A LYOPHILISED RT-qPCR DIAGNOSTIC KIT FOR DIAGNOSTICS OF TOBRFV VIRUS

Abstract

The invention relates to a lyophilised RT-qPCR (real time- polymerase chain reaction) diagnostic kit developed against a virus called ToBRFV (Tomato brown rugose fruit virus), particularly in tomato plants. The invention also covers the production and application method of the diagnostic kit subject to the invention.

Description

[0001] A LYOPHILISED RT-qPCR DIAGNOSTIC KIT FOR DIAGNOSTICS OF TOBRFV VIRUS

[0002] Technical Field of The Invention

[0003] The invention relates to a lyophilized RT-qPCR (real-time polymerase chain reaction) diagnostic kit developed specifically against the virus named ToBRFV (Tomato brown rugose fruit virus) occurring in tomato plants.

[0004] Background of The Invention

[0005] Nowadays, it is known that various viruses affect the growth / fruiting periods of especially annual crops, causing them to spread rapidly in production areas and lead to significant yield losses.

[0006] Annual plants are those that complete their life cycle within one year. In other words, these plants grow, bloom, produce seeds, and eventually die within a single season. The production of annual plants holds a significant place in agriculture, and various species of plants fall into this category. Tomato (Solanum lycopersicurr) and pepper (Capsicum spp.) are among the widely cultivated annual plants.

[0007] Annual plants have important advantages. Firstly, because they grow quickly, products can be obtained in a short time. Additionally, their fast cycles from planting to harvest allow for quick adaptation to market demands. However, they also have significant disadvantages. Risks stemming from pests and climate changes can negatively affect annual plants. Viruses, in particular, can be quite effective on tomato and pepper plants, causing severe damage. These plants are susceptible to various viral infections, which can adversely affect their growth, yield, and overall health. Some types of viruses that have been commonly observed in tomato and pepper plants from past to present are already known, and various methods have been developed over time to prevent the damage caused by these viruses. In recent times, various tomato viruses have become effective, with the Tomato Brown Rugose Fruit Virus (ToBRFV), which emerged in 2019, posing a new threat. This virus primarily affects tomato and pepper plants, significantly impacting the quality and yield of fruits and thus creating high economic risk. Like all viral diseases, ToBRFV does not have a direct chemical control method, and if not detected early, it can spread rapidly in production areas, causing substantial yield losses.

[0008] In particular, in Turkey, the quality and yield of products such as tomatoes and peppers, which are annual plants, are of great importance for export. Additionally, for exports to many countries such as Russia and European Union countries, it is required that the Plant Health Certificate (PHC) specifies that the production areas for tomatoes and peppers are free from this virus. Samples from the products subject to export must be analyzed using PCR (Polymerase Chain Reaction) and qPCR (Quantitative Polymerase Chain Reaction). Especially for products like tomatoes and peppers, which are exported as fresh vegetables, the most critical stage is to quickly analyze the product during the waiting period for export, ensuring it remains fresh until delivery.

[0009] Therefore, it is crucial that both fresh vegetable and seed production companies, as well as the relevant organizations of the Ministry of Agriculture, analyze this virus using rapid and sensitive methods at every stage of production. Due to the stable nature of ToBRFV, its rapid spread in production areas not only directly affects production but also significantly hinders exports. This important viral agent has the potential to cause substantial economic problems and losses on a national scale.

[0010] Many different plant viruses can produce common or similar symptoms on the same plant. ToBRFV can also exhibit different disease symptoms in different climate conditions or in plants with different genetic structures. Sometimes, viruses may not even show symptoms on plants due to environmental factors and genetic structures. Therefore, visually detecting ToBRFV is very difficult and risky. Currently, molecular analyses such as the DAS-ELISA (Double Antibody Sandwich-Enzyme Linked Immunosorbent Analysis) method and the PCR (Polymerase Chain Reaction) method are widely used to diagnose viruses. The most reliable, sensitive, and standardized method for identifying ToBRFV at the species level is molecular diagnosis. The qPCR (Quantitative Polymerase Chain Reaction) method is among the most sensitive and rapid molecular diagnostic methods. The working principle of molecular methods is based on the amplification of nucleic acid (RNA or DNA) of the harmful organism, depending on enzymatic activity and temperature cycles. The enzymes used in the analysis typically need to be stored at -20 °C. Analysts generally need to work with multiple samples on ice to prevent enzyme activation from deteriorating. Ready-made "master mixes," which are mixtures of the enzymes and other chemicals used in the analysis, are also quite common nowadays. However, these mixtures are provided as stock and need to be diluted to a 1x reaction mixture for analysis. Additionally, since distributing the mixture into tubes or plates takes time, maintaining a cold environment is absolutely necessary to preserve conditions such as enzyme activation.

[0011] The current alternative products on the market include kits that work with the ELISA method and various brands of master mixes combined with primer-probe. Diagnosis using the current ELISA method takes 2 days. Additionally, these kits can be procured from abroad over extended periods through cold chain logistics. Although analyses performed with master mix and primer-probe mixtures yield results within 1 -2 hours, the commercial master mixes used in primer-probe studies require a cold chain for procurement and have import processes that can take 6-8 weeks. In these applications that require a cold chain, prolonged waiting periods can lead to a decrease in enzymatic activity.

[0012] Therefore, to address the aforementioned problems, there is a need for the development of a practical single-step RT-qPCR diagnostic kit that allows for the rapid and easy detection of the ToBRFV virus and enables the user to work comfortably at room temperature conditions. In the state of the prior art, there are existing methods and devices developed for the detection of different viruses. Conducting this detection quickly and accurately is crucial both during production and in the post-production clearance phase.

[0013] The Chinese patent CN 111363856B can be cited as an example of the state of the prior art. This document describes a method for simultaneously detecting five major viral pathogens affecting tomato plants, one of which is the Tomato Brown Rugose Fruit Virus (ToBRFV).

[0014] Another example of the state of the prior art is the Chinese patent application CN113481329A. Similar to the previous one, this document discusses a method for detecting five major viral pathogens affecting tomato plants, including ToBRFV.

[0015] A further example of the state of the prior art is the Chinese patent application CN115820930A. This document discusses an RT-qPCR (real-time quantitative polymerase chain reaction) detection method and its application for Tomato Spotted Wilt Virus (TSWV), which is carried by a single tomato seed.

[0016] In the examples given for the state of the prior art, there is no mention of a single-step, rapid diagnostic kit developed specifically for the ToBRFV virus that allows for operation at room temperature conditions. Therefore, there remains a need for the development of a diagnostic kit with these features in the field.

[0017] Additionally, specific primers for the detection of ToBRFV exist in the the prior art; however, these primers were developed in previous years and based on the viruses prevalent in the regions where they were created. There is a need to design more current primers specific to the viral genetic material, tailored to the isolates present in our country.

[0018] In conclusion, to overcome the disadvantages present in the prior art, there is a need for the development of a single-step diagnostic kit for the detection of ToBRFV. This kit should enable rapid and sensitive analysis and allow for operation at room temperature.

[0019] Detailed Description of The Invention

[0020] The invention relates to a lyophilized RT-qPCR (real-time polymerase chain reaction) diagnostic kit developed specifically against the ToBRFV (Tomato Brown Rugose Fruit Virus) occurring in tomato plants.

[0021] An aim of the invention is to develop a single-step RT-qPCR diagnostic kit for ToBRFV that recognizes all isolates of ToBRFV identified worldwide to date, does not require cold chain storage, and eliminates the need for laboratory specialists to work with lengthy protocols such as preparing primer-probe and master mix mixtures.

[0022] Another aim of the invention is to eliminate variations in application conditions by using a sensitive and stable method for diagnosing the ToBRFV virus. This ensures the most accurate detection regardless of environmental conditions.

[0023] The invention generally pertains to a diagnostic kit developed for the detection of ToBRFV in plants, comprising a master mix and a primer-probe mixture. The master mix and primer-probe mixtures are obtained through a lyophilization process.

[0024] In one embodiment of the invention, the master mix comprises the following components: i. Tris-HCI (Tris(hydroxymethyl)) (pH 8.4) ii. (NH4)aSO4 (Ammonium Sulfate) iii. KCI (Potassium Chloride) iv. dNTP (Deoxynucleoside triphosphate) v. MgSC (Magnesium Sulfate) vi. RT enzyme (RT, Reverse Transcriptase) vii. Taq polymerase (Thermus aquaticus (Taq) DNA polymerase) viii. Trehalose and / or qPCR protector ix. qPCR dye In another configuration of the invention, the master mix composition is more detailed as follows: In one embodiment of the invention, the specific nucleotide sequences of the primerprobe set are provided below. These sequences are referred to as SEQ NO:1 for the forward primer, SEQ NO:2 for the reverse primer, and SEQ NO:3 for the probe.

[0025] Primer-forward: AATGATAAAGAGGCGTG****** Primer-reverse: CGTCGATAAATAACCTTTT******

[0026] Probe: TCAAGCCACAAGAGATAA******

[0027] During the selection of the primer-probe set, tools such as Primer3 were used. Initially, approximately two hundred complete ToBRFV genomes were collected, analyzed, and conserved regions of ToBRFV were identified from the NCBI (National Center for

[0028] Biotechnology Information) database. Additionally, related species such as ToMV, TMV, and ToMMV were analyzed to prevent the primers from binding to unintended targets.

[0029] Here are the parameters and their values that were adjusted in Primer3: The design positions of the forward and reverse primers and the probe in the RdRp (RNA dependent RNA polymerase) region of the ToBRFV genome are shown in Figure 1.

[0030] During the validation phase of the selected primer-probe set, a Limit of Detection (LOD) study was conducted (Figure 2). The primers subject to the invention can detect ToBRFV at concentrations as low as 10’5in an experiment with 40 ng RNA concentration from an infected sample.

[0031] In a preferred embodiment of the invention, the process of quantifying and / or detecting the target RNA includes RT-PCR, preferably real-time RT-PCR (also known as "quantitative RT-PCR" or q RT-PCR).

[0032] In summary, the invention is a diagnostic kit for the detection of ToBRFV in plants, comprising a master mix and a ToBRFV-specific primer-probe mixture, with this mixture being subjected to a lyophilization process.

[0033] The scope of protection of the invention also includes the production method of the diagnostic kit. The process steps of this production method are as follows:

[0034] -developing of virus-specific forward, reverse, and probe primer sequences for the virus to be diagnosed and selecting of a primer-probe set that does not work incorrectly on different organisms and has high PCR efficiency,

[0035] -preparing the master mix,

[0036] -mixing the selected primer / probe set with the master mix in predetermined ratios.

[0037] -subjecting this mixture to a lyophilization process with the following steps:

[0038] • Drying at -60°C temperature and under pressure at 2.2 x 10A-2 mbar for at least two hours.

[0039] • Drying at -60°C temperature and under pressure at 1.1 x 10A-3 mbar for at least four hours. • Drying at -60°C temperature and under pressure at 1.5 x 10A-2 mbar for at least one hour.

[0040] • Treating with nitrogen under low pressure at -60°C temperature.*

[0041] *ln the final treatment mentioned above, the pressure is cut off.

[0042] In one configuration of the invention, the predetermined ratios of the selected primer / probe set to the master mix are as follows:

[0043] The total volume of the primer / probe mixture and the master mix mixture is 2.5 pL, with the master mix being 2 pL and the primer mixture being 0.5 pL (25 pM). In other words, the volume ratio of the primer mixture to the master mix mixture is 1 :4.

[0044] Additionally, a method for the diagnosis of ToBRFV using the diagnostic kit subject to the invention is also within the scope of protection. This method includes the steps of adding water and a genetic material (RNA) sample obtained from the plant to be diagnosed for the virus into the diagnostic kit subject to the invention, and running this sample under predetermined temperature and cycle conditions in a real-time PCR device. The flowchart related to this method is provided in Figure 4. In this figure, the lyophilized tube (1) with added nucleic acid is shown, as well as the steps of adding the nucleic acid (RNA) to each lyophilized tube (2) and performing the qPCR analysis on these tubes (3). In one embodiment of the invention, the volume ratio of the water to the RNA sample added to the diagnostic kit is 3:1.

[0045] In one embodiment of the invention, 15 pL of water and 5 pL of genetic material (RNA) sample are added. The predetermined temperature and cycle conditions specified in the mentioned method are as follows:

[0046] The diagnostic kit subject to the invention can be transported at room temperature and stored for 6 months. The single-step RT-qPCR efficiency value is at least 90%. The diagnostic kit subject to the invention is specific only to ToBRFV and does not detect other viruses from the same family, such as ToMV (Tomato mosaic virus), TSWV (Tomato spotted wiit virus'), and ToMMV (Tomato mottie mosaic virus).

[0047] The qPCR mixture in the tubes within the diagnostic kit contains a lyophilized master mix. Since all necessary consumables for the diagnostic experiment are included in the ready-to-use tubes within the kit, users only need to add the genetic material (RNA) to these tubes and run the temperature and cycle conditions specified in the kit's protocol on a real-time PCR device, all while working at room temperature. This simple application makes it feasible to use in any laboratory. Thanks to the use of this developed diagnostic kit, diagnostic laboratories will be able to quickly analyze products subjected to testing and detect the presence of ToBRFV with this highly sensitive method. The detection of ToBRFV, which is of great importance in production and export, will be carried out in a very fast, sensitive, and practical manner. This will accelerate the export process and help prevent the spread of the virus in production areas through swift actions.

[0048] At the national level, ELISA, PCR, and qPCR primer probe methods are commonly used in quarantine and research institute laboratories. The diagnostic kit subject to the invention differs from these methods with its primer sensitivity and the aim to detect widespread national isolates, its requirement of no cold chain, and its simple applicability by directly applying the RNA of the pathogen to strip tubes for analysis.

[0049] Internationally available ToBRFV diagnostic kits are currently in the form of ELISA kits. The single-step RT-qPCR diagnostic kit for ToBRFV, subject to this invention, stands out from the existing products of international kit manufacturers by being more sensitive and capable of detecting geographically specific isolates in Turkey and worldwide due to its primer-probe content.

[0050] The developed single-step RT-qPCR diagnostic kit also differs from the ELISA kits and primer-probe mixtures used in the current technology by its ease of application in the laboratory and its independence from the cold chain.

[0051] Since the product subject to the invention is optimized to operate and be transported at room temperature, it provides fast (within approximately 1 hour) and reliable results.

[0052] The geographical specificity of the diagnostic kit subject to the invention includes strains from America, Europe, Turkey, Africa, and Asia.

[0053] The diagnostic kit subject to the invention offers sensitive detection at the nucleic acid level and allows for virus quantification.

[0054] The primers included in the invention provide sensitive detection down to 10 copies, making it 10,000 times more sensitive than ELISA. The ability to perform sensitive detection even at low viral concentrations is due to the primer set contained in the invention.

[0055] With the diagnostic kit subject to the invention, application support can be provided to the manufacturing company.

[0056] The technological gain achieved with this kit can serve as a foundation for the development of other important diagnostic kits for viruses, bacteria, and fungal diseases.

[0057] The developed diagnostic kit is intended to be used in the quarantine laboratories and research institute laboratories of the Ministry of Agriculture and Forestry, in private seed and vegetable production companies, in universities, and in private laboratories for the detection of ToBRFV. It is used for the detection of ToBRFV as required by the Plant Quarantine Regulation during inspections by tomato and pepper vegetable producers and seed companies.

[0058] The following tests are provided to ensure product suitability:

[0059] 1. Logistics Test: Testing the product after transportation in cargo for 1 -14 days.

[0060] 2. Shelf Life Test: Testing the product after being kept at room temperature for 1, 3, and 6 months.

[0061] 3. Efficiency Test: Testing the PCR efficiency value of the product.

[0062] The previously mentioned LOD (limit of detection) tests fall within this scope. Accordingly, in the LOD study of the invention, 7 sub-dilution series (1 / 10, 1 / 100, 1 / 1000, 1 / 100000, etc.) were created, and the limit of detection was determined up to a sensitivity of 10A-5.

[0063] 4. Accuracy Test: Testing whether it works with different types of viruses.

[0064] The primer-probe included in the kit is specific to ToBRFV, and a cut-off value of 33 Cq (quantification cycle) is required. Accordingly, the kit subject to the invention was determined not to detect any viral agents from the Tobamovirus group, including ToBRFV, in in-silico analyses conducted on isolates registered with NCBI (National Center for Biotechnology Information). Additionally, in laboratory studies, the lyophilized diagnostic kit subject to the application did not detect other agents when a cut-off of 33 Cq was applied, while it detected the ToBRFV isolate at high intensity (Ct: 7.35). The graph demonstrating this is provided in Figure 3.

[0065] Table 1. Isolates known to be closely related to ToBRFV and their source codes:

[0066] 5. Sensitivity Test: Determining the capacity to detect at minimum virus load.

[0067] This has been determined through the LOD study. In conclusion, thanks to the invention subject to the application, a single-step RT-qPCR diagnostic kit for ToBRFV has been developed that recognizes all isolates of ToBRFV identified worldwide to date, does not require cold chain storage, and eliminates the need for laboratory specialists to work with long protocols such as preparing primerprobe and master mix mixtures, aiming to solve the problems in the current technology. Description of Figures

[0068] Figure 1. Appearance of primers and probes in the RdRp (RNA-dependent RNA polymerase) region.

[0069] Figure 2. qPCR results of our specific primers in the LOD study conducted in the wet laboratory run.

[0070] Figure 3. qPCR graph visual of the inclusivity analysis performed with the ToBRFV diagnostic kit.

[0071] Figure 4. A flowchart related to the ToBRFV diagnostic kit subject to the invention.

[0072] Reference List for Figure 4:

[0073] 1. Lyophilized tube with added nucleic acid.

[0074] 2. Illustration of adding nucleic acid (RNA) to each lyophilized tube.

[0075] 3. Conducting qPCR analysis by placing the lyophilized tube with added nucleic acid into the device.

Claims

Claims1. A diagnostic kit for the detection of ToBRFV in plants, characterized by comprising: a master mix comprising; i. Tris-HCI (pH 8.4) ii. (NH4)2SO4iii. KCI iv. dNTP v. MgSO4vi. RT enzyme vii. Taq polymerase viii. Trehalose and / or qPCR protector ix. qPCR dye and a primer-probe mixture, and wherein the mixture is obtained through a lyophilization process.

2. The diagnostic kit according to claim 1, wherein the master mix mixture comprises the following amounts, by volume (mM / pL / mg): i. Tris-HCI (pH 8.4)- 60 mM ii. (NH4)2SO4- 25 mM iii. KCI - 10 mM iv. dNTP- 10 mM v. MgSO4-1 mM vi. RT enzyme- 0.1 pL vii. Taq polymerase - 0.2 pL viii. Trehalose and / or qPCR protector -0.8 mg ix. qPCR dye- 0.025 pL3. The diagnostic kit according to claim 1 or 2, wherein the primer-probe set comprising:a) a primer-forward with the sequence SEQ ID NO:1, b) a primer-reverse with the sequence SEQ ID NO:2, and c) a probe with the sequence SEQ ID NO:3.

4. The diagnostic kit according to claim 3, wherein the primer-probe set comprising: a) a forward primer with the sequence SEQ ID NO:1, with a volume of 25 pM, b) a reverse primer with the sequence SEQ ID NO:2, with a volume of 25 pM, and c) a probe with the sequence SEQ ID NO:3, with a volume of 12,5 pM.

5. The diagnostic kit according to any of claims 1 to 4, wherein the lyophilization process comprising the steps of:- drying at -60°C temperature and 2.2 x 10’2mbar pressure for at least two hours,- drying at -60°C temperature and 1.1 x 10’3mbar pressure for at least four hours,- drying at -60°C temperature and 1.5 x 10’2mbar pressure for at least one hour,- treating with nitrogen at -60°C temperature.

6. A method for producing a diagnostic kit according to any of the preceding claims, for the detection of ToBRFV in plants, characterized by comprising the steps of:- developing virus-specific forward, reverse, and probe primer sequences for the virus to be diagnosed, and selecting a primer-probe set with high PCR efficiency that does not yield false results on different organisms,- preparing the master mix,- mixing the selected primer-probe set with the master mix in predetermined ratios,- subjecting this mixture to a lyophilization process comprising the steps of:- drying at -60°C temperature and 2.2 x 10’2mbar pressure for at least two hours,- drying at -60°C temperature and 1.1 x 10’3mbar pressure for at least four hours,- drying at -60°C temperature and 1.5 x 10’2mbar pressure for at least one hour,- treating with nitrogen at -60°C temperature.

7. The method according to claim 6, wherein the volume ratio of the primer-probe set to the master mix is 1:4.

8. A method for using the diagnostic kit according to any one of claims 1 to 5, for the detection of ToBRFV, characterized by comprising the steps of:- adding water and a sample of genetic material (RNA) obtained from the plant to be diagnosed for the virus into the diagnostic kit,- running the obtained sample in a real-time PCR device under predetermined temperature and cycle conditions.

9. The method according to claim 8, wherein the volume ratio of the water to the RNA sample added to the diagnostic kit is 3:1.

10. The method according to claim 8 or 9, wherein 15 pL of water and 5 pL of RNA sample are added to the diagnostic kit.

11. The method according to any one of claims 8 to 10, wherein the predetermined temperature and cycle conditions are as follows:

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