCGAGACCTCGGCGAAGGCCATGCATCATCTAATGCAGCATCAGGAACGAATGAAGCT TCAGACTTTGAACAAACAAACAGCTGTTGTAAACAATGGCCAGACCGGCAAGAAAGCGCCAATCAAGAATTC GAATCAGTTGAATCAGAAGGGCGCTTCTGAAGGCCTTCCGGCATCGTTTAGACAAGCGACGCAGCCTTCGGG GAAAGgtgagaaatatcttttgatgaaggtgttttgattttttgagacattttatacgacaaatttgaaaaa ttttaaaaaagtatcaaaaagtatcaataatcagaaatgagactttttttgtttgaaacgttatttggtgga ggtattttgattttttgagacattttatacgaccaaatgtttcaaatcaccaaaattttggaaaggtatcaa aagctatcagtgatcactttggacgctttttgctaatttttttgaaagcttgtttggtggaggtatttgatt tttttgggacattttatacggtcaaatcttcaaaagcatcaaaatttctaaaaagtatcaaaaagcatcaat agtcaaaattgagaaaaaaaaatttgtttgaaatttcactacattttgatcttttgtgacattttacacgaa caaaaatttggaaaaagtatcaaaagtgttaatgaccaaaggtgggaaaaaaatttttgccttaaaaatttg tccggtagagacatttgattttttgtgacattttatacgatcaattcaaaaaattttgaaaagtatcataga gtatcaatgatcacttttgccgttttttccttttttgtttctgatggtatgtagaaaacctcgaacccacca tataaccaatttattgtaatttttactcatattcttcaatttccagACAAATCCGCAACCGGCAGTAAAGCG CACGGCTACCAGACCCAACAATACGCCTCCACTGGTGGTTGTGCGGGCGGACAATGTGCGAATGGCGTTAAT GGCGCCTACGGAGGCCAGGGATACTCGAGTGTGGACTACAATCCATTGTTGGCGGGAGGTGTGCGCGGAGGG CCCGGAATCAAGGCGAGGATCAATTCGCGTGGATTCCAGTACGCTTCGGCCTTGATTGCTCCGATTTTGGAT CAAGCAATCCGCAAAGCGAGAATCCCACCGATCACACAGAGAATCGCAGAGgtaggttgagattttttgatg gaaaaatttggaaaatataaatgatattagaaaaatgacgatttttttgttatttttgctatatcttgtaag aaaattaatattttttcttgaaagtcataccaagtgaggctatatggcctctactttttatctttataagtt ttttgaaaatggccttgaattttcgaaggaaaaatttggaaaaatctgaaaaatatggaaaaatgccatttc ttcttatttttgccatatctcatcgggaaattcatattttttcttaaaagatctactaggatagtctataag accttggctttacagtcatatgggtttttggaaacttctctgcgctaaagtacgactttttttaagaaaaac ggctgtttccttgaactttcgagctcttgaagacttttatttttcctgaaacccttcgggcataaaatattt tttcactgatttatccaaacaatagcatccatgacggttaagttgatatatcatttagcatgatccatcaac tgtatcaaaagatacgatcaaaataaaattgccaccttttactgaaattcacaaaatttcaaaatcatcatc ccgtattttacccgcaattttttcagGTAAATGGTTGTATTCAAGTCTACAATCTCTACGTTAGTCGCTACC GTTGCCCTCAACGAGTTGTCCTTTATCCAGCGCCTCCAAATCAAATTGTTTTGGCTGTCCAGAATCTGGATG TTGggtaagttgatattatggctttttgcggtgtcatcattatacgtatattgcaaagaaagttatagttag cgcaaatttcaatacaaaagttttcatcgacactcttcgatactttttgcggaaaaatttagaaaaatttat tttttccacatattactcctattttttgagccacttcgtattttaccctccaactttcaGAGTGACCGGAAA CTTGGCCGGAACGGCCAACATCTTGATCCCAATTCCAATCAGTGGAATCATCGAGGTGAACGCGCATCAAGT CTCGATTACCGTGGCGTTGGTTGTGGATCGCGCGGCTAGCGGTGGAATTAGTTTGAGAGTTGCAAGTTGTCA AGCCTACATCGGATACCTTGATGCGTACATCGTGAATGGCGGTCTGATCGGCGATTTGGCCAATGGAATGTT TAGAGtgagcgtttggtgggggttttaggggttgttgcggaatgccaagaatttgaaaattttggcgggaat tgaaattattgaaattttttaagatttttgagctagctgtgattctgtaaaggattttgactaattcaacat ttcttgtgaaattcctgacctttgcaacggaatgccaagattttttgaagagtgccaaatttggcgggaaat ttgaattttaaaagattttgtgaagaaaaccgtgaaaattttcacgtcatttgacagtttttgacattttac aacacttgaaacagaatgccaaaaactttatttttggcgggaaatttgaacttaaaaagattttgggtggaa attcaaagaatctctaatttttacgtcattttgactgtttttgatattttacagcacttgaaacagaatgcc aaaaattttatttttggcgggaaatttaaatttccaaatttttgctcgttcaggtatctaacacaatatccg atttaactaacataagaccactcttttcaggGCCGAATTTCCAGCCAAATCCAGCAAATGTTGCCGGGCGAG CTCTGCAACCGACTGCCCGGAATCCTCGACTCGGAAGTCAACTCCAAACTCGGCGCCATTCCTCAGTCCATC TCGTTGACGCAAATCCTGAATGCGGCTGGTGGTGCGCTCGGAATTCAGAACCTTCTGGCTGGCGCCGCATCC GGCGGTCAATGTCCCTCGACCTGTGGTGGAGCTCCAGCTCAAGTGCCAACGCCCGTTGTGGCGCCGCCAGTG CCAGTTGGGCCGGCGCCAGCTCCGTTGCCGCCAGTCGGTGCGGCGTTGCCATATCAGGCCGGTCCGGTGGCT AGACCGGTAGCCGGGCCTGTAGGATTGCCTGTCGGGGCTGCTTTGCCTTATAATGTTGGGCCAGCCGCggtg agtagggattttaagcaaaatttttgaagaaaaagagagctatgagccttacttatataggtgatatttttg gctttaaaacttctatagcccttctattttcaGCGCGCCGCTCCCAAAGCCCAATCCATTCCGATCCCAACG GACCATGGCCATAATGGAGACAACTTGGCCACACTCAACTCCCGAAAGGTCCAAAGATACCATGTGGCGGGA GTCAGTGCTGTGcaggtaaatactttcgcccagcattaacatttagcttcaaaccagccaaaaattccgtct gcaacgaagcgacagctaaaattttcagcggttgataggcgggctagacggtttatcatggcttagttttac gctacgtctagatgcttctatatatttcgggttgccgtgcgtcttctcgggtcaacaacagttcggtcgatt ttactttgggtttctccttgatttttcgttgtatgcatggctaggagaaggcatagttaatagaaactgtaa gattgttcgggccctagggtagtttgtcacctattctgtggtattttacaggtattacggcactaatttcga gataaaactcacaggtctttgtcttttggaaaaagttttggttttgctgggccaagtctccgccgagctcca gcagctcttaccggctttgcaggcacttcctacaaccttgttttttagctctcccacttgtattgaactata gtcgacctgaaatgtcgggacgcgaaaatatttatttattcgcctttccaagcctctaaattttcgaatttt tCAGCAAGCCCGTCCCCTTCCCCGACCCGAAGGCTTCGACGCTCGCGGCCGCCGGATCGCCCGCGCCGCCGC TCCAAAAGCAACGATCCCGGTTGTGGTGGAGAGCCGAAACAAGACCAAAGCCAAAGCCTTTGGAAGTGGATC TGTGCAGTTTGGCGGAGCCGGTGGTGCCGGTGATGTTTGCTCTGGTTGCCCAAGCAGCGGCGGTGGAAGTCA GCTGGGATTCTTGTCGACGCTGGTTCAGTCGTTGGATTTGgtaagtaattttttgggggaaacgtaaaattt tcaagtcaagaatgttgatttgagctactttcagccttagttttgtagtgttagaaccttgtagggggacat tttatacgatgtgatgattaaaacttttttgaaaagatcgaaaagcgatgcataaagtatctcagaatttag tcttcagctttgagacgaggtaaaaagacgaattgttggcggaaatttatgtttggtgacattttatacgat gaaagctgcatttgtgcaaaacttaaaatttagcgtataaaatgtcgccaatagcgaagattaaaattcttc gattagaacaaggtctttaggacatatgaataagattttcggtaaaatagactttgcttgagacattttata tgatgaaaactggatttgcatgaaaattaaaattttgcgtataagatgtcgccaaagtctaaaaaaacaatt acccataaaggcaaggtagttaggtcaaacgaaagcattttctttaatcatagtgtcaaattatacgatgaa ttgcaaattttttacgaaatccaaaatttttcgtataaaatgtcacccaccattttttcgtcaaaaatcgat atttttagAACAAACTCAGCACGTTGGCGATCACCACTCAACTTCTTCAAAGCTACGCCACCAGCAATGACT ACACGATCGAGATCAACGGAGAGTTCTCGCCGAATGGCCAAGGCGGCACTCCGTTCGGCCCATTCCCCATGT ATTTCCCGTATTCGCCGGGCCGCAAAATGGCCGAAGTGTTGGTCAGCGACTACACCATCAACTCGCTGTTCT ACTGGCTTCATCGGACTGGATTCCTGACCTTCCGCATCGGGCCCGAGACTCCGAAAATTGGGGAGTTGTTGA GAACAACCTGTGCTGACGCCGACGAAACCCTGGAAGATCATGGTGTGGAGGTGGATGAGGAGCTGAGAAAAC GAAGAaggaagcataagagagaggcggggtaggtcgaataattgaaaaattaaaaaaaaaaacaaattttga aatttttttcgaaaaatatggatttttcataaaaaaacgatgcAGGAAGCATAAGAGAGAGGCGGGgtaggt ctgaaaattgaaaaaacaaaaaatttttttttttttgaaaactgaaaaataacaaaaacaaccgagattttt aggtcaaaaaatggggaaaattataattttttttacaagtttgggcaataagaatataaaaaaatgttttcc taagacggaaattttcaaaatttgagaatcgatttaattttttgtacattttttagcaaaaaaaattttttt attgctttattgcagtctgtcgggaaaataatgagggcaatgtcgttgcgccggttgcatcgctgaaacgaa aaaaaaatcgattttctccgcgacacaattcattttctcggcagcgattcgatttttggtgcgacagagggc tcattattttcccgacagacaaattacaaaaaatttttatttattttttaaaatttttttgctttatataat ttggaagtaaaaaaattaatagatttttttttattttttccccatttttttacatttttttgcattttattt ttttactttacaaataaaaaaaatcgatttttccaaaaatttttcgtattttaataattttttttcaatttc agACTCTCTCCCTACGACTTCACCATCATCCGTCAGAAGCGGCAAGACACCTCGTTGACGGATCTCGGAATT TGCTTCGGCGACATTCTCCCAGCAATCCGAGAGCGCTACCCCAATCAACGAATCGTAATCGCGATAAAAACT CGACAAGCACCGTCGATTCTTTTGTCGCAACGGAATGGAGGCACTGTGACGTTGGACTTGGTCGCCGACGCC GAAATTTACATTGAATCGACGAATCAACGAGTTGGGGCCATCACAATCTCAGCTGCGGCTGATATTGTCGTG TCGACTTATggtgagttggtttggaaagtttagaaaaattttttggaaaatcgaaaaagataaaaaaaaaat tattttcgcaaaatttaaaatacgcaaattcttcgccatttcactaaatcaaaaaaaaaataaaacaaataa taaaataaagatttttttttgatccaagaaaacttaaaatacgcaatctcctccgccattttacttaagcaa aaaaataataaagaataaaaaaaaaacaaaaaaaaaaacaaaatgaaaaagaaaatcaaaaaaaaaaaaaaa aaaataataaaacaagaatttttaatccagaaaaaaaattttatttttttttcagcatttttccaaagtaaa aaaaaaattaaaaacaaattcgaaaaattcgaaaaattcgcatcaaaaatttcttcaaacaacttctctaaa aatatttttttgtccagtgaaaattttaaaattcgaaccgttacatttatatctttgtttttcaGGTGGCCG GATCAGTGGAAGCGCTCAAATTACCCGATTGCAACTCAATGATTACGAAGGTACCCTCGGATTGCCTCAAGA TGCTCTTGACAACTTGGGTTCGCTGGGAAAAGAAGTCATCCAGAaggtaaggaggccttagaattttcggaa aataattttcaaaaaaaaattttttgttgtctcacacagtgcagtttactcttttccgcacggtgcggagca tttcgaaacactttgcactgtgcaagcgtctgttgcgaccgcaaaaaaataagcttgcaccgtgcgttattt cgattgcacagtgcattttttgcgaaccattttgcactgtgcaagcgtctgtcgcaaccgcacagtgcaaaa aagatcgcttttttctgcaccctgcggatcttttcgaaccattttgcactgtgcaagcgtctgtcgcaaccg cacagtgcaaaaaagatcgcttttttctgcaccctgcggatcttttcgaaccatttgcactgtgcagaggtc tgtcgccaccgcacagtgcaaaaaagatcgcttttttatgcaccgtgcggatcttttcgaaccattttgcac tgtgcagacgtttgtcgcaaccgcacagtgcaaaaaagatcgcttttttatgcaccgtgcggatcttttcga accattttgcactgtgcaagctttgtcgtcatcgcacagtgcaaaaaagatcgcttttttatgcaccgtgcg gatcttttcgaaccattttgcactgtgcaagctttgtcgtcatcgcacagtgcaaaaaagatcgctttttta tgcaccgtgcggatcttttcgaaccattttgcactgtgcaagctttgtcgtcatcgcacagtgcaaaaaaga tcgtttttttctgcactttgcggatcttttcgaaccattttgcactgtgcagacgtctgtcgccaccgcaca gtgcaaaaaagatcgcttttttatgcaccgtgcggatcttttcgagccattttgcactgtgcagacgtctgt cgcaaccgcacagtgcaaaaaagatcgcttttttatgcaccgtgcggatcttttcgaaccattttgcactgt gcaagctttgtcgtgatcgcacagtgcaaaaaattaaaaaaaaaatctttcaaatctcaatttttcAGGCCG CCAACGACGCGCTCCAAAACGGAATTGCCCTCAACATTCCCAGTGGAATTGGTGGACTTCCAATCAACTTTG TGCAGCCCGAATTCCACATTCTGGAGCATGCGTTGCACATTGAAAGCGACTTCACCGTGGATCCGGCGGGTC TTCAGGGCCTGTTGGGAGGCGGTGGCGGCGGATTTGGAGGCGCGTGTCGACGGTAG SEQ ID NO: 30 is the genomic nucleic acid sequence for the BUXY 3172 gene located on chromosome 2 [(+ / + strands) 1,738,090-1,739,030]. The sequence includes 3 exons.

[0034] ATGAAGGTTGCGGTGCTTCTGTTTATGTTGGTGGTCGCTGCTGCGGCTATTGAGAAGAAGAAGAGCATCACG ATCCCTTTCCCTTTCCATGCCAACAGAGCCAAGCTGATCAACAAGTTGAAGCTGATCGTTGACGACGCGGGA GTTCATGTCAGTCATCGAAATGTCGCCAAAATCACTGACAAGATCAGAAATGGggtaagtttacatgccggt aatctctgtttatagtcgcataattattttatgcctatattatcatatttttaaatttctagCTAGCAAATA TCAAAGCAATCCACAAGTTGCCCAGTCTTGTCGGGCTCGTCAACGATTTGGAGAGCAGCATCCTCGCTCTCC CTGACACCAACCCAAGAGGTCCCGCCTTCCAAGCGGTCGTCAAGAAAGCCGATGAACTCCACGCCGTCTTGT TCACCGTCCTCAATGACGTCAAGAGAATCCCATACGACCTCGAAAAGCTCAACTTGCACTTGTTGGAGAAGg taggtagctgaggtcaacttgcaaccctgactcgtttatccgccggcttataatcaacctttcagAGCGAAG AAAACGATCAAATCCGCAAGAACCTCAAAAGTCTTAAGCTAAACTTTGGCCTAACGTTCTTGCAGCTGAACC ACTTTTACAGGCGTGGAGCACTCAAGCGTATTAATATTCACCTTGAAGTCCAAGAACTCGTGGACCTGCTCA GCCGGCCTTCCGTCTCGAAACAACAATATTCTGCGGCTATTAAAAAGATTTTAAAAAAGCTGGGTAGGATTG AGCCACTCCCCACAAAGGAACCAGTCAAAAAAAACACAGACGTACCATTTATCACATTCGATCCAGTCCACA TTTTCACAAAGGGACCAATCCACATTCGCACGAAGAACCCACACCAGAAGATCACAAAGAAACCAAGAGAGG AT TAG SEQ ID NO: 31 is an exemplary amino acid sequence of the BUXY 280 protein.

[0035] MSQQRNTVAINRSLWLLLFSAILVHADSRNSNVPQAKSPPLPAPKIQTIDGHSETSAKAMHHLMQHQERMKL QTLNKQTAVVNNGQTGKKAPIKNSNQLNQKGASEGLPASFRQATQPSGKDKSATGSKAHGYQTQQYASTGGC AGGQCANGVNGAYGGQGYSSVDYNPLLAGGVRGGPGIKARINSRGFQYASALIAPILDQAIRKARIPPITQR IAEVNGCIQVYNLYVSRYRCPQRWLYPAPPNQIVLAVQNLDVGVTGNLAGTANILIPIPISGIIEVNAHQV SITVALWDRAASGGISLRVASCQAYIGYLDAYIVNGGLIGDLANGMFRGRISSQIQQMLPGELCNRLPGIL DSEVNSKLGAIPQSISLTQILNAAGGALGIQNLLAGAASGGQCPSTCGGAPAQVPTPWAPPVPVGPAPAPL PPVGAALPYQAGPVARPVAGPVGLPVGAALPYNVGPAARAAPKAQSIP IPTDHGHNGDNLATLNSRKVQRYH VAGVSAVQQARPLPRPEGFDARGRRIARAAAPKATIPWVESRNKTKAKAFGSGSVQFGGAGGAGDVCSGCP SSGGGSQLGFLSTLVQSLDLNKLSTLAITTQLLQSYATSNDYTIEINGEFSPNGQGGTPFGPFPMYFPYSPG RKMAEVLVSDYTINSLFYWLHRTGFLTFRIGPETPKIGELLRTTCADADETLEDHGVEVDEELRKRRRKHKR EAGLSPYDFTIIRQKRQDTSLTDLGICFGDILPAIRERYPNQRIVIAIKTRQAPSILLSQRNGGTVTLDLVA DAEIYIESTNQRVGAITISAAADIVVSTYGGRISGSAQITRLQLNDYEGTLGLPQDALDNLGSLGKEVIQKA ANDALQNGIALNIPSGIGGLPINFVQPEFHILEHALHIESDFTVDPAGLQGLLGGGGGGFGGACRR SEQ ID NO: 32 is an exemplary amino acid sequence of the BUXY 3172 protein.

[0036] MKVAVLLFMLWAAAAIEKKKSITIPFPFHANRAKLINKLKLIVDDAGVHVSHRNVAKITDKIRNGVANIKA IHKLPSLVGLVNDLESSILALPDTNPRGPAFQAVVKKADELHAVLFTVLNDVKRIPYDLEKLNLHLLEKSEE NDQIRKNLKSLKLNFGLTFLQLNHIFRRGALKRINIHLEVQELVDLLSRPSVSKQQYSAAIKKILKKLGRIE PLPTKEPVKKNTDVPFITFDPVHIFTKGPIHIRTKNPHQKITKKPRED SEQ ID NO: 33 is an exemplary nucleic acid sequence of the BUXY 3172 coding sequence (representing the mRNA sequence).

[0037] ATGAAGGTTGCGGTGCTTCTGTTTATGTTGGTGGTCGCTGCTGCGGCTATTGAGAAGAAGAAGAGCATCACG ATCCCTTTCCCTTTCCATGCCAACAGAGCCAAGCTGATCAACAAGTTGAAGCTGATCGTTGACGACGCGGGA GTTCATGTCAGTCATCGAAATGTCGCCAAAATCACTGACAAGATCAGAAATGGGGTAGCAAATATCAAAGCA ATCCACAAGTTGCCCAGTCTTGTCGGGCTCGTCAACGATTTGGAGAGCAGCATCCTCGCTCTCCCTGACACC AACCCAAGAGGTCCCGCCTTCCAAGCGGTCGTCAAGAAAGCCGATGAACTCCACGCCGTCTTGTTCACCGTC CTCAATGACGTCAAGAGAATCCCATACGACCTCGAAAAGCTCAACTTGCACTTGTTGGAGAAGAGCGAAGAA AACGATCAAATCCGCAAGAACCTCAAAAGTCTTAAGCTAAACTTTGGCCTAACGTTCTTGCAGCTGAACCAC ATTTTCAGGCGTGGAGCACTCAAGCGTATTAATATTCACCTTGAAGTCCAAGAACTCGTGGACCTGCTCAGC CGGCCTTCCGTCTCGAAACAACAATATTCTGCGGCTATTAAAAAGATTTTAAAAAAGCTGGGTAGGATTGAG CCACTCCCCACAAAGGAACCAGTCAAAAAAAACACAGACGTACCATTTATCACATTCGATCCAGTCCACATT TTCACAAAGGGACCAATCCACATTCGCACGAAGAACCCACACCAGAAGATCACAAAGAAACCAAGAGAGGAT TAG SEQ ID NO: 34 is an exemplary nucleic acid sequence of the BUXY 280 coding sequence (representing the mRNA sequence).

[0038] ATGTCTCAACAGCGGAACACTGTCGCAATAAATCGGAGCTTATGGCTCCTCCTCTTCTCCGCAATTTTGGTG CACGCCGACTCCCGCAACAGCAATGTCCCTCAGGCAAAGTCGCCGCCGTTGCCGGCGCCCAAAATCCAAACC ATCGATGGGCACAGCGAGACCTCGGCGAAGGCCATGCATCATCTAATGCAGCATCAGGAACGAATGAAGCTT CAGACTTTGAACAAACAAACAGCTGTTGTAAACAATGGCCAGACCGGCAAGAAAGCGCCAATCAAGAATTCG AATCAGTTGAATCAGAAGGGCGCTTCTGAAGGCCTTCCGGCATCGTTTAGACAAGCGACGCAGCCTTCGGGG AAAGACAAATCCGCAACCGGCAGTAAAGCGCACGGCTACCAGACCCAACAATACGCCTCCACTGGTGGTTGT GCGGGCGGACAATGTGCGAATGGCGTTAATGGCGCCTACGGAGGCCAGGGATACTCGAGTGTGGACTACAAT CCATTGTTGGCGGGAGGTGTGCGCGGAGGGCCCGGAATCAAGGCGAGGATCAATTCGCGTGGATTCCAGTAC GCTTCGGCCTTGATTGCTCCGATTTTGGATCAAGCAATCCGCAAAGCGAGAATCCCACCGATCACACAGAGA ATCGCAGAGGTAAATGGTTGTATTCAAGTCTACAATCTCTACGTTAGTCGCTACCGTTGCCCTCAACGAGTT GTCCTTTATCCAGCGCCTCCAAATCAAATTGTTTTGGCTGTCCAGAATCTGGATGTTGGAGTGACCGGAAAC TTGGCCGGAACGGCCAACATCTTGATCCCAATTCCAATCAGTGGAATCATCGAGGTGAACGCGCATCAAGTC TCGATTACCGTGGCGTTGGTTGTGGATCGCGCGGCTAGCGGTGGAATTAGTTTGAGAGTTGCAAGTTGTCAA GCCTACATCGGATACCTTGATGCGTACATCGTGAATGGCGGTCTGATCGGCGATTTGGCCAATGGAATGTTT AGAGGCCGAATTTCCAGCCAAATCCAGCAAATGTTGCCGGGCGAGCTCTGCAACCGACTGCCCGGAATCCTC GACTCGGAAGTCAACTCCAAACTCGGCGCCATTCCTCAGTCCATCTCGTTGACGCAAATCCTGAATGCGGCT GGTGGTGCGCTCGGAATTCAGAACCTTCTGGCTGGCGCCGCATCCGGCGGTCAATGTCCCTCGACCTGTGGT GGAGCTCCAGCTCAAGTGCCAACGCCCGTTGTGGCGCCGCCAGTGCCAGTTGGGCCGGCGCCAGCTCCGTTG CCGCCAGTCGGTGCGGCGTTGCCATATCAGGCCGGTCCGGTGGCTAGACCGGTAGCCGGGCCTGTAGGATTG CCTGTCGGGGCTGCTTTGCCTTATAATGTTGGGCCAGCCGCGCGCGCCGCTCCCAAAGCCCAATCCATTCCG ATCCCAACGGACCATGGCCATAATGGAGACAACTTGGCCACACTCAACTCCCGAAAGGTCCAAAGATACCAT GTGGCGGGAGTCAGTGCTGTGCAGCAAGCCCGTCCCCTTCCCCGACCCGAAGGCTTCGACGCTCGCGGCCGC CGGATCGCCCGCGCCGCCGCTCCAAAAGCAACGATCCCGGTTGTGGTGGAGAGCCGAAACAAGACCAAAGCC AAAGCCTTTGGAAGTGGATCTGTGCAGTTTGGCGGAGCCGGTGGTGCCGGTGATGTTTGCTCTGGTTGCCCA AGCAGCGGCGGTGGAAGTCAGCTGGGATTCTTGTCGACGCTGGTTCAGTCGTTGGATTTGAACAAACTCAGC ACGTTGGCGATCACCACTCAACTTCTTCAAAGCTACGCCACCAGCAATGACTACACGATCGAGATCAACGGA GAGTTCTCGCCGAATGGCCAAGGCGGCACTCCGTTCGGCCCATTCCCCATGTATTTCCCGTATTCGCCGGGC CGCAAAATGGCCGAAGTGTTGGTCAGCGACTACACCATCAACTCGCTGTTCTACTGGCTTCATCGGACTGGA TTCCTGACCTTCCGCATCGGGCCCGAGACTCCGAAAATTGGGGAGTTGTTGAGAACAACCTGTGCTGACGCC GACGAAACCCTGGAAGATCATGGTGTGGAGGTGGATGAGGAGCTGAGAAAACGAAGAAGGAAGCATAAGAGA GAGGCGGGACTCTCTCCCTACGACTTCACCATCATCCGTCAGAAGCGGCAAGACACCTCGTTGACGGATCTC GGAATTTGCTTCGGCGACATTCTCCCAGCAATCCGAGAGCGCTACCCCAATCAACGAATCGTAATCGCGATA AAAACTCGACAAGCACCGTCGATTCTTTTGTCGCAACGGAATGGAGGCACTGTGACGTTGGACTTGGTCGCC GACGCCGAAATTTACATTGAATCGACGAATCAACGAGTTGGGGCCATCACAATCTCAGCTGCGGCTGATATT GTCGTGTCGACTTATGGTGGCCGGATCAGTGGAAGCGCTCAAATTACCCGATTGCAACTCAATGATTACGAA GGTACCCTCGGATTGCCTCAAGATGCTCTTGACAACTTGGGTTCGCTGGGAAAAGAAGTCATCCAGAAGGCC GCCAACGACGCGCTCCAAAACGGAATTGCCCTCAACATTCCCAGTGGAATTGGTGGACTTCCAATCAACTTT GTGCAGCCCGAATTCCACATTCTGGAGCATGCGTTGCACATTGAAAGCGACTTCACCGTGGATCCGGCGGGT CTTCAGGGCCTGTTGGGAGGCGGTGGCGGCGGATTTGGAGGCGCGTGTCGACGGTAG SEQ ID NOs: 35-42 are exemplary nucleic acid sequences of primers, probes, and amplicons for the IntegritE-DNA® or IntegritE-RNA™ assays.

[0039] DETAILED DESCRIPTION

[0040] I. Introduction

[0041] The pinewood nematode (PWN), Bursaphelenchus xylophilus, is endemic to North America and lives on conifers, such as pines [Mamiya et al., Nematologica, 1972. 18: p. 120-124; Kiyohara et al., J Jap Forestry Soc, 1971. 53: p. 210-218]. However, PWN causes devastating pine wilt disease in Asia and Europe, leading it to be classified as a quarantine pest [Pimentel et al., CABI Compendium, 2022. CABI Compendium: p. 10448]. This necessitates careful phytosanitation and surveillance procedures on wood products from North American regions known to contain PWN for export to outside markets.

[0042] Traditionally, pinewood nematode detection involves morphological analysis of species-specific characteristics after nematodes are extracted from wood using a Baermann funnel. However, this analysis is time-consuming and requires specialized morphological expertise. Moreover, morphological distinction of egg and juvenile stages of pinewood nematode from other related species is problematic, thereby prompting the need for genomic DNA (gDNA)-based discrimination methods [Cardoso et al., European Journal of Plant Pathology, 2011. 133(2): p. 419-425]. However, genomic DNA methods can only detect the presence of the pest, not viability. Since only living organisms are regulated as quarantine concerns, molecular tools that can distinguish between live and dead pinewood nematodes will provide increased confidence in the effectiveness of new wood treatments.

[0043] Determining the viability of target taxa within environmental samples has important regulatory implications because live organisms are often the focus of attention for environmental protection and biosurveillance. Recently, the concept of eRNA has been discussed as a means for accomplishing this [Cristescu et al., Trends in Ecology & Evolution, 2019. 34(8): p. 694-697], yet the concept has not been extensively explored. Unlike DNA, RNA half-life is relatively short and therefore may be helpful to confidently ascribe recent presence and differentiate living and dead organisms.

[0044] There have been attempts to develop a molecular approach utilizing the exon / intron differentiation between mRNA and gDNA of mRNA from the PWN expansin gene [Leal et al., Forest Pathology, 2015.

[0045] 45(2): p. 134-148], however, the isothermal amplification method that was used lacked the sensitivity needed to reliably detect and distinguish the nematode pest Bursaphelenchus xylophilus from other nematodes, particularly its closest relative, B. mucronatus [Ye et al., Molecular Phylogenetics and Evolution, 2007. 43: p. 1185-1197]. This method was also only validated on adult worms, not on other life stages, and the gene choice was solely based on containing a relatively large intron, not on gene expression pattern. Additional challenges lie in the ability to perform well and not give false positive results with environmental (e.g., wood) samples. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) did not have the required sensitivity in its detection method that a probe-based detection method has. Moreover, in contrast to gel-, intercalating agent-, or colorimetric assay-based readouts, the probe-based detection provides additional sequence specificity for complex environmental samples [Langlois et al., Environmental DNA, 2021. 3: p. 519-527]. Given the variable nature of environmental samples, the probe-based detection method is preferred for examining environmental samples [Abbott et al., National standard of Canada, CSA 2023. W219:23: p. 23].

[0046] The methods described herein use the basic premise of selecting mRNA that is consistently expressed in high abundance that includes at least one intron to distinguish between mRNA and gDNA sources. This can be accomplished using knowledge of post-transcriptional splicing, which removes intronic sequence during the maturation of the transcript. Thus, one can distinguish between a nuclear gene and its processed transcript by designing primer / probe combinations that detect the processed transcript and not the intron-containing gene. While any number of mRNA transcripts could be selected to fulfill the role of viability sensor, another important factor for mRNA transcript selection is identifying a transcript or transcripts whose expression rapidly decreases upon exposure to known kill conditions such as incubation for 30 minutes at 56°C rather than waiting several days for testing.

[0047] Two assays per target taxon are described herein - one to detect the mRNA (erBUXY), and the other to detect a segment of nuclear genomic DNA (eBUXY) such that a relative viability index may be generated as a ratio of the relative mRNA to gDNA qPCR product intensities. These paired assays can be incorporated into a kit format that includes controls to establish sample integrity to comprise the BxCheck assay. The IntegritE-DNA® and IntegritE-RNA™ assays check for sample inhibition and degradation of gDNA and mRNA, respectively [Hobbs et al., Environmental DNA, 2020. 2: p. 350-361; Veldhoen et al., PLoS One, 2016. 11(11): p. e0164907; Imberry et al., Ecotoxicol Environ Saf, 2024. 279: p. 116463],

[0048] Genes for the BxCheck assay components (FIG. 1) are identified herein that have the desired properties above. The BxCheck assay can be used to monitor phytosanitary effectiveness and evaluate potential alternative fumigants as replacements for methyl bromide, which is being phased out due to its damage to the ozone layer, for the phytosanitary treatment of wood products.

[0049] II. Summary of Terms

[0050] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found Benjamin Lewin, Genes XII, published by Jones & Bartlett Publishers, 2018; and Krebs et al. (eds.) ISBN:9781284104493; The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; Remington’s Pharmaceutical Sciences, Adejare (Ed.), Academic Press, 23rd Edition, 2020, ISBN: 9780128223895 / 9780128200070; and other similar references.

[0051] In case of conflict, the present specification, including explanations of terms, will control. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a protein” includes singular or plural proteins and can be considered equivalent to the phrase “at least one protein.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects of the disclosure, the following explanations of terms are provided: About: Refers to 10% of a referenced value unless context clearly indicates otherwise. For example, “about 100” refers to a range of 90 to 110.

[0052] Amplifying a Nucleic Acid Molecule: To increase the number of copies of a nucleic acid molecule, for example using PCR or qPCR. The resulting amplification products are called “amplicons.” An example of in vitro amplification is the polymerase chain reaction (PCR), in which a nucleic acid molecule, for example DNA or RNA (or cDNA thereof), is obtained from an environmental sample, is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to the nucleic acid molecule. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule.

[0053] Other examples of in vitro amplification techniques that can be used with the disclosed methods include real-time PCR, reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR), reverse transcriptase semi-nested PCR, strand displacement amplification (see USPN 5,744,311), transcription-free isothermal amplification (see USPN 6,033,881), repair chain reaction amplification (see WO 90 / 01069), ligase chain reaction amplification (see EP-A-320308), gap filling ligase chain reaction amplification (see USPN 5,427,930), coupled ligase detection and PCR (see USPN 6,027,889), and NASBA™ RNA transcription-free amplification (see USPN 6,025,134).

[0054] Contacting: Placing in direct physical association, includes both in solid and liquid form. For example, contacting can occur in vitro with isolated nucleic acid molecules in solution, for example in a qPCR reaction mixture.

[0055] Detect: To determine the existence or presence of an agent of interest, for example, determining the absence or presence of a target nucleic acid molecule (e.g., a rapid kill RNA marker) or amplicon thereof in an environmental sample.

[0056] Environmental Sample: A sample obtained from an environment. In some aspects, the environment is one in which an organism of interest (e.g., Bursaphelenchus xylophilus) is present or is suspected of being present. Environmental samples include, but are not limited to, water samples (fresh water, salt water, brackish water, etc.), soil samples, plant samples (e.g., wood samples), or other substrate samples from an environment.

[0057] Fluorophore: A chemical compound that when excited by exposure to a particular stimulus, e.g.. a defined wavelength of light, emits light (fluoresces), for example at a different wavelength. The disclosed primers and probes can be labeled with a fluorophore (e.g., have a fluorophore covalently attached thereto).

[0058] Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescent molecules eliminates the need for an external source of electromagnetic radiation, such as a laser. Examples of chemiluminescent molecules include, but are not limited to, aequorin or luciferase. Hybridization: To form base pairing between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. For example, the primers and probes disclosed herein can form a duplex molecule with a target nucleic acid molecule or amplicon generated from such a target. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid molecules. Generally, the temperature of hybridization and the ionic strength (such as the Na+concentration) of the hybridization buffer will determine the stringency of hybridization. However, for hybridization conditions related to PCR, the salt concentration is generally fixed by the buffer conditions and stringency of hybridization controlled by temperature (for example 42°C low stringency, 48-50°C medium stringency, and 55-60°C high stringency).

[0059] Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Green and Sambrook (2014) Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, NY. For purposes of this disclosure, “stringent conditions” encompass conditions under which hybridization only will occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. “Moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of “very high stringency” are those under which sequences with more than 5% mismatch will not hybridize. In some aspects, a primer and / or probe disclosed herein is hybridized to a target nucleic acid under conditions for moderate, medium, high, or very high stringency. In some aspects, a primer and / or probe disclosed herein is hybridized to a target nucleic acid under conditions for high or very high stringency.

[0060] Isolated: An “isolated” biological component (such as a nucleic acid molecule, organism, or cell) has been substantially separated or purified away from other components of an environment and / or cell of an organism in which it naturally occurs. Nucleic acid molecules that have been “isolated” include molecules (such as DNA or RNA) purified by standard purification methods. The term also embraces nucleic acid molecules, proteins and peptides prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. In some aspects, an isolated nucleic acid molecule is one that is substantially separated from protein, lipids, or other types of nucleic acid molecules, in a cell.

[0061] Isolated does not require absolute purity, and can include nucleic acid molecules that are at least 50% pure, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% pure.

[0062] Kill Treatment: A condition that kills the target organism. In some aspects, the kill treatment is exposure of the target organism to a temperature of at least 56°C for at least 30 minutes.

[0063] Label: An agent capable of detection. In some aspects, a label can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Green and Sambrook (2014) Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, NY.

[0064] Oligonucleotide: A polynucleotide sequence (such as DNA or RNA), typically of at least 6 nucleotides, for example at least 9, at least 15, at least 18, at least 24, at least 30, or at least 50 nucleotides long. In some aspects, an oligonucleotide is 12-50 nucleotides, for example, 18 to 35 nucleotides, 18 to 30 nucleotides, 19 to 30 nucleotides, 19 to 29 nucleotides, or 20 to 29 nucleotides. An oligonucleotide can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Oligonucleotides can include modified or artificial nucleotides.

[0065] Primer: A short nucleic acid molecule that can be used to initiate the synthesis of a longer nucleic acid sequence. In some examples, a primer includes (e.g., has attached thereto) a label (e.g., a detachable label). Primers can be annealed to a complementary target nucleic acid strand by nucleic acid hybridization to form a hybrid between the primer and the target. Then, the sequence is extended from the primer along the target by a polymerase (e.g., DNA polymerase or reverse transcriptase). Primer pairs can be used to amplify a nucleic acid sequence, for example by PCR or other nucleic-acid amplification methods, or can be used for other purposes, such as converting RNA to cDNA. Typically, primers are short nucleic acids (e.g., 10-50 bp).

[0066] Probe: A nucleic acid molecule which can be used to detect (for example by hybridizing to) the presence of a target nucleic acid molecule. Probes can include a detectable label (e.g., a fluorophore), and in some examples may also include a fluorescence quencher. Typically, probes are short nucleic acids (e.g., 10-50 bp).

[0067] Rapid Kill RNA Marker: An RNA molecule that is expressed or abundant when an organism is living, but rapidly degrades when the organism is deceased and / or when exposed to conditions that the organism would not survive. In some aspects, a rapid kill RNA marker is not detectable by 1 hour of the organism becoming deceased, or by 1 hour following exposure to conditions that the organism would not survive. Non-limiting examples of rapid kill RNA markers for B. xylophilus include contig 280 and contig 3172, which are described herein.

[0068] Sample: A specimen, which may contain DNA, RNA (e.g., mRNA), proteins, or combinations thereof. Examples include, but are not limited to, water (such as freshwater, brackish water, saltwater) soil, plant material (e.g., wood), and air. In some examples, samples are used directly in the methods provided herein. In some examples, samples are manipulated prior to analysis using the disclosed methods, such as through concentrating, filtering, centrifuging, diluting, desalting, denaturing, reducing, alkylating, proteolyzing, or combinations thereof. In some examples, components of the samples are isolated or purified prior to analysis using the disclosed methods, such as isolating cells, proteins, and / or nucleic acid molecules from the samples. Sequence Identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.

[0069] Methods of alignment of sequences for comparison have been described. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res.

[0070] 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0071] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biomedical Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

[0072] BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options can be set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (such as C:\seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (such as C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (such as C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2.

[0073] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 19 matches when aligned with a test sequence having 20 nucleotides is 95.0 percent identical to the test sequence (19 -20*100=95.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing 21 nucleotides that aligns with 19 consecutive nucleotides from an identified sequence as follows contains a region that shares 90 percent sequence identity to that identified sequence (that is, 194-21*100=90).

[0074] One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions (such as high or very high stringency), as described above. Under Conditions Sufficient For: A phrase that is used to describe an environment that permits the desired activity. A non-limiting example includes contacting primers (and in some examples also a nucleic acid probe) with a nucleic acid molecule (such as one from an environmental sample) with reagents and temperature conditions sufficient for amplification of the target nucleic acid molecule in the sample. Another non-limiting example includes contacting a probe with a nucleic acid molecule (e.g., amplicons) under conditions sufficient for hybridization between the probe and a sequence the probe is complementary to.

[0075] Viable or Living: A viable or living organism is an organism that is alive. Conversely, a non-viable organism is a deceased organism.

[0076] III. Methods

[0077] Disclosed herein are methods of detecting a target organism from an environmental nucleic acid sample. In some aspects, the methods detect the presence of a living or deceased target organism. The methods include detecting a rapid kill RNA marker of the target organism in the environmental nucleic acid sample. The rapid kill RNA marker is low or not detectable when the target organism is deceased. In some aspects, the methods further include detecting a nuclear genomic DNA molecule of the target organism in the environmental nucleic acid sample. The genomic DNA molecule indicates presence of the target organism (dead or alive). The methods are useful, for example, for differentiating the presence of living or deceased organisms in the sampled environment. The nucleic acid sample can include, for example, RNA, genomic DNA, or both RNA and genomic DNA.

[0078] The target organism can be any organism of interest. In some aspects, the target organism is a pest or an invasive species. In some aspects, the target organism is a nematode. In some aspects, the target organism is Bursaphelenchus xylophilus (commonly known as pine wood nematode or pine wilt nematode).

[0079] The methods disclosed herein can include a step of obtaining a nucleic acid sample from an environment of interest. In such examples, the methods include, for example, (i) obtaining a nucleic acid sample from an environment (e.g., isolating or extracting nucleic acids from an environmental sample), thereby obtaining an environmental nucleic acid sample; and (ii) detecting a rapid kill RNA marker of the target organism in the environmental nucleic acid sample. An “environment” is the surroundings in which an organism lives or could be present. Environmental nucleic acid samples typically include nucleic acids that have been shed by a target organism (e.g., Bursaphelenchus xylophilus) in their environment, and may or may not also include tissues or cells from the target organism itself. In some aspects, an environmental nucleic acid sample does not include cells or tissue of the target organism. Environmental samples include, but are not limited to, water samples (fresh water, salt water, brackish water, etc.), soil samples, plant samples (e.g., wood samples), artificial substrates, or other samples from an environment of interest. In some aspects, the environment is one in which a target organism (e.g., Bursaphelenchus xylophilus) is present or is suspected of being present. In some aspects, the environmental sample is wood that is infested with, or suspected of being infested with, Bursaphelenchus xylophilus. In some aspects, the wood is pine. In some aspects, the wood is from a plant of the Pinus genus.

[0080] In some aspects, an environmental sample is collected (e.g., a piece of wood) and nucleic acids (e.g., RNA and / or DNA) are isolated or extracted from the environmental sample. The isolated or extracted nucleic acids can also be concentrated, for example, by precipitation, using a membrane filter that binds nucleic acids, or by using a nucleic acid purification column, which includes a substrate that binds nucleic acids. In some aspects, the environmental sample is processed to help remove PCR inhibitors or other contaminants (e.g., by using a nucleic acid column or clean-up kit, for example, NEB® Monarch® RNA Cleanup kit). Techniques for extracting, concentrating, and / or purifying nucleic acids (e.g., DNA and RNA) from environmental samples that are of use in the methods disclosed herein are known and a practitioner can readily select a suitable method. In some aspects, an environmental sample is passed through a membrane filter to remove contaminants and / or to capture nucleic acid molecules present in the sample.

[0081] In some aspects, a commercial kit is used to extract DNA and / or RNA from an environmental sample, for example, QIAgen® AllPrep® DNA / RNA kit (catalog no: 80204), QIAgen® RNeasy kit, QIAgen® DNeasy kit, Thermo Fisher® RiboPure® RNA extraction kit, Thermo Fisher® PureLink® RNA extraction kit, Thermo Fisher® MagMAX® nucleic acid extraction kit, Zymo Research® ZymoBIOMICS® kit (e.g., RNA miniprep kit, MagBead RNA kit DNA / RNA miniprep kit, or MagBead DNA / RNA kit) Zymo Research® Quick-RNA® kit (e.g., fecal / soil microbe microprep kit, fungal / bacterial miniprep or microprep kit, tissue / insect kit, DNA / RNA water kit, or plant kit), or other commercial kit.

[0082] In some aspects, nucleic acids are extracted using TRIzol® organic reagent. TRIzol® is a monophasic solution of phenol and guanidine isothiocyanate, which facilitates the breakdown of cells and the denaturation of proteins. When a sample is mixed with TRIzol®, cells are lysed, releasing cellular components into solution. The phenol and guanidine isothiocyanate in TRIzol® disrupt the cell membrane and denature proteins, protecting RNA from degradation by RNases. Following homogenization, chloroform is added, and the mixture is centrifuged, resulting in three distinct phases: an aqueous phase containing RNA, an interphase containing DNA, and an organic phase containing proteins and other cellular components. Thus, TRIzol® is useful for isolating RNA, DNA, and / or proteins from a single sample. RNA from the aqueous phase can be precipitated by mixing with isopropanol and collected via centrifugation. The resulting RNA pellet is typically washed with ethanol to remove impurities, dried, and redissolved in a RNase- and / or DNAase-free water or buffer for further use.

[0083] Following extraction or isolation, the nucleic acids are optionally further processed to remove impurities (e.g., RNA / DNA clean-up) and / or to concentrate the nucleic acids. In some aspects, isolated nucleic acids are cleaned and / or concentrated by precipitation with 70%-100% (e.g., 70%-95%, 75%-95%, 80%-95%, 85%-95%, 70%-98%, 75%-98%, 80%-98%, 85%-98%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, etc.) isopropanol followed by one or more pellet washes with 70-100% (e.g., 70%-95%, 75%-95%, 80%-95%, 85%-95%, 70%-98%, 75%-98%, 80%-98%, 85%-98%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, etc.) ethanol. The pellet is then allowed the dry and is redissolved in a solution (e.g., in RNase- and / or DNAase-free water or buffer). In other aspects, a nucleic acid purification column is used to remove impurities and / or concentrate the nucleic acids. Purification columns typically use a bind-wash-elute process based on the selective binding of nucleic acids to a substrate (e.g., a silica membrane) under certain conditions. During the binding step, buffer conditions cause nucleic acids to attach to the substrate, while other contaminants flow through and are washed away. A low-salt buffer is typically used to elute the purified nucleic acids from the substrate for collection. Other suitable known methods and commercial kits are also available for nucleic acid clean-up.

[0084] When the environmental nucleic acid sample includes RNA, the RNA can be converted into cDNA by reverse transcription. Methods of synthesizing cDNA from samples of RNA are known. In general, the RNA is mixed with primers (e.g., oligo(dT), random primers, or gene-specific), dNTPs, and reverse transcriptase, and run in a thermocycler under conditions that allow for primer annealing and reverse transcriptase activity. Following cDNA synthesis, the reverse transcriptase enzyme is inactivated (e.g., heat inactivated). In a non-limiting example, RNA is reverse transcribed into cDNA using a cDNA reverse transcription kit (Applied Biosystems® Inc., CAT No. 4374967). cDNA can also be further purified and / or concentrated as described above.

[0085] In some aspects, the methods include a quality control step that assays the integrity of the environmental nucleic acid sample. The quality control step can occur before, or simultaneously with, a step of detecting the rapid kill RNA marker or genomic DNA molecule. In some aspects, the assay is the ePlant5 assay, also known as IntegritE-DNA® (for genomic samples; detects genomic chloroplast DNA) or IntegritE-RNA™ (for cDNA samples; detects chloroplast RNA). IntegritE assays target endogenous chloroplast DNA in order to assess the presence of any residual PCR inhibitory compounds and to identify whether any samples are degraded or otherwise compromised. When a nucleic acid sample fails an IntegritE test, the sample is further processed to remove impurities (for example, using a purification / clean-up method described herein), and the IntegritE assay is performed again on the purified sample. IntegritE assays are described, for example, in US Patent No. 11,479,813. In brief, the environmental nucleic acid sample is contacted with a forward and reverse primer specific to endogenous algae or plant chloroplast DNA under conditions sufficient for amplification. If endogenous algae or plant chloroplast amplicons are detected, the sample passes quality control. If endogenous algae or plant chloroplast amplicons are not detected, then the environmental nucleic acid sample is purified / cleaned-up (e.g., precipitated and washed or run on a nucleic acid purification column), to remove potential PCR inhibitors, and the sample is then reanalyzed. If algae or plant chloroplast amplicons are detected in the cleaned-up sample, then sample passes quality control. If the algae or plant chloroplast amplicons are not detected in the cleaned-up sample, then sample is considered a false negative due to insufficient quality for amplification (e.g., degraded or low concentration of nucleic acids).

[0086] The methods disclosed herein include detecting a rapid kill RNA marker and / or genomic DNA molecule of the target organism. A rapid kill RNA marker and / or genomic DNA molecule can be detected by any suitable method, including but not limited to, a sequencing-based method (e.g., sanger sequencing, high-throughput sequencing, pyrosequencing, etc.), amplification-based method (e.g., PCR, qPCR, etc.), or hybridization-based method (e.g., Southern or Northern blotting). In some aspects, a qPCR method (e.g., SYBR® green or TaqMan®) is used to detect the rapid kill RNA marker and / or genomic DNA molecule of the target organism.

[0087] Examples of in vitro amplification techniques that can be used with the disclosed methods include real-time PCR, reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR), reverse transcriptase semi-nested PCR, strand displacement amplification (see, e.g., US Patent No. 5,744,311), transcription-free isothermal amplification (see, e.g., US Patent No. 6,033,881), repair chain reaction amplification (see, e.g., WO 90 / 01069), ligase chain reaction amplification (see, e.g., EP-A-320308), gap filling ligase chain reaction amplification (see, e.g., US Patent No. 5,427,930), coupled ligase detection and PCR (see, e.g., US Patent No. 6,027,889), and NASBA™ RNA transcription-free amplification (see, e.g., US Patent No. 6,025,134).

[0088] When an amplification-based method of detection is used, the methods disclosed herein can include a step of contacting the environmental nucleic acid sample with a forward and reverse primer specific to the rapid kill RNA marker or genomic DNA molecule; ii) amplifying the rapid kill RNA marker or genomic DNA molecule (e.g., PCR amplification), thereby producing rapid kill RNA marker or genomic DNA amplicons, and iii) detecting the amplicons, thereby detecting the rapid kill RNA marker or genomic DNA molecule. In some aspects, a probe complementary to a target rapid kill RNA marker and / or genomic DNA molecule is used to facilitate detection. Probes are typically 10 to 50 nucleotides in length, for example, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 12-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 18-50, 18-45, 18-35, 18-30, 18-25, 18-20, 20-50, 20-45, 20-40, 20-35, or 20-35 nucleotides. In some aspects, the probe is a fluorescent probe (e.g., a fluorophore is attached to the probe). In some aspects, the probe is a TaqMan® qPCR probe. TaqMan® probes utilize a dual-labeled format, with a fluorophore at the 5' end and a quencher at the 3' end. During amplification, the probe is cleaved by the DNA polymerase, resulting in the separation of the fluorophore and quencher, leading to a fluorescent signal. The signal is directly proportional to the amount of target nucleic acid present in the sample, allowing for quantification of the target nucleic acid.

[0089] In some aspects, an amplification-based method includes contacting (e.g., incubating) the environmental nucleic acid sample with a forward and reverse primer set specific to the rapid kill RNA marker or genomic DNA molecule, as well as other reagents necessary for amplification (e.g., polymerase, dNTPs, fluorescent dye or probe, buffer, etc.). The resulting mixture is incubated under conditions sufficient for amplification of the rapid kill RNA marker or genomic DNA molecule, thereby generating amplicons. In some examples, at least 16 (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, etc.) amplification cycles are performed. In some examples, 16-75 (e.g., 16-70, 16-60, 16-50, 16-45, 16-40, 20-75, 20-70, 20-60, 20-50, 20-45, 20-40, 25-75, 25-70, 25-60, 25-50, 25-45, 25-40, 30-70, 30-60, 30-50, 30-40, 40-70, 40-60, 40-50, etc.) amplification cycles are performed. Resulting amplicons are determined or measured, and in some examples quantified. The presence of amplicons indicates the presence of the rapid kill RNA marker or genomic DNA molecule in the environmental nucleic acid sample. In some aspects, determining or measuring the amplicons includes contacting the amplicons with a labeled nucleic acid probe complementary to the rapid kill RNA marker or genomic DNA molecule. In a non-limiting example, quantitative real-time PCR (qPCR) reagents and reaction conditions are used.

[0090] The rapid kill RNA marker is an RNA molecule that is expressed or abundant when the target organism is alive, but rapidly degrades (e.g., within 30 minutes) when the target organism is deceased or is otherwise exposed to conditions the organism would not survive. In some aspects, a rapid kill RNA marker is not detectable within 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours of an organism becoming deceased. In some aspects, a rapid kill RNA marker is not detectable within 10 minutes of an organism becoming deceased. In some aspects, a rapid kill RNA marker is not detectable within 5 minutes of an organism becoming deceased. In some aspects, a rapid kill RNA marker is not detectable within 1 minute to 12 hours of an organism becoming deceased, for example, 1 minute to 6 hours, 1 minute to 2 hours, 1 minute to 1 hour, 1 minute to 30 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 10 minutes to 12 hours, 10 minutes to 6 hours, 10 minutes to 2 hours, 10 minutes to 1 hour, 10 minutes to 30 minutes, 10 minutes to 15 minutes, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, 1 hour to 12 hours, 1 hour to 6 hours, 1 hour to 2 hours, 2 hours to 12 hours, 2 hours to 6 hours, 4 hours to 12 hours, 4 hours to 6 hours, or 6 hours to 12 hours of an organism becoming deceased. In some aspects, the rapid kill marker is not detectable immediately after applying a successful kill treatment (e.g., exposure to a temperature of at least 56°C for at least 30 minutes).

[0091] In some aspects, the kill treatment is exposure to at least 50°C, for example, at least 52°C, at least 54°C, at least 56°C, at least 58°C, at least 60°C, at least 62°C, at least 64°C, at least 66°C, at least 68°C, at least 70°C, at least 80°C, or higher temperature for at least 10 minutes, for example, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 60 minutes, or longer. In some aspects, the kill treatment is exposure to about 56°C for about 30 minutes. In some aspects, the kill treatment is exposure to at least 56°C for at least 30 minutes. In some aspects, the kill treatment is exposure to 56-66°C for 30-60 minutes.

[0092] In some aspects, detection of the rapid kill RNA marker indicates that the target organism is alive. In some aspects, no detection of the rapid kill RNA marker indicates that the target organism is deceased.

[0093] In some aspects, the rapid kill RNA marker is low or not detectable in the environmental nucleic acid sample when the target organism is deceased. A rapid kill RNA marker is “low” when the expression level of the rapid kill RNA marker is decreased relative to the expression of the rapid kill RNA marker when the organism is alive. Expression level can be determined, for example, by a method that quantifies nucleic acids in a sample (e.g., qPCR or quantitative RNA-seq) or by comparing a signal that correlates to nucleic acid abundance in a sample (e.g., fluorescent or chemiluminescent signal). In some aspects, the rapid kill RNA marker is low when expression of the rapid kill RNA marker is 50% or less (e.g., 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.05%, or less) relative to expression of the rapid kill RNA marker when the target organism is alive.

[0094] In some aspects, the methods further include determining a relative viability index. The relative viability index is a ratio of the relative rapid kill RNA marker relative to the genomic DNA molecule. In some aspects, amplicons of the rapid kill RNA marker and genomic DNA molecule are quantified (e.g., using qPCR), and an expression ratio is calculated (the relative viability index). A cut-off value distinguishing “living” samples from “deceased” samples is set. Comparison of the relative viability index to the established cut-off value determines whether the target organism is living or decreased. In some aspects, the cut-off value for the relative viability index is (rapid kill RNA marker / genomic DNA molecule) 0.5, 0.25, 0.1, 0.075, 0.05, 0.025, 0.01, 0.0075, 0.005, 0.0025, 0.001, etc. wherein when the ratio is higher (e.g., 1.0) then the organism is determined to be alive and when the ratio is at or below the cut-off value, the organism is determined to be deceased. In some aspects, the cut-off value is a value at least 70% accurate for determining a “living” organism, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% accurate. In some aspects, the cut-off value is determined by processing control samples of known status (e.g., it is known whether the target organism is alive or deceased).

[0095] Non-limiting examples of rapid kill RNA markers for B. xylophilus include sequences specific to mRNA of contig 280 or contig 3172 (see, e.g., SEQ ID NOS: 34 and 33, respectively) of B. xylophilus.

[0096] In some aspects, the rapid kill RNA marker is a sequence specific to mRNA of contig 280. In some aspects, the rapid kill RNA marker is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 8, 12, 16, or 20. In some aspects, the rapid kill RNA marker includes a sequence at least 95% identical to any one of SEQ ID NOS: 8, 12, 16, or 20. In some aspects, the rapid kill RNA marker includes a sequence at least 98% identical to any one of SEQ ID NOS: 8, 12, 16, or 20. In some aspects, the rapid kill RNA marker includes or consists of any one of SEQ ID NOS: 8, 12, 16, or 20. In some aspects, the rapid kill RNA marker includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In some aspects, the rapid kill RNA marker includes a sequence at least 95% identical to SEQ ID NO: 8. In some aspects, the rapid kill RNA marker includes a sequence at least 98% identical to SEQ ID NO: 8. In some aspects, the rapid kill RNA marker includes or consists of SEQ ID NO: 8.

[0097] In some aspects, the rapid kill RNA marker is a sequence specific to mRNA of contig 3172. In some aspects, the rapid kill RNA marker includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 28. In some aspects, the rapid kill RNA marker includes a sequence at least 95% identical to SEQ ID NO: 28. In some aspects, the rapid kill RNA marker includes a sequence at least 98% identical to SEQ ID NO: 28. In some aspects, the rapid kill RNA marker includes or consists of SEQ ID NO: 28.

[0098] In some aspects, the methods disclosed herein include contacting the environmental nucleic acid sample with a forward and reverse primer specific to the rapid kill RNA marker. In some aspects, the forward or reverse primer is about 10 to 50 contiguous nucleotides, for example about 12 to 50 nucleotides, 15 to 40 nucleotides, 15 to 30 nucleotides, 12 to 40 nucleotides, 18 to 35 nucleotides, 18 to 30 nucleotides, 18 to 25 nucleotides 19 to 30 nucleotides, 19 to 29 nucleotides, or 20 to 29 nucleotides.

[0099] In some aspects, the forward primer includes a sequence at least 90% e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 5, 9, 13, 17, or 25. In some aspects, the forward primer includes a sequence at least 95% identical to any one of SEQ ID NOS: 5, 9, 13, 17, or 25. In some aspects, the forward primer includes any one of SEQ ID NOS: 5, 9, 13, 17, or 25. In some aspects, the reverse primer includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 6, 10, 14, 18, or 26. In some aspects, the reverse primer includes a sequence at least 95% identical to any one of SEQ ID NO: 6, 10, 14, 18, or 26. In some aspects, the reverse primer includes any one of SEQ ID NOS: 6, 10, 14, 18, or 26.

[0100] In some aspects, the forward and reverse primers include a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to (i) SEQ ID NOS: 5 and / or 6; (ii) SEQ ID NOS: 9 and / or 10; (iii) SEQ ID NOS: 13 and / or 14; (iv) SEQ ID NOS: 17 and / or 18; or (v) SEQ ID NOS: 25 and / or 26, respectively. In some aspects, the forward and reverse primers include (i) SEQ ID NOS: 5 and / or 6; (ii) SEQ ID NOS: 9 and / or 10; (iii) SEQ ID NOS: 13 and / or 14; (iv) SEQ ID NOS: 17 and / or 18; or (v) SEQ ID NOS: 25 and / or 26, respectively. In some aspects, the forward and reverse primers include or consist of (i) SEQ ID NOS: 5 and 6; (ii) SEQ ID NOS: 9 and 10; (iii) SEQ ID NOS: 13 and 14; (iv) SEQ ID NOS: 17 and 18; or (v) SEQ ID NOS: 25 and / or 26, respectively. In some aspects, the forward and reverse primers include or consist of SEQ ID NOS: 17 and 18, respectively.

[0101] In some aspects, the methods further include using a probe to detect rapid kill RNA marker amplicons. Probes are typically 10-50 nucleotides in length. In some aspects, the probe is 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 12-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 18-50, 18-45, 18-35, 18-30, 18-25, 18-20, 20-50, 20-45, 20-40, 20-35, or 20-35 nucleotides in length. In some aspects, the probe includes at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 8, 12, 16, 20, or 28. In some aspects, the probe includes at least 95% sequence identity to any one of SEQ ID NOS: 8, 12, 16, 20, or 28. In some aspects, the probe includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 7, 11, 15, 19, or 27. In some aspects, the probe includes or consists of any one of SEQ ID NOS: 7, 11, 15, 19, or 27.

[0102] In some aspects, the methods include using the following primer and probe combinations:

[0103] a) a forward primer comprising or consisting of SEQ ID NO: 5, a reverse primer comprising or consisting of SEQ ID NO: 6, and a probe comprising or consisting of SEQ ID NO: 7;

[0104] b) a forward primer comprising or consisting of SEQ ID NO: 9, a reverse primer comprising or consisting of SEQ ID NO: 10, and a probe comprising or consisting of SEQ ID NO: 11;

[0105] c) a forward primer comprising or consisting of SEQ ID NO: 13, a reverse primer comprising or consisting of SEQ ID NO: 14, and a probe comprising or consisting of SEQ ID NO: 15; d) a forward primer comprising or consisting of SEQ ID NO: 17, a reverse primer comprising or consisting of SEQ ID NO: 18, and a probe comprising or consisting of SEQ ID NO: 19; or

[0106] e) a forward primer comprising or consisting of SEQ ID NO: 25, a reverse primer comprising or consisting of SEQ ID NO: 26, and a probe comprising or consisting of SEQ ID NO: 27.

[0107] The methods disclosed herein can include detecting a genomic DNA sequence. In some aspects, the genomic DNA sequence is a genomic DNA sequence of B. xylophilus, for example, a sequence specific to genomic DNA of contig 280 or contig 3172 (see, e.g., SEQ ID NO: 29 and 30, respectively). In some aspects, the genomic DNA sequence is specific to genomic DNA of contig 280. In some aspects, the genomic DNA molecule is a control. In some aspects, detection of the genomic DNA molecule indicates presence of an alive or deceased target organism.

[0108] In some aspects, the genomic DNA molecule includes a sequence at least 90% e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. In some aspects, the genomic DNA molecule includes a sequence at least 95% identical to SEQ ID NO: 4. In some aspects, the genomic DNA molecule includes a sequence at least 98% identical to SEQ ID NO: 4. In some aspects, the genomic DNA molecule includes or consists of SEQ ID NO: 4. In some aspects, the genomic DNA sequence is specific to genomic DNA of contig 3172. In some aspects, the genomic DNA molecule includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 24. In some aspects, the genomic DNA molecule includes a sequence at least 95% identical to SEQ ID NO: 24. In some aspects, the genomic DNA molecule includes a sequence at least 98% identical to SEQ ID NO: 24. In some aspects, the genomic DNA molecule includes or consists of SEQ ID NO: 24.

[0109] In some aspects, the methods disclosed herein include contacting the environmental nucleic acid sample with a forward and reverse primer specific to the genomic DNA molecule. In some aspects, the forward or reverse primer for detecting the genomic DNA sequence is about 10 to 50 contiguous nucleotides, for example about 12 to 50 nucleotides, 15 to 40 nucleotides, 15 to 30 nucleotides, 12 to 40 nucleotides, 18 to 35 nucleotides, 18 to 30 nucleotides, 18 to 25 nucleotides, 19 to 30 nucleotides, 19 to 29 nucleotides, or 20 to 29 nucleotides.

[0110] In some aspects, the forward primer for detecting the genomic DNA molecule includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 1 or 21. In some aspects, the forward primer includes a sequence at least 95% identical to any one of SEQ ID NOS: 1 or 21. In some aspects, the forward primer includes or consists of any one of SEQ ID NOS: 1 or 21. In some aspects, the reverse primer for detecting the genomic DNA molecule includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 2 or 22. In some aspects, the reverse primer includes a sequence at least 95% identical to any one of SEQ ID NOS: 2 or 22. In some aspects, the reverse primer includes or consists of any one of SEQ ID NOS: 2 or 22.

[0111] In some aspects, the forward and reverse primers for detecting the genomic DNA molecule include a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to (i) SEQ ID NOS: 1 and / or 2; or (ii) SEQ ID NOS: 21 and / or 22, respectively. In some aspects, the forward and reverse primer include (i) SEQ ID NOS: 1 and / or 2; or (ii) SEQ ID NOS: 21 and / or 22, respectively. In some aspects, the forward and reverse primer include or consist of (i) SEQ ID NOS: 1 and 2; or (ii) SEQ ID NOS: 21 and 22, respectively. In some aspects, the forward and reverse primers include or consist of SEQ ID NOS: 1 and 2.

[0112] In some aspects, the methods further include using a probe to detect genomic DNA molecule amplicons. In some aspects, the probe is 10-50 contiguous nucleotides, for example, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 12-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 18-50, 18-45, 18-35, 18-30, 18-25, 18-20, 20-50, 20-45, 20-40, 20-35, or 20-35 nucleotides. In some aspects, the probe includes at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 4 or 24. In some aspects, the probe includes at least 95% sequence identity to any one of SEQ ID NOS: 4 or 24. In some aspects, the probe includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 3 or 23. In some aspects, the probe includes or consists of any one of SEQ ID NOS: 3 or 23.

[0113] In some aspects, the methods include using the following primer and probe combinations for detecting the genomic DNA molecule:

[0114] a) a forward primer comprising or consisting of SEQ ID NO: 1, a reverse primer comprising or consisting of SEQ ID NO: 2, and a probe comprising or consisting of SEQ ID NO: 3; or

[0115] b) a forward primer comprising or consisting of SEQ ID NO: 21, a reverse primer comprising or consisting of SEQ ID NO: 22, and a probe comprising or consisting of SEQ ID NO: 23.

[0116] In some examples, more than one rapid kill RNA marker and / or genomic DNA molecule are detected in the sample, for example, at least two (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some aspects, one rapid kill RNA marker and one genomic DNA molecule are detected in the environmental nucleic acid sample. In some aspects, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) rapid kill RNA marker and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) genomic DNA molecule are detected in the environmental nucleic acid sample.

[0117] Sequence variants of any primer or probe disclosed herein are also provided. Sequence variants include, for example, sequences having one or a few nucleotide variations (e.g., up to 2, up to 3, up to 4, or up to 5 nucleotide variations) that do not significantly affect the ability of the primer or probe to function as intended (e.g., amplify the rapid kill RNA marker or genomic DNA sequence).

[0118] Any of the primers or probes disclosed herein can include modifications, for example, one or more labels, which can be used to detect amplification of a nucleic acid molecule, a resulting amplicon, or both. In some aspects, the label is covalently attached to the primer or probe. In some examples, the disclosed primers and / or probes include a fluorophore. Exemplary fluorophores that could be used with the primers and probes disclosed herein are provided, for example, in U. S. Patent No. 5,866,366 to Nazarenko et al., for example, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, Yakima Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QF1TC (XRITC); fluorescamine; IR144; IR1446;

[0119] Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B -phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron. RTM. Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N, N, N', N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other fluorophores known to those skilled in the art can also be used, for example those available from Molecular Probes (Eugene, OR).

[0120] In some aspects, the fluorophore is a xanthene dye (e.g., fluorescein (e.g., carboxyfluorescein, hexachlorofluorescein (HEX), tetrachlorofluorescein), rhodamine etc.), NED™ NHS ester dye, ABY™ dye, JUN™ dye, etc. In some aspects, the fluorophore is covalently attached to the 5’-end of the probe (e.g., 6-FAM (6-carboxyfluorescein), 6-TET (6-tetrachlorofluorescein), 6- VIC, 6-HEX (6-hexachlorofluorescein), 6-NED, and 6-PET). In some aspects, the probes include a quencher (e.g., nonfluorescent quencher-minor groove binder (MGB-NFQ), QSY® fluorescein analog quencher, black hole quencher 1 (BHQ1), or tetramethylrhodamine (TAMRA)), for example, at the 3’-end of the probe.

[0121] Any of the primers or probes disclosed herein can be, for example, DNA, RNA, XNAs (xenonucleic acids), or chimeras thereof. In some aspects, a primer or probe disclosed herein is DNA. The primers or probes disclosed herein can also include modified bases, for example, locked nucleic acids (LNAs), 2’-O-methoxy-ethyl modified bases (2’ -MOE), 2'-O-methyl RNA bases, 2' fluoro bases (which have a fluorine modified ribose to increase binding affinity (Tm)), puromycin, 8-oxo dG, N6-methyl-2'-deoxyadenosine, 5-bromo-deoxyuridine, deoxyuridine, 2,6-diaminopurine (2-Amino-dA), Dideoxycytidine (ddC), deoxyinosine, hydroxymethyl dC, inverted dT, iso-dG, iso-dC, inverted dideoxy-T, 5-methyl deoxycytidine, 5 -nitro indole, 5-hydroxybutynl -2’ -deoxyuridine, or 8-aza-7-deazaguanosine.

[0122] Primer pairs and / or probes can be multiplexed such that the identification of one target nucleic acid molecule occurs simultaneously or contemporaneously with one or more other targets. In some aspects, detection of 1-10 (e.g., 1-8, 1-6, 1-4, 1-3, 1-2, 2-10, 2-8, 2-6, 2-4, 4-10, 4-8, or 4-6) RNA molecules (e.g., rapid kill RNA markers or IntegritE-RNA™ target) is performed in a single sample. In some aspects, detection of 2-6 RNA molecules is performed in a single sample. In some aspects, detection of 2-4 RNA molecules is performed in a single sample. In some aspects, detection of 1-10 (e.g., 1-8, 1-6, 1-4, 1-3, 1-2, 2-10, 2-8, 2-6, 2-4, 4-10, 4-8, or 4-6) genomic molecules (e.g., nuclear genomic DNA molecules or IntegritE-DNA® targets) is performed in a single sample. In some aspects, detection of 2-6 genomic molecules is performed in a single sample. In some aspects, detection of 2-4 genomic molecules is performed in a single sample.

[0123] IV. Kits

[0124] Also provided are kits including a forward and reverse primer specific to a rapid kill RNA marker, for example, a forward and reverse primer that amplify a sequence that is specific to mRNA of contig 280 or contig 3172 of B. xylophilus. In some aspects, the forward or reverse primer is about 10 to 50 nucleotides, for example about 12 to 50 nucleotides, 15 to 40 nucleotides, 15 to 30 nucleotides, 12 to 40 nucleotides, 18 to 35 nucleotides, 18 to 30 nucleotides, 18 to 25 nucleotides 19 to 30 nucleotides, 19 to 29 nucleotides, or 20 to 29 nucleotides. In some aspects, the forward and reverse primer is a primer set disclosed herein (see, e.g., Table 3).

[0125] In some aspects, the forward primer includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 5, 9, 13, 17, or 25. In some aspects, the forward primer includes a sequence at least 95% identical to any one of SEQ ID NOS: 5, 9, 13, 17, or 25. In some aspects, the forward primer includes any one of SEQ ID NOS: 5, 9, 13, 17, or 25. In some aspects, the reverse primer includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 6, 10, 14, 18, or 26. In some aspects, the reverse primer includes a sequence at least 95% identical to any one of SEQ ID NO: 6, 10, 14, 18, or 26. In some aspects, the reverse primer includes any one of SEQ ID NOS: 6, 10, 14, 18, or 26.

[0126] In some aspects, the forward and reverse primers include a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to (i) SEQ ID NOS: 5 and / or 6; (ii) SEQ ID NOS: 9 and / or 10; (iii) SEQ ID NOS: 13 and / or 14; (iv) SEQ ID NOS: 17 and / or 18; or (v) SEQ ID NOS: 25 and / or 26, respectively. In some aspects, the forward and reverse primers include (i) SEQ ID NOS: 5 and / or 6; (ii) SEQ ID NOS: 9 and / or 10; (iii) SEQ ID NOS: 13 and / or 14; (iv) SEQ ID NOS: 17 and / or 18; or (v) SEQ ID NOS: 25 and / or 26, respectively. In some aspects, the forward and reverse primers include or consist of (i) SEQ ID NOS: 5 and 6; (ii) SEQ ID NOS: 9 and 10; (iii) SEQ ID NOS: 13 and 14; (iv) SEQ ID NOS: 17 and 18; or (v) SEQ ID NOS: 25 and / or 26, respectively. In some aspects, the forward and reverse primers include or consist of SEQ ID NOS: 17 and 18, respectively.

[0127] In some aspects, the kit further includes a probe for detecting rapid kill RNA marker amplicons. In some aspects, the probe is 10-50 nucleotides, for example, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 12-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 18-50, 18-45, 18-35, 18-30, 18-25, 18-20, 20-50, 20-45, 20-40, 20-35, or 20-35 nucleotides. In some aspects, the probe includes at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 8, 12, 16, 20, or 28. In some aspects, the probe includes at least 95% sequence identity to any one of SEQ ID NOS: 8, 12, 16, 20, or 28. In some aspects, the probe includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOS: 7, 11, 15, 19, or 27. In some aspects, the probe includes or consists of any one of SEQ ID NOS: 7, 11, 15, 19, or 27.

[0128] In some aspects, the kit includes one or more of the following primer / probe combinations:

[0129] a) a forward primer comprising or consisting of SEQ ID NO: 5, a reverse primer comprising or consisting of SEQ ID NO: 6, and a probe comprising or consisting of SEQ ID NO: 7;

[0130] b) a forward primer comprising or consisting of SEQ ID NO: 9, a reverse primer comprising or consisting of SEQ ID NO: 10, and a probe comprising or consisting of SEQ ID NO: 11;

[0131] c) a forward primer comprising or consisting of SEQ ID NO: 13, a reverse primer comprising or consisting of SEQ ID NO: 14, and a probe comprising or consisting of SEQ ID NO: 15;

[0132] d) a forward primer comprising or consisting of SEQ ID NO: 17, a reverse primer comprising or consisting of SEQ ID NO: 18, and a probe comprising or consisting of SEQ ID NO: 19; or

[0133] e) a forward primer comprising or consisting of SEQ ID NO: 25, a reverse primer comprising or consisting of SEQ ID NO: 26, and a probe comprising or consisting of SEQ ID NO: 27.

[0134] The kits disclosed herein can further include a forward and reverse primer for detecting a genomic DNA sequence. In some aspects, the genomic DNA sequence is a genomic DNA sequence of B. xylophilus, for example, a sequence specific to genomic DNA of contig 280 or contig 3172 (see, e.g., SEQ ID NO: 29 and 30, respectively). In some aspects, the genomic DNA sequence is specific to genomic DNA of contig 280 or contig 3172. In some aspects, the genomic DNA molecule includes a sequence at least 90% e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4 or 24. In some aspects, the genomic DNA molecule includes a sequence at least 95% identical to SEQ ID NO: 4 or 24. In some aspects, the genomic DNA molecule includes a sequence at least 98% identical to SEQ ID NO: 4 or 24. In some aspects, the genomic DNA molecule includes or consists of SEQ ID NO: 4 or 24.

[0135] In some aspects, the forward or reverse primer for detecting the genomic DNA sequence is about 10-50 nucleotides, for example about 12 to 50 nucleotides, 15 to 40 nucleotides, 15 to 30 nucleotides, 12 to 40 nucleotides, 18 to 35 nucleotides, 18 to 30 nucleotides, 18 to 25 nucleotides 19 to 30 nucleotides, 19 to 29 nucleotides, or 20 to 29 nucleotides. In some aspects, the forward and reverse primer for detecting the genomic DNA sequence is a primer set disclosed herein (see, e.g., Table 3). In some aspects, the forward primer for detecting the genomic DNA sequence includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1 or 21. In some aspects, the forward primer for detecting the genomic DNA sequence includes a sequence at least 95% identical to SEQ ID NO: 1 or 21. In some aspects, the forward primer for detecting the genomic DNA sequence includes or consists of SEQ ID NO: 1 or 21. In some aspects, the reverse primer for detecting the genomic DNA sequence includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2 or 22. In some aspects, the reverse primer for detecting the genomic DNA sequence includes a sequence at least 95% identical to SEQ ID NO: 2 or 22. In some aspects, the reverse primer for detecting the genomic DNA sequence includes or consists of SEQ ID NO: 2 or 22.

[0136] In some aspects, the forward and reverse primers specific to the genomic DNA molecule include a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to (i) SEQ ID NOS: 1 and / or 2; or (ii) SEQ ID NOS: 21 and / or 22, respectively. In some aspects, the forward and reverse primer include (i) SEQ ID NOS: 1 and / or 2; or (ii) SEQ ID NOS: 21 and / or 22, respectively. In some aspects, the forward and reverse primer include or consist of (i) SEQ ID NOS: 1 and 2; or (ii) SEQ ID NOS: 21 and 22, respectively. In some aspects, the forward and reverse primers include or consist of SEQ ID NOS: 1 and 2.

[0137] The kits disclosed herein can further include a probe for detecting genomic DNA molecule amplicons. In some aspects, the probe includes at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 4 or 24. In some aspects, the probe includes at least 95% sequence identity to SEQ ID NO: 4 or 24. In some aspects, the probe includes a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3 or 23. In some aspects, the probe includes a sequence at least 95% identical to SEQ ID NO: 3 or 23. In some aspects, the probe includes or consists of SEQ ID NO: 3 or 23.

[0138] In some aspects, the methods include using the following primer and probe combinations for detecting the genomic DNA molecule:

[0139] a) a forward primer comprising or consisting of SEQ ID NO: 1, a reverse primer comprising or consisting of SEQ ID NO: 2, and a probe comprising or consisting of SEQ ID NO: 3; or

[0140] b) a forward primer comprising or consisting of SEQ ID NO: 21, a reverse primer comprising or consisting of SEQ ID NO: 22, and a probe comprising or consisting of SEQ ID NO: 23.

[0141] The kits can further include, for example, one or more containers for holding or collecting a sample, one or more nucleic acid isolation reagents, one or more qPCR reagents, one or more filters, one or more DNA or RNA purification columns, or combinations thereof.

[0142] Exemplary nucleic acid isolation reagents include, but are not limited to, lysis buffers, proteinases, RNases, DNases, isopropanol, ethanol, phenol, chloroform, TRIzol® organic acid-guanidinium-phenol based reagent, silica substrates, magnetic beads, buffers, RNase and / or DNase-free water or buffer, etc. Exemplary qPCR reagents include, but are not limited to, polymerases, dNTPs (nucleotides), probes, primers, fluorescent dyes, buffers, etc. Exemplary filters include, but are not limited to, glass fiber filters, surface-modified mesh filters, silica-based filters, or other chemically-treated filter papers. In some aspects, the filter binds nucleic acids.

[0143] V. Clauses

[0144] Clause 1. A method of detecting a living target organism from an environmental nucleic acid sample, comprising: detecting a rapid kill RNA marker of the target organism, wherein the rapid kill RNA marker is low or not detectable when the target organism is deceased, thereby detecting the living target organism. Clause 2. A method of detecting a living target organism in an environment, comprising: obtaining a nucleic acid sample from the environment; and detecting a rapid kill RNA marker of the target organism; wherein the rapid kill RNA marker is low or not detectable when the target organism is deceased, thereby detecting the living target organism.

[0145] Clause 3. The method of any one of the prior clauses, wherein the method further comprises detecting a genomic DNA molecule of the target organism.

[0146] Clause 4. The method of any one of the prior clauses, wherein the nucleic acid sample comprises RNA, genomic DNA, or both RNA and genomic DNA.

[0147] Clause 5. The method of any one of the prior clauses, wherein the nucleic acid sample is prepared from an environmental sample.

[0148] Clause 6. The method of any one of the prior clauses, wherein the environmental sample is wood.

[0149] Clause 7. The method of any one of the prior clauses, wherein the organism is a pest or invasive species. Clause 8. The method of any one of the prior clauses, wherein the organism is Bursaphelenchus xylophilus. Clause 9. The method of any one of the prior clauses, wherein the environmental sample is wood infested with or suspected of being infested with Bursaphelenchus xylophilus.

[0150] Clause 10. The method of any one of the prior clauses, wherein the rapid kill RNA marker is low or not detectable following a heat treatment of at least 56°C for at least 30 mins.

[0151] Clause 11. The method of any one of the prior clauses, wherein detecting the genomic DNA molecule and / or detecting the rapid kill RNA marker comprises: i) contacting the nucleic acid sample with a forward and reverse primer specific to the genomic DNA molecule or the rapid kill RNA marker; ii) amplifying the genomic DNA molecule or the rapid kill RNA marker, thereby producing amplicons, and iii) detecting the amplicons, thereby detecting the genomic DNA molecule or the rapid kill RNA marker.

[0152] Clause 12. The method of clause 11, wherein the forward and / or reverse primer comprise a primer sequence disclosed in Table 3.

[0153] Clause 13. The method of any one of the prior clauses, wherein detecting the genomic DNA molecule and / or the rapid kill RNA marker comprises detection by a molecular probe, optionally wherein the probe comprises a probe sequence disclosed in Table 3.

[0154] Clause 14. The method of any one of the prior clauses, wherein the rapid kill RNA marker is contig 280 or contig 3172 of Bursaphelenchus xylophilus. Clause 15. The method of any one of the prior clauses, wherein the rapid kill RNA marker comprises at least 95% sequence identity to SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 33, or 34.

[0155] Clause 16. The method of any one of the prior clauses, wherein the rapid kill RNA marker comprises SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 33, or 34.

[0156] Clause 17. A kit comprising a forward and reverse primer specific to a rapid kill RNA marker.

[0157] Clause 18. The kit of clause 17, comprising: a) a forward primer comprising SEQ ID NO: 1 and a reverse primer comprising SEQ ID NO: 2; b) a forward primer comprising SEQ ID NO: 5 and a reverse primer comprising SEQ ID NO: 6; c) a forward primer comprising SEQ ID NO: 9 and a reverse primer comprising SEQ ID NO: 10; d) a forward primer comprising SEQ ID NO: 13 and a reverse primer comprising SEQ ID NO: 14; e) a forward primer comprising SEQ ID NO: 17 and a reverse primer comprising SEQ ID NO: 18; f) a forward primer comprising SEQ ID NO: 21 and a reverse primer comprising SEQ ID NO: 22; or g) a forward primer comprising SEQ ID NO: 25 and a reverse primer comprising SEQ ID NO: 26.

[0158] Clause 19. The kit of clause 17 or 18, further comprising a probe.

[0159] Clause 20. The kit of clause 19, wherein the kit comprises: a) a forward primer comprising SEQ ID NO: 1, a reverse primer comprising SEQ ID NO: 2, and a probe comprising SEQ ID NO: 3; b) a forward primer comprising SEQ ID NO: 5, a reverse primer comprising SEQ ID NO: 6, and a probe comprising SEQ ID NO: 7; c) a forward primer comprising SEQ ID NO: 9, a reverse primer comprising SEQ ID NO: 10, and a probe comprising SEQ ID NO: 11; d) a forward primer comprising SEQ ID NO: 13, a reverse primer comprising SEQ ID NO: 14, and a probe comprising SEQ ID NO: 15; e) a forward primer comprising SEQ ID NO: 17, a reverse primer comprising SEQ ID NO: 18, and a probe comprising SEQ ID NO: 19; f) a forward primer comprising SEQ ID NO: 21, a reverse primer comprising SEQ ID NO: 22, and a probe comprising SEQ ID NO: 23; or g) a forward primer comprising SEQ ID NO: 25, a reverse primer comprising SEQ ID NO: 26, and a probe comprising SEQ ID NO: 27.

[0160] Clause 21. The kit of any one of clauses 17-20, further comprising: a container for holding or collecting a sample; reagents for isolating DNA; reagents for qPCR; a filter; or combinations thereof.

[0161] EXAMPLES

[0162] The following examples are provided to illustrate particular features or aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0163] EXAMPLE 1

[0164] Materials and Methods

[0165] Nematode Isolates. Five isolates of B. xylophilus, two isolates of B. mucronatus and five isolates of other Bursaphelenchus species from various locations were used (Table 1). Isolates were grown on a lawn of Botrytis cinerea cultured on 2% malt extract agar plates at room temperature for 1 month before subculturing. One percent MEA plates were inoculated with Botrytis cinerea and incubated at 24°C until the Botrytis fully covered the plate. The PWN isolate was then transferred on to the plates with sterile distilled water. One ml of sterile water was pipetted onto the lid of the plate with active growing PWN. The PWN adhering to the lid were collected in the water. The water / PWN slurry was then pipetted onto two Botrytis plates (0.5ml each). The plates were sealed with Parafilm and placed in the incubator for 1-2 weeks. Once the water has been absorbed onto the plate the plate was stored upside down to aid in PWN collection.

[0166] Table 1. PWN and other isolates and origins

[0167]

[0168] PWN Stage Collection and Heat Treatment Regimes. To generate the required RNA and gDNA samples, PWN strain Q14 was used. Eggs, Jill, and adult stages were subjected to four different temperature treatment regimes with five biological replicates each (Table 2). The highest temperature is the kill temperature and time currently used for phytosanitary measures.

[0169] Table 2. Number of biological replicates per treatment

[0170] regime.

[0171]

[0172]

[0173] Fifty grams unhulled barley grains were placed in an Erlenmeyer flask containing 100 mL water. A foam stopper was placed in the flask opening and covered with aluminum foil. The flask was autoclaved for 40 minutes and then allowed to cool in a laminar flow hood. Once cooled the grains were inoculated with Botrytis cinerea and incubated for 7-14 days. When the grains were fully covered with Botrytis, the inoculation water / PWN slurry, as described above, was added to the flask. All inoculations were done under sterile conditions. The grains were incubated at 24°C for at least two weeks until the sides of the flasks were visibly covered with PWN and the white fluffy Botrytis had all been consumed. This method yielded approximately 20 pg of the desired stage PWN for extraction after approximately four weeks. Details are below.

[0174] Egg Stage

[0175] Vigorously growing PWN cultures were collected from grains. Sterile distilled water was applied to the grains and the slurry was strained to remove grains and collected in a 50 mL centrifuge tube. Cultures grown on grains had a much higher number of PWN present, but also more debris.

[0176] The tubes were centrifuged at 1,000 rpm for 2 minutes to help separate out some debris, the water and debris was decanted off and more distilled water added. This was repeated until nematodes did not centrifuge out and stayed in solution. This process was repeated until the water was clear after centrifuging. The nematodes were allowed to settle for 5 minutes and the water was decanted off.

[0177] To estimate the amount of PWN collected, the clump of PWN at the bottom of the centrifuge tube into a weighed 1.5 mL tube and centrifuged at 14,000 rpm for 2 minutes. The excess water was removed, and the tube weighed to determine PWN weight.

[0178] The water bath was preheated to the desired temperature. Six glass chamber plates were then cleaned and sterilized (FIG. 2). 150 pg PWN was collected in 3 mL water. PWN solution (100 pL) was added to each sample chamber. Sterile water (200 pL) was added and the chambers were placed in the incubator at room temperature for 30 minutes. After 30 min the chambers were removed and PWN slurry was collected into the 15 mL centrifuge tube. Each chamber was rinsed a few times with clean sterile water to remove any PWN that remain. There was about 200 eggs left in each chamber, the odd PWN may also be present.

[0179] Sample introduction to the water bath was staggered 10-15 minutes apart to accommodate for the time needed to collect samples after the heat treatment experiment. Sterile water (100 pL) was added at the test temperatures (25 °C, 30 °C, 40 °C and 56 °C) to each chamber and the chamber was sealed with a cork stopper (FIG. 3). The chamber was placed in the water bath tray and water was added to the plate to weigh it down, after which the timer was started. Six glass plates were added to one water bath. After test hold time (2 hours / 30 minutes) the first plate was removed, one sample consisted of 5 chambers. The water from each chamber was aspirated off and checked under the microscope to ensure little to no eggs were removed. 600 pL RPL lysis solution was used for each sample. 100 pL RPL solution were pipetted into each chamber. Then, a rubber policemen or curved needle was used to remove the eggs from the bottom of the plate. The lysate was transferred to a sterile 1.5 mL tube with 200 mg micro beads present. This was repeated for all 5 chambers and the implement was rinsed with the final 100 pL. The tube was placed in the bead mill and shaken at 6 m / sec for 30 seconds two times. The tube was then incubated in a heat block at 65°C for 5 minutes shaking intermittently. After 5 minutes, the sample was pipetted onto a Qiashredder™ column and centrifuged. The supernatant was collected, and flash frozen in liquid nitrogen and stored at -80°C until nucleic acid extraction.

[0180] Adult Stage

[0181] Dav 1

[0182] The PWN slurry used for egg collection above was collected and the excess water collected. The slurry was brought up to 1 mL with sterile water. This was then added to a burette with 0.3% CMC at the end of the day (FIG. 4). This was left for 16-24 hours.

[0183] Day 2

[0184] Two mL were collected from the bottom of the burette in a 15 mL centrifuge tube. Then sterile water was added to bring the volume to 10 mL. The tube was centrifuged at max speed for 2 minutes, and the water / CMC was decanted. This was repeated a few times to remove all the CMC. The PWN were allowed to sit a minimum of 30 minutes sterile water to reinvigorate them.

[0185] The PWN were resuspended in 600 pL water and 100 pL of the suspension was aliquoted into each chamber. 300 pL of test temperature water was pipetted to each chamber and the chamber was sealed with a cork stopper. The plates were added to the bath and the dish was filled to weigh it down. The timer was started at this point.

[0186] At the end of test, the slurry was pipetted into a 1.5 mL centrifuge tube and centrifuged at max speed for 1 minute. The supernatant was removed, and the pellet was flash frozen in liquid nitrogen and stored at -80°C until nucleic acid extraction.

[0187] Jill Stage

[0188] Dav 1

[0189] Wood was chopped into small pieces at ~ 15 g for every 15 pg desired per funnel. The chopped wood was placed into two squares of Kimwipes® tissue, the top twisted closed and placed in a Baermann funnel setup (FIG. 5). The funnel was filled with water and left for 24 hours.

[0190] Dav 2 After 24 hours, 1.5 - 2 rnL of water solution was collected from the tubes into one centrifuge tube, which was then centrifuged at 1,000 rpm for 2 minutes to help separate out any debris that may be present. The water was decanted off and fresh distilled water added. The washes were repeated until the nematodes did not centrifuge out and stayed in solution. The nematodes were allowed to settle for 5 minutes and the water decanted off.

[0191] The PWN were resuspended in 600 pL water and 100 pL of the suspension was aliquoted into each chamber. The PWN were distributed and handled as described for the adults above.

[0192] Isolation of Total RNA and Genomic DNA. Total RNA and genomic DNA (gDNA) isolation was performed according to the manufacturer’ s instructions using the QIAgen All-Prep kit. All isolations were performed on ice, with sterile, DNase-, RNase-free plasticware. A 20 min DNAse I digestion step at 20°C was included between two RW 1 washes. RNA quality and quantity was assessed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Ottawa, Canada).

[0193] cDNA Synthesis. RNA was reverse transcribed to cDNA using an applied high-capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems Inc., CAT No. 4374967) following the manufacturer’s protocol. RNA was diluted to approximately 1 pg RNA per reaction prior to cDNA synthesis. Reverse transcription was carried out on a MyCycler™ Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA). The reverse transcription reaction consisted of a 10 minute primer ligation step at 24°C, followed by a 2 hour DNA polymerization step at 37°C, and a 5 minute enzyme deactivation step at 85 °C. Synthesized cDNA was stored at -20°C. A 1 / 20 dilution was performed to produce working stocks of cDNA for qPCR. Diluted cDNA was also stored at -20°C.

[0194] RNA-Seq Analyses. In preparation for RNA-Seq, RNA sample integrity was analyzed using a Bioanalyzer 2100 (Agilent Technologies), and samples with RNA integrity number (RIN) of >7 were used for RNA-Seq analyses (five biological replicates per treatment). After collection, RNA samples were transported on dry ice to the Michael Smith Genome Sciences Centre (GSC, BC Cancer Research, Canada), where strandspecific mRNA libraries were constructed and sequenced using Illumina NovaSeq (paired-end platform generating 2 x 150 base pair reads for each sample).

[0195] The quality of reads was evaluated using FastQC to ensure data accuracy across all samples. Reads were aligned to the B. xylophilus BXY Jv5 GenBank assembly GC A_904066235.2 genome assembly using STAR two-pass alignment (version 2.6.1). Mapped reads were assembled and counted using StringTie (version 1.3.4) with a minimum read coverage of 1 for most transcripts, and 4.75 for single-exon transcripts. Gene counts were exported as transcript count tables comparing individual transcript counts from the control (25°C) condition compared to each of the other conditions. Transcripts were annotated with BXY Jv5 GenBank assembly annotations. Differential gene expression analysis was performed using DESeq2 (version 1.28.1) as described previously [Imbery et al., Ecotoxicol Environ Saf, 2024. 279: p. 116463 ] (FDR < 0.01, fold-change greater than 2 or less than 0.5).

[0196] The predicted protein sequence was obtained using the EXPASY protein translation tool and a predicted tertiary structure was obtained using Alphafold2.

[0197] Sample Viability Analyses. Two pL gDNA or cDNA converted from the RNA were tested for sample integrity using the ePlant5 assay known as the IntegritE-DNA® when used on gDNA and IntegritE-RNA™ when used on cDNA. IntegritE targets endogenous chloroplast DNA and was used to assess the presence of any residual PCR inhibitory compounds and to identify any samples that may be degraded or otherwise compromised as described previously [Hobbs et al., PLOS ONE, 2019. 14(3): p. e0213849; Veldhoen et al., PLoS One, 2016. 11(11): p. e0164907; Lopez et al., Global Ecology and Conservation, 2024.

[0198] 54: p. e03040]. All samples passed the IntegritE tests.

[0199] eBUXY and erBUXY assay designs and validation. All assays were designed and validated according to the previously suggested workflow [Langlois et al,. Environmental DNA, 2021. 3: p. 519-527]. Contig sequences were identified that had the desired expression profile and also contained suitable gene structure containing introns. Primer and probe design was accomplished with Beacon Designer™ 8.21 (PREMIER Biosoft, California, USA). Primer and probe sequences for each assay used in the present study are listed in Table 3. For in vitro specificity validation, SYBR™ green qPCR (QIAcuity EG PCR Kit [Cat. 250111, Qiagen, Hilden, Germany]) validation was run using several primer pairs together with target and non-target gDNA or cDNA as a template. The thermocycler profile used was: initial denaturation of 2 minutes at 95°C followed by 50 cycles of 15 seconds at 95°C, 30 seconds at 60°C, and 45 seconds at 72°C, followed by a melt curve in 0.5°C increments from 65°C to 95°C. Subsequently, high-end specificity validation (primer and probe) was performed using TaqMan™ qPCR (QIAcuity™ Probe PCR Kit [Cat. 250101, QIAgen, Hilden, Germany]). This validation employed the same set of gDNA and cDNA used during the initial primer validation, with 25 technical replicates per sample. To characterize assay sensitivity, serial dilutions of synthetic DNA amplicon (gBlocks™, IDT) were prepared to construct a standard curve.

[0200] Based on the constructed standard curve, eLowQuant was used to calculate the limit of detection (LOD) and limit of quantification (LOQ) based on a modified Binomial-Poisson distribution model [Lesperance et al., Environmental DNA, 2021. 3: p. 970-981]. These measurements generally describe the smallest concentration of DNA that can be reliably measured by eDNA assays with reasonable statistical certainty. The LOD from continuous data (LODcontinuous) was also determined as the lowest copy number where there is a >95% detection [Abbott et al., National standard of Canada, CSA 2023. W219:23: p.

[0201] 23]. This LODcontinuous indicates the breakpoint for continuous and discontinuous data defining the computational approaches for determining sample copy number. Lastly, the PCR assay efficiency measures the ability of the designed primers and probe to amplify the target DNA region for every PCR cycle. EXAMPLE 2

[0202] Assay Design for Rapid Detection of Live Pinewood Nematodes BxCheck is a four-part assay that is designed to reduce the risk of false positives and negatives. Plant chloroplast gDNA and cDNA is tested to ensure that the sample can support amplification of DNA and RNA (FIGs. 7A-7D). To identify the appropriate gene and transcript for selection for Bx eDNA and Bx eRNA assay design, RNA-Seq data was analyzed and obtained from different heat treatment regimes.

[0203] For this study, 15,859 genes and 30,795 transcripts from the B. xylophilus genome assembly were investigated. About 14,000 contigs were investigated per sample. To narrow it down, the contigs were sorted for samples that had high expression at live temp (30°C and 40°C) and had low expression levels at 56°C (kill temp) across all three developmental stages: adult, juvenile and egg. No data were available for eggs at 56°C as no RNA was isolatable from any egg sample at this temperature.

[0204] Of the thousands of data points, only two contigs presented the desired characteristics in the RNA expression profile (FIGs. 8A-8C, 9A-9C). These contigs were named Contig 280 and Contig 3172 (see SEQ ID NOs: 29-30; FIG. 17). Contig280 corresponds to an 8,408 nt gene on the - strand of Chromosome 1 with 12 exons (SEQ ID NO: 29). The open reading frame is 2,793 nucleotides long giving rise to a putative protein of 930 amino acids with an estimated size of -102 kDa (SEQ ID NO: 31). Contig 3172 corresponds to a three-exon gene located on Chromosome 2 of 941 nucleotides whose open reading frame is 795 nucleotides (FIG. 17) giving rise to a putative protein of 264 amino acid and 29 kDa (SEQ ID NO: 32). Neither putative protein is currently annotated, and no conserved domains have been identified from the National Center for Biotechnology Information (NCBI) databases. While Contig280 protein did align with 82% identity to B. okinawaensis sequence using Blastp, no alignments were observed with the NCBI nr protein database with Contig3172.

[0205] The eBUXY5 Contig280 and eBUXY9 Contig 3172 were designed to reliably detect gDNA from only B. xylophilus (Table 4 and FIG. 10). To detect cDNA from B. xylophilus, we designed three robust assays for Contig 280 - erBUXYl, erBUXY5, erBUXY6, and erBUXY7 (Table 3) and one for Contig 3172 - erBUXY4. These assays show the desired specificity for only PWN cDNA (Table 4 and FIG. 10).

[0206] eBUXY5: primers / probe are in exon 11 of Contig 280.

[0207] erBUXY5: primers / probe span exons 3-5 of Contig 280.

[0208] erBUXYl: primers / probe span exons 3-5 with the forward primer split between exons 3 and 4 (the assay is similar as erBUXY5 but with a different forward primer) of Contig 280.

[0209] erBUXY6: forward primer spans exons 3 and 4, probe is in exon 4, reverse primer is in exon 5 of Contig 280.

[0210] erBUXY7: forward primer spans exons 3 and 4, probe is in exon 4, reverse primer is in exon 5 of Contig 280.

[0211] Nematode mRNA was isolated from inoculated or mock-inoculated wood using established protocols followed by infected wood subjected to phytosanitary measures [Leal et al., Forest Pathology, 2015. 45(2): p. 134-148]. To do this, existing soil extraction techniques or swabbing natural and artificial cover objects to detect eDNA were modified [Matthias et al., Ecological Indicators, 2021. 131]. Wood samples were directly infected with pinewood nematode at known quantities and then subjected to a variety of treatments aimed at killing the pest [Uzunovic et al., 2018, FPInnovations; Uzunovic et al,. Journal of Wood Science, 2013. 59(160-170)].

[0212] The eRNA / eDNA results can be directly compared with conventional funnel collection and manual counting methods as described previously [Leal et al., Forest Pathology, 2015. 45(2): p. 134-148].

[0213] FIG. 14 shows an experiment involving wood block samples inoculated with the Nel2 / 02 strain of Bursaphelenchus xylophilus and then heated to a kill temperature of 56°C for 30 minutes in a Humble water bath (Heat-treated) or kept at 25°C as a control (Control). Sapwood from healthy, freshly cut lodgepole pine (Pinus contorta var. latifolia Engelm) logs was cut into 2.5 x 3.8 x 0.64 cm samples and sterilized with 25 kiloGrays ionizing radiation (Sterigenics, Port Coquitlam, BC, Canada) and frozen at -20°C. Wood blocks were thawed, aseptically inoculated with a hyphae / spore mixture of the blue stain fungi, Leptographium terebrantis, Leptographium longiclavatum, Ophiostoma montium and Ophiostoma clavigerum, as well as the wood decay fungus, Trichaptum abietinum, and incubated in glass jars sealed with breathable membranes for seven days, following previously established methods (Hoover, K., A. Uzunovic, B. Gething, A. Dale, K. Leung, N. Ostiguy, and J. J. Janowiak. 2010. Lethal temperature for pinewood nematode, Bursaphelenchus xylophilus, in infested wood using microwave energy', J Nematol, 42: 101-10). After blue staining was present on the wood blocks the samples were aseptically inoculated with the PWN strain Nel2 / 02. Each individual block was inoculated with approximately 500 nematodes from a solution of 5,000 nematodes / mL prepared as described previously (Id.). The inoculated blocks were held at room temperature until heat treatment. Inoculated wood blocks were treated in a Humble water bath (Noseworthy et al. 2023 “Determination of specific lethal heat treatment parameters for pests associated with wood products using the Humble water bath,” Journal of Pest Science, 96: 1187-97) at 56°C for 30 min, held at room temperature for 5.5 days, then stored at -80°C. Control blocks were not heat treated and kept at room temperature after inoculation and before storage at -80°C.

[0214] To extract nucleic acids from inoculated wood blocks, a previously described method for coextracting DNA and RNA from recalcitrant woody tissues (Zeng et al. 2018 “Co-extraction of genomic DNA & total RNA from recalcitrant woody tissues for next-generation sequencing studies,” Future Sci OA, 4: Fso309) was combined with Qiagen’s AllPrep® DNA / RNA Mini kit. Briefly, wood samples were ground to a fine powder in 50 mL Retsch® metal cannisters with three 12 mm diameter stainless steel ball bearings. Samples were ground 3 times at 30 Hz for 1 min using a Retsch® Mixer Mill MM400 (Fisher Scientific), re-freezing the cannisters between each grind cycle. Frozen wood powder (1.5 g) was immediately added to a 50 mL conical tube containing 15 mL Extraction Buffer (100 mM Tris-HCl pH 8.0, 25 mM EDTA pH 8.0, 2M NaCl, 2% CT AB, 2% polyvinylpyrrolidione K 30) preheated to 65°C. Tubes were vortexed briefly, filter- sterilized dithiothreitol (DTT, IM) was added to a final concentration of 10 mM, tubes were vortexed again, and the slurry was incubated for 15 min at 65°C with manual, intermittent shaking. Tubes were centrifuged at 12,000 xg at 4°C for 15 min to pellet the woody tissue, the supernatant was transferred to a new tube, and two extractions with equal volumes of chloroform: isoamyl alcohol (24:1) were performed. One-tenth volume of 3 M sodium acetate (pH 5.2) and 0.75 volumes of 4°C isopropanol were added to the final aqueous phase, mixed by inverting the tube several times, and the solution was stored for 2 h at 4°C to allow precipitation of nucleic acids. Samples were centrifuged at 12,000 xg at 4°C for 15 min, the supernatant discarded, and the pellet washed twice with 70% ethanol. All traces of ethanol were removed from the tube using a fine pipette tip, and the pellet was suspended in 60 pL SSTE buffer (IM NaCl, 0.5% SDS, WmM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) pre-heated to 65°C. Ten volumes Buffer RLT Plus (Qiagen) was added, then the All-Prep DNA / RNA Mini kit method was followed to complete the protocol. A 20 min DNAse I digestion step at 20°C was included between two RW 1 washes. Total RNA was eluted using 40 pL RNase-free water, and the first eluate was re-applied to the column to increase yield. Genomic DNA was eluted from the All-Prep DNA spin column using 30 pL Buffer EB (56°C). The first eluate was re-applied to the column to increase yield.

[0215] RNA and DNA quality and quantity were assessed using Qubit® High Sensitivity RNA and DNA assays, respectively (Thermo Fisher) and on 1% agarose TAE gel. RNA was stored at -80°C. cDNA was synthesized as described above, using a constant volume of 10 pL RNA in each reaction.

[0216] Three biological replicates of control and heat-treated Nel2 / 02-inoculated blocks were used to assess assay performance. The IntegritE-DNA® and IntegritE-RNA™ assays were run first. Four technical replicates of each sample were run on each of the IntegritE assays, alongside eight technical replicates of notemplate controls (NTCs) prepared with UltraPure DNase / RNase-free distilled water (Invitrogen) and two positive controls (ePlant5 gBlock™; 10 copies / pL) consisting of gBlocks™ synthetic DNA fragments (IDT). An additional negative control, no reverse transcriptase (Minus RT) control, was used in duplicate for each of the cDNA samples (FIG. 14).

[0217] Samples that passed IntegritE-DNA® and IntegritE-RNA™ testing were assayed with the eDNA assays, eBUXY5, eBUXY9, and the eRNA assays, erBUXY7 and erBUXY4. Eight technical replicates were tested for every sample. Each qPCR plate included eight technical replicates of no-template controls (NTCs) prepared with UltraPure DNase / RNase-free distilled water (Invitrogen) and two appropriate Bx assay positive controls (10 copies / pL) consisting of gBlocks™ synthetic DNA fragments (IDT). RNA assays included the Minus RT control run in duplicate for each sample (FIG. 14).

[0218] DNA was analyzed using the IntegritE-DNA® test to confirm the integrity of the genetic material. As this assay is based on the detection of expected contaminating chloroplast DNA, it serves as an indicator of general sample integrity and demonstrates the assay’s reliability in detecting DNA regardless of treatment conditions. eDNA assays, eBUXY5 and eBUXY9, targeting Contigs 280 and 3172, respectively, were used on DNA samples. Amplification was consistently observed across all samples, confirming the presence of nematode DNA and demonstrating the assay’s reliability in detecting genetic material regardless of treatment conditions. RNA was analyzed using the IntegritE-RNA™ test to confirm RNA integrity after it had been reverse-transcribed to cDNA. As this assay is based on the detection of expected contaminating chloroplast RNA, it serves as an indicator of general sample integrity and demonstrates the assay’s reliability in detecting RNA derived from RNA regardless of treatment conditions. eRNA assays, erBUXY4 and erBUXY7, targeting Contigs 3172 and 280, respectively, was used to analyze cDNA reverse transcribed from RNA samples to assess whether the nematodes were killed by the heat treatment. While the control samples always gave a signal detection, the heat-treated samples never gave a signal detection. This indicates that while there were nematodes present, and the integrity of the DNA and RNA samples were confirmed, that the tool can distinguish live nematodes from dead ones.

[0219] EXAMPLE 3

[0220] Assay Validation

[0221] Lodgepole pine lumber (30.5” long 2x4s) infected with Bursaphelenchus xylophilus (Q 14-26 strain) was treated to meet the following Canadian Food Inspection Agency (CFIA) schedule: Heat Treatment Schedule for Option A for a Lumber Thickness of up to 60 mm. The treatment included the following parameters: Minimum Heat Treatment Run Time - 6 h and 26 min, Wet Bulb Temperature Continuous Run Time > 60°C (140°F) - 2 h and 3 min and Minimum Final Wet Bulb Temperature - 63°C (145°F). This treatment kills all nematodes at all stages of life in the lumber. Similarly infected lumber was not heat-treated but maintained at 25 °C as a control.

[0222] Nucleic acids were extracted from wood shavings from infected, heat-treated and control lumber at 0, 24, 72, and 144 hours after kiln heat treatment and then were assessed using assays that target DNA (IntegritE-DNA®, eBUXY5, and eBUXY9) and assays that target RNA via cDNA (IntegritE-RNA™, erBUXY7). Four technical replicates per sample were run for the IntegritE tests while eight technical replicates were run per sample for the other assays.

[0223] The IntegritE-DNA® test confirms the integrity of the genetic material (FIG. 15 A). As this assay is based on the detection of expected contaminating chloroplast DNA, it serves as an indicator of general sample integrity and demonstrates the assay’s reliability in detecting genetic material regardless of treatment conditions. eDNA assays, eBUXY5 (FIG. 15C) and eBUXY9 (FIG. 15E), target genomic DNA of Contigs 280 and 3172, respectively. Amplification was consistently observed across all DNA samples, confirming the presence of nematode DNA and demonstrating the assay’s reliability in detecting genetic material regardless of treatment conditions.

[0224] RNA was analyzed using the IntegritE-RNA™ test (FIG. 15B) to confirm RNA integrity after reverse transcription to cDNA. As this assay is based on the detection of expected contaminating endogenous chloroplast RNA, it serves as an indicator of general sample integrity and demonstrates the assay’s reliability in detecting genetic material regardless of treatment conditions. eRNA assay erBUXY7 (FIG. 15D), targeting Contig 280, was used to analyze cDNA reverse transcribed from RNA samples to assess whether the nematodes were killed by the heat treatment. While the control samples always gave a signal detection, the heat-treated samples never gave a signal detection. This indicates that while there were nematodes present, and the integrity of the DNA and RNA samples were confirmed, that the tool can distinguish live nematodes from dead ones almost immediately after lethal heat treatment. In a follow-up test, nucleic acids were extracted from wood shavings from lumber as described above except using Bursaphelenchus xylophilus (Nel2 / 02 strain), and then assessed using assays that target DNA (IntegritE-DNA®, eBUXY5, and eBUXY9) and assays that target RNA via cDNA (IntegritE-RNA™, erBUXY7, and erBUXY4). Four technical replicates per sample were run for the IntegritE tests while eight technical replicates were run per sample for the other assays.

[0225] The IntegritE-DNA® test (FIG. 16 A) confirms integrity of the genetic material. eDNA assays, eBUXY5 (FIG. 16C) and eBUXY9 (FIG. 16E), targeting Contigs 280 and 3172, respectively, were used on DNA samples. Amplification was consistently observed across all DNA samples, confirming the presence of nematode DNA and demonstrating the assay’s reliability in detecting genetic material regardless of treatment conditions. RNA was analyzed using the IntegritE-RNA™ test (FIG. 16B) to confirm RNA integrity after reverse transcription to eDNA. As this assay is based on the detection of expected contaminating endogenous chloroplast RNA, it serves as an indicator of general sample integrity and demonstrates the assay’s reliability in detecting eDNA derived from RNA regardless of treatment conditions. eRNA assays, erBUXY7 (FIG. 16D) and erBUXY4 (FIG. 16F), targeting Contigs 280 and 3172, respectively, were used to analyze eDNA reverse transcribed from RNA samples to assess whether the nematodes were killed by the heat treatment. While the control samples always gave a signal detection, the heat-treated samples never gave a signal detection. This indicates that while there were nematodes present, and the integrity of the DNA and RNA samples were confirmed, that the tool can distinguish live nematodes from dead ones almost immediately after lethal heat treatment.

[0226] Table 3. Assay details for both Bx eDNA and eRNA qPCR designs.

[0227]

[0228]

[0229]

[0230] Table 4. Assay specificity results. +, detected; not detected

[0231]

[0232] It will be apparent that the precise details of the methods or compositions described herein may be varied or modified without departing from the spirit of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

Claims:

1. A method of detecting a living target organism from an environmental nucleic acid sample, comprising:detecting a rapid kill RNA marker of the target organism in the environmental nucleic acid sample, wherein the rapid kill RNA marker is low or not detectable when the target organism is deceased, thereby detecting the living target organism.

2. The method of claim 1, further comprising obtaining a nucleic acid sample from the environment prior to the detecting step.

3. The method of claim 1 or claim 2, wherein detecting the rapid kill RNA marker comprises:i) contacting the environmental nucleic acid sample with a forward and reverse primer specific to the rapid kill RNA marker;ii) amplifying the rapid kill RNA marker, thereby producing rapid kill RNA marker amplicons, and iii) detecting the rapid kill RNA marker amplicons, thereby detecting the rapid kill RNA marker.

4. The method of any one of the prior claims, wherein the method further comprises detecting a genomic DNA molecule of the target organism.

5. The method of claim 4, wherein detecting the genomic DNA molecule comprises:i) contacting the environmental nucleic acid sample with a forward and reverse primer specific to the genomic DNA molecule;ii) amplifying the genomic DNA molecule, thereby producing genomic DNA amplicons, and iii) detecting the genomic DNA amplicons, thereby detecting the genomic DNA molecule.

6. The method of any one of the prior claims, wherein the nucleic acid sample comprises RNA, genomic DNA, or both RNA and genomic DNA.

7. The method of any one of the prior claims, wherein the environmental nucleic acid sample is prepared from a sample of wood.

8. The method of any one of the prior claims, wherein the target organism is a pest or invasive species.

9. The method of any one of the prior claims, wherein the target organism is Bursaphelenchus xylophilus.

10. The method of claim 7, wherein the sample of wood is infested with, or suspected of being infested with, Bursaphelenchus xylophilus.

11. The method of any one of the prior claims, wherein the rapid kill RNA marker is low or not detectable following a kill treatment, optionally wherein the kill treatment is a heat treatment of at least 56°C for at least 30 mins.

12. The method of any one of claims 3-11, wherein the forward and / or reverse primer specific to the rapid kill RNA marker comprise or consist of:i) SEQ ID NOS: 17 and / or 18;ii) SEQ ID NOS: 25 and / or 26;iii) SEQ ID NOS: 9 and / or 10;iv) SEQ ID NOS: 5 and / or 6; orv) SEQ ID NOS: 13 and / or 14.

13. The method of any one of claims 3-12, wherein detecting the rapid kill RNA marker amplicons comprises using a molecular probe, optionally wherein the molecular probe comprises or consists of SEQ ID NO: 19, 27, 11, 7, or 15.

14. The method of any one of claims 5-13, wherein the forward and / or reverse primer specific to the genomic DNA molecule comprise or consist of:i) SEQ ID NOS: 1 and / or 2; orii) SEQ ID NOS: 21 and / or 22.

15. The method of any one of claims 3-14, wherein detecting the genomic DNA amplicons comprises using a molecular probe, optionally wherein the molecular probe comprises or consists of SEQ ID NO: 3 or 23.

16. The method of any one of the prior claims, wherein the rapid kill RNA marker is a sequence in Contig 280 or Contig 3172 of Bursaphelenchus xylophilus.

17. The method of any one of the prior claims, whereini) the rapid kill RNA marker comprises at least 95% sequence identity to SEQ ID NO: 20, 28, 12, 8, or 16; orii) the rapid kill RNA marker comprises or consists of SEQ ID NO: 20, 28, 12, 8, or 16.

18. A kit comprising a forward and reverse primer specific to a rapid kill RNA marker.

19. The kit of claim 18, comprising:a) a forward primer comprising or consisting of SEQ ID NO: 17 and a reverse primer comprising or consisting of SEQ ID NO: 18;b) a forward primer comprising or consisting of SEQ ID NO: 25 and a reverse primer comprising or consisting of SEQ ID NO: 26;c) a forward primer comprising or consisting of SEQ ID NO: 9 and a reverse primer comprising or consisting of SEQ ID NO: 10;d) a forward primer comprising or consisting of SEQ ID NO: 5 and a reverse primer comprising or consisting of SEQ ID NO: 6; ore) a forward primer comprising or consisting of SEQ ID NO: 13 and a reverse primer comprising or consisting of SEQ ID NO: 14.

20. The kit of claim 18 or 19, further comprising a probe.

21. The kit of claim 20, wherein the kit comprises:a) a forward primer comprising or consisting of SEQ ID NO: 17, a reverse primer comprising or consisting of SEQ ID NO: 18, and a probe comprising or consisting of SEQ ID NO: 19;b) a forward primer comprising or consisting of SEQ ID NO: 25, a reverse primer comprising or consisting of SEQ ID NO: 26, and a probe comprising or consisting of SEQ ID NO: 27;c) a forward primer comprising or consisting of SEQ ID NO: 9, a reverse primer comprising or consisting of SEQ ID NO: 10, and a probe comprising or consisting of SEQ ID NO: 11;d) a forward primer comprising or consisting of SEQ ID NO: 5, a reverse primer comprising or consisting of SEQ ID NO: 6, and a probe comprising or consisting of SEQ ID NO: 7; ore) a forward primer comprising or consisting of SEQ ID NO: 13, a reverse primer comprising or consisting of SEQ ID NO: 14, and a probe comprising or consisting of SEQ ID NO: 15.

22. The kit of any one of claims 18-21, further comprising:a container for holding or collecting a sample;one or more DNA isolation reagents;one or more qPCR reagents;a filter;or combinations thereof.

Images