US20180216168A1 - Diagnostic kits - Google Patents

Diagnostic kits Download PDF

Info

Publication number
US20180216168A1
US20180216168A1 US15/747,424 US201615747424A US2018216168A1 US 20180216168 A1 US20180216168 A1 US 20180216168A1 US 201615747424 A US201615747424 A US 201615747424A US 2018216168 A1 US2018216168 A1 US 2018216168A1
Authority
US
United States
Prior art keywords
nucleic acid
seq
nos
diagnostic kit
plant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/747,424
Other languages
English (en)
Inventor
David Dent
Dhaval Patel
Gary DEVINE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AZOTIC TECHNOLOGIES Ltd
Original Assignee
AZOTIC TECHNOLOGIES Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AZOTIC TECHNOLOGIES Ltd filed Critical AZOTIC TECHNOLOGIES Ltd
Assigned to AZOTIC TECHNOLOGIES LTD reassignment AZOTIC TECHNOLOGIES LTD ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DENT, DAVID, DEVINE, Gary
Publication of US20180216168A1 publication Critical patent/US20180216168A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/06—Coating or dressing seed
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/02—Acetobacter

Definitions

  • the present invention relates to a diagnostic kit, able to identify particular strains of the nitrogen-fixing bacteria Gluconacetobacter diazotrophicus (Gd) that have good utility in agriculture, in terms of their ability to colonise plant cells intracellularly, giving rise to particularly effective nitrogen fixation, as well as to reagents for use in the kits. Novel strains that are identified by the kits form the subject of a co-pending application.
  • Gd nitrogen-fixing bacteria
  • Novel strains that are identified by the kits form the subject of a co-pending application.
  • Gluconacetobacter diazotrophicus has been well studied for its nitrogen fixing and plant growth promoting activities as reviewed in Eskin et al. International Journal of Agronomy (2014):1-13. Certain strains of Gd however have been shown to be particularly advantageous in the treatment of plants since they are able to establish themselves intracellularly within plant cells along with exhibiting species and tissue independence (Cocking et al., In vitro Cellular & Developmental Biology Plant (2006) 42 (1). These properties, combined with their ability to travel throughout a range of plant tissues, make such strains better able to deliver the benefits to the target crop plants.
  • a diagnostic kit comprising means to determine the presence in a sample of at least one nucleic acid sequence selected from SEQ ID NOS 1-10 shown in attached Table 1 hereinafter.
  • SEQ ID NOs 1-10 represent unique and novel sequences, which appear in a preferred sub-species or strain of Gd. Strains having these unique characteristics have been found to be particularly effective in intracellular colonization of plant cells resulting in beneficial nitrogen fixing. In particular, it has been found that use of a stain of the invention leads to yield enhancements in crops such as cereal crops like maize and wheat. Alternatively, similar yields can be achieved even with reduction in traditional nitrogen fertiliser applications.
  • sequences provide a means for identifying these beneficial strains.
  • they have been found to be amenable to detection using primers which do not cross-react with plant species.
  • the kit of the invention comprises means for determining the presence of more than one of the nucleic acid sequences of SEQ ID NOs 1-10, for example up to 10, such as up to 5, or up to 3 of said nucleic acid sequences of SEQ ID NOs 1-10.
  • a reliable diagnostic kit would be provided which will ensure that the strain detected is the one which is similar or substantially similar to the beneficial strain. Similar strains would be detected even if one or more of the sequences differs for example as result of mutation.
  • Preferred strains appear to contain a plasmid, and so plasmid detection, for example by isolation using a commercially-available plasmid isolation kit, may provide further confirmation of the identification of a beneficial strain.
  • any plasmid identified should be less than 27455 bp in size, for example about 17566 bp.
  • the presence of a plasmid, in particular of this size differs from a previously known strain of Gd, UAP5541, which has been reported as lacking in plasmids (Luis E. Fuentes-Ramièrez et al., FEMS Microbiology Ecology 29 (1999) 117-128).
  • the size of the plasmid is smaller than that reported previously in respect of PAL5 strains containing a single plasmid (Giongo et al. Standards in Genomic Sciences, May 2010, Volume 2, Issue 3, pp 309-317 doi: 10.4056/sigs.972221).
  • the plasmid of beneficial strains may also be characterized in that it is restricted into two fragments by the restriction enzyme EcoRI, wherein the fragments are about 12 Kb and about 5.6 kb in size respectively.
  • the kit of the invention may further comprise means for determining the presence and nature of the plasmid, including the provision of a restriction enzyme such as EcoR1, to allow for the detection of fragments of the above-mentioned sizes.
  • the plasmid lacks a number of key sequences which have been previously identified as being present in the plasmid of PAL5. These sequences are shown as SEQ ID Nos 65, 66, 67 and 68 in the attached sequence listing. Thus the absence of these particular sequences may provide a further characterizing feature of the strains.
  • the kit may comprise means for detecting these SEQ ID Nos 65, 66, 67 and 68 to provide a negative control in the sense that the absence of positive results for these sequences may be used to confirm the presence or identity of preferred strains, or the presence of the these sequences may be used to reject less preferred strains in strain selection.
  • the diagnostic kit further comprises means for determining the presence of at least one nucleic acid sequence which is characteristic of Gd species, for example 2, or 3 nucleic acid sequences which are characteristic of Gd species. In this way, the kit would provide confirmation that some Gd is present in the sample, thus confirming the accuracy of the test. If the sample is known to contain Gd, a positive result in this determination would act as a ‘control’ confirming that the test has been carried out effectively.
  • Suitable specific strains will be sequences found in Gd species generally but not in other species, and in particular not in other microbial species or in at least some plants, in particular plants which may be targeted for Gd treatments.
  • nucleic acid sequence which is used to detect Gd species have been found to be amendable to detection of Gd species present in a range of crops, without cross-reacting with plant species.
  • the kit comprises means for detecting the presence of a plant specific nucleic acid sequence, such as a chloroplast specific nucleic acid which amplifies universally from plant DNA.
  • a plant specific nucleic acid sequence such as a chloroplast specific nucleic acid which amplifies universally from plant DNA.
  • the inclusion of such means will act as a control when the kit is used in the context of detection of Gd within a plant species, as this means will produce a detectable signal, even in the absence of any Gd. If this signal fails, this would indicate failure in the test rather than necessarily that there is no Gd present.
  • a particular chloroplast primer set which is available commercially from Thermo Scientific. Product as Phire Plant Direct PCR Master Mix, is based on the disclosure by Demesure B et al (1995) Molecular Ecology 4:129-131.
  • the means for detecting the presence of the nucleic acid sequences may take various forms as would be understood in the art.
  • the kit of the invention comprises means for detecting specific nucleic acids themselves.
  • nucleic acids may be detected using any of the available techniques including nucleic acid binding assays and immunoassays using antibodies raised to haptenised forms of the nucleic acids.
  • the detection involves nucleic acid amplification reactions, and thus in particular, the kits will comprise one or more amplification primers which target the or each nucleic acid sequence being detected.
  • the size of the sequence that may be amplified would be in the range of from 10-3070 base pairs, and suitably from 20-2000 base pairs, for example from 50-500 base pairs, such as from 100-300 base pairs.
  • the size of the fragment will depend upon the nature of the detection reaction being used, but it should be sufficient to ensure that the product is characteristic of SEQ ID Nos 1-10 or variants as defined above, and so is not present in other sequences, in particular plant sequences.
  • they may be selected to be of differing sizes, so that they may be easily differentiated during detection, using techniques such as separation on the basis of size, or melting point analysis.
  • the kit may further comprise one or more additional reagents necessary to carry out a nucleic acid amplification reaction.
  • additional reagents necessary to carry out a nucleic acid amplification reaction.
  • it may include enzymes such as polymerases, salts such as magnesium or manganese salts, buffers and nucleotides as would be understood in the art.
  • kits may, if required, include means for detecting the products of the amplification.
  • means for detecting the products of the amplification may include dyes or probes, in particular labelled probes that bind the target sequence intermediate the primers.
  • the primers themselves may be labelled to facilitate detection.
  • the amplification is LAMP reaction.
  • LAMP assays utilise at least four and suitably six primers which are designed to target six different regions of the target sequence. There will always be two outer primers (F3 and B3) and two inner primers (FIP and BIP). Optionally, in addition there are two loop primers (FLoop and BLoop). The use of the Loop primers usually reduces the amplification time and increases the specificity of the assay.
  • FIP and BIP primers consist of F2, complementary to the F2c region of the template sequence, and F1c, identical to the F1 region of the template.
  • Tm melting temperature of the primers
  • the presence of the products of amplification reactions may be determined using any available technology. Thus they may include techniques where products are separated on a gel on the basis of size and/or charge and detected such as agarose gel electrophoresis. Alternatively, they may be detected in situ, using for example using intercalating dyes or labelled probes or primers.
  • the detection of amplification products using a wide variety of signalling and detection systems is known. Many of these systems can be operated in ‘real-time’, allowing the progress of amplification to be monitored as it progresses, allowing for quantification of the product.
  • fluorescent energy transfer FET
  • FRET Förster resonance energy transfer
  • a major example of such a process used commercially is the TaqMan® process, in which a dual-labelled probe, carrying both a first label comprising a fluorescent energy donor molecule or reporter and a second label comprising a fluorescent energy acceptor molecule or quencher, is included in a PCR system.
  • these molecules interact so that the fluorescent signal from the donor molecule is quenched by the acceptor.
  • the probe binds to the target sequence and is digested as the polymerase extends primers used in the PCR. Digestion of the probe leads to separation of the donor and acceptor molecules, so that they no longer interact. In this way, the quenching effect of the acceptor is eliminated, thus modifying emissions from the molecule. This change in emission can be monitored and related to the progress of the amplification reaction.
  • the kit may comprise components sufficient to carry out multiple separate amplification reactions, such as individual sets of primers.
  • the kit is set up to carry out a multiplex reaction, where multiple targets may be detected in a single reaction.
  • the primers are suitably selected so that each amplified product has a significantly different size or charge so that they may be readily separated and identified on an agarose gel or by melting point analysis using a signalling reagent such as a DNA intercalating dye.
  • any labels provided for example on primers or probes will provide a different and distinguishable signal from other primer sets, for example on the basis of the wavelength of the emitted signal and/or the fact that the product has a different melting point or annealing temperature, which may be distinguished by carrying out a melting point analysis of products.
  • Suitable amplification primers in particular for PCR amplification, together with the approximate size of the products they generate are selected from those set out in Table 3 below:
  • Primer sets represented by SEQ ID NOS 14-33 have been found to act as useful strain-specific primers for beneficial strains of Gd, while primer sets represented by SEQ ID NOS 34-39 act as useful Gd species-specific primers.
  • the invention provides a method for determining the presence in a sample of a strain of Gluconacetobacter diazotrophicus (Gd) able to intracellularly colonise plant cells, said method comprising detecting in said sample at least one nucleic acid sequence selected from SEQ ID NOS 1-10.
  • Gd Gluconacetobacter diazotrophicus
  • up to 10 for example up to 5 such as about 3 of said nucleic acid sequences of SEQ ID NOs 1-10 are detected.
  • the method further comprises detecting at least one nucleic acid which is characteristic of Gd species, as described above, such as a nucleic acid sequence of SEQ ID NOS 11-13. Again, more than one such species specific nucleic acid sequence may be detected if required.
  • the method further comprises detecting a plant specific nucleic acid sequence, which may be characteristic of the particular Gd colonised plant being examined or may be universally present in plants, such as a chloroplast specific nucleic acid sequence, as a control for the reaction.
  • the method comprises a nucleic acid amplification reaction, such as the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Suitable primers are as described above.
  • the sample which may be used in the method of the invention may be any sample that contains or is suspected of containing a strain of Gd.
  • This may include cultures or laboratory samples, which may contain the desired strain of Gd.
  • they may comprise plant samples, including leaf, stem, or root samples, from which nucleic acid has been released, for example by causing cell lysis, for example using mechanical, chemical or sonic means.
  • the sample is from a plant to which a strain of Gluconacetobacter diazotrophicus (Gd) has previously been applied. In this way, the successful colonisation of the plant by Gd can be confirmed.
  • Gd Gluconacetobacter diazotrophicus
  • tests will be carried out in a laboratory, although mobile testing, for example, in field conditions, may be carried out if suitable equipment, such as mobile PCR machines, are available, or if detection of targets in particular protein targets, using techniques such as ELISAs, which may be carried out on lateral flow devices are employed.
  • FIG. 1A is a gel showing PCR products from a range of designed to be strain or species specific, including primer sets A-H in Table 3 (shown in lanes 4, 5, 6, 7, 8, 9, 12 and 13 respectively).
  • FIG. 1B is a gel showing PCR products from a range of designed to be strain or species specific, including primer sets I-M in Table 3 (shown in lanes 4, 5, 9, 12 and 13 respectively).
  • FIG. 2 is a gel illustrating PCR products using Primer set E following inoculation of reactions with 100 ng of (1) OSR var. Ability, (2) OSR var. Extrovert, (3) rice var. Valencia, (4) wheat var. Willow, (5) grass var. Aberglyn, (6) grass var. Dickens, (7) maize, (8) quinoa , (9) Arabidopsis var. Columbia, (10) barley var. Chapeaux, (11) grass var. Twystar, (12) grass var. J Premier Wicket, (13) potato, and (14) tomato. Lane (15) contains amplicon produced from 10 ng genomic DNA from Gd, (16) contains the no template PCR control, and the molecular weight marker at each end of the gel is Hyperladder 1 kb plus (Bioline).
  • FIG. 4A is a graph showing positive amplification of Gluconacetobacter diazotrophicus by fluorescent LAMP using the Genie II real-time machine and a primer set embodying the invention. Positive DNA amplification is detected by a fluorescence signal.
  • FIG. 4B is an anneal curve for the Gluconacetobacter diazotrophicus samples, following amplification by LAMP; the reaction was put through an anneal analysis and the temperature at which the dsDNA reanneals is detected as a burst of fluorescence.
  • FIGS. 5A-C are graphs showing representative results of QPCR experiments carried out using primers designed to amplify sequences according to the method of the invention, when carried out using serial dilutions of samples containing GD DNA.
  • FIG. 5A shows the results for primer set designated P5 for SEQ ID NOs 58 and 59.
  • FIG. 5B shows results for a primer set designated P8 for SEQ ID NOs 60 and 61.
  • FIG. 5C shows results for a primer set designated P17 for SEQ ID NO 62 and 63 as defined hereinafter.
  • FIGS. 6A-F are graphs showing representative results of QPCR experiments carried out using primers designed to amplify sequences that may be detected using a kit of the invention, when carried out using serial dilutions of samples containing GD DNA and plant genomic DNA.
  • FIG. 6A shows the results for primer set designated P5 in the presence of wheat DNA.
  • FIG. 6B shows melt peak graphs of the products of FIG. 6A for all the samples (i.e. dilutions of Gd in presence of wheat genome and relevant controls).
  • FIG. 6C shows melt peak graph of the controls from FIG.
  • FIG. 6A shows results for a primer set designated P17 as defined hereinafter in the presence of maize DNA.
  • FIG. 6E shows the melt peak graph of the products of FIG. 6D for all the samples tested (i.e. dilutions of Gd in presence of Maize genome and relevant controls).
  • FIG. 6F shows the melt peak graphs of the controls from FIG. 6D where a positive control comprising Gd DNA only resulted in giving signal, and negative controls comprising plant DNA only and QPCR negative samples only (NTC—no transcript control), both did not resulted in giving signal.
  • FIG. 7 shows a resolved 1% agarose gel showing plasmid DNA extracted from a particular strain of GD (IMI504958).
  • FIG. 8 shows resolved 1% agarose gel restriction digestion product of plasmid DNA with EcoRI from strain of Gd (IMI504958).
  • the restricted fragments are mentioned as 1) ⁇ 12 Kb and 2) ⁇ 5.6 Kb when run alongside 1 kb ladder where the nearest fragment from the ladder is highlighted for the size comparison.
  • FIG. 9 shows an agarose gel obtained from a PCR amplification to detect Gd from the seedlings of wheat obtained during a field trial.
  • FIG. 10 is a graph showing the chlorophyll index of wheat treated with Gd in accordance with the invention compared to a control.
  • a set of 25 primer sets were designed based upon the sequences identified in the analysis of the genome.
  • the specificity of these 25 primer sets (20 designed to be strain-specific and 5 designed to be species-specific) were first tested by carrying out a conventional PCR reaction using genomic DNA of IMI504958 and PAL5.
  • the results with IMI504958 are illustrated in FIGS. 1A-B .
  • Results showed that 16 strain-specific primer sets delineated IMI504958 from PAL5 as obtained from three different collections (ATCC49037, DSM5601 and LMG7603). However, 4 putative strain-specific primer sets cross-reacted with at least one PAL5 and hence were removed from strain-specific study.
  • strain- and species-specific primers were tested against two other strains of Gd, one originally isolated in India (IMI 502398) and the other from Mauritius (IMI 502399), as well as a revived 2001 culture of UAP5541 strain (stored in glycerol at ⁇ 80° C.), using the method described above.
  • the data was in agreement with the 16 strain specific primers and 4 species specific primer sets as one of the species-specific primer sets produced a higher molecular weight band. This was a surprise result.
  • RNA molecules were degraded using RNase A treatment. Following the removal of insoluble cellular debris using chloroform:isoamyl mix (24:1), deoxynucleic acids were precipitated in ethanol using sodium acetate, washed using diluted ethanol, and resuspended in molecular grade water.
  • LAMP primers were designed to amplify regions of SEQ ID NOS 6, 7 and 9 and are shown in Table 4 below as follows:
  • SEQ ID NOS 40-45 were designed to amplify SEQ ID NO 6 above
  • SEQ ID NOS 46-51 were designed to amplify SEQ ID NO 7 above
  • SEQ ID NOS 52-57 were designed to amplify SEQ ID NO 9 above.
  • Real-time LAMP was carried out on a Genie II instrument (OptiGene), and 1 ⁇ l of sample was added to a 24 ⁇ l reaction mix containing 15 ⁇ l Isothermal Master Mix ISO-001 (OptiGene), 200 nM of each external primer (F3 and B3), 2 ⁇ M of each internal primer (FIP and BIP) and 1 ⁇ M of loops primer (FLoop and BLoop).
  • RT-LAMP reaction consisted of 30 minutes of isothermal amplification at 63° C. To evaluate the annealing temperature of the products, reactions were then subjected to a slow annealing step from 95 to 68° C. (0.05° C./s) with fluorescence monitoring.
  • the third primer set specific for SEQ ID NO 9 gave amplification in nine and a half minutes with an anneal at 89.2° C. (see FIG. 4A ).
  • the primer set specific for SEQ ID NO 6 amplified the positive control at around 11 minutes with an anneal of approximately 88° C.
  • the primer set specific for SEQ ID NO 7 was the slowest, amplifying the positive control at around 23 minutes with an annealing temperature around 90° C.
  • COX cytochrome-oxidase gene
  • a range of QPCR reactions were carried out on samples comprising known quantities of DNA from Gd (IMI504958) and also from a range of crop species including maize, barley and wheat genomic DNA.
  • QPCR reaction mixtures were prepared to a volume of 20 ⁇ L volume per reaction.
  • Gd DNA alone, these consisted of 10 ⁇ L iTaqTM Universal SYBR Green® Supermix (2 ⁇ ) (Bio-Rad), 1 ⁇ L each of forward and reverse primers (final concentration of 10 ⁇ mol), 7 ⁇ L SDW (sterile distilled water) and 1 ⁇ L DNA template at the required concentration.
  • the primer set represented by SEQ ID NOs 58 and 59 was aimed at amplifying a 149 base pair region of SEQ ID NO 3
  • the primer set represented by SEQ ID NOS 60 and 61 was designed to amplify a 104 base pair region of SEQ ID NO 6 above
  • the primer pair represented by SEQ ID NO 62 and 63 was designed to amplify a 130 base pair region of SEQ ID NO 10 above.
  • Thermocycling was carried out using a CFX96 TouchTM Real-Time PCR Detection System from Bio-rad. Initial denaturation was performed at 95° C. for 3 minutes; amplification was performed using 40 cycles of denaturation at 95° C. for 5 seconds followed by 60° C. for 30 seconds (plate read post each amplification).
  • the quantitative amplification was carried out in the presence of genomic plant DNA in order to determine whether there was any cross reactivity. It was found that whilst there was cross reactivity with some plants species, the primer pairs P5 showed no cross-reactivity to wheat and barley genomes, P8 showed no cross-reactivity to wheat barley and maize and P17 showed no cross-reactivity to wheat and maize genomes making these potentially suitable primer sets for detecting Gd in crop species.
  • composition was varied in that 6 ⁇ L SDW (sterile distilled water) was used together with 1 ⁇ L relevant Gd dilution DNA template and 1 ⁇ L plant genomic DNA template.
  • SDW sterile distilled water
  • Gd DNA (92 ng/ ⁇ l) was serially diluted from 10 ⁇ 1 to 10 ⁇ 7 with either wheat DNA (70.6 ng/ ⁇ l) or maize DNA (111 ng/ ⁇ l) and amplification reactions run as described above.
  • primers will maintain efficiency in the presence of plant genomes and thus may form the basis of a detection kit.
  • FIGS. 6(B) and 6(E) Representative examples of the results are shown in FIGS. 6(B) and 6(E) . Clear melt curves are visible for amplified Gd DNA, without plant genomic DNA.
  • Plasmid DNA extraction from Gd was performed using Qiagen mini prep kit (Cat. No. 69104). The low copy number plasmid extraction protocol was followed using 5 ml and 10 ml 48 hour bacterial culture. The extracted plasmid was run on 1% agarose gel flanked by a 1 kb ladder ( FIG. 7 ) and imaged.
  • genomic DNA of Gd was also included on lane-1.
  • the results, shown in FIG. 7 indicate the presence of a single plasmid of about 17.5 Kb in size, which is smaller than that reported previously for plasmids found in PAL5.
  • the plasmid DNA was sequenced and a primer was designed using Primer3 to cover the start and end sites of linear sequence data (P_End_Fw—CCAAATCTCTGGAACGGGTA (SEQ ID NO 64). Sangar sequencing was performed using this primer (SEQ ID NO 64) and the sequenced data was aligned to confirm the plasmid sequence was complete.
  • plasmid DNA in its natural form is circular and can form secondary and tertiary structures, this may impact on the accuracy of size measurements using agarose gels.
  • a restriction map of plasmid was studied using NEBcutter. The restriction digestion will linearize the plasmid providing only a single conformational structure. Also, the restriction enzyme selection is done after studying the sequence, thus allowing the plasmid sequence to be validated as well.
  • the NEB-cutter showed the restriction enzyme EcoRI to digest the plasmid DNA at 3864-9461 bp and 9462-3863 bp producing a DNA fragment of 5598 bp and 11968 bp. Both the size can be studied using a 1 Kb ladder available in the lab, removing the limitation of the reference ladder's maximum size detection as well.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Soil Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Pretreatment Of Seeds And Plants (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US15/747,424 2015-07-28 2016-07-27 Diagnostic kits Abandoned US20180216168A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB1513277.2 2015-07-28
GBGB1513277.2A GB201513277D0 (en) 2015-07-28 2015-07-28 Diagnostic kits
PCT/GB2016/052288 WO2017017439A1 (en) 2015-07-28 2016-07-27 Diagnostic kits

Publications (1)

Publication Number Publication Date
US20180216168A1 true US20180216168A1 (en) 2018-08-02

Family

ID=54106731

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/747,424 Abandoned US20180216168A1 (en) 2015-07-28 2016-07-27 Diagnostic kits
US15/747,425 Active 2037-08-08 US10793484B2 (en) 2015-07-28 2016-07-27 Microorganisms and their use in agriculture

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/747,425 Active 2037-08-08 US10793484B2 (en) 2015-07-28 2016-07-27 Microorganisms and their use in agriculture

Country Status (16)

Country Link
US (2) US20180216168A1 (https=)
EP (2) EP3328990A1 (https=)
JP (1) JP2018522559A (https=)
CN (1) CN108291193A (https=)
AU (1) AU2016300161A1 (https=)
BR (1) BR112018001507A2 (https=)
CA (2) CA2992800A1 (https=)
DK (1) DK3328989T3 (https=)
EA (1) EA201890232A1 (https=)
ES (1) ES2817004T3 (https=)
GB (3) GB201513277D0 (https=)
MX (1) MX382661B (https=)
PH (1) PH12018500191A1 (https=)
PL (1) PL3328989T3 (https=)
WO (2) WO2017017439A1 (https=)
ZA (1) ZA201801302B (https=)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201413333D0 (en) 2014-07-28 2014-09-10 Azotic Technologies Ltd Plant inoculation
GB201513277D0 (en) 2015-07-28 2015-09-09 Azotic Technologies Ltd Diagnostic kits
US12365868B2 (en) 2023-06-20 2025-07-22 Tenfold Technologies, LLC Systems for production of products to promote nitrogen use efficiency in plants
CN117511806B (zh) * 2023-11-14 2024-07-30 内蒙古昆明卷烟有限责任公司 一种嗜糖土地芽孢杆菌b37及其应用

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10010614A1 (de) 2000-03-03 2001-09-13 Gsf Forschungszentrum Umwelt Oligonukleotidsonden zum art- und/oder gattungsspezifischen Nachweis von das Pflanzenwachstum fördernden Bakterien
WO2002045513A2 (en) * 2000-12-04 2002-06-13 Wisconsin Alumni Research Foundation Bacterial inoculants for enhancing plant growth
GB0121126D0 (en) * 2001-08-31 2001-10-24 Univ Nottingham Systemic non-nodular endosymbiotic nitrogen fixation in plants
CA2735269C (en) * 2008-08-29 2018-10-16 Saint Mary's University Use of gluconacetobacter with reduced use of nitrogen fertilizer to improve beet crop production
JP5965104B2 (ja) * 2010-03-03 2016-08-03 三星ディスプレイ株式會社Ｓａｍｓｕｎｇ Ｄｉｓｐｌａｙ Ｃｏ．，Ｌｔｄ． 高収率のセルロース産生活性がある新規なグルコンアセトバクター属菌株
MX2012013434A (es) 2010-05-21 2013-01-22 Syngenta Participations Ag Inyeccion bacteriana en la caña de azucar.
CN103052311B (zh) * 2010-07-15 2016-05-18 Fb科技控股公司 微生物组合物和方法
KR101291669B1 (ko) * 2011-05-09 2013-08-01 서울대학교산학협력단 미코박테리아―유래된 ｄｎａ 오류 복구 뉴클레오타이드 서열 및 이의 용도
WO2013141815A1 (en) * 2012-03-21 2013-09-26 Temasek Life Sciences Laboratory Limited Nitrogen-fixing bacterial inoculant for improvement of crop productivity and reduction of nitrous oxide emission
US11408013B2 (en) * 2013-07-19 2022-08-09 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
GB201413335D0 (en) 2014-07-28 2014-09-10 Azotic Technologies Ltd Agricultural methods
GB201413333D0 (en) * 2014-07-28 2014-09-10 Azotic Technologies Ltd Plant inoculation
CN104744129B (zh) * 2015-03-03 2017-11-07 中国烟草总公司广东省公司 一种香蕉专用生物活性有机肥及其应用
US9790134B2 (en) * 2015-06-02 2017-10-17 Koch Agronomic Services, Llc Agricultural microbial inoculant compositions and uses thereof
GB201513277D0 (en) 2015-07-28 2015-09-09 Azotic Technologies Ltd Diagnostic kits

Also Published As

Publication number Publication date
GB2556001A (en) 2018-05-16
JP2018522559A (ja) 2018-08-16
GB2558094A (en) 2018-07-04
CA2992800A1 (en) 2017-02-02
CA2992809A1 (en) 2017-02-02
WO2017017440A1 (en) 2017-02-02
GB201513277D0 (en) 2015-09-09
WO2017017439A1 (en) 2017-02-02
PL3328989T3 (pl) 2021-02-08
ZA201801302B (en) 2019-07-31
GB201801538D0 (en) 2018-03-14
EP3328989A1 (en) 2018-06-06
GB201801537D0 (en) 2018-03-14
GB2556001B (en) 2021-04-21
DK3328989T3 (da) 2020-09-28
BR112018001507A2 (pt) 2018-09-18
ES2817004T3 (es) 2021-04-06
AU2016300161A1 (en) 2018-02-15
US10793484B2 (en) 2020-10-06
CN108291193A (zh) 2018-07-17
GB2558094B (en) 2020-04-22
MX2018001200A (es) 2018-07-06
US20180215675A1 (en) 2018-08-02
MX382661B (es) 2025-03-13
EP3328990A1 (en) 2018-06-06
EP3328989B1 (en) 2020-07-08
PH12018500191A1 (en) 2018-07-30
EA201890232A1 (ru) 2018-08-31

Similar Documents

Publication Publication Date Title
Morgan et al. Improved real-time PCR detection of ‘Candidatus Liberibacter asiaticus’ from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes
Aroca et al. A biomarker for the identification of four Phaeoacremonium species using the β-tubulin gene as the target sequence
US20180216168A1 (en) Diagnostic kits
Aggarwal et al. Gene-based analysis of Puccinia species and development of PCR-based marker to detect Puccinia striiformis f. sp. tritici causing yellow rust of wheat
CN108676910A (zh) 一种层出镰刀菌的lamp检测引物及其应用
Prasannakumar et al. Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay
Buddhachat et al. Rapid detection of two pathogenically important Xanthomonas in rice using a loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD)
CN106399511A (zh) 一种实时荧光定量pcr检测土壤西瓜枯萎病菌的方法及引物
Liu et al. A rapid PCR-LAMP assay for the early detection of Lasiodiplodia theobromae from basal stem rot-infected passion fruit plants
Fu et al. Rapid and quantitative detection of Leifsonia xyli subsp. xyli in sugarcane stalk juice using a real‐time fluorescent (TaqMan) PCR assay
CN104611420A (zh) 一种结核菌检测试剂盒
KR102212379B1 (ko) 포도에서 발생하는 3종의 바이러스 검출을 위한 프라이머 세트 및 이를 이용한 바이러스 검출 방법
KR101490614B1 (ko) 산토모나스 아르보리콜라 저글란디스 검출용 프라이머 세트 및 이를 이용한 산토모나스 아르보리콜라 저글란디스 검출 방법
KR101493910B1 (ko) 딸기잠재원형반점 바이러스 검출용 프라이머 세트 및 이의 용도
KR101457275B1 (ko) 사탕무황화바이러스를 검출하기 위한 등온증폭 반응용 프라이머 조성물, 및 이의 이용
Smith et al. An internal reaction control for routine detection of Clavibacter michiganensis subsp. sepedonicus using a real-time TaqMan PCR-based assay
KR101024493B1 (ko) 사과 바이로이드 감염 진단을 위한 프라이머 및 이를이용한 진단방법
Nabi et al. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria
Bagherian et al. A new sensitive method for detection of viroids
KR101444005B1 (ko) 귀리에서 Ｈａｌｏ ｂｌｉｇｈｔ 병을 일으키는 슈도모나스 코로나파시엔스 검출용 프라이머 세트 및 이를 이용한 슈도모나스 코로나파시엔스 검출 방법
KR101482376B1 (ko) 사과에서 blister spot병을 일으키는 슈도모나스 시린개 피브이 파퓰란스 검출용 프라이머 세트 및 이를 이용한 슈도모나스 시린개 피브이 파퓰란스 검출 방법
Yan et al. Rapid and simple detection of alfalfa mosaic virus using RT-RPA-CRISPR-Cas12a based lateral flow assay in alfalfa crops in China
CN120905214B (zh) 基于RPA-CRISPR/Cas12a体系可视化检测小麦链格孢的方法及试剂盒
JP6381771B1 (ja) 萎黄病菌由来の核酸を増幅するためのプライマーセットおよび萎黄病菌の検出方法
Yadav et al. Development of rapid novel detection assay for Dickeya dadantii

Legal Events

Date Code Title Description
AS Assignment

Owner name: AZOTIC TECHNOLOGIES LTD, UNITED KINGDOM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DENT, DAVID;DEVINE, GARY;REEL/FRAME:046431/0568

Effective date: 20180213

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION