A PAIR OF OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF GyrA GENE DNA IN Mycobacterium tuberculosis BACTERIAL CELLS

Abstract

This invention is a sequence of nucleotide bases that make up the primer to detect and identify the gyrA gene region in Mycobacterium tuberculosis (MTB) bacteria. This primer can detect MTB bacterial cells as the cause of tuberculosis and is expected to be one of the efforts to establish the diagnosis of tuberculosis (TB). The gyrA primer consists of 2 nucleotide sequences called forward primers and reverse primers where the nucleotide base sequences are respectively Forward primer: CGAGGAATACCCGGTACAG and Reverse primer: GCATTATCGTCGCCACTTTC. This gyrA primer can be used on DNA samples of virulent MTB H37Rv and DNA samples from the blood of patients suspected of new cases of pulmonary TB. The gyrA primer has been proven to be usable on DNA extracted using commercial kit methods and boiling methods. The designed gyrA primer is also expected to be usable on DNA samples extracted using other DNA extraction methods.

Description

PRIMER-COMPOSING NUCLEOTIDE BASE SEQUENCE FOR DETECTING AND IDENTIFYING THE GYRA GENE REGION OF MYCOBACTERIA tuberculosis Invention Engineering Field The present invention relates to a diagnostic method based on the presence of Mycobacterium tuberculosis bacteria in blood samples Patients suspected of having Tuberculosis using gyrA primers on Polymerase Chain Reaction (PCR) method. Background of the Invention Indonesia is still in second place as a country with the highest burden of tuberculosis (TB) after India. India, Indonesia, and China are the three countries with the largest number of TB cases in the world. world according to WHO estimates. Data on the percentage of new cases Tuberculosis Multi-Drug Resistance (TB-MDR) acquired from Some regions in Indonesia are subnational data. This is possibly due to TB diagnostic facilities that are not yet evenly with accurate methods. In 2022, ten countries accounts for 718 of the global gap between estimated numbers of people affected by TB and the number of people diagnosed with TB and officially reported as TB cases. To date, there are still there are problems in the diagnosis and treatment of TB, as well as difficulties related to the effectiveness of TB prevention and methods TB screening. Pulmonary tuberculosis is a contagious infectious disease that caused by Mycobacterium tuberculosis (MTB). Diagnosis of TB accurate and fast is an important basis for immediate determine the appropriate treatment and management of TB complete with accurate prevention of the spread of cases. World The Health Organization (WHO) has launched the End TB program Strategy that will be implemented with enthusiasm to 2016-2035. Another obstacle is the slow growth of MTB in clinical specimen cultures, up to several weeks to 2 or 3 months. In addition, microscopic examination of Acid Fast staining Bacilli (AFB) are also reported to have limited sensitivity. and specificity, but has the advantage of being able to detect pathogens from the specimen, which means that there is still a replication process active from MTB pathogens. Accurate microscopic detection techniques has also been developed, which is also useful for monitoring the results TB treatment. Currently expensive commercial diagnostic kits are not affordable to the general public. Until now, this method continues to be developed to improve the accuracy of TB diagnosis. Increased multiplication of mycobacterial cells in ongoing TB cases the disease process involves multiplication of bacterial cells including nucleic acids can be found in the bloodstream, this can increasing the detection sensitivity of nucleic acid amplification methods in blood specimens of patients suspected of having tuberculosis. MTB identification specifications are based on the detection of areas conserved and specific genes that represent roles survival of Mycobacteria, especially those that play a role in active multiplication of Mycobacteria cells. Cell multiplication played primarily by the gyrase gene complex. One of the The gene complex is the gyrA gene. Accurate specimens are also is a potential determinant of the accuracy of the diagnosis and method correct diagnosis. Patient's blood specimen from venous blood Peripheral can be an easy method for accurate specimens in patients with various conditions, including cases of pulmonary TB difficulty obtaining sputum and Extra-Pulmonary Tuberculosis (EPTB) which is difficult to obtain specimens. In this patent description, the gyrA primer will be explained. and use in the PCR nucleic acid amplification method, with DNA samples from the virulent MTB strain H37Rv as a positive control and in DNA from 14 blood samples of suspected pulmonary TB patients new. It is expected that the gyrA primer of this invention can also be used for Nucleic Acid Amplification (NAA) PCR method with DNA samples originating from specimens other than blood, such as sputum, fluid pleura, trunk wash, tissue biopsy and other specimens. In addition can be used with a wide variety of DNA sample origins, gyrA primers This designed tool can be used on DNA samples that have been processed by various methods. In this patent description, the primary gyrA was shown to be usable on extracted DNA. with the commercial kit method and the boiling method. It is expected This gyrA primer can be used on extracted DNA samples. with various other methods for the PCR method for the purpose establishing a TB diagnosis is useful as a determinant of appropriate treatment appropriate. Brief Description of the Invention The main objective of this invention is to overcome the problem that have existed before, especially in detection methods the presence of infection from MTB bacteria in pulmonary TB patients. Invention This is a pair of primers consisting of a forward primer and a reverse primer. reverse primer. The designed primer is a specific primer to detect the presence of the gyrA gene in MTB. The purpose and benefits and a more complete understanding of the invention This is explained with reference to the pictures. Short Description of Image To facilitate understanding of the essence of this invention, hereinafter will describe the embodiment of the invention through the attached drawings, Where: Figure 1, is a flow diagram of the primary design process. Figure 2, is the electrophoresis result of the amplicon. using the gyrA primer generated by optimization through nucleic acid amplification procedure, namely: annealing temperature. With the description: Description: M —- Marker 100bp?» A - 55.0 Sc (Positive): B —- 54.9c (Positive): C — 54.6c (Positive): D - 54.2” (Positive): E —- 53.89c (Positive): F — 53.49c (Positive): G — 53.2” (Positive): H — 53.00C (Positive): K(-) - Aguadest is sterile (Negative). At temperatures ranging from 53.0 - 55.00c it can produce specific and conserved amplicons 1008. Figure 3, is the result of amplicon electrophoresis of the gene region. gyrA of MTB detected from blood specimens of pulmonary TB patients / new cases with description M — marker, 1 - 14 — Amplification results Nucleic Acid (NAA) from DNA of blood specimens of pulmonary TB patients new: Kt - positive control using DNA from MTB H37Rv, K- — negative control using sterile aguades. Complete Description of the Invention The sequence of nucleotide bases that make up the primers for detecting and identifying the gyrA gene region of MTB bacteria is an invention which is expected to help the development of diagnostic enforcement early stages of TB disease. Based on Figure 1, the formation of primary The gyrA gene originated from a template search at the National Center for Biotechnology Information (NCBI) (https: / / blast.ncbi.nlm.nih.gov / ). Genes used as The template for this primer is the gyrA gene region taken from NCBI references genomic DNA of virulent MTB strain H37Rv (accession number: CP003248.2). This gyrA gene region primer targets AAN with the result 270 nucleotide base pairs long. The selected region of the gyrA gene is a specific and conserved part of MTB bacteria, then it can be implied that the detected gene has high specificity and sensitivity to detect the presence of MTB bacteria in suspected pulmonary tuberculosis patients and TB patients extra-pulmonary. Based on Figure 1, the next process is to design gyrA gene region primer using GENETYX ver. 10 software. input the full gyrA gene sequence template into the GENETYX software ver. 10 and set the desired criteria. Primary criteria are regulated include: the length of the primary nucleotide base sequence, primary melting temperature (Tm), percentage of Guanine and Cytosine bases primary ($GC). The desired primary length is between 18 - 30 nucleotide bases: melting temperature (Tm) of the primer desired is 55 - 80 “”C: and $8GC primary desired is 50 - 608. After setting the primary criteria, the GENETYX software ver. 10 will generate several primer pairs. Then select the pair of primers that most closely matches the set criteria. After selecting a pair of primers, the next step is verify the primer pair with Clone software Manager Professional ver. 9.2. Clone Manager Professional Software will verify the previously selected primer pair based on previously desired criteria. For verify the primer pair further, Verification is done using the BLAST-N program available at NCBI website to find out which primer pair has been selected is actually part of the gyrA gene region of MTB bacteria. The NCBI BLAST-N results of the primer pair are shown in Table 1. Table 1 explains that a pair of primers that have been selected is the region of the MTB gyrA gene. Table 1. NCBI BLAST-N results of the selected primer pairs Species Order Name Region Accession Link Primer Primer Detected Gen Number gyrA- CGAGGAAT | Mycobacterium | 9512 — | CPOO3248 https: / / www. 2301-F ACCCGGTA Im 9530 ncbi.nlm.nih CAG tuberculosis (gyrA).gov / nucleot H37Rv ide / CP003248 .2?report-—ge nbankslog$-n uclaligngbla st rank—2s8RI D-9PUMXJIDOL 6&from—95126 to—9530 gyrA- GCATTATC | Mycobacterium | 9762 — | CPOO3248 https: / / www. 2307-R GTceccac |m 9781 ncbi.nlm.nih TTTC tuberculosis (gyrA).gov / nucleot H37Rv ide / CP003248 .2?report-—ge nbankslog$-n uclaligngbla st rank—2s8RI D—-9PV2HKZ001 3&from—-97628 to—-9781 After verifying the selected primer pair, The next step is to optimize the primer attachment temperature. (annealing) in the Nucleic Acid Amplification (NAA) process. Optimization This uses a temperature variation of 53.0 - 55.08cC. AAN was carried out with the stages outlined in Table 2. Carrying out the stages Denaturation, Annealing, and Extension 35 times. Table 2. Stages of Nucleic Acid Amplification (NAA) with gyrA Primer Stages Temperature Duration Pre-denaturation 940 10 minutes Denaturation 940C 15 seconds Annealing 53 -— 550 20 seconds Extension 1200 45 seconds Final extension 1200 2 minutes Elongation 40C oo Primary verification of gyrA was performed with AAN and using DNA template from MTB cell culture of virulent strain H3 / Rv. MTB DNA H37Rv 1 was extracted using the commercial kit OiAmp Oiagen which produced in Germany. The resulting amplicons were visualized using electrophoresis stage to see the success of region amplification MTB gyrA gene. Electrophoresis is declared successful if there is fluorescent band whose size corresponds to the target region of the gyrA gene on each agarose gel well previously contained the resulting amplicon AAN. In Figure 2 it can be seen that there is one fluorescent band on eight agarose wells located under the 300bp marker and in above the 200bp marker so it can be implied that the band This is the AAN target which is 27 / 0bp in size. The size This corresponds to the primary target that detects the gene region. gGYrA in MTB bacteria that has been designed. Figure 3 shows that the designed gyrA primer can used to detect the presence of MTB bacteria in the blood new case of pulmonary TB patient. DNA extraction has been carried out with blood samples of new cases of pulmonary TB patients with modifications to blood sample preparation. The first stage is carried out by adding 10 ml of sterile aguabidest in 3 ml of the patient's blood sample. Then mix with a vortex machine until the mixture is homogeneous. This mixing aims to lyse the blood cells so that DNA and microbiota in white blood cells can come out and The DNA is extracted. The next process is centrifuging mix at a speed of 4000 rpm for 15 minutes then continued with the DNA extraction procedure of Mycobacterium sp. cells. in the blood suspension of suspected pulmonary TB patients, a solution was taken TE 50mM / 5mM solution volume 400 ul, then added in 100 bl blood suspension (ratio 4:1). DNA extraction reaction is made effective by stirring the sputum suspension and Tris-EDTA solution 50mM / 5mM until homogeneous, then continue the heating process 90”C, while doing it until homogeneous, then heated again at 90”C. After that, it is left and maintained at a temperature of 90”C, so that the cell fragment components precipitate, while the DNA is in the liquid above the sediment (supernatant). Next 500ul of the supernatant liquid directly above the sediment was taken separated and placed in an Ependorf tube as a result DNA extract solution (crude DNA). In Figure 3, the AAN results of the gene region have been visualized. gyrA using DNA samples from new cases of pulmonary TB patients. Results Positive is indicated by a fluorescent band in each well. From The results of the electrophoresis showed that out of 14 people patients, gyrA primers successfully detected MTB bacteria in 12 people patients. Thus, it can be seen that the gyrA primer that has been designed to have a sensitivity of 865. The gyrA primer that has been designed is a primer that comes from from the template region of the gyrA gene and not the entire gyrA gene. Template the selected region is a specific and conserved region in MTB bacteria. Because the template used is part of from genes and not all genes, then the AAN process requires shorter time. Even though it has more time In short, the designed gyrA primers have specificity high. This specificity has been demonstrated in Figure 2 and Figure 3 each explains that the gyrA primer can 1008 detects MTB with MTB H3 / Rv DNA samples and 868 can detect MTB with blood samples from patients suspected of new cases Pulmonary TB. With a shorter duration of NAA, TB diagnosis can be It is hoped that it will be faster and more precise so that it can be done appropriate treatment.

Claims

1. A pair of primer sequences to detect DNA from cells Mycobacterium tuberculosis bacteria in the blood of patients who suspected pulmonary tuberculosis, primary consisting of primary forward and reverse primers consisting of nucleotide bases in the following order: A A. Primary forward: CGAGGAATACCCGGTACAG b. Reverse primer: GCATTATCGTCGCCACTTTC.

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