Real-Time PCR Kit for the Diagnosis of Factor V Leiden Thrombotic Disease with FIT Probe

Abstract

Real-Time PCR kit for the diagnosis of thrombotic Factor V Leiden disease with the help of FIT Probe. FVL mutation leads to clot formation and thrombosis in various vessels of the body, and has a high risk of mortality, especially in the homozygous state. In Iran, it is frequently molecularly examined in cases such as recurrent miscarriage and stroke. The lack of diagnostic kits with high accuracy and sensitivity and low cost, the difficult and complicated implementation protocol, and the time-consuming nature of testing in some imported kits that operate with a different mechanism than the currently designed kit are among the challenges facing medical diagnostic laboratories. The designed kit is based on specific PNA-FIT probes, which specifically identify single nucleotide mutations. According to Figure 2, the structure of these probes is designed in such a way that, upon binding or non-binding to the target site, fluorescent light is detected by the thermocycler, and the presence or absence of the mutated base is determined, respectively.

Description

Description of the invention Title of the invention (as stated in the declaration) Real-Time PCR Kit for the Diagnosis of Thrombotic Factor V Leiden Disease with FIT Probe Real-Time PCR Kit for the Detection of Factor V Leiden thrombotic disease by the FIT Probe Technical background of the relevant invention This invention relates to the Real-Time PCR technique as a laboratory molecular diagnostic method in the research-diagnostic fields of laboratory hematology, genetics, and medicine. Technical problem and stating the objectives of the invention Coagulation and fibrinolysis are two important arms of the hemostasis process, to establish proper blood flow and prevent bleeding and thrombosis. When this balance is disrupted due to hereditary or acquired disorders, the patient will be susceptible to symptoms and complications of bleeding or thrombosis. Excessive increase in coagulation or decrease in fibrinolysis causes stagnation of blood flow in the vessels. Thrombotic disorders are a group of multifactorial diseases that are influenced by environmental and genetic factors. Among the important and common genetic factors that are among the initial requests of doctors in molecular examination of patients is the polymorphism associated with factor V Leiden (rs6025), which has been confirmed to be associated with disorders such as recurrent miscarriage (RPL), preeclampsia, myocardial infarction (MI), stroke (IS), pulmonary embolism (PE), deep vein thrombosis (DVT), Budd-Chiari syndrome (BCS), some cancers, etc. The prevalence of FVL in the general population is 5% and in cases of venous thromboembolism (VTE) is 20-40%. In Iran, the prevalence of this polymorphism is high due to consanguineous marriages.Heterozygous individuals for this mutation are 3 to 10 times more likely to develop venous thrombosis than normal individuals, and homozygous individuals are 50 to 100 times more likely to develop venous thrombosis, so identifying this mutation in the treatment process, preventing subsequent recurrences, reducing the risk of mortality during surgery, and providing pre-pregnancy genetic counseling in susceptible individuals will be helpful. Most of the kits currently in laboratories are based on CVD StripAssay, RFLP-PCR, and QRT-PCR methods, which respectively reduce the complexity of the testing process and increase the accuracy of the results obtained. Each of these methods has challenges in the diagnosis process, which are stated separately (prices requested from companies are as of November 1402): CVD StripAssay Diagnostic Panel Kit for 9 Common Thrombosis Mutations: After DNA extraction, it has four complex steps and is designed based on reverse hybridization and requires a skilled technician to interpret the results. This kit is imported from Australia and the time it takes to perform this test is at least two days and the cost of purchasing it from intermediary companies is about 8 million. Each kit is used for 20 patients. This kit is less expensive because it evaluates several thrombotic factors simultaneously, so it is more cost-effective for laboratories, but it is very time-consuming and challenging. The StripAssay kit is widely used in laboratories. QRT-PCR kit: It is a single mutation and is designed based on specific TaqMan probes labeled with two different fluorescent colors and, similar to the previous method, is able to identify homozygous and heterozygous forms from each other. It costs about 11 million for 24 reactions and is more accurate than the StripAssay and RFLP kits. The test process is much easier, but due to the colors labeled on the probe, it is more expensive than other kits. The QPCR method is more popular due to its ease of operation and interpretation of the results. The test takes about four hours without the DNA extraction process. However, in some tests, it has false negative and positive results. Because designing TaqMan probes for the site where this mutation is located (gene plot as shown in Figure 1) is challenging, and the possibility of error in identifying target SNPs and failure of the enzyme to break these types of probes cannot be ignored. Some of these kits are designed specifically for specific real-time devices such as Rotor-Gene and cannot be used in other QPCR devices. RFLP-PCR kit: This kit consists of two processes: the amplification process, which is carried out by two forward and reverse primers. Next, to observe the PCR products, they must be subjected to enzymatic digestion by restriction enzymes and finally these products must be run on an electrophoresis gel. Interpretation of the results obtained is based on observing the band on the gel, and the test duration without the DNA extraction process is about 10 hours. The total cost of these processes for 50 reactions is about 20 million. The problem with this method is that the enzymes are imported and very expensive, and this technique also requires a larger amount of DNA and product to observe the real results. The process is time-consuming and requires a skilled technician to perform the test and interpret the results. Other challenges include the possibility of not having an enzymatic cut or not finding the appropriate enzyme for digestion in the desired gene region, as a result of which the error rate in this method is high. Given the length of time these imported kits spend at customs until they reach the laboratory, there is a possibility of losing many patients. Also, the ability to provide and maintain cooling conditions for the kits to maintain the effective performance of the components in them, such as limited-effect enzymes, is very poor in the customs warehouse until they reach domestic companies, and this has reduced the effectiveness of the enzymes and other components in the kits, which can result in false results after testing. Considering the molecular diagnostic kits available on the market for FVL in terms of the complexity of the process and the need for special skills, as well as their high cost and time-consuming nature, we decided to design this diagnostic kit. In fact, the goals of designing this molecular diagnostic kit for FVL polymorphism based on the PNA-FIT probe are to increase the accuracy and precision in detecting target mutations, reduce testing time, reduce patient costs, given the great need for investigating thrombotic disorders and recurrent miscarriage, and ultimately, to be usable in conventional types of QPCR devices.The specific PNA-FIT probes used in this kit have the desired sensitivity and specificity in identifying true positives and negatives, and can be useful and practical for screening and diagnosing these mutations. Considering the country's needs, to achieve the fastest and most accurate results and help the doctor to identify and treat the patient more effectively, the design of this kit can be valuable. A description of the state of the prior art and the history of developments related to the claimed invention. After searching the databases related to patents in Iran and internationally, no similar results were found for the present invention, and finally we designed this diagnostic kit. Diagnostic methods close to the present invention are as follows: 1) Application No. WO1995021938A1 was filed in 1995 by Rogier Maria Bertina, Pieter Hendrik Reitsma, regarding "A method for screening for the presence of a genetic defect associated with thrombosis / or a poor anticoagulant response to activated protein C". This invention uses a cloning technique. The homozygous mutant and normal FVL RNAs were cloned separately into the pG30 vector. They were then digested with the restriction enzymes EcoRI and Casp451. The resulting fragments were amplified after a transcription step using primers and probes labeled with ECL, ELGA, HRP and Biotin. Difference: Cloning technique is a very challenging, time-consuming and expensive method, so it is not cost-effective for medical diagnostic laboratories with a large number of patients. On the other hand, cloning technique is generally used for research studies (and not medical diagnosis). QPCR method based on FIT probe design is a cost-effective, less challenging and high-precision technique, which can be used routinely in medical diagnostic laboratories. FIT probes have high sensitivity and specificity in identifying single nucleotide mutations. 2) Application number DE19840897A1 in Germany, 2000, by Dirk Happich, Dirk Hoernschemeyer, entitled "Detection of factor V mutation by PCR using probes labeled with reporter / quencher dyes", in which the specific TaqMan probe for detecting FVL is labeled with two dyes: FAM and TAMRA. Difference: The probes designed in this kit are based on TaqMan. The accuracy of the TaqMan probe in identifying the FVL single nucleotide polymorphism is lower due to the location of the mutation in the gene, because if the probe is designed for the mutant state, there is a possibility that it will bind to the normal complementary sequence and in that case it will still emit light, but the PNA-FIT probe used in our target kit, even if it binds, because it does not completely pair with the target sequence, the emitted light cannot be detected. Also, the synthesis cost of TaqMan probes is more expensive than PNA-FIT because they are labeled with two fluorescent dyes. 3) Application No. ES2169756T3 was filed by John Lister-James in Spain in 2002, entitled “Tyrosine derivatives substituted with piperidile at position four as a scintigraphic agent for imaging in the detection of thrombosis.” In this invention, radioactive materials are attached to the GPIIB / IIIA receptor on the surface of platelets, and these platelets are then injected into a patient with thrombosis to identify the location of the clot. Difference: A relatively non-invasive diagnostic imaging method of nuclear medicine and requires the patient to be present in the hospital. This method requires a skilled technician and special specialized devices that not all medical centers have, and it also does not identify the main factor causing the clot at the molecular level. This is while the method used for the present kit is QPCR, which has the corresponding thermocycler devices found in every laboratory and investigates the cause of thrombosis at the DNA level. 4) Application number WO2005078129A2 entitled "Method for the diagnosis / prognosis of thrombosis" was introduced in 2005 by inventors Aurélie Brion, Philippe Leissner and Cécile Berard, who carried out the molecular study of two mutations associated with the coagulation factors F2-G20210A and FVL. This method is based on qPCR and uses Molecular Beacon probes. MBs are a type of hybridization probes, which are stem-loop in shape. Like TaqMan probes, they are made of nucleic acid and have two fluorescent dyes, reporter and quencher, at their 5' and 3' ends respectively. Difference: MB probes with stem-loop structure have a higher number of nucleotides than other types of probes (at least 15 to 25 nucleotides are required in the loop region). On the other hand, it is labeled with two fluorescent dyes, so both of these things increase the cost of synthesis of this type of probes. In the present invention, we use PNA-FIT probes, which, due to their peptide-acidonucleic structure, can reduce the number of nucleotides but still provide the appropriate Tm temperature for the reaction, which in turn reduces the cost of synthesis. Also, unlike nucleic acid probes, they are not sensitive to the ionic concentrations of the reaction medium and identify SNPs with high accuracy. FIT probes have higher sensitivity and specificity than other types of probes, including MB probes. 5) Application No. US7312034B2 in the United States, 2007, was registered by Carmen Virgos, Maureen Cronin on "Universal e-tag primer and probe compositions and methods". In this invention, electrophoretic tag (e-tag) probes and primers are used to detect FVL, MTHFR and ApoE in multiplex. In this method, first, forward and reverse primers labeled with e-tag are attached to the target sequences and the extension process occurs. Then, probes labeled with e-tag are added to the resulting amplicon and each is attached to the complementary target region and finally, the e-tag part is separated by the cleavage enzyme and the peak associated with the movement of the e-tag separated from the probe is identified by the device and electropherogram. Difference: In this invention, despite the value of multiplex identification of several SNPs in a reaction environment, it has a complex and expensive process. It requires several special devices such as microfluidics to identify separated e-tags, which are currently not recognized in Iran as microfluidic devices, and generally, medical diagnostic laboratories do not use these chips due to their high cost and the need for knowledge of how to use and interpret their results. However, the diagnostic method of the present invention is widely used in laboratories with the help of conventional Real-Time PCR devices, which are very user-friendly. On the other hand, the probe used in the present kit is of the PNA-FIT type, which has high accuracy and precision in identifying single nucleotide mutations. It also does not have the complexity and challenges of the design mechanism of other probes. 6) Application No. CN105907857 in China, 2016, investigated "Molecular marker for arterial thrombosis, reagent and application thereof". In this invention, Atf3 is introduced as a blood molecule biomarker in thrombosis development, which was detected using Real-Time PCR technique and Cybergreen fluorescent dye. Difference: The purpose of this invention is to confirm the effect of the Atf3 gene in the occurrence of thrombosis, while the purpose of the present invention is to detect FVL point mutations in individuals at risk of thrombosis using the PNA-FIT probe, which reduces costs and increases the accuracy of diagnosis. 7) Application No. CN105925714 in China, 2016, entitled "Molecular marker in the diagnosis of cerebral ischemic thrombosis", which evaluated the expression of the CTAGE5 gene in stroke patients using QPCR and Cybergreen dye. Difference: The target gene and the method of investigation are different from the claimed invention. Cybergreen dye binds to any double-stranded DNA and is not suitable for detecting single nucleotide mutations at all, while PNA-FIT probes have the desired specificity and sensitivity to detect only one mutated base. The aim of the present kit is to detect only one mutated nucleotide, so specific probes must be used for detection. 8) Application No. CN108977527 in China, 2018, refers to the investigation of the effect of the "TMEM63C gene" on the occurrence of "DVT" using QPCR and Cybergreen. Difference: The target gene and assay method are different from the claimed invention. Cybergreen dye binds to any double-stranded DNA and is not suitable for detecting single nucleotide mutations, while PNA-FIT probes have the desired specificity and sensitivity to detect a single mutated base. 9) Application No. CN106337089B in China, 2018, is about "a type of LncRNA for the diagnosis of cerebral artery thrombosis" using real-time PCR technique and using Cybergreen dye as a target gene marker. Difference: Fluorescent dyes such as EVA Green and SYBR Green bind to the minor grooves of any double-stranded DNA molecule, so they have very low specificity in identifying single nucleotide polymorphisms. This invention only wants to confirm the presence of a type of LncRNA (LOC105376505 in patients with cerebral artery thrombosis and does not seek to identify point mutations. Therefore, the forward and reverse primers are designed in such a way that they can cover the entire target gene region and no specific probe is designed for point mutations, because firstly, the purpose of this invention is not to identify SNPs and secondly, the fluorescent dye SYBR Green does not act specifically. However, the purpose of the present kit is to detect FVL single nucleotide polymorphism, and to identify this single base, specific probes must be used, among which PNA-FITs are the most suitable type of probe in terms of high sensitivity and specificity. 10) Application No. CN106319089B in China, 2018, entitled "Applications of LncRNA in the Diagnosis of Cerebral Artery Thrombosis", which investigates the presence of a type of LncRNA (LOC101929707) using the QPCR technique and detection by the fluorescent dye Cybergreen. Difference: Similar to the previous invention, it aims to investigate the presence of this LncRNA in patients with cerebral artery thrombosis, which does not require the design of a specific probe to identify point mutations. Unlike our invention, which investigates the SNP of factor V Leiden, which requires specific probes with high accuracy and sensitivity. PNA-FITs are probes with a high ability to identify single base mutations. 11) Application No. CN108384848A in China, 2021, "Use of circ_0021132 as a marker for the diagnosis of deep vein thrombosis in serum" using Real-Time PCR techniques with Cybergreen dye and genetic chip, which chips include nucleic acid hybridizing probes. This invention investigated the expression of circ_0021132 in the serum of patients with DVT. Difference: One of the important techniques in DNA microarray-based expression analysis is microarrays, which are very laborious and expensive and require high knowledge in interpreting the results. Generally, these types of techniques are used to identify and confirm the biomarker power of a protein or gene product. Therefore, in the invention under consideration, FVL has been previously confirmed as a biomarker, and the purpose of designing this kit is to reduce costs and increase the accuracy of point mutation detection in the shortest time by FIT probes. PNA-FIT has high sensitivity and specificity in identifying SNPs. 12) Application No. 389090250 in Iran, 2011, by Mehdi Ebrahimi Fakhari and Hamidreza Ebrahimi Fakhari, regarding the invention of a "device for preventing foot drop and venous thrombosis." Which helps improve the quality of life of patients who suffer from brain injuries and subsequent complications such as DVT and foot drop. Difference: This invention is a rehabilitation device to improve the quality of life of patients suffering from foot drop and venous thrombosis. Therefore, it is not a diagnostic invention. The present kit is a laboratory diagnostic invention at the molecular level. 13) Application No. 139350140003002201 by Ali Maleki, in Iran, 2014, is "Smart device for preventing varicose veins and deep vein thrombosis based on electrical stimulation." One of the complications associated with thrombosis is varicose veins. Inactivity, especially in people prone to it due to their jobs, can cause a decrease in blood flow in the vessels and, as a result, an increase in thrombosis. This device, independent of the person's physical activity, helps prevent clot formation in the vessels by providing continuous electrical stimulation. Difference: A device to assist patients prone to thrombosis when they are immobile for long periods of time, such as during long trips, hospitalizations, and desk jobs. Therefore, it is not a diagnostic invention. The present kit is a laboratory diagnostic invention at the molecular level. 14) Application No. 139550140003007465 by Shafiqeh Shojaei and Amir Rastegari Kiriani, in Iran, 2016, on the subject of "Natural medicine for the prevention and treatment of various types of thrombosis." It is a herbal-based anticoagulant drug. Difference: The above invention is in the field of thrombosis treatment, not diagnosis. The present kit is a laboratory diagnostic invention at the molecular level. 15) Application No. 139950140003007935 in Iran, 2010, by Mehdi Azarafraz, Yasman Payandeh, Frank Rahmani, and Sajjad Mohammad Ali, an invention was filed regarding an "electromechanical device for preventing bedsores and thrombosis in patients" that helps improve and enhance the quality of life of patients who are immobile for a long time, such as people who are unable to move, overweight patients, and people who have suffered from cerebral coma. Difference: This invention is a device that helps prevent bedsores in susceptible patients who are immobile for long periods of time. Therefore, it is not a diagnostic invention. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention The subject of this invention, considering the problems raised above regarding the current molecular detection methods of common single nucleotide polymorphisms (SNPs) in the occurrence of thrombosis, can be a solution to improve and upgrade the diagnostic kits available on the market. The present invention is a molecular detection kit for Factor V Leiden (FVL), which is one of the most common and important mutations in the occurrence of thrombosis (Tables 1 and 2 of the technical map show the polymorphism and bioinformatics characteristics of FVL), by designing specific probes based on peptide nucleic acid (PNA) labeled with fluorescent dye thiazole orange (TO). The key and distinguishing feature of this kit is the design of PNA-FIT probes, which help to identify the target SNP with higher accuracy than existing probes. In general, the components of this kit include: Forward and Reverse primer pairs PNA-FIT probe Other parameters required for the polymerization and amplification process: MgCl2, dNTPs, 10x-Buffer, Taq DNA Polymerase and H2O (Distilled Water) Design of primers and probes specific for FVL: The coagulation factor V gene is located on chromosome 1q24.2 and is produced from the translation of the negative strand transcript, FV. The rs6025 mutation is located in exon 10. After preparing the coagulation factor V gene plot and finding the point mutation site using Ensembl and NCBI sites, the appropriate position of the primers and probes was manually determined, and then the sequence characteristics and the presence of secondary structures such as primer and probe dimers were evaluated using offline software GeneRunner, Oligo7, and Oligo Analyzer, and the specificity of these primers and probes was examined by Primer Blast (Figure 1 of the technical map). PNA-FIT probe: Peptide nucleic acid is a type of synthetic analog that mimics the behavior and properties of natural nucleic acids and proteins. PNA is a polymer of N-2-aminoethyl-glycine (AEG) units, to each of which a natural purine or pyrimidine base is attached by a methyl-carbonyl bond. The connection of each PNA monomer to each other is done by an amide bond. Therefore, PNA is structurally similar to peptides, but functionally and behaviorally similar to nucleic acids. The following are some of the unique properties of PNA-FIT probes that distinguish them from other types of probes (Figures 2, 1-3, and 2-3 from the technical drawing): 1) PNA-FITs, due to their physical, chemical and biological properties, have higher specificity and binding affinity to the target DNA than other specific probes such as TaqMan, Beacon, Scorpion and FRET which have a nucleic acid structure, as well as CyberGreen and EvaGreen which are non-specific fluorescent dyes. Therefore, in addition to the desired sensitivity in identifying true positives of SNPs, they also have high specificity in identifying true negatives. 2) PNA-FIT probes have a non-cyclic, non-chiral polyamide backbone that reduces the electrostatic repulsion between PNA-DNA, PNA-RNA, and PNA-PNA hybrids, thereby increasing the binding strength compared to other types of probes. This increased stability of the hybrids increases the Tm temperature (an important indicator in probe design and PCR temperature regulation) by about 15 to 20 °C. 3) On the other hand, due to the uncharged peptide backbone of PNAs, their hybridization with DNA, RNA, or PNA is not dependent on the ionic strength of the environment. This is while natural double-stranded nucleic acid molecules have stronger bonds in environments with higher ionic strength. The lower the ionic strength, the looser the double-stranded nucleic acid hybrid becomes. 4) These features provide advantages over probes with nucleic acid structures, because in regions of the gene that have a high content of consecutively repeated bases, which leads to challenges in probe and primer design, such as undesirable secondary structures, failure to achieve the appropriate Tm temperature, and not being within the Tm range of other primers and probes used in a microtube for the same gene. Therefore, by reducing the length of the PNA-FIT probe even to 10 nucleotides, the sensitivity and specificity of detection can still be increased and the challenges encountered in the design of other types of probes can be overcome. 5) PNA strands cannot be used as a substrate for the DNA Polymerase enzyme, i.e. as a primer, so unlike other types of probes, 3'OH end blockers cannot be used. 6) The half-life of PNA (at least 48 hours) is longer than that of primers and probes with natural nucleic acid structure and of equal length (less than 15 minutes). PNAs are also resistant to heat and acidic environment, so their long-term storage and preservation is easier. 7) PNA-FIT probes have high sensitivity to detect mismatches between PNA and DNA. The decrease in Tm per unpaired base in the hybrid with PNA is greater than that of natural paired DNA-DNA duplexes, so it will be very helpful in interpreting QPCR results. Therefore, these types of probes are much more efficient in identifying SNPs. 8) The designed PNA-FITs are labeled with the fluorescent dye TO at one of the internal thymidine bases. When the matching between the probe and the target sequence is correct, the TO dye emits light upon excitation. Also, based on the Ct given by the thermocycler, heterozygosity and homozygosity can be distinguished to some extent (under certain conditions) (if the concentrations and wavelength ratios of 280 / 260 and 230 / 260 are normalized). 9) The orange fluorescent dye thiazole is detected in the FAM channel due to its Maximum emission (λMax=533 nm) and Maximum excitation (λMax=514 nm) wavelengths. Based on studies conducted on this type of probe, it was stated that when TO is labeled with the thymine base, Tm increases further. Because it is possible that the appropriate specifications of the primers and probe designed in in-silico conditions may be slightly different from those in-vitro, after designing and ordering the synthesis of the primers and probe (Table 3 of the technical plan) for the FVL mutation, to confirm the specificity of the primers for the target gene, we first performed Conventional PCR and then electrophoresis to observe a single specific band of 137 bp for the region where we designed the primers (Tables 4 and 6 of the technical plan show the compounds used and the temperature and time conditions in Conventional PCR, respectively, and Figure 4 of the technical plan shows the schematic view of the agarose gel electrophoresis image). After confirming the specificity of the primers in-vitro, we set up the conditions for performing QPCR (Tables 5 and 7 of the technical plan show the compounds used and the temperature and time conditions in QPCR, respectively). To set up the conditions, first a temperature gradient was set to determine the best annealing temperature and a concentration gradient was set to find the best concentration of primers, probe, and MgCl2. (Because we could not include actual images of the electrophoresis gel, actual graphs related to QPCR, and sequence results in the technical drawing file, if necessary, the esteemed referee may request that the original images of the results be submitted as well.) Note: To validate the results obtained, we used the GAPDH gene as the internal control of the design. The primers and TaqMan probe used for the internal control were also designed by us. The specifications of the primers and internal control probe are listed in Table 3 of the technical drawing. Note: To ensure the results obtained, we also included a microtube without the target DNA sample as NTC in each run of the device, along with the internal control, normal sample, and patient sample. Note: Because we used samples validated by the CVD StripAssay kit, we also sent the samples for sequencing to ensure the results obtained. Ingredients and equipment required for horizontal electrophoresis: 1) Agarose powder 2) Tris Basic 3) Borate (Boric Acid) 4) EDTA 5) Distilled Water 6) Ladder 50 bp 7) EtBr 8) Sampler and sampler head 9) Electrophoresis device (Submarine Electrophoresis System Mupid-exU, Japan) 10) Gel Documentation Device (Syngene™ Ingenius 3 Manual Gel Documentation System, UK) How to prepare 2% agarose gel and perform electrophoresis: First, we prepared TBE buffer according to Table 8, then poured 60 ml of 1x TBE buffer with 1.2 gr of agarose powder into an Erlenmeyer flask, closed its lid with foil to prevent evaporation, and placed it on a heater to obtain a uniform and transparent solution. Then, we let the solution reach a temperature of about 50 °C, and after adding diluted EtBr dye in a very small amount (about 300 to 400 microliters) to the solution, and slowly poured it into a well-sealed electrophoresis cast or tray, without creating bubbles, and let the gel set. When the gel set, we slowly removed the comb. First, fill the electrophoresis tank with TBE buffer, and then place the gel with the cassette in the tank. The gel should be completely covered with buffer. (Due to the fluorescent dye, the process from the step where we used EtBr to the end was carried out in a dark environment.) In the next step, we mixed 5µl of each of the three microtubes FVL+, IC, and NTC with 1µl of 5x Loading Dye, and then loaded the total 6µl into the wells. A 50 bp ladder or size marker was also loaded into one of the wells. After that, we put the lid on the electrophoresis tank and ran it for 40 minutes at a voltage of 100 amps. After the electrophoresis was completed, we removed the gel from the cassette and placed it in the gel documentation device. Finally, with its help, we were able to observe two bands of 137 and 352 bp for FVL+ and IC, or GAPDH, respectively. Therefore, the designed primers are completely specific to the target sequence and no nonspecific or primer-dimer binding was observed. We sent the remaining contents of the FVL+ microtube for sequence confirmation. In the next step, we performed QPCR according to the defined conditions. The QPCR results, based on Ct analysis and the fluorescence intensity graph of the device, showed that the samples were heterozygous, which was completely consistent with the Sanger sequencing results. Note: The DNA concentrations were taken in equal amounts for the initial setup for all reactions. The concentrations were measured in ng / µl using a spectrophotometer (DS-11 Series Spectrophotometer / Fluorometer from DeNovix). Note: The QPCR results of the positive and negative samples of the target kit were consistent with the results of the StripAssay kit and the sequence. Another point worth mentioning is that a number of samples were also analyzed with other common Real-Time PCR devices such as StepOne Plus (product of Applied Biosystems, USA) and MIC (Magnetic Induction Cycler, product of Bio Molecular Systems, Australia), and their results were also consistent. Note: Also, a total of 11 positive and 24 negative samples were examined, of which all patient samples were positive and all normal samples were negative. Then, according to the formulas related to sensitivity, specificity, positive predictive value and negative predictive value, the values ​​of Sensitivity, Specificity, PPV and NPV were obtained respectively as described in Table 9 from the technical map. Explanation of shapes, maps and diagrams (Figure 1 of the technical drawing): Summary of the steps for designing primers and a specific probe. To construct the coagulation factor V gene plot, we first obtained the sequence of exon 10 along with a few nucleotides before and after it from the Ensembl website and saved it in a word file, and finally found the best position of the primers and probe. The green highlight shows exon 10, the gray highlights show the sequences of the designed forward and reverse primers, and the yellow highlight shows the sequence of the PNA-FIT probe. The sequences of the reverse primer and probe were ordered in complementary and reverse order. The G base number 1691 highlighted in red is the same C in the complementary strand that is converted to T due to mutation, and the A base in the complementary sequence is T, which is labeled with the fluorescent dye TO. The length of the amplified fragment is 137 bp. Then, the presence of secondary structures and unwanted dimers was checked by GeneRunner, Oligo7 and Oligo Analyzer software, where loose secondary structures were insignificant and negligible due to positive G∆ and low Tm. Finally, the specificity of the designed primers and probe was evaluated by Primer Blast. All the properties required for performing a reliable QPCR in-silico conditions were suitable. (Figure 2 from the technical drawing): Schematic view of the DNA-PNA-FIT hybrid structure in two normal open and mutant open states, assuming that the probe is designed for the mutant state. a. Matched mode: When the DNA has a point mutation, it is fully coupled to the PNA-FIT probe, so the orange thiazole fluorescent dye is excited and the light is detected by the detector of the device. b. Mismatched mode: When the DNA has not undergone a point mutation, it is not fully coupled to the PNA-FIT probe, so the orange thiazole fluorescent dye is not excited and as a result the light is not detected by the device. The accuracy of this coupling is such that even if the flanking nucleotides are also coupled, the intensity of the TO excitation in the state of complete non-coupling is still reduced. (Figure 1-3 from the technical drawing): Identifying the SNP on the DNA strand and pairing it with the mutant PNA-FIT probe. a: The upper strand shows the PNA-FIT probe and its components. The probe is labeled with the fluorescent dye TO at the T base. The terminal DABCYL acts as a quencher, so that optical noise from the unexcited TO is not emitted before the probe binds to the target, reducing interference between the signals. The lower strand shows the DNA sequence that has a single nucleotide mutation at the C base, so since the probe is designed for the mutant state, it is paired with the complementary DNA strand that contains the SNP. b: As the probe is fully positioned on the DNA strand, the fluorescent dye TO is excited and as a result a peak is shown in the thermocycler. The diagram shows an example of a comparison between the homozygous and heterozygous states of the patient. The homozygous patient sample (CC) has a lower Ct than the heterozygous patient (AC) (provided the quality of the extracted DNA is the same). (Figure 2-3 from the technical drawing): Failure to identify the healthy DNA strand and its pairing with the mutant PNA-FIT probe. a: The upper strand shows the PNA-FIT probe designed for the mutant state and the lower strand shows the normal DNA sequence without the mutation. b: The lack of excitation of the TO fluorescent dye, due to the incomplete pairing of the probe sequence and the DNA strand, results in the thermocycler not showing an acceptable peak. The diagram shows an example of a comparison between the healthy homozygote and heterozygote states. In the heterozygote sample (AC), the instrument shows the peak and Ct, while for the normal sample (AA) it is reported as undetectable and most of the peaks are below the threshold line. (Figure 4 of the technical drawing): Schematic view of the steps and conditions of Conventional PCR and QPCR. a: First, to confirm the specificity of the primers, a PCR and electrophoresis step is performed. If the primers bind only to the target fragment and no primer-dimer is formed, only one band is seen. In conventional PCR, it is not possible to distinguish between homozygous and heterozygous states, and only the specificity of the primers is examined in the In-Vitro state. b: Then, according to the above conditions, QPCR is performed to quantitatively examine and analyze the presence / absence of mutations. In QPCR, we can evaluate specificity, homozygosity, and heterozygosity by analyzing the amplification curve and, to some extent, the Ct value. The Ct values ​​and the height of the graph (fluorescence light intensity) ∆Rn / Ct in homozygotes are lower and higher than in heterozygotes, respectively (if the concentrations and wavelength ratios of 230 / 260 and 280 / 260 are normalized for DNA samples). (Table 1 of the technical map): Characteristics of the Factor V Leiden polymorphism. This table provides information about the FVL mutation, including its location on chromosome 1 and a portion of the sequence surrounding the mutated region (the mutant site is shown in red). Each mutation is also identified in various databases with a specific rs or reference SNP, which is listed in the corresponding rs table. (Table 2 of the technical map): Bioinformatic characteristics related to the Factor V Leiden polymorphism. This table lists FVL information recorded in various biological databases, including the type of amino acid altered by the mutation, its type of inheritance (autosomal recessive), clinical complications, the prevalence of the mutated allele in the population, and its location on DNA and mRNA. (Table 3 of the technical map): Specifications of primers and probes associated with the five Leiden mutation genes and the internal control GAPDH. This table shows information about the designed primers and probes, including the nucleotide sequence of each primer and probe, the number of nucleotides, the melting temperature, the GC content of each sequence, and the length of the product obtained by amplification. (Table 4 of the technical drawing): Conventional PCR reaction compositions for 1 rxn= 30 µl. This table shows information about the amounts in each PCR reaction, which has a volume of 30 microliters, depending on the concentration of each consumable (calculated according to the formula below). Where C and V are the concentration and volume of the substance used, respectively. For example, the purchased MgCl2 substance has an initial (concentrated) concentration of 50 mM, which must be brought to a concentration of 1.5 mM in a volume of 30 μL, so 0.9% should be removed from the original vial: They are calculated similarly for other substances in the reaction. (Table 5 of the technical drawing): QPCR reaction compositions for 1 rxn= 30 µl. This table shows the amounts required for each 30 μL QPCR reaction, depending on the concentration of each reagent (calculated using the formula below). (It is worth noting that the concentrations used for cPCR and QPCR are different because QPCR is more efficient than conventional PCR.) Where C and V are the concentration and volume of the substance used, respectively. For example, the purchased MgCl2 substance has an initial (concentrated) concentration of 50 mM, which must be brought to a concentration of 2 mM in a volume of 30 μL, so 1 / 2 of the original vial must be removed: They are calculated similarly for other substances in the reaction. (Table 6 of the technical drawing): Temperature and time conditions in Conventional PCR. The cPCR setup is different from QPCR, and consists of three stages: initial denaturation, 30 cycles, and final extension. cPCR was performed with an American brand Biosystems thermocycler. (Table 7 of the technical drawing): Temperature and time conditions in Three-step QPCR. The cPCR setup is different from QPCR, and consists of two stages: initial denaturation and 40 cycles. Fluorescence detection is performed by the device at the primer-probe annealing stage. QPCR was performed using a Chinese brand Hilforce thermocycler. (Table 8 of the technical drawing): Ingredients required to prepare 1X TBE Buffer. According to the values ​​in the table, the TBE buffer required for preparing agarose gel and performing electrophoresis was prepared. (Table 9 of the technical map): Results related to sensitivity, specificity, positive and negative predictive value for the Factor V Leiden mutation. Out of the total 35 samples, 11 positive and 24 confirmed negative samples were evaluated with the CVD Strip Assay kit. The results of all samples tested by our kit were in perfect agreement with the Strip Assay and sequence results. Therefore, the kit has 100% accuracy and precision according to the sensitivity and specificity formulas. A clear and precise statement of the advantages of the claimed invention over prior inventions. 1) PNA-FIT probes have much higher accuracy and sensitivity than the widely used non-specific Cybergreen dye, because these dyes bind to any type of double-stranded DNA and the device reports false positives. However, PNA-FIT probes, like other nucleic acid-based probes such as TaqMan, Scorpion, FRET, and Beacon, have higher specificity and sensitivity for identifying SNPs (fluorescent dyes such as Cybergreen cannot be used to detect SNPs because they bind to any dsDNA and require a melting curve to analyze their results). When specific PNA-FIT probes are used, we do not need a Melt Curve diagram, which reduces run time and device wear and tear, and allows for more accurate analysis of SNPs. 2) The PNA-FIT probe has a higher ability to identify SNPs than the TaqMan, FRET, Scorpion, and Beacon probes, because several points must be considered when designing these probes: A_ First, in order to optimize the %GC and Tm length, they are higher than PNA. This increase in the number of nucleotides increases the probability of secondary structures forming, especially considering Figure 1 of the technical map, which shows a plot of part of the coagulation factor V gene, the region around the mutated repeat has a very high %GC, so increasing the length of the probe increases the %GC and Tm. As a result, the distance between the Tm of the probe and the Tm of the forward and reverse primers increases, which can make the setup of the QPCR reaction difficult. B_ On the other hand, increasing the length of the probe in this mutation creates undesirable secondary structures that cannot be ignored. PNA probes also have fewer undesirable secondary structures due to their reduced length. C_ Increasing the length of the probe increases the cost of its synthesis. The cost of PNA probes is also lower than other probes due to their shorter length. D_ TaqMan and FRET probes must be designed to be about ten nucleotides away from the primer. This requirement makes the design of such probes more challenging. Z_ On the other hand, the property of TaqMan probes is such that the condition of binding the probe to the target fragment and the distance between the two labeled reporter and quencher dyes after binding causes the reporter dye to be excited. Therefore, because the probes differ from the target fragment in only one nucleotide, the possibility of their binding to the target is very high, as a result, they cause false positive results, and reduce the specificity or specificity of the test. However, if the PNA-FIT probe is bound to the target, due to the same nucleotide that does not pair, the intensity of the excitation of the thiazole orange dye is greatly reduced, and this increases the specificity of this type of probes compared to others. R_ In the design of the aforementioned nucleic acid probes, as mentioned, two fluorescent dyes, a reporter and a quencher, must be used. These dyes significantly increase the cost. However, in the PNA-FIT probe, only one internal dye, thiazole orange, is used, which is more cost-effective. G_ In designing various probes, the Tm temperature of the probe should be about 10 degrees higher than the Tm temperature of the primers, which is not possible except by increasing the length of the probe. However, due to the physicochemical properties of PNA-FIT probes, this temperature increase can be achieved even at short lengths. Sometimes, without increasing the length of the probe, by increasing the %GC of the probe in regions rich in guanine-cytosine bases, the Tm temperature can be increased, but on the other hand, the possibility of forming strong undesirable secondary structures, as well as the tight binding of the probe to the template strand and its non-dissociation during the amplification reaction, causes errors (especially in our target sequence, which had a high GC content around the mutant base). G_ Some of the Real-Time PCR kits for FVL mutation available on the market are specific to certain real-time devices, including Rotor-Gene. 3) Advantages of using this kit over the existing imported PCR-RFLP kit: A_ PCR-RFLP is a two-step technique, so the test takes longer than QPCR (about ten hours). B_ The use of cutting enzymes in this method has increased the cost of this kit. C_ Due to the importation of this kit and its stay at customs until it reaches the laboratory, the quality of the enzyme is reduced due to the failure to observe the cold chain. D_ The accuracy of this test is less than that of QPCR, because the probability of error in cleavage by the enzyme is high. Z_ Primer design and selection of the appropriate enzyme for cutting are challenging in this technique. R_ Requires a larger amount of DNA than the QPCR method. 4) Advantages of using this kit over the imported CVD StripAssay kit: A_ The duration of testing with this kit is at least 2 days, while the expected duration for the current kit is about 2 hours. B_ It is very laborious and requires high skill. 5) In general, the present invention is much more cost-effective and time-efficient than existing kits. The cost we calculated for a 20-unit FVL kit (including the costs of primers, probes, enzymes, buffers, MgCl2, dNTPs, distilled water, vials, various samplers, and costs related to labor, electricity, and water consumption, positive and negative controls of the kit, and packaging) is estimated to be about two million Tomans (as of November 1402). 6) The specificity and sensitivity of this kit are highly desirable compared to existing kits due to the presence of PNA-FIT probes. 7) All types of thermocyclers have a detection channel (FAM) of this color, and given that it has been tested in various devices, it can also be used in conventional types of QPCR devices. 8) Due to the high frequency of FVL polymorphism in the country, by designing and manufacturing this kit, the need to import the aforementioned kits with a different method is eliminated. Also, the costs of import and ancillary intermediaries are eliminated. In addition, according to the above-mentioned materials, the method used in the design of this kit has high sensitivity and specificity in identifying the above mutation. 9) This Master mix kit consists of Primer Forward, Primer Reverse, Probe PNA-FIT, dNTPs, MgCl2, 10x buffer, DNA Polymerase and DW. Therefore, it is sufficient to add the DNA sample extracted from the patient to an appropriate volume of the master mix according to the setup protocol, mix well and finally place it in the thermocycler according to the specified time and temperature program. Therefore, it is very easy to use. Description of at least one implementation method for implementing the invention The present invention is a molecular diagnostic kit that private laboratories and hospitals can apply for. The method of using this kit is as follows: it is sufficient to extract the patient's DNA sample and then, according to the protocol in the kit, mix a volume of extracted DNA with a volume of Master mix well, and then set up the thermocycler according to the time and temperature program specified in the kit's data sheet. Finally, by observing or not observing the graph and Ct, it is determined whether the patient is FVL+ or FVL-, respectively. Explicit mention of the industrial application of the invention In order to produce this kit in the industrial phase and commercialize it, the sequences of forward and reverse primers and the specific PNA-FIT probe must be synthesized on a large scale, then, along with other required compounds such as: dNTPs, MgCl2, 10x buffer, DNA Polymerase and DW, a 30 µl master mix of these compounds must be made according to the volume guidelines stated in this specification. Of course, these master mixes are generally produced in several reactions or reactions according to the needs of the target population, such as, 20 rxn, 50, 96, 100, 400. The data sheet of the present kit should also include information related to the time and temperature setup of the thermocycler stated in this specification. According to the above explanations, the goals of designing and manufacturing this kit are to use a method to increase the accuracy and sensitivity in identifying the factor V Leiden mutation, reduce the time of testing, reduce the costs of patients, given the great need in investigating thrombotic disorders and recurrent miscarriage, and finally, the ability to be used in conventional types of QPCR devices. Therefore, based on the results obtained from the design of this PNA-FIT probe specific for the FVL-G1691A mutation, and their high sensitivity and specificity in identifying true positive and negative cases, the design of this kit can be valuable in order to achieve the fastest and most accurate results and help the doctor to identify and treat the patient more effectively. Also, with the possibility of exporting this product if domestic standards and efficiency are observed, this design can be introduced as a valuable investment opportunity and even an export to other countries. This invention can be used in any medical diagnostic laboratory and hospital that is equipped with the simplest QPCR thermocycler device. Brief description of the invention Design and manufacture of Real-Time PCR kit for the diagnosis of thrombotic disease Factor V Leiden with the help of FIT Probe. FVL mutation leads to clot formation and thrombosis in various vessels of the body, and has a high risk of mortality, especially in the homozygous state. In Iran, it is frequently molecularly examined in cases such as recurrent miscarriage and stroke. The lack of diagnostic kits with high accuracy and sensitivity and low cost, the difficult and complex implementation protocol, and the time-consuming nature of testing in some imported kits that operate with a different mechanism than the currently designed kit are challenges for medical diagnostic laboratories. The designed kit is based on specific PNA-FIT probes, which specifically identify single nucleotide mutations. According to Figure 2 of the technical drawing, the structure of these probes is designed in such a way that, upon binding or non-binding to the target site, fluorescent light is detected by the thermocycler, and the presence or absence of the mutated residue is determined, respectively.

Claims

Claims What is claimed: Claim 1) The Factor V Leiden Molecular Detection Kit for the detection of the common thrombotic SNP rs6025, using the QRT-PCR technique, includes products called specific primers and a PNA-FIT probe labeled with the fluorescent dye thiazole orange (TO), which is capable of detecting the Factor V Leiden mutation. Claim 2) According to claim 1, the PNA-FIT probe is a synthetic analog of DNA that behaves like nucleic acids and acts like peptides in terms of structure. The sequences of the primers and probe are as follows: Primer Forward FVL: 5'-AGGACTACTTCTAATCTGTAAGAGCA-3' Primer Reverse FVL: 5'-CCCATTATTTAGCCAGGAGACCTA-3' Probe PNA-FIT FVL: 5'-CCT T GCC T (Thiazole orange) GTCCA- DABCYL Claim 3) According to claims 1 and 2, the designed PNA-FITs are labeled with the fluorescent dye TO at one of the internal thymidine bases. When the matching between the probe and the target sequence is correct, the TO dye emits light upon excitation. Also, heterozygosity and homozygosity can be distinguished from each other based on the height of the graph (fluorescent light intensity) and the Ct given by the thermocycler. Claim 4) According to claim 1, this kit contains a master mix of dNTPs, MgCl2, 10x Buffer, DNA Polymerase enzyme, PNA-FIT Probe, Forward Primer and Reverse Primer specific for the FVL mutation. Claim 5) According to claims 1 and 2, PNA-FIT probes have a polyamide backbone, which is non-cyclic and non-chiral, which reduces the electrostatic repulsion between the PNA-DNA and PNA-PNA hybrids, thereby increasing the binding strength compared to other types of probes. This increase in the stability of the hybrids increases the Tm temperature by about 15 to 20 degrees Celsius.

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