Portable laboratory for detecting phytopathogenic microorganisms

Abstract

The present invention relates to a portable laboratory for detecting phytopathogenic microorganisms using a plant sample, comprising at least the following elements: a thermocycler suitable for qPCR; a minicentrifuge; a scale; a manual homogeniser suitable for breaking the plant sample, and a kit for extracting and detecting DNA specific to pathogens of plant species. The manual homogeniser comprises a handgrip for holding a shaft terminating in a surface with bearings for breaking the plant sample and the thermocycler suitable for qPCR has the ability to analyse up to 16 samples simultaneously.

Description

[0001] PORTABLE LABORATORY FOR THE DETECTION OF PHYTOPATHOGENIC MICROORGANISMS

[0002] DESCRIPTIVE MEMORANDUM

[0003] Field of technology

[0004] The present invention falls within the sector of detection of diseases caused by phytopathogenic microorganisms.

[0005] Background

[0006] Currently, an average of 25-35% of agricultural production is lost due to diseases caused by phytopathogenic microorganisms. To reduce this loss, chemical pesticides are widely applied, decreasing soil quality and increasing fertilizer use.

[0007] This excessive use of pesticides has not only environmental but also economic repercussions. Therefore, farmers need innovative techniques that allow them to maintain sanitary control over their crops, enabling them to profitably provide healthy products to an increasingly competitive international market.

[0008] The accurate and early detection of plant pathogens plays a crucial role in phytosanitary control. The lack of rapid, accurate, and reliable means for detecting and monitoring plant pathogens is a major limitation of integrated disease management.

[0009] Today, the use of molecular tools for identifying microorganisms and diagnosing diseases is widespread. Quantitative PCR analysis has proven highly effective for diagnosing and quantifying bacterial and fungal pathogens, both of which cause diseases in plants and soil. Therefore, applying this technique allows for monitoring the presence of pathogens in both plants and soil and, consequently, could be a good approach to reducing pesticide use.

[0010] The detection and identification of phytopathogens that cause crop diseases is mainly carried out in qualified laboratories, which means a delay of several days in obtaining the result of the analysis, which can be considerably increased depending on the location of the laboratory.

[0011] This fact can result in a deterioration of the sample or a late diagnosis, causing loss of harvests and / or crops with the consequent economic loss.

[0012] Therefore, the objective of the present invention is to transfer disease diagnostic technologies from a laboratory environment directly to the sampling site.

[0013] To this end, the present invention discloses a portable laboratory for the diagnosis of phytopathogenic microorganisms, which includes all the equipment and reagents necessary to successfully perform an in situ diagnosis of a plant sample by real-time polymerase chain reaction (qPCR).

[0014] Description

[0015] Specific detection of phytopathogenic microorganisms can be performed using qPCR. This technique has proven to be the most reliable, rapid, and specific for diagnosing pathogenic microorganisms, allowing for the acquisition of high-quality, real-time information regarding the phytosanitary status of crops.

[0016] Early diagnosis allows for rapid decisions that can be crucial in many aspects, such as the timely application of a specific plant protection product. This also benefits the environment by reducing the need for soil treatments and their frequency of application. Therefore, the real-time information provided by the portable laboratory of the invention, without the need to transport samples to distant laboratories, will reduce the number of applications of plant protection products to the soil and ensure their correct use. Decisions regarding their application will be based on concise and reliable data derived from DNA techniques to identify the specific causative microorganism and select the most appropriate product.

[0017] The present invention relates to a portable, self-contained laboratory comprising elements, equipment, instruments, and reagents for the specific, in situ detection of phytopathogenic microorganisms using the qPCR technique, from a plant sample without the need to transport the sample to a laboratory. In the present invention, the term “in situ” means that it is performed at the same location or in close proximity to the site where the plant sample is obtained.

[0018] Both the equipment and the protocol included in the portable laboratory of the invention are very easy to use, allowing unqualified personnel, unfamiliar with the laboratory, to perform the molecular analysis of the samples in a total of 1 hour and 30 minutes maximum.

[0019] The invention relates to a portable laboratory for the detection of phytopathogenic microorganisms from a plant sample comprising the following elements: at least one thermocycler suitable for qPCR, preferably with an internal battery, for example, Liberty16; at least one mini centrifuge, preferably with an external battery; at least one balance; at least one manual homogenizer, a homogenizer being any tool known in the prior art suitable for manually breaking up the plant sample; and at least one kit for the extraction and detection of pathogen-specific DNA from plant species.

[0020] In one particular embodiment, the thermocycler suitable for qPCR can analyze up to 16 samples simultaneously; for example, up to 16 phytopathogenic microorganisms can be analyzed per sample. Or, one phytopathogenic microorganism can be analyzed in 16 samples simultaneously. Other combinations of the number of samples analyzed and pathogens analyzed per sample are also possible.

[0021] In a particular embodiment, the portable laboratory of the invention may comprise one or more of the following elements: at least one portable electronic device adapted to work in conjunction with the thermocycler and analyze the data generated by the latter, for example, an iPad or tablet with the appropriate application for operating the thermocycler software; at least one external battery; at least one homogenization stand; at least one timer; at least one pair of scissors; at least one sampler and / or calibrated inoculation loop; at least one plastic tube holder, preferably Eppendorf tubes, preferably 2 ml; and / or

[0022] 70% alcohol in water for disinfection

[0023] In a further embodiment, the portable laboratory may comprise at least one Pasteur pipette.

[0024] The kit for the extraction and detection of pathogen-specific DNA from plant species can be any commercial kit that includes all the reagents necessary for DNA extraction from plant samples, such as Quaagen, phytAlert, Macherey Nagel, or Omega, and for the specific detection of DNA from phytopathogenic microorganisms, such as phytAlert. Specific DNA detection can also be performed using a mixture, in 0.2 ml reaction tubes, of all the reagents, obtained separately, required for real-time qPCR. These reagents include primer and probe assemblies labeled with a fluorophore, such as TacMan; qPCR premixes, such as Takara Zymotaq, phytAlert, or True Allele; and molecular-grade water (BSA).

[0025] The portable laboratory is housed in a case suitable for on-site fieldwork, which is sturdy enough to protect the internal components and allows for convenient and safe transport.

[0026] The components of the portable laboratory must be of a suitable size and weight so that they can be placed in the same carrying case and transported by one person. For example, the centrifuge, thermocycler, and balance must be portable, meaning they fit inside the portable laboratory case. Furthermore, all components that require it must be battery-operated, allowing their use anywhere without needing to be plugged into an electrical outlet.

[0027] The thermocycler is an essential component of the portable laboratory of the invention, as it enables the identification of microorganisms. In one particular embodiment, the thermocycler of the invention allows for the simultaneous detection of up to 16 microorganisms in a single sample of plant material, or the detection of one microorganism in 16 samples of plant material in a single analysis, since it has at least 16 wells for performing 16 qPCR reactions in the same analysis, for example, Liberty16 (Ubiquitome). In another particular embodiment, the portable laboratory of the invention allows for the simultaneous analysis of at least two plant samples, as it has space for at least two qPCR tubes; preferably, the thermocycler allows for at least eight reactions, for example.

[0028] The inventors of the present invention have determined that a freeze dryer is not necessary to carry out the process of identifying phytopathogenic microorganisms. As will be shown later in the embodiments, the kits for the extraction and detection of pathogen-specific DNA from plant species in the present invention are used at room temperature without affecting the detection capability.

[0029] The manual homogenizer allows for the breaking and lysis of the plant sample to extract the DNA onto which the extraction and detection kit will be applied. The manual homogenizer comprises a handle for holding a shaft that ends in a bearing surface for crushing the plant material sample.

[0030] In one particular embodiment, the extraction and detection kit comprises a bag containing a DNA extraction buffer; any DNA extraction buffer known in the state of the art and suitable for plant samples that have been previously homogenized with the hand homogenizer included in the portable laboratory can be used.

[0031] On the other hand, the portable laboratory of the invention may also include any other additional element known in the prior art and commonly used in the field of detecting phytopathogenic microorganisms that is portable and suitable for fieldwork, that is, for on-site application. For example, for a specific detection kit, the portable laboratory may include additional supports and containers necessary for using that particular kit.

[0032] Phytopathogenic microorganisms can be fungi, bacteria, and / or viruses. They are the main causes of disease in the following sectors: turf cultivation, fruit and vegetable production, post-harvest handling, and seedbeds and nurseries.

[0033] The portable laboratory of the invention allows for the simultaneous evaluation of two or more different plant samples. The plant sample can be obtained from any suitable part of the plant, for example, bulbs, seeds, leaves, follicles, styles, stigmas, flowers, and whole aerial parts of plants.

[0034] Plant samples can include, for example, turfgrass, seedlings from nurseries / seedbeds, post-harvest crops, vegetables, and / or fruit trees. An expert in the field would be able to identify the specific range of phytopathogenic microorganisms present in each plant species. In a particular implementation, when the plant sample is turfgrass, the portable laboratory will include a kit suitable for detecting one or more phytopathogenic microorganisms in turfgrass, for example: Colletotrichum spp., Microdochium bolleyi, Bipolaris sorokiniana, Ophiosphaerella Korrai, Drechslera poae, Curvularia spp., Gaeumannomyces graminis, Pythium spp., Magnaporthe poae, Fusarium spp., Pyricularia grisea, Rhizoctoria solani, Phialophora graminicola, Phytophthora spp., and Pythium aphanidermatum.

[0035] In another particular embodiment, when the plant sample comes from seedlings from seedbed / nursery the portable laboratory will comprise a kit suitable for detecting one or more phytopathogenic microorganisms in young seedlings, for example: Phytophthora spp., Phytophthora parasitica, Pythium spp. I, Pythium spp. II, Rhizoctonia solani, Fusarium oxysporum, Elsinoe fawceti, Phyllosticta citricarpa, Mycosphaerella citri, Diaporthe citri, Colletotrichum acutatum, Colletotrichum gloeosporioides, Xanthomonas axonopodis pv., Citri Xylella fastidiosa, Candidatus liberibacter subs.Asiaticus.

[0036] In another specific embodiment, when the plant sample comes from post-harvest crops—that is, for crops that have been harvested and are to be stored and / or transported—the portable laboratory will include a kit suitable for detecting one or more phytopathogenic microorganisms from these crops. Examples include Alternaria alternata, Monilinia fructicola, Botrytis cinerea, Monilinia laxa, Cladosporium spp., Mucor piriformes, Colletotrichum spp. I., Neofabraea spp., Colletotrichum spp. II., Penicillium expansum, Geotrichum candidum, Penicillium digitatum, Geotrichum citri-aurantii, Penicillium italicum, Fusarium spp., and Rhizopus stolonifer.

[0037] The portable laboratory of the invention may further include instructions and the necessary plastic for detecting phytopathogenic microorganisms from a plant sample. The elements of the portable laboratory may be contained in separate containers, or several elements may be contained in a single container. A suitable container includes a single tube (e.g., a vial), one or more wells of a plate (e.g., a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate, etc.), or the like.

[0038] Instructions for using the portable laboratory of the invention may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the portable laboratory in the form of a leaflet, on the labeling of the portable laboratory container or its components (i.e., associated with the container or sub-container), etc. In other embodiments, the instructions are present as an electronic storage data file on a suitable computer-readable storage medium, for example, a portable flash drive, DVD, CD-ROM, floppy disk, etc. In still other embodiments, the actual instructions are not present in the portable laboratory, but means are provided for obtaining the instructions from a remote source, for example, via the Internet.An example of this implementation is a portable laboratory that includes a web address where the instructions can be viewed and / or downloaded. Like the instructions themselves, the means of obtaining them are engraved on a suitable substrate.

[0039] The advantages of the portable laboratory of the invention are as follows:

[0040] It allows real-time qPCR molecular analysis to be performed at any location, without the need for a laboratory (in situ) using a suitable DNA detection kit.

[0041] It does not require an external power supply to perform the analyses, so these can be carried out in any location.

[0042] Virtually all use of laboratory equipment (benchtop centrifuge, benchtop thermocycler, thermoblock, automatic homogenizer, micropipettes) is eliminated, requiring only a manual homogenizer, a thermocycler and a mini-centrifuge, all three of which are portable and can operate at 12v with a rechargeable battery.

[0043] It eliminates the need to send samples to the laboratory, and therefore, the sample is analyzed under the best possible conditions, minimizing the risk of deterioration during transport, as well as cross-contamination with other pathogens.

[0044] It greatly simplifies the processing, homogenization and lysis steps of the starting plant material by using the sealed homogenization or lysis bag with DNA extraction buffer inside.

[0045] The invention can be used by technical personnel unfamiliar with a laboratory environment or molecular biology.

[0046] The economic cost of the portable laboratory of the invention is considerably lower than that used in a laboratory for conventional sample analysis.

[0047] The analysis time of the portable laboratory of the invention is approximately 1 hour and 30 minutes. This analysis can be performed in just a few steps, at any location, and at the same time as sample collection, improving analysis turnaround times and allowing for quick and early objective decisions. This can help prevent crop losses and, therefore, save time and money for the company or technician responsible for the crop. In the present invention, the terms "a" or "an" may refer to one or more of the elements being modified (for example, "an element" may mean one or more elements).

[0048] The invention is illustrated by the following examples, which describe in detail the objects of the portable laboratory. These examples should not be considered as limiting the scope of the invention but rather as illustrative of it.

[0049] The conventional protocol for detecting phytopathogenic microorganisms is as follows: sample collection, sending the sample to the laboratory (minimum 1 day);

[0050] DNA extraction using purification columns (1 h protocol): o Minimum 10 steps o Fixed equipment:

[0051] ■ I. Automatic homogenizer

[0052] ■ II. Thermoblock

[0053] ■ III. bench centrifuge

[0054] ■ Automatic micropipettes

[0055] Sample analysis (1 h protocol): or Fixed benchtop thermocycler or Need for automatic micropipettes

[0056] The protocol for detecting phytopathogenic microorganisms using the portable laboratory of the invention is shown in the following diagram:

[0057] Sample collection

[0058] DNA extraction: or Portable equipment (5 min protocol):

[0059] ■ I. Manual Homogenizer

[0060] ■ II. Portable centrifuge

[0061] Sample analysis (1 h protocol) or Portable thermocycler or Eliminates the need for automatic micropipettes

[0062] The protocol for detecting phytopathogenic microorganisms using the portable laboratory of the invention is shown in more detail in the following diagram:

[0063] 1- Take 5 to 10 whole plants; remove any soil clinging to the roots; cut off the aerial part, leaving a little of the base. 2- Cut all the plants into small pieces with scissors and mix.

[0064] 3- Weigh 0.3 g and place in the homogenization bag

[0065] 4- Crush for 20 seconds.

[0066] 5- Transfer the liquid from the bag to the 2 ml tube using the pipette.

[0067] 6- Centrifuge 1 min

[0068] 7. Load the sample into the PCR tubes. Use a separate loop for each tube; close the tubes, ensuring they are completely sealed.

[0069] 8- Insert the tubes into the PCR

[0070] 9- Open the PCR program; select the protocol and plate according to the kit used. Results in 1 hour.

[0071] Brief Description of the Figures

[0072] Figure 1 Portable laboratory case of the invention, showing its contents

[0073] EXAMPLES OF IMPLEMENTATION

[0074] SAMPLE COLLECTION AND SHIPPING

[0075] By using the portable laboratory of the invention, it is not necessary to send the sample to the laboratory, since it is analyzed on-site with the portable laboratory of the invention (Fig. 2).

[0076] DNA EXTRACTION FROM THE SAMPLE

[0077] The conventional DNA extraction protocol of a laboratory, which consists of a minimum of 10 steps and 1h duration, and the use of specific and fixed laboratory equipment Fig. 1 A.

[0078] The optimized protocol allows reducing those 9 steps to a total of 5 steps and eliminating the use of fixed equipment (Fig. 1 B and C). It also eliminates the need for laboratory micropipettes and their disposable tips, which are replaced by 1 ml Pasteur pipettes and calibrated 1 pl inoculating loops:

[0079] 1. Homogenization of the sample and cell lysis with a ball mortar inside a homogenization bag with DNA extraction buffer and filter for filtration of the lysate.

[0080] 2. Clarification of the lysate by centrifugation of the filtrate 3. Fixation of the DNA in a column with a silica membrane

[0081] 4. Washing and drying the membrane.

[0082] 5. Transfer the liquid resulting from homogenization to a 2 mL Eppendorf tube.

[0083] In one particular example, the commercial PhytAlert Portable kit was used because it allows for DNA extraction from the sample, helps inactivate polymerase inhibitors, and does not interfere with the qPCR reaction. Furthermore, the kit's buffer is incorporated directly into a sealed homogenization bag for easy transport and use.

[0084] Using a Pasteur pipette (which replaces an automatic micropipette), the contents of the bag are transferred to a 2 mL Eppendorf tube. This step is necessary because the sample needs to be centrifuged before loading it into the qPCR wells.

[0085] 3. To ensure that the qPCR reaction is not inhibited regardless of the nature of the starting plant material, a rapid purification of the DNA extract is performed. Using a Pasteur pipette, a portion of the liquid sample from the previous step is mixed with a binding buffer and passed through a column included in the detection kit. After passing the sample, 6 drops of wash buffer (the buffer included in the extraction kit comes in a dropper bottle, eliminating the need for an additional pipette) are added to remove any impurities.

[0086] 4. Place the column in a clean Eppendorf tube and elute the fully purified sample by adding 3 drops of elution buffer (the buffer included in the extraction kit comes in a dropper bottle so you don't have to use any extra pipettes to dispense the buffer) to the column.

[0087] The efficiency of extraction using the phytAlert Portable kit was compared to a commercial laboratory DNA extraction kit (non-portable) by analyzing DNA extractions performed with both methods using qPCR. Threshold cycles (CTs) were similar, with fully comparable and satisfactory results.

[0088] qPCR ANALYSIS OF THE PLANT SAMPLE

[0089] The extraction and detection kit allows for the analysis of up to 16 pathogens per sample, or the simultaneous analysis of one pathogen in 16 samples. Other combinations of samples and pathogens analyzed per sample are also possible. Laboratory thermocyclers for qPCR are typically bulky and delicate instruments. In this extraction and detection kit, the benchtop thermocycler has been replaced with a portable unit that operates on a 12V power supply. The disadvantage of the portable thermocycler is that it can perform only 16 reactions compared to the 96 reactions of the benchtop thermocycler. However, by using multiplex reactions, it is possible to analyze two microorganisms per reaction, ultimately allowing for the analysis of 32 different pathogens per sample with the portable thermocycler.

[0090] It was also verified that the portable and laboratory thermocyclers had the same precision and sensitivity, this result being totally satisfactory, obtaining a variation of less than 1 CT between both thermocyclers.

[0091] The thermocyclers compared were the Liberty16 Thermocycler included in the invention kit, and the laboratory 7500 Fast Real Time thermocycler.

[0092] For validation, one of the phytAlert qPCR kits, already validated and optimized by the inventors for the detection of a specific phytopathogenic microorganism, was used as a reference. The DNA detection and extraction kit used for equipment validation was the phytAlert monospecific kit for the identification of Alternaria spp.

[0093] VALIDATION OF THE LIBERTY16 PORTABLE THERMOCYCLER

[0094] The validation of the Liberty16 portable real-time system thermocycler was satisfactory, meeting the criteria of accuracy, reproducibility and limit of detection compared to the 7500 Fast Real-Time PCR System benchtop thermocycler.

[0095] To verify accuracy and reproducibility, five biological replicates were performed, each with three technical replicates of DNA extracted from a seedling infected with Alternaria spp. The standard deviation of the CT (Threshold of Detection) was then calculated between technical replicates belonging to each biological replicate, as well as the standard deviation between the biological replicates. In all cases, there was a variation of less than 1 CT between the two thermocyclers.

[0096] To verify the limit of detection, serial 1 / 10 dilutions were performed on a pure culture DNA extract of Alternaria spp., with an initial amount of 10 ng. The experiment was performed in triplicate. The limit of detection, as expected, was the same for both thermocyclers, achieving amplification of DNA as low as 10⁻⁴ ng. ELIMINATION OF THE NEED FOR A BENCHTOP CENTRIFUGE

[0097] DNA clarification involves the use of benchtop centrifuges; however, the possibility of performing this step using a small portable centrifuge that does not require a power connection was verified.

[0098] The objective was to verify that, even though the extract from the homogenized plant had considerable viscosity, the portable centrifuge would have sufficient power to pass the liquid through the porous matrix of the clarification column.

[0099] To do this, 20 different samples of turfgrass were homogenized in a bag with a mortar and pestle and passed through the clarification column as follows:

[0100] - 10 samples were centrifuged in the benchtop centrifuge

[0101] - 10 samples in the portable mini-centrifuge (Fisherbrand™)

[0102] It was verified that all 20 samples had passed through the porous matrix of the column successfully. Therefore, the portable minicentrifuge was validated as equipment for the portable laboratory of the invention.

[0103] OPTIMIZATION OF THE METHOD FOR PRESERVING THE MIXTURE OF REAGENTS NECESSARY FOR THE qPCR REACTION.

[0104] The objective of this task was to determine whether or not lyophilization was necessary for the preservation and transport of the qPCR reagents to be used. Lyophilization was performed in strips of 8-well tubes, as this is the format used by the Min¡8 real-time thermocycler.

[0105] For this activity the LM_Lyomycron 55 freeze dryer (Coolvacuum) was used, although any other freeze dryer with similar physical characteristics and properties would be equally valid.

[0106] For the development of the trial, 20 strips of qPCR tubes were prepared according to the corresponding mixture of probe / primers, for the detection of the phytopathogenic microorganisms of interest in each crop: turf, post-harvest and seedbed, totaling 60 strips of tubes.

[0107] TRIAL I: Comparison of Lyophilization vs Standard Reaction

[0108] In this assay, 30 of the 60 prepared test tube strips were used. These were used to analyze the microorganisms whose probes / primers had been inserted into the different wells of the strips, using DNA extracted from the pure culture of the corresponding pathogen as a template. The experiments were performed in duplicate. The CT value obtained for each detection of pure culture was established as a reference.

[0109] At the same time as this experiment was carried out, a replica of it was performed but with qPCR reaction mixtures prepared at the time, without lyophilization, in order to corroborate that there were no differences in the amplification threshold, since it was considered possible that lyophilization might affect the reaction mixture in some way.

[0110] The result of this test was positive, confirming that there was no deviation in the data obtained from the amplification thresholds of the different reactions, establishing that the reaction mixtures had not been affected by lyophilization.

[0111] TRIAL II: Study of the preservation of the freeze-dried strips

[0112] The remaining freeze-dried tube strips were stored at room temperature in a sealed bag.

[0113] Each week, two strips of tubes were analyzed in duplicate, under the same conditions as the first week, verifying that the CT data corresponded to the reference values.

[0114] The trial lasted a total of 9 weeks, with no significant differences observed in the CT corresponding to each reaction compared to the reference values ​​established in week 1.

[0115] OPTIMIZATION OF A SIMPLE SYSTEM FOR ADDING THE DNA SAMPLE TO THE REACTION WITHOUT THE NEED FOR MICROPIPETTES.

[0116] First, the amount of DNA extraction sample that could be added to the qPCR reaction mixture was optimized. The result of this trial was that only a small amount of DNA extracted from each culture could be added, since larger amounts produced almost complete inhibition of the qPCR reaction.

[0117] Since the amount of liquid (pl) is extremely small for an inexperienced operator to handle, a solution was sought that did not involve using a micropipette and that would also facilitate the handling of this small quantity. The result was the replacement of the 0.5 pl to 10 pl micropipette with a calibrated inoculating loop. This sterile loop is inserted into the Eppendorf tube containing the DNA extract from the sample, and then transferred to the reaction tube. In one particular embodiment, the portable kit of the invention consists of a strip of 8 tubes with 8 different mixtures; therefore, a total of 8 inoculating loops are required, using one loop per tube.

[0118] It was verified that the results obtained using this method showed no discrepancies with the results obtained using and adding 1 ul of sample with a micropipette, these being totally satisfactory, with a deviation of 0.25 CTs.

[0119] OPTIMIZATION OF MULTIPLEX AMPLIFICATION REACTIONS, THAT IS, DETECTION IN A SINGLE qPCR REACTION OF MORE THAN ONE MICROORGANISM.

[0120] In this case, the qPCR reaction was optimized to detect two microorganisms per well, since this would increase the performance of the portable qPCR thermocycler from 16 identifications to 32. Thus, in this way, twice as many microorganisms could be identified in the same time it would take to analyze one.

[0121] The duplex reactions were optimized according to the following scheme:

[0122] 1. Amplification of the pure cultures corresponding to each microorganism to be detected in each well. This analysis verifies that, under ideal conditions, the microorganisms are correctly detected in duplex.

[0123] 2. Secondly, it was verified that microorganisms present in a sample previously analyzed in single-well assays are correctly detected in duplex assays. In other words, performing the reactions in a well where two microorganisms are detected does not affect the results compared to identifying only one.

Claims

CLAIMS 1. A portable laboratory for the detection of phytopathogenic microorganisms from a plant sample comprising the following elements: at least one thermocycler suitable for qPCR, at least one minicentrifuge, at least one balance, at least one manual homogenizer suitable for breaking up the plant sample, and at least one kit for the extraction and detection of DNA specific to plant pathogens.

2. The portable laboratory, according to claim 1, wherein the manual homogenizer comprises a handle for holding a shaft terminated in a bearing surface for breaking the plant sample.

3. The portable laboratory, according to claim 1, wherein the thermocycler suitable for qPCR has the capacity to analyze up to 16 samples simultaneously.

4. The portable laboratory, according to any one of the preceding claims, comprising one or more of the following elements: at least one portable electronic device adapted to work in conjunction with the thermocycler and analyze the data generated by the latter, at least one external battery, at least one homogenization stand, at least one timer, at least one pair of scissors, at least one calibrated sampler and / or inoculation loop, at least one 2 ml tube holder 70% alcohol in water for disinfection.

5. The portable laboratory according to any one of the preceding claims, comprising a calibrated inoculation loop 6. The portable laboratory according to any one of the preceding claims, comprising a case conditioned for the transport of the elements of any one of claims 1 to 5.

7. The portable laboratory according to any one of the preceding claims, wherein the phytopathogenic microorganisms are fungi, viruses and / or bacteria.

Images