Design of a primer set for the diagnosis of foot-and-mouth virus (FMD) a, o, asia1 serotypes in turkey by loop-mediated isothermal amplification (RT-lamp)

Abstract

The present invention relates to primer sets for the diagnosis of foot-and-mouth disease and to a method for the diagnosis of foot and mouth disease using said primer sets by the Loop Mediated Isothermal Amplification (RT-LAMP) method. With the primers of the present invention and the diagnostic method using these primers, A, O and Asia 1 serotypes of foot and mouth virus can be diagnosed quickly and with high sensitivity.

Description

[0001] DESIGN OF A PRIMER SET FOR THE DIAGNOSIS OF FOOT-AND-MOUTH VIRUS (FMD) A, O, ASIA1 SEROTYPES IN TURKEY BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (RT-LAMP)

[0002] Field of the Invention

[0003] The present invention relates to a primer set for the diagnosis of foot-and-mouth disease and to a method for the diagnosis of foot-and-mouth disease using said primer sets by the Loop Mediated Isothermal Amplification (RT-LAMP) method. With the invention, fast and high sensitivity diagnosis of foot and mouth disease is provided.

[0004] State of the Art

[0005] Foot-and-mouth disease is a viral disease that can be seen in all cloven-hoofed animals. The disease occurs in the presence of the Aphtovirus subgroup (genus) of the Picornaviridae family and can be transmitted to humans in rare cases [1]. FMD has high morbidity and mortality, especially in young animals. It may persist in the esopharynx in animals that have experienced the disease. In this case, in cases where immunity decreases in old outbreaks, the epidemic may re-emerge without transmitting the virus from outside. The disease is transmitted between animals through respiration and oral secretions, and the rate of transmission appears to be much faster in herds. Lesions occurring in the nail, mouth and udder areas of animals exposed to the disease are among the first symptoms of the disease. Owners of animals showing the above-mentioned symptoms are obliged to make relevant notifications regarding the existence of suspicion of foot-and-mouth disease. The reason why reporting FMD is mandatory is that the disease can cause poor condition in adult animals and death in puppies [2], Although there is no treatment method for animals with the disease, antibiotics and antiviral drugs are used to prevent secondary infection since they are susceptible to other infections. The biggest preventive measure in the fight against foot and mouth disease is to ensure herd immunity through vaccination and control of animal movements. For this reason, inactive vaccination is applied to cattle in Turkey twice a year. Vaccination of small cattle is not mandatory, but optional businesses can do so. In addition, in regions where outbreaks occur, outbreak vaccination is carried out within a radius of 3-10 km. A dominant vaccination decision has been taken in 2024 and vaccination will be carried out 3 times a year. The currently used FMD vaccines are tetravalent vaccines suitable for all 3 serotypes. However, antibody response / immunity may not occur in every vaccinated animal, or the antibody response that occurs may decrease rapidly. Newborn animals and animals without an antibody response remain susceptible to infection until the next vaccination. This disease, which has no cure, can easily spread due to animal movements at points where vaccination fails. Loss of meat and milk in infected animals causes serious economic losses. Therefore, rapid and accurate detection of the presence of FMD is of great importance to prevent the spread of the disease and to quickly perform outbreak vaccinations.

[0006] The virus that causes the disease has 7 known serotypes named A, O, C, Sat 1 , Sat 2, Sat 3 and Asia 1. Among these serotypes, A, O and Asia 1 are commonly seen in our country. Detecting these serotypes in animals is the first step in dealing with the disease. Many tests are now used to determine which animals in the rest of the herd are infected by animals with foot-and-mouth disease symptoms.

[0007] In the state of the art, virus isolation test methods are used to detect foot and mouth disease. These test methods require a laboratory, special devices and technical personnel. Virus isolation procedures performed during the tests take at least 48 hours, and expensive equipment such as a BSL-2 security cabin is needed during this process. However, as a result of the tests, only the presence of viruses that can cause cytopathogenic effects (CPE) can be detected.

[0008] The detection of foot and mouth virus is carried out with the PCR test used in the state of the art. Since these PCR tests are molecular testing methods, expensive equipment is required to obtain RNA / DNA before the test. Prepared samples are analysed by technical personnel in PCR devices. Preparation of samples before analysis takes approximately 1 hour, and while real-time PCR tests take 3 hours, this time can be up to 5 hours in conventional PCR tests. This is because electrophoresis and expensive imaging devices are needed to visualize the results of convectional PCR assays. In the antigen ELISA tests used in the state of the art, the test results performed by technical personnel are obtained within 4 hours at best. In addition, these results need to be evaluated with devices that are large and portable.

[0009] The limitations and inadequacies of the state of the art solutions, the expensive equipment required for the tests, the need for trained technical personnel for the tests, the fact that it takes more than three hours to obtain test results, and the fact that these tests can only be performed in a laboratory environment have made it necessary to make development in this field.

[0010] Brief Description and Objects of the Invention

[0011] The present invention describes primer sets to be used in the diagnosis of foot-and- mouth disease and the method that enables the diagnosis of foot and mouth disease by the Loop-Mediated Isothermal Amplification (RT-LAMP) method using these primer sets. With this diagnostic method, foot and mouth disease is detected quickly, with high sensitivity and without the need for expensive equipment.

[0012] In the invention, foot and mouth disease serotypes are diagnosed by RT-LAMP using the A4 primer set with the nucleotide sequence SEQ ID NO: 1 -6 of the A serotype, the ASIA 1 serotype using the AS5 primer set with the nucleotide sequence SEQ ID NO: 7-12 and the O serotype using the ARYA4 primer set with the nucleotide sequence SEQ ID NO: 13-18 or the OL1 primer set with the nucleotide sequence SEQ ID NO: 19-24.

[0013] The object of the present invention is to provide primer sets that enable the rapid detection of foot and mouth disease. With the primers used in the method described in the invention, A, O, ASIA1 serotypes that cause foot and mouth disease are detected quickly and with high sensitivity. In this way, unlike the state of the art tests, the presence of foot and mouth disease in animal samples and the serotype of the disease can be quickly determined. With the help of the rapid detection of the disease, the necessary quarantine measures can be taken immediately.

[0014] Another object of the present invention is to provide field and rapid diagnosis of foot and mouth virus serotypes commonly seen in Turkey. With the primer sets of the present invention, A, O and ASIA 1 serotypes, which are the most common foot and mouth virus serotypes in Turkey, can be detected quickly in the field. With the rapid monitoring of the presence of these serotypes in the field, necessary measures can be taken and possible problems can be prevented.

[0015] Another object of the present invention is to ensure the detection of foot and mouth disease with high sensitivity and specificity. With the large number of primers used in the method of the present invention, a method with higher specificity is provided compared to other PCR tests in the state of the art.

[0016] Another object of the present invention is to use monovalent or bivalent vaccines appropriate to the detected serotype instead of tetravalent vaccines in outbreak vaccinations, which can reduce the raw material cost in vaccine production by 2 to 4 times.

[0017] Another object of the present invention is that when serotype determination is made quickly in the farm where foot and mouth disease occurs, the traceability and control of animal movements will increase, even if the farm owner is not open to sharing information about anamnesis and animal movements. Additionally, operators who see that they can be tracked may become more sensitive and open to sharing information over time.

[0018] Description of the Figures

[0019] Figure 1 : Method scheme

[0020] Figure 2: Diagnostic comparison of 88 serotype O samples (OL1 and ARYA4 primers)

[0021] (Vl*(+ / -): Virus Isolation (With this test, only CPE is detected.), RT-PCR / O: O serotype conventional reverse transcriptase PCR, qRT-PCR / O: O serotype real-time reverse transcriptase PCR, AR4 qRT-LAMP / O: Real-time reverse transcriptase LAMP, OL1 qRT-LAMP / O with O serotype AR4 primers: Real-time reverse transcriptase LAMP with O serotype OL1 primers)

[0022] Figure 3: Diagnostic comparison of 43 serotype A samples (A4 primers) (Vl*(+ / -): Virus Isolation (With this test, only CPE is detected.), RT-PCR / A: A Serotype conventional reverse transcriptase PCR, qRT-PCR / A: A Serotype real-time reverse transcriptase PCR, A4 qRT-LAMP / A: Real-time reverse transcriptase LAMP with A Serotype A4 primers) Figure 4: Diagnostic comparison of 37 Asia serotype samples (AS5 Primers) (RT-PCR / As: Asia serotype conventional reverse transcriptase PCR, qRT-PCR / As: Asia serotype real-time reverse transcriptase PCR, AS5 qRT-LAMP / As: Real-time reverse transcriptase LAMP with Asia serotype AS5 primers)

[0023] Detailed Description of the Invention

[0024] The present invention discloses primers for rapid and highly sensitive detection of foot and mouth disease-causing serotypes A, O, ASIA1 by Loop-Mediated Isothermal Amplification (RT-LAMP) and a method using these primers.

[0025] In the invention, foot and mouth disease serotypes are diagnosed by RT-LAMP using the A4 primer set with the nucleotide sequence SEQ ID NO: 1 -6 of the A serotype, the ASIA 1 serotype using the AS5 primer set with the nucleotide sequence SEQ ID NO: 7-12 and the O serotype using the ARYA4 primer set with the nucleotide sequence SEQ ID NO: 13-18 or the OL1 primer set with the nucleotide sequence SEQ ID NO: 19-24. In addition to being fast and highly sensitive, the diagnostic method of the invention also provides convenience because it can be applied in the field.

[0026] The primers that enable the diagnosis of the invention were produced in amounts of 50 nmol. Primers were designed using the free Primer Explorer V5 primer design software with target genome sequences of specific lengths. Following in silico checks of the primers, those found suitable were ordered and tested with known positive samples. Specificity and sensitivity checks were performed in comparison with PCR. The process steps are detailed below. A4 primer set with SEQ ID NO: 1 -6 nucleotide sequence for serotype A, AS5 primer set with SEQ ID NO: 7-12 nucleotide sequence for ASIA 1 serotype and ARYA 4 and OL1 primers with SEQ ID NO: 13-18 and SEQ ID NO: 19-24 nucleotide sequences for O serotype are used in the diagnosis of foot and mouth disease with RT-LAMP. Each internal primer of a set of 5 primers designed with each target genome was checked in silico with the MEGA 6.06 genome sequencing and phylogenetic analysis program. In addition, the delta G values of the 3' and 5' ends of the primers were examined and the primers were evaluated to preliminary examine whether they would work or not. Primers were tested with a specially prepared positive control (with PK specific to each serotype) and a negative control virus with known starting titers close to the virus for which they were designed or the genome of that virus. It has been optimized to run more samples with the A4, AS5, AR4, OL1 primer sets, which were found to work the fastest and most specifically (with negative control results).

[0027] For analytical sensitivity tests, each primer set was reconstituted at Iog10 of its positive control virus with a real-time Optigene Genie III LAMP device and tested in comparison with real-time RT-PCR (qRT-PCR) and conventional RT-PCR.

[0028] Table 1. 0L1 Primer Analytical Sensitivity Tests Table 2. ARYA4 Primer Analytical Sensitivity Tests

[0029] Table 3. A4 Primer Analytical Sensitivity Tests

[0030] Table 4. AS5 Primer Analytical Sensitivity Tests

[0031] Each primer was tested with positive field samples of previously known serotype in order to determine the diagnostic sensitivities of its own serotype. The same samples specific to each serotype were subjected to comparative serotyping testing with realtime RT-PCR and conventional RT-PCR, and the test results are shown in Figures 2- 4. Figure 2 describes the diagnosis of 88 archive samples known to be serotype O with qRT-LAMP and their comparison with qRT-PCR, RT-PCR and Virus Isolation test. According to Figure 2, 82 samples were detected with the qRT-PCR test, 61 samples with the RT-PCR test, 76 samples with the OL1 primer set in the qRT-LAMP test, and all 88 samples with the ARYA4 primer set. With virus isolation, 57 samples showed CPE. Figure 3 describes the diagnosis of 43 archive samples known to be A serotype with qRT-LAMP and their comparison with qRT-PCR, RT-PCR and Virus Isolation test. According to Figure 3, 43 A serotype samples were detected with the qRT-PCR test, 39 with the RT-PCR, and 41 with the A4 primer set in the qRT-LAMP test. With virus isolation, 27 samples showed CPE. Figure 4 describes the diagnosis of 37 archive samples known to be Asia serotype with qRT-LAMP and their comparison with qRT-PCR, RT-PCR and Virus Isolation test. According to Figure 4, 30 Asia serotypes were detected with the qRT-PCR test, 37 with the RT-PCR, and 37 with the AS5 primer set in the qRT-LAMP test. CPE presence / absence analysis was also detected with virus isolation tests.

[0032] Each invention-specific primer set, exotic, local and vaccine virus prototypes of different serotypes and subtypes, isolates from the archive of the Foot and Mouth Disease Institute and IBR, BVD, PPR, BT inactivated viruses representing other animal diseases from Etlik VKAE for differential diagnosis were used in originality / specifity tests. It was determined that all primer sets were specific for their own serotype and other diseases. In addition, 0 / lnd2001d(Bhryn) exotic strain with OL1 , OI Manisa vaccine prototype with ARYA4 primer, A-05(2019) exotic strain with A4, Asial / Shamir exotic strain with AS5 primer could be identified. In addition, the ability to detect strains that are unique to its own serotype but exotic strains of its own serotype supports the rapid detection of an outbreak that may occur with uncontrolled animal movements. Table 5. Tests Between Primer Sets (A4, AS5, ARYA4 and OL1 ) and Viruses (IBR,

[0033] BVD, PPR and BT)

[0034] It was determined that the invention primers did not form primer-dimers and the specificity was tested comparatively with qRT-PCR and RT-PCR tests with negative samples. Table 6. Testing of negative samples with Primer Sets (A4, AS5, ARYA4 and OL1 )

[0035] For the preparation and extraction of the samples to be tested; recent field samples (epithelium-heart etc.) for each serotype were obtained from the archive and homogenized in a sterile mortar with sterile sand and PBS. The supernatants (inoculum) of the samples centrifuged at 3000 rpm for 10 minutes were portioned and stored at -80 °C for use in the tests. In addition, different strains, IBR, BVD, PPR, BT inactive viruses used in the specificity test were taken as isolates from the Alum Institute archives and Etlik VKAE, portioned for tests and stored at -80 °C. Nucleic acid extraction was performed with the ready -to-use Total RNA / DNA Extraction kit specific to the Techstar Extraction device, by applying the kit protocols.

[0036] Made for comparison and control purposes; presence-absence analysis of foot and mouth virus was performed by conventional PCR using literature primers. Presenceabsence analysis is performed by qRT-PCR and serotype determination is performed by mpx-qRT-PCR using primer-probes developed in TAGEM / HSGYAD / 13 / A02 / 902 / 19 ‘Development and Validation of Multiplex Molecular Methods for Differentiation of Foot and Mouth Virus A, O, and Asial Serotypes / 2016' project supported by TAGEM and used in routine diagnosis of Foot and Mouth Institute. Probes labeled with serotype O ROX, serotype A with JOE, and serotype Asia with FAM were used. AgPath-IDTM One-step RT-PCR Kit PCR mastermix enzyme was used according to its own protocol. For PCR, mastermix, instrument and primer concentrations are the optimized amounts used in the routine. Qiagen Rotorgene and Roche LightCycler480 II PCR devices were used for real-time PCR reactions. The content of the PCR Mixture A / O / Asia (Monoplex) mixture prepared for the real-time RT-PCR test is given in Table 7 and the content of the PCR Mixture Serotype / Universal (Multiplex) mixture is given in Table 8. 2.5 pl of nucleic acid is added to each of the mixtures given in Table 7 and Table 8. Then, real-time RT-PCR test is applied to these mixtures with the parameters specified in Table 9. In this test, both multiplex and monoplex primers and mastermixes are prepared in the amounts indicated in the figure and the nucleic acids (RNA) of the samples are added and real-time PCR devices are started according to the protocol indicated in the figure. Since the study is carried out with a probe, mastermixes are prepared in an environment without direct fluorescent light. Positive samples give sigmoid graph and CT (cycle time) value. Table 7. PCR Mix A / O / Asia (Monoplex) Content for Real-Time RT-PCR Testing

[0037] (*Serotype-specific qRT-PCR primer is added, **Probes are marked with A-JOE, O- ROX, Asia-FAM, Presence-absence(+ / -)-FAM)

[0038] Table 8. PCR Mix Serotype / Universal (Multiplex) Content for Real-Time RT-PCR Testing

[0039] (*Serotype-specific qRT-PCR primer is added)

[0040] Table 9. Real-Time RT-PCR Test Parameters

[0041] (NOTES: The Rotorgene device has a small tube option that can give the same results with half the PCR mixture. In this case, the same result is obtained when the test is performed by adding half of the PCR mixture and 1 .25 ul of nucleic acid.)

[0042] PCR Mixture A / O / Asia (Monoplex) prepared for the conventional RT-PCR test is given in Table 10 and PCR Mixture Serotype / Universal (Multiplex) is given in Table 11. 2,5 pl of nucleic acid is added to each of the mixtures given in Table 10 and Table 11. Then, conventional RT-PCR test is applied to these mixtures with the parameters specified in Table 12. In this test, both multiplex and monoplex primers and mastermixes used routinely are prepared as indicated in the figure and nucleic acids (RNA) of the samples are added and PCR products are stained with Looding Dye (amplicon dye) and loaded into the wells in the gel prepared with 2% agarose after 2-2,5 hours of protocol. It is carried out in an Electrophoresis device containing TBE Buffer at 145 volts for 30-45 minutes. After the imaging is taken, the band sizes are measured and the results are evaluated. Table 15 shows the comparison results of serotype determination detection times of qRT-LAMP, qRT-PCR and RT-PCR. For this comparison, 88, 43 and 37 positive samples were tested for serotypes O, A and Asia, respectively. Although the qRT-LAMP test directly shows the minutes of detection, for qRT-PCR this needs to be calculated based on CT values. In RT-PCR, the results are not known until imaging is performed. qRT-LAMP can determine serotypes for all serotypes in half the time compared to qRT-PCR, and results are obtained 7-8 times faster than RT-PCR.

[0043] Table lO.Content of PCR Mix A / O / Asia (Monoplex) for conventional RT-PCR testing

[0044] (*Serotype-specific RT-PCR primer is added)

[0045] Table 11.PCR Mixture Serotype / Universal (Multiplex) Content for Conventional RT- PCR test

[0046] (*Serotype-specific RT-PCR primer is added) Table 12. Conventional RT-PCR test parameters

[0047] (Execution in Gel: The PCR product stained with loading dye was electrophoresed with BIO-RAD Model 1000 / 500 Power provider at 145 volts from negative to positive in TBE buffer solution from negative to positive by loading 3-5pl in Gel wells prepared with 2% agarose and electrophoresed for 30-45 minutes and Imaging: Taken in 10 seconds with the GEL LOGIC 1500 Imaging system and UV lamp.)

[0048] Table 13. Real Time PCR Protocol Used in the Study

[0049] For conventional PCR, Apliied Biosystems MiniAmp Plus Thermal Cycler PCR device, Canon machine imaging cabinet and electrophoresis device are used.

[0050] Table 14. Conventional RT-PCR Protocol Used in the Study

[0051] Studies were carried out in separate tubes at separate times for the determination of each serotype. In the reaction consisting of 6 primers; front forward (SEQ ID NO:1 , SEQ ID NO:7, SEQ ID NO:13 and SEQ ID NO:19) and reverse back (SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14 and SEQ ID NQ:20) primers are prepared as 5 pmol per reaction, loop forward (SEQ ID NO:5, SEQ ID NO:11 , SEQ ID NO:17 and SEQ ID NO: 23) and reverse loop (SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18 and SEQ ID NO:24) primers were prepared as 25 pmol, and the front inner (SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:21) and reverse outer (SEQ ID NO:4, SEQ ID NQ:10, SEQ ID NO:16 and SEQ ID NO:22) primers were prepared as 50 pmol.

[0052] Optigene ISQ-004 mastermix was used in mastermix selection because it is specific for RNA viruses. 15 pl mastermix, 5 pl primer mix, 5 pl nucleic acid were used in the reactions.

[0053] Optigene Genie III LAMP device was used for the reaction. Although the reaction was carried out for 60 minutes at a constant temperature of 65 °C, the reactions were terminated early in cases where they responded faster. The reaction was terminated by applying a temperature of 95 °C for 1 minute, and the annealing temperature was measured by decreasing to 80 °C with a rate of 0,05 °C / sec. The fluorescent radiation in Mastermix is read from the blue channel and shows a peak on the device screen in case of a positive reaction. The device measures these radiations every 15 seconds and creates a sigmoid curve graph.

[0054] While evaluating the results; the similarity of the annealing temperature is strictly observed to ensure its authenticity. Samples giving different annealing temperatures are considered negative even if they give a peak. It is repeated even if it gives different temperatures in places close to the annealing temperature (0.5-2 °C less or more). Samples showing a characteristic sigmoid curve are considered positive if their annealing temperatures are compatible. Table 15. Comparison of serotype determination detection times of qRT-LAMP, qRT- PCR and RT-PCR

[0055] REFERENCES

[0056] [1] Ince, O.B., Kanat, 0. “§ap Hastaligi”. Etlik Vet Mikrobiyol Derg, 2015; 26 (2): 45-

[0057] 51 2022. p.33-7.

Claims

CLAIMS1. Primer sets for the diagnosis of foot and mouth disease A, O and ASIA 1 serotypes by Loop Mediated Isothermal Amplification (RT-LAMP) method, comprising;• A4 primer set with nucleotide sequence SEQ ID NO: 1 -6 for A serotype,• AS5 primer set with nucleotide sequence SEQ ID NO: 7-12 for ASIA 1 serotype,• ARYA4 primer set with nucleotide sequence SEQ ID NO: 13-18 or OL1 primer set with nucleotide sequence SEQ ID NO: 19-24 for O serotype.

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