Primers for diagnosis of foot and mouth disease sat2 serotype topotype-xiv virus by lamp method

Abstract

The invention relates to a primer set to be used in the diagnosis of foot and mouth disease and a method for diagnosing foot and mouth disease using the LAMP (Loop Mediated Isothermal Amplification) method using said primer set. By means of the use of said primer set with the LAMP method, the diagnosis of foot and mouth disease SAT2 serotype XIV genotype can be achieved within 15-20 minutes.

Description

[0001] PRIMERS FOR DIAGNOSIS OF FOOT AND MOUTH DISEASE SAT2 SEROTYPE TOPOTYPE-XIV VIRUS BY LAMP METHOD

[0002] Technical Field

[0003] The invention relates to a primer set to be used in the diagnosis of foot and mouth disease and a method for diagnosing foot and mouth disease using the LAMP (Loop Mediated Isothermal Amplification) method using said primer set. By means of the use of the primer set with the LAMP method in the invention, the diagnosis of foot and mouth disease can be achieved in a very short time.

[0004] State of the Art

[0005] Foot and mouth disease is an acute, highly contagious and economically highly destructive viral infection of cloven-hoofed animals such as cattle, sheep, goats and pigs

[0001] . The disease was widespread in Europe, Asia, Africa, North and South America during the 19thcentury, and was seen only once in Australia. The development of industrial farming has led to attention being focused on foot and mouth disease, and the consequences of the disease have been found to be more important than the acute disease. The disease causes a cessation of milk yield (lactation) in dairy herds and a 25% decrease in milk production, often as a result of complications with mastitis, and a delay in development in beef herds. In various countries, the disease has been eradicated by slaughtering infected animals or effective vaccination programs. Although America, Australia, New Zealand, Japan and European countries are free from foot and mouth disease, no country is safe from disease because the disease is enzootic all the world. The causative agent of the disease is foot and mouth virus, which is in the Aphtovirus subgroup of the Picornaviridae family. Foot and mouth virus has 7 antigenically distinct serotypes (A, O, Asial , C, SAT1 -2-3) and many subtypes. The virus shows varying sensitivity to physical factors. It is heat-resistant and is destroyed and inactivated in 12 hours at 37°C, in 1 / 2 hour at 60-65°C, and in a few minutes at 85°C. However, it is quite resistant to low temperatures and sudden freezing and thawing. In infected carcasses, it is rapidly inactivated due to lactic acid formation in 24-48 hours at +4°C, while it can persist for a long time in blood, bone marrow, lymph nodes and internal organs, and can remain active for a long time in skeletal muscles in sudden freezing. In addition, the rate of transmission of the disease is high and can reach up to 100% in sensitive and young unvaccinated animal populations. For this reason, the disease is of great economic, political and commercial importance.

[0006] Foot and mouth disease spreads directly and indirectly. The most obvious way of spreading the disease is by taking the virus in the air in through the respiratory system. Infected or incubating animals spread the virus through respiration, skin, secretions and extracts, milk and sperm. The disease can also be transmitted through contaminated animal products, contaminated vehicles and tools, humans, wild animals, birds, wind and transportation vehicles. In cattle, fever, loss of appetite, depression and decreased milk yield are the first clinical findings. Saliva flow begins within 24 hours and vesicles form on the tongue and gums. Vesicles can be found in the interdigital region, coronary region, udder skin, mouth and nasal mucosa. Extensive ulcerative wounds can form with the rupture of vesicles. While wounds (lesions) on the tongue usually heal in a few days, lesions on the feet and nasal region are often exposed to secondary bacterial infections. Pneumonia and mastitis may occur as a result of secondary bacterial infections, and nails may fall off. While the disease is milder in sheep and goats, it is usually characterised by lameness in sheep and lameness is persistent. Lesions in the mouth are smaller and shorter-lasting than lesions in cattle. Generally, the economic losses caused by the disease are lower than in cattle and clinical findings can only be determined with careful observation. Although the mortality rate of foot and mouth disease is low, cases of myocarditis resulting in death can be seen in young animals due to the localisation of the virus in the heart. The morbidity rate of the disease is high, and economic losses due to the rapid decrease in meat and milk yields are important.

[0007] There are 3 main strategies applied for the control of foot and mouth disease: slaughter, quarantine and vaccination. The slaughter process involves slaughtering animals infected with foot and mouth disease and animals that have come into contact with them. The main aim here is to eliminate the main virus source and break the life cycle of the virus. The disadvantage of this method is that it causes a large loss of animals. This method is applied in countries that have discontinued the vaccination program where foot and mouth disease is sporadic. The desired result with the vaccination method is to provide high antibody levels in the animal population in countries where the disease is widespread. To this end, it is aimed to provide resistance to the virus in the population with regular and intensive vaccination programs. However, quarantine and prophylactic measures are needed to achieve success. Factors such as the epidemiology of the disease, environmental factors, and the culture level of farmers are also important in determining the vaccination strategy. The general procedure applied in the state of the art is that the animal owner / keeper informs the official authorities about the presence of a disease, the farm visit and examination by the official authorities, and if the suspicion of disease persists, the quarantine process begins. Then, samples are taken and sent to the reference laboratory. In the laboratories, the presence-absence analysis is followed by serotype and subtype determinations. These can take up to 5-10 days including the cargo and laboratory procedures. The 3-10 km radius area where the quarantined enterprise is located is vaccinated with the outbreak vaccination procedure.

[0008] In the diagnosis of foot and mouth disease, clinical findings raise suspicion of the disease, but definitive diagnosis is made using virological or serological methods. In differential diagnosis, infections that cause lameness, mucosal erosions, salivation, nasal discharge and breast lesions are also taken into consideration. If epizootiological and clinical findings raise suspicion of foot and mouth disease, diagnosis is made in the laboratory by agent isolation or serological methods. In the state of the art, foot and mouth antigen is detected from incoming pathogenic materials with ELISA and Multiplex polymerase chain reaction (PCR) tests and the serological type of the virus is determined. However, while the virus isolation process takes 48 hours, ELISA takes 4-5 hours, and PCR takes at least 2 hours taking the extraction process (30-45 minutes) into account. In addition, the ELISA method does not have high sensitivity and is not suitable for certain types of clinical samples. Although the PCR method has higher sensitivity compared to ELISA, it can give false positive results [2],

[0009] The patent application WO2019151764A1 in the state of the art relates to seven types of foot and mouth disease virus serotype diagnostic primers and their use. In said document, the RT-LAMP method is used with a primer set targeting seven serotypes of foot and mouth disease virus to increase the efficiency of the amplification reaction. It is stated that it is used to detect seven serotypes of foot and mouth disease virus with only a low concentration of the sample to verify the high sensitivity and excellent specificity of foot and mouth disease virus. However, the method described in said document analyses the presence or absence of foot and mouth disease virus without distinguishing between serotypes.

[0010] Due to the limitations and inadequacies of the solutions in the state of the art, the inability to determine the type-specific serotype of foot and mouth disease, which leads to economic losses, the long time it takes to diagnose the disease, the low sensitivity of the primers and methods used in diagnosis, and the high probability of giving falsepositive results, it has become necessary to make improvements in the diagnosis of foot and mouth disease

[0011] Brief Description and Aims of the Invention

[0012] In the invention, a primer set to be used in the diagnosis of foot and mouth disease and a method for diagnosing foot and mouth disease using said primer set using the LAMP (Loop Mediated Isothermal Amplification) method is described. By means of the use of said primer set with the LAMP method, the diagnosis of foot and mouth disease can be achieved within 15-20 minutes. The primer set of the invention comprises primers with the nucleotide sequence of SEQ ID NO: 1 -6, primers with the nucleotide sequence of SEQ ID NO: 7-12, primers with the nucleotide sequence of SEQ ID NO: 13-18, primers with the nucleotide sequence of SEQ ID NO: 19-24 or primers with the nucleotide sequence of SEQ ID NO: 25-30.

[0013] The first aim of the invention is to provide a primer set that provides fast and sensitive results for use in the diagnosis of foot and mouth disease.

[0014] Another aim of the invention is to diagnose foot and mouth disease in a short time. With the method of the invention, foot and mouth disease can be diagnosed within 15- 20 minutes.

[0015] Another aim of the invention is to determine the serotype of foot and mouth disease. By means of the primer set of the invention and the use of the primer set of the invention with the LAMP method, it is possible to diagnose foot and mouth disease according to serotype, rather than the presence-absence analysis. By means of the primer set of the invention, SAT2 serotype can be detected. Description of Drawings

[0016] Figure 1. Comparison of RNAs of IRAQI samples with LAMP and RT-PCR performed with the primers of the invention

[0017] Figure 2. Comparison of the primers of the invention from field samples in Turkiye and results obtained after the LAMP method with RT-PCR.

[0018] Detailed Description of the Invention

[0019] The invention relates to a primer set to be used in the diagnosis of foot and mouth disease and a method for diagnosing foot and mouth disease using the LAMP (Loop Mediated Isothermal Amplification) method using said primer set. By means of the use of said primer set with the LAMP method, the diagnosis of foot and mouth disease can be achieved within 15-20 minutes. The primer set of the invention comprises primers with the nucleotide sequence of SEQ ID NO: 1 -6, primers with the nucleotide sequence of SEQ ID NO: 7-12, primers with the nucleotide sequence of SEQ ID NO: 13-18, primers with the nucleotide sequence of SEQ ID NO: 19-24 or primers with the nucleotide sequence of SEQ ID NO: 25-30.

[0020] The primers designed specifically for the SAT2 serotype XIV topotype according to said method to be used in the diagnosis of foot and mouth disease were produced in 50 nmol test quantities using the HPLC purification method. After the sample was homogenised into the reaction mixture prepared with the primer set and mastermix, its supernatant or RNA was added and tested at 65°C for 60 minutes in a real-time LAMP device (Optigene Genie III). As the reaction progressed, the reaction in the tubes was measured fluorometrically with the blue reading channel of the two-channel device. The amplicons formed in the tubes multiply and the fluorometric measurement appears on the screen of the device in real-time as a sigmoid curve graph. At the end of the reaction, the device measures the annealing temperature of the formed amplicons and the minutes, seconds and annealing temperatures at which the first sigmoid curve was initiated are found on the result screen of the device.

[0021] When the results are evaluated, it is considered positive in the presence of both the sigmoid curve graph and the compatible annealing temperature. When the results obtained with the positive control, dilutions and field samples studied in this developed test are examined, the earliest diagnosis was seen in the 7thminute and the latest diagnosis was seen in the 57thminute. The lowest binding temperature was determined as 86.75°C and the highest binding temperature as 89.35°C. Accordingly, the results corresponding to this range are evaluated as positive, and the results that are close are evaluated as suspicious / repeated. When there is an air bubble in the reaction tube, it may give a result in 1 minute and 30 seconds or it may give a binding temperature result at 80-81 °C. In this case, the results are evaluated as non-specific and are considered negative. The test is also evaluated in the same way with other primer sets, which are the subject of the invention.

[0022] For the aim of the primer design, in order to obtain the target genome, RNA was obtained from the sample known to be SAT2 with the extraction device. After the sample was homogenised with PBS and the inoculum was prepared, total RNA / DNA was collected from 200 pL of inoculum in 100 pL of elution buffer according to the kit protocol and stored at -80°C. Since the foot and mouth disease virus is an RNA virus, complementary DNA was obtained using a conventional PCR device and PCR was performed using the primers with the nucleotide sequences SEQ ID NO: 31 and 32 listed in the table below, which are used in the SAT2 virus sequence recommended by WOAH.

[0023] Table 1. Primer sets used in the SAT2 virus sequence

[0024] Following the PCR process that produces a 1279-base product, the presence of the product was confirmed by gel electrophoresis imaging, and then sequence analysis was performed. The chromatogram of the sample that underwent Sanger sequencing was aligned and edited by creating a database with similar subtypes in NCBI (locating and placing the region similar to itself, and then checking, reading and correcting individual nucleotides). As a result, the sequence of the region with the nucleotide sequence of SEQ ID NO: 33, consisting of 642 bases, including the VP1 region, was obtained. In addition, the VP1 region and the VP1 and VP3 regions from the full genome sequence of the 2022 Ethiopian SAT2 virus were also used together.

[0025] A modification was made by adding the TTTT nucleotide to the middle of all FIP (F1 c- F2) and BIP (B1 c-B2) primers in the primer sets subject to the invention. This modification aims to speed up the test and increase looping properties.

[0026] The foot and mouth virus used in the design of the invention primer was isolated with LFBK and / or BHK21 monolayer cell cultures. The strong virus numbered IRAK41 , which is also used in vaccine production, was adapted in the BHK21 vaccine production cell line and used as a positive control, and the virus isolate was taken in the plaque test and its infective titre was measured 3 times. The average titre was determined as 8.85 pfu. This virus, whose titre was determined, was also used in the analytical sensitivity test.

[0027] Each primer set consisting of 6 primers was tested separately with a positive control. The forward and reverse primers (F3-B3) in the primer sets were prepared as 5 pmol, the forward loop and reverse loop primers as 25 pmol, and the front inner and back outer primers as 50 pmol. 15 pl mastermix (Optigene 004 Isothermal mastermix), 5 pl primer mix and 5 pl RNA were used in the reactions. In the tests performed, all primer sets were able to detect the positive control and did not give false positives in the negative controls. The Iraq-Turkey field samples were tested with the S3 primer set (SEQ ID NO: 13-18) that identified the positive control sample the fastest and analytical sensitivity and specificity tests were performed.

[0028] The other serotypes and their subtypes viruses selected for use in authenticity / specificity tests were previously sequenced routinely used portioned -80°C vaccine strain or non-vaccine strain isolates. For the specificity test of the LAMP test against the SAT2 serotype XIV genome with the S3 discovery primer, samples from different serotypes and their subtypes were tested and all were found negative except for the positive control virus. The test results are given in Table 2. The serotype, topotype, genotype and subgroups / strains of the viruses are given in the table. Table 2. Specificity tests with other serotypes and subtypes

[0029] The field samples, which are the diagnostic material used in the invention, were homogenised with PBS and RNA was extracted from their supernatants / inocula as previously mentioned. The remaining inocula were portioned to be used in the test.

[0030] The RNA and portioned inocula of the samples were stored at -80°C.

[0031] In the invention, the real-time RT-LAMP reaction was performed at 65°C constant temperature for 60 minutes on the Optigene Gene III device. In cases where the reactions responded faster, the test was terminated earlier. The reaction was terminated by applying 95°C heat for 1 minute and the binding heat was measured with the real-time Optigene III device by decreasing to 80°C with 0.05°C / sec. The fluorescence in the mastermix gives a peak on the device screen in a positive reaction. The device measures these radiations every 15 seconds and instantly indicates the starting minute of the sigmoid curve in the result section. It also gives the binding heat of the amplicon formed in each reaction tube at the end of the test. This value, which is important in the evaluation phase of the test, shows whether a positive sample gives a unique result. In order to compare the results of the LAMP test used in the method in the invention, all samples were tested with the real-time RT-PCR test. In the real-time RT-PCR test, the SAT2_XIV_AS_P probe published by the OIE-FAO Reference Laboratory with its preliminary study, the SAT2_XIV_AS_F forward primer, the SAT2_XIV_AS_R reverse primer and the ABI AMBION PCR mastermix used in routine were tested using the mastermix’s user manual and routine use. Although the test progressed in a single step, cDNA was made in the first stage of the PCR device procedure and then the PCR cycles were started. The PCR protocol was performed in the PCR device as follows: 10 minutes at 50°C for complementary DNA, 10 minutes at 95°C for denaturation, and 35 PCR cycles (denaturation; 15 seconds at 95°C, annealing; 45 seconds at 60°C).

[0032] The comparison of the primers with the nucleotide sequence SEQ ID NO: 13-18, the RNAs of the field samples and IRAQI samples, and the inocula of the field samples with the LAMP method results and the qRT-PCR test is given in Figure 1 and Figure 2. The numbers in the graphs represent the number of samples found to be positive. In said invention, the inoculum and RNA of 14 IRAQI samples known to be SAT2 serotype were tested with RT-LAMP and compared with real-time RT-PCR (Figure 1 ). While 10 out of 14 samples were detected with real-time RT-PCR, all were detected with real-time RT-LAMP performed with the S3 primer set from both RNA and inoculum (Figure 1 ). With the start of SAT2 outbreaks in the country in March, the inoculum and RNA of 65 field samples known to have SAT2 serotype were tested with RT-LAMP and compared with real-time RT-PCR (Figure 2). While 64 out of 65 samples were detected with real-time RT-PCR, all were detected with real-time RT-LAMP and also 63 of the samples taken for testing without extraction from the inoculum were detected with realtime RT-LAMP (Figure 2).

[0033] In addition, the analytical sensitivity test in the invention was performed by diluting the RNA of the positive control virus with a titre of 8.85 pfu / ml 3 times at the Iog10 base and testing 3 times with qRT-LAMP and qRT-PCR, and the results are given in Table 4. In addition, adapted positive control virus diluted at Iog10 base was also tested in LFBK cell line. Virus isolation test, which is the gold standard in diagnostic methods, was also added to the sensitivity test. It is shown that the qRT-LAMP test optimised with the designed primer set can diagnose the presence of viruses 10 times more than real-time RT-PCR and 100 times less than virus isolation. Table 3. Comparison of Analytical Sensitivity Tests

[0034] REFERENCES

[0035] [1] Grubman, M. J., & Baxt, B. (2004, April). Foot-and-mouth disease. Clinical microbiology reviews.

[0036] [2] Sharma, G. K., Mahajan, S., Matura, R., Subramaniam, S., Ranjan, R., Biswal, J., Rout, M., Mohapatra, J. K., Dash, B. B., Sanyal, A., & Pattnaik, B. (2015, August 12). Diagnostic assays developed for the control of foot-and-mouth disease in India. World journal of virology.

Claims

CLAIMS1. A primer set for use in the diagnosis of foot and mouth disease SAT2 serotype by the LAMP (Loop Mediated Isothermal Amplification) method, comprising• primers with SEQ ID NO: 1 -6 nucleotide sequence or,• primers with SEQ ID NO: 7-12 nucleotide sequence or,• primers with SEQ ID NO: 13-18 nucleotide sequence or,• primers with SEQ ID NO: 19-24 nucleotide sequence or,• primers with SEQ ID NO: 25-30 nucleotide sequence.

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