DNA Probe Design for Detection Mutation at Codon 315 In katG Gene of Mycobacterium Tuberculosis to Real-Time Polymerase Chain Reaction
Abstract
High-level resistance to isoniazid as a first-line tuberculosis drugs can be caused by mutations in codon 315 katG
Mycobacterium tuberculosis . Mutation at codon 315 is the most frequent mutation with the highest amino acid variation,
compared to other codons in the Mycobacterium tuberculosis katG gene. Therefore, a specific probe is required for rapid and
proper detection of mutations at codon 315. In this study, the design of a nucleotide sequence probe with TaqMan labeling was
performed using Clone Manager Suite 6 software . The mutant probe obtained was analysed in two stages. The initial analysis
is based on the length of the probe (22-30 bases), Tm (70ºC), %GC (35-65%), not in hairpin form, dimer (< 5 bases), runs and
repeat (? 4 for base A, T, C, and < 3 for base G). Furthermore the final analysis was carried out with no G base in 2 bases at
the end of the 5’ probe and the amount of base C ? G.
The study resulted in 260 probe mutants. After the initial analysis, 11 mutant probes were obtained to recognize mutations in
the codon of 315 katG Mycobacterioum tuberculosis genes. The probe consists of 2 probes for the S315T mutation, 6 probes
for S315N mutation, and 3 probes for S315V mutation. The criteria of the 11 mutant probes are 22-23 bases long, Tm 70ºC, %
GC 56-63%, 4 dimer , 2 runs , and does not have repeats and does not form hairpin at a temperature of 56ºC. Based on the final
analysis, 3 mutant probes were obtained fulfilling the TaqMan probe labeling criteria, namely K315MT1 for specific detection
of mutation S?T and K315MN5, then K315MN23 for specific detection of mutation S?N.
The conclusion of this study shows that the best mutant nucleotide sequence probes for the detection of mutations at codon of
315 KatG Mycobacterium tuberculosis genes are 5’-FAM-CC ACC GGC ATC GAG GTC GTA TG-TAMRA-3’; 5’FAM-ATC
ACC AAC GGC ATC GAG GTC G-TAMRA-3’; dan 5’FAM-C ACC AAC GGC ATC GAG GTC GTA T-TAMRA-3’. The
design of the mutant probe according to the TaqMan probe criterion for real-time PCR was obtained by 3 probes from the 11
selected mutant probes in the initial analysis.
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References
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Tuberculosis Report 2014. Perancis: World Health
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Mycobacterium tuberculosis Strains Derived from
Tuberculosis Patients in Mazandaran, Iran, 2013. Int J
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Shi. 2015. The Mutations of katG and inhA Genes of
Isoniazid-Resistant Mycobacterium tuberculosis
Isolates in Taiwan. Journal of Microbiology,
Imunology and Infection. Volume 48: 249-255.
[7] Murray, J. L., P. Hu, dan D. A. Shafer. 2014. Seven
Novel Probe System for Real-Time PCR Provide
Absolute Single-Base Discrimination, Higher
Signaling, and Generic Components. The Journal of
Molecular Diagnostics. Vol. 16 (6): 627-638.
[8] Suryadi, T. 2013. Identifikasi Mutasi Gen katG Pada
Isolat P10 dan 151 Mycobacterium tuberculosis
Multidrug Resistance di Bali Dengan Metode
Polymerase Chain Reaction. Skripsi. Universitas
Udayana, Bukit Jimbaran: 1-39.
[9] Deniariasih, N. W. 2013. Deteksi Mutasi Pada Gen
katG (Fragmen 0,7 kb) Isolat 86 dan P11
Mycobacterium tuberculosis Multidrug Resistance
Dengan Teknik Polymerase Chain Reaction (PCR).
Skripsi. Universitas Udayana, Bukit Jimbaran: 1-39.
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Nojoumi. 2008. Detection of Mutation in Isoniazid-
Resistant Mycobacterium tuberculosis Isolates From
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Medical Microbiology. Volume 26(2): 143-147.
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Napiorkowska, E. A. Kopec, Z. Zwolska, dan J.
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With Isoniazid Resistance in Multidrug-Resistant
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K. Kobayashi.2004. Dual-Probe Assay For Rapid
Detection of Drug-Resistant Mycobacterium
tuberculosis by Real-Time PCR. Journal of Clinical
Microbiology. Volume 42(11): 5277-5285.
[19] Kurreck, J. dan C. A. Stein. 2015. Molecular
Medicine. Jerman: Wiley-VCH. 37-51.
[20] Chou, C.C., C. C. H. Chen, T. T. Lee, dan K. Peck.
2004. Optimization of Probe Length and The Number
of Probes Per Gene for Optimal Microarray Analysis
of Gene Expression. Nucleic Acid Research. Volume
32(12): 99-107.
[21] Kalendar, R., D. Lee, dan A. H. Schulman. 2011. Java
Web Tools for PCR, In Silico PCR, and
Oligonucleotide Assembly and Analysis. Genomics.
Volume 98: 137-144.
[22] Yilmaz, L. S., S. Parnerkar, dan D. D. R. Noguera.
2011. mathFISH, A Web Tool That Uses
Thermodynamics-Based Mathematical Models for In
Silico Evaluation of Oligonucleotide Probes for
Fluorescence In Situ Hybridization. Applied And
Environmental Microbiology. Volume 77(3): 1118-
1122.
[23] Zhang, Y. dan W. W. Yew. 2009. Mechanisms of
Drug Resistance in Mycobacterium tuberculosis. Int.
J. Tuberc Lung Dis. Volume 13(11): 1320-1330.
[24] Lina, M., B. Bela, dan A. Yasmon. 2009. Deteksi
Mutasi Gen katG Mycobacterium tuberculosis Dengan
Metode PCR (Polymerase Chain Reaction)-
Hibridisasi Dot Blot Menggunakan Pelacak
Oligonukleotida Bertanda 32P. Jurnal ilmiah Aplikasi
Isotop dan Radiasi. Volume 5 (1): 54-67.
[25] Smith, J. E. 2009. Biotechnology 5th Edition. New
York: Cambridge University Press. 41-44.
[26] Anonim a. 2004. Assay Formats for Use in Real-Time
PCR. Technical Note. Jerman: Roche Applied
Science. 1-13. Available at: https://lifescience.
roche.com/wcsstore/RASCatalogAssetStore/Articles/A
ssay%20Format%20for%20use%20in%20Real-
Time%20PCR.pdf. (Cited March 17, 2016).
[27] Alvandi, E. dan F Koohdani. 2014. Zip Nucleic Acid:
A New Reliable Method To Increase The Melting
Temperature of Real-Time PCR Probes. Journal of
Diabetes & Metabolic Disorders. Vol. 13 (26): 1-4.
[28] Livak, K. J., S. J. A. Flood, J. Marmaro, W. Giusti,
dan K. Deetz. 1995. Oligonucleotides With
Fluorescent Dyes At Opposite Ends Provide A
Quenched Probe System Useful For Detecting PCR
Product and Nucleic Acid Hybridization. Genome
Research. Vol. 4: 357-362.
[29] Anonim d. 2001. Allelic Discrimination Using The 5’
Nuclease Assay. USA: Applied Biosystems. 1-8.
http://www.austincc.edu/mlt/mdfund/mdfund
_Unit11AllelicDiscrimination.pdf. (Cited March 14,
2016).
[30] Logan, J., K. Edwards, dan N. Saunders. 2009. Real-
Time PCR: Current Technology and Applications.
London: Caister Academic Press. 1-27. Available
at:www.horizonpress.com/realtimepcr.
http://citeseerx.
ist.psu.edu/viewdoc/download?doi=10.1.1.261.223&re
p=rep1&type=pdf. (Cited April 23, 2016).
[31] Anonim b. 2006. Real-Time PCR Applications Guide.
Bulletin 5279. USA: Bio-Rad Laboratories Inc. 1-100.
Available at: www.bio-rad.com/webroot/
web/pdf/lsr/literature/Bulletin_5279.pdf. (Cited
February 15, 2016).
[32] Holland, P. M., R. D. Abramcon, R. Watson. Dan D.
H. Gelfand. 1991. Detection of Spesific Polymerase
Chain Reaction Product By Utilizing The 5’→3’
Exonuclease Activity of Thermus aquaticus DNA
Polymerase. Proc Natl Acad Sci Biochemistry. Vol.
88: 7276-7280.
[33] Liu, H., H. Wang, Z. Shi, H. Wang, C. Yang, S. Silke,
W. Tan, dan Z. Lu. 2006. Taqman Probe Array For
Quantitative Detection of DNA Targets. Nucleic Acids
Research. Vol. 34(1): 1-8.
[34] Handoyo, D., dan A. Rudiretna. 2000. Prinsip Umum
dan Pelaksanaan Polymerase Chain Reaction
(PCR).Unitas. Vol. 9 (1): 17-29.
[35] Patel, N. K., dan N. Prakash. 2013. Principle and
Tools For Primer Design. Atmiya Spandan Biological
Sciences. Vol. 1 (1): 79-95.
[36] Bishop, J. L., S. A. Campbell, P. Farrell, M.
Fitzgerald, M. Haugen, W. Kocmond, D.E. Madden,
W. E. Murray, dan D. H. Persing. 2015. Designing
Real-Time Assays on the SmartCycler® II System.
United State: Cepheid Technical Support. 1-
8.Available at: http://www.cepheid.com/us/
component/phocadownload/category/2-
support?download=8:smart-note-6-1. (Cited April 20,
2016).
[37] Proudnikov, D., V. Yuferov, Y. Zhou, K. S. Laforge,
A. Ho, M. J. Kreek. 2003. Optimizing Primer-Probe
Design for Fluorescent PCR. Journal of Neuroscience
Methods. Vol 123: 31-45.
[38] Lazaro, R. D. dan M. Hernandez. 2013. Real-Time
PCR in Food Science Current Technology and
Applications. Norfolk, UK: Caister Academic Press. 5-
12.
[39] Mulle, J. G., V. C. Patel, S. T. Warren, M. R. Hegde,
D. J. Cutler, dan M. E. Zwick.2010. Empirical
Evaluation of Oligonucleotide Probe Selection for
DNA Microarrays. Plos One. Vol. 5 (3): 1-7.
[40] Anonim c. 2006. Beacon Designer 5.10 Manual.
Corona Way: USA: Premier Biosoft International. 22-
89. Available at:
http://211.69.128.172/tlli/upfile/doc/7584c8_Beacon%
20Designer%20510Manual.pdf. (Cited February 25,
2016).
[41] Kubista, M., J. M. Andrade, M. Bengtsson, A.
Forootan, J. Jonak, K. Lind, R. Sindelka, R. Sjoback,
B. Sjogreen, L. Strombom, A. Stahlberg, dan N. Zoric.
2006. The Real-Time Polymerase Chain Reaction.
Molecular Aspects of Medicine Review. Vol. 27: 95-
125.
[42] Yuwono, T. 2008. Biologi Molekuler. Jakarta:
Penerbit Erlangga. 49-74.
[43] Borah, P. 2011. Primer Designing For PCR.Sci Vis.
Vol. 3: 134-136.
[44] Rychlik, W. 2008.Oligo Primer Analysis Software
Version 7. USA: Molecular Biology Insights Inc. 90-
123.
[45] Nazarenko, I., R. Pires, B. Lowe, M. Obaidy, dan A.
Rashtchian. 2002. Effect of Primary and Secondary
Structure of Oligodeoxyribonucleotides on The
Fluorescent Properties of Conjugated Dyes. Nucleic
Acids Research. Volume 30 (9): 2089-2195.
[46] Mackay, I. M. 2007. Real-Time PCR in Microbiology
From Diagnosis to Characterization. Norfolk, Uk:
Caister Academic Press. 1-64
Tuberculosis Report 2014. Perancis: World Health
Organization.
[2] World Health Organization(WHO). 2015. Global
Tuberculosis Report 2015. Perancis: World Health
Organization.
[3] Lisdawati, V., N. Puspandari, L. Rif’ati, T. Soekarno,
M, Melatiwati, K. Syamsidar, L. Ratnasari, N.
Izzatun, dan I. Parwati. 2015. Molecular
Epidemiology Study of Mycobacterium tuberculosis
and Its Susceptibility to Anti-TuberculosisDrugs in
Indonesia. BMC Infectious Disease. Volume 15: 366-
373.
[4] Babamahmoodi, F., M. R. Mahdavi, H. Jalali, B.
Talebi, P. Roshan, dan M. Mahdavi. 2014. Evaluation
of Gene Mutations Involved in Drug Resistance in
Mycobacterium tuberculosis Strains Derived from
Tuberculosis Patients in Mazandaran, Iran, 2013. Int J
Mol Cell Med Summer. Volume 3(3): 191-195.
[5] Silva, P. E. A. D., dan J. C. Palomino. 2011.
Molecular Basis and Mechanisms of Drug Resistance
in Mycobacterium tuberculosis: Classical and New
Drugs. Journal of Antimicrobial Chemotherapy.
Volume 66(7): 1417-1430.
[6] Tseng, S. T., C. H. Tai, C. R. Li, C. F. Lin, dan Z. Y.
Shi. 2015. The Mutations of katG and inhA Genes of
Isoniazid-Resistant Mycobacterium tuberculosis
Isolates in Taiwan. Journal of Microbiology,
Imunology and Infection. Volume 48: 249-255.
[7] Murray, J. L., P. Hu, dan D. A. Shafer. 2014. Seven
Novel Probe System for Real-Time PCR Provide
Absolute Single-Base Discrimination, Higher
Signaling, and Generic Components. The Journal of
Molecular Diagnostics. Vol. 16 (6): 627-638.
[8] Suryadi, T. 2013. Identifikasi Mutasi Gen katG Pada
Isolat P10 dan 151 Mycobacterium tuberculosis
Multidrug Resistance di Bali Dengan Metode
Polymerase Chain Reaction. Skripsi. Universitas
Udayana, Bukit Jimbaran: 1-39.
[9] Deniariasih, N. W. 2013. Deteksi Mutasi Pada Gen
katG (Fragmen 0,7 kb) Isolat 86 dan P11
Mycobacterium tuberculosis Multidrug Resistance
Dengan Teknik Polymerase Chain Reaction (PCR).
Skripsi. Universitas Udayana, Bukit Jimbaran: 1-39.
[10] Hazbon, M. H., M. Brimacombe, M. B. D.Valle, M.
Cavatore, M. I. Guerrero, M. V. Basil, H. B. Jacobe,
C. Lavender, J. Fyfe, L. G. Garcia, C. I. Leon, M.
Bose, F. Chaves, M. Murray, K. D. Eisenach, J. S.
Osornio, M. D. Cave, A. P. D. Leon, dan D. Alland.
2006. Population Genetics Study of Isoniazid
Resistance Mutations and Evolution of Multidrug-
Resistant Mycobacterium tuberculosis. Antimicrobial
Agents and Chemotherapy. Volume 50(8): 2640-2649.
[11] Bostanabad, S. Z., L. P. Titov, A. Bahrmand, S. A.
Nojoumi. 2008. Detection of Mutation in Isoniazid-
Resistant Mycobacterium tuberculosis Isolates From
Tuberculosis Patient in Belarus. Indian Journal of
Medical Microbiology. Volume 26(2): 143-147.
[12] Jagielski, T., Z. Bakuta, K. Roeske, M. Kaminski, A.
Napiorkowska, E. A. Kopec, Z. Zwolska, dan J.
Bielecki. 2014. Detection of Mutations Associated
With Isoniazid Resistance in Multidrug-Resistant
Mycobacterium tuberculosis Clinical Isolates. J.
Antimicrob Chemother. Volume 69(4): 2369-2375.
[13] Navarro, E., G. S. Heras, M. J. Castano, dan J. Solera.
2015. Real-Time PCR Detection Chemistry. Clinica
Chimica Acta. Volume 439: 231-250.
[14] Espy, M. J., J. R. Uhl, L. M. Sloan, S. P. Buckwalter,
M. F. Jones, E. A. Vetter, J. D. C. Yao, N. L.
Wengenack, dan J. E. Rosenblatt. 2006. Real-Time
PCR in Clinical Microbiology: Applications for
Routine Laboratory Testing. Journal of Clinical
Microbiology Review. Volume 19(1): 165-256.
[15] Dorak, M. T. 2006. Real-Time PCR. New York:
Taylor and Francis Group.1-30.
[16] McPherson, M., dan S. Moller. 2006. PCR Edisi 2.
New York: Taylor & Francis Group. 209-305.
[17] Walker, J.M. dan Rapley, R. 2008.Medical
Biomethods Handbook.Totowa, New Jersey: Humana
Press Inc. 13-341.
[18] Wada, T., S. Maeda, A. Tamaru, S. Imai, A. Hase, dan
K. Kobayashi.2004. Dual-Probe Assay For Rapid
Detection of Drug-Resistant Mycobacterium
tuberculosis by Real-Time PCR. Journal of Clinical
Microbiology. Volume 42(11): 5277-5285.
[19] Kurreck, J. dan C. A. Stein. 2015. Molecular
Medicine. Jerman: Wiley-VCH. 37-51.
[20] Chou, C.C., C. C. H. Chen, T. T. Lee, dan K. Peck.
2004. Optimization of Probe Length and The Number
of Probes Per Gene for Optimal Microarray Analysis
of Gene Expression. Nucleic Acid Research. Volume
32(12): 99-107.
[21] Kalendar, R., D. Lee, dan A. H. Schulman. 2011. Java
Web Tools for PCR, In Silico PCR, and
Oligonucleotide Assembly and Analysis. Genomics.
Volume 98: 137-144.
[22] Yilmaz, L. S., S. Parnerkar, dan D. D. R. Noguera.
2011. mathFISH, A Web Tool That Uses
Thermodynamics-Based Mathematical Models for In
Silico Evaluation of Oligonucleotide Probes for
Fluorescence In Situ Hybridization. Applied And
Environmental Microbiology. Volume 77(3): 1118-
1122.
[23] Zhang, Y. dan W. W. Yew. 2009. Mechanisms of
Drug Resistance in Mycobacterium tuberculosis. Int.
J. Tuberc Lung Dis. Volume 13(11): 1320-1330.
[24] Lina, M., B. Bela, dan A. Yasmon. 2009. Deteksi
Mutasi Gen katG Mycobacterium tuberculosis Dengan
Metode PCR (Polymerase Chain Reaction)-
Hibridisasi Dot Blot Menggunakan Pelacak
Oligonukleotida Bertanda 32P. Jurnal ilmiah Aplikasi
Isotop dan Radiasi. Volume 5 (1): 54-67.
[25] Smith, J. E. 2009. Biotechnology 5th Edition. New
York: Cambridge University Press. 41-44.
[26] Anonim a. 2004. Assay Formats for Use in Real-Time
PCR. Technical Note. Jerman: Roche Applied
Science. 1-13. Available at: https://lifescience.
roche.com/wcsstore/RASCatalogAssetStore/Articles/A
ssay%20Format%20for%20use%20in%20Real-
Time%20PCR.pdf. (Cited March 17, 2016).
[27] Alvandi, E. dan F Koohdani. 2014. Zip Nucleic Acid:
A New Reliable Method To Increase The Melting
Temperature of Real-Time PCR Probes. Journal of
Diabetes & Metabolic Disorders. Vol. 13 (26): 1-4.
[28] Livak, K. J., S. J. A. Flood, J. Marmaro, W. Giusti,
dan K. Deetz. 1995. Oligonucleotides With
Fluorescent Dyes At Opposite Ends Provide A
Quenched Probe System Useful For Detecting PCR
Product and Nucleic Acid Hybridization. Genome
Research. Vol. 4: 357-362.
[29] Anonim d. 2001. Allelic Discrimination Using The 5’
Nuclease Assay. USA: Applied Biosystems. 1-8.
http://www.austincc.edu/mlt/mdfund/mdfund
_Unit11AllelicDiscrimination.pdf. (Cited March 14,
2016).
[30] Logan, J., K. Edwards, dan N. Saunders. 2009. Real-
Time PCR: Current Technology and Applications.
London: Caister Academic Press. 1-27. Available
at:www.horizonpress.com/realtimepcr.
http://citeseerx.
ist.psu.edu/viewdoc/download?doi=10.1.1.261.223&re
p=rep1&type=pdf. (Cited April 23, 2016).
[31] Anonim b. 2006. Real-Time PCR Applications Guide.
Bulletin 5279. USA: Bio-Rad Laboratories Inc. 1-100.
Available at: www.bio-rad.com/webroot/
web/pdf/lsr/literature/Bulletin_5279.pdf. (Cited
February 15, 2016).
[32] Holland, P. M., R. D. Abramcon, R. Watson. Dan D.
H. Gelfand. 1991. Detection of Spesific Polymerase
Chain Reaction Product By Utilizing The 5’→3’
Exonuclease Activity of Thermus aquaticus DNA
Polymerase. Proc Natl Acad Sci Biochemistry. Vol.
88: 7276-7280.
[33] Liu, H., H. Wang, Z. Shi, H. Wang, C. Yang, S. Silke,
W. Tan, dan Z. Lu. 2006. Taqman Probe Array For
Quantitative Detection of DNA Targets. Nucleic Acids
Research. Vol. 34(1): 1-8.
[34] Handoyo, D., dan A. Rudiretna. 2000. Prinsip Umum
dan Pelaksanaan Polymerase Chain Reaction
(PCR).Unitas. Vol. 9 (1): 17-29.
[35] Patel, N. K., dan N. Prakash. 2013. Principle and
Tools For Primer Design. Atmiya Spandan Biological
Sciences. Vol. 1 (1): 79-95.
[36] Bishop, J. L., S. A. Campbell, P. Farrell, M.
Fitzgerald, M. Haugen, W. Kocmond, D.E. Madden,
W. E. Murray, dan D. H. Persing. 2015. Designing
Real-Time Assays on the SmartCycler® II System.
United State: Cepheid Technical Support. 1-
8.Available at: http://www.cepheid.com/us/
component/phocadownload/category/2-
support?download=8:smart-note-6-1. (Cited April 20,
2016).
[37] Proudnikov, D., V. Yuferov, Y. Zhou, K. S. Laforge,
A. Ho, M. J. Kreek. 2003. Optimizing Primer-Probe
Design for Fluorescent PCR. Journal of Neuroscience
Methods. Vol 123: 31-45.
[38] Lazaro, R. D. dan M. Hernandez. 2013. Real-Time
PCR in Food Science Current Technology and
Applications. Norfolk, UK: Caister Academic Press. 5-
12.
[39] Mulle, J. G., V. C. Patel, S. T. Warren, M. R. Hegde,
D. J. Cutler, dan M. E. Zwick.2010. Empirical
Evaluation of Oligonucleotide Probe Selection for
DNA Microarrays. Plos One. Vol. 5 (3): 1-7.
[40] Anonim c. 2006. Beacon Designer 5.10 Manual.
Corona Way: USA: Premier Biosoft International. 22-
89. Available at:
http://211.69.128.172/tlli/upfile/doc/7584c8_Beacon%
20Designer%20510Manual.pdf. (Cited February 25,
2016).
[41] Kubista, M., J. M. Andrade, M. Bengtsson, A.
Forootan, J. Jonak, K. Lind, R. Sindelka, R. Sjoback,
B. Sjogreen, L. Strombom, A. Stahlberg, dan N. Zoric.
2006. The Real-Time Polymerase Chain Reaction.
Molecular Aspects of Medicine Review. Vol. 27: 95-
125.
[42] Yuwono, T. 2008. Biologi Molekuler. Jakarta:
Penerbit Erlangga. 49-74.
[43] Borah, P. 2011. Primer Designing For PCR.Sci Vis.
Vol. 3: 134-136.
[44] Rychlik, W. 2008.Oligo Primer Analysis Software
Version 7. USA: Molecular Biology Insights Inc. 90-
123.
[45] Nazarenko, I., R. Pires, B. Lowe, M. Obaidy, dan A.
Rashtchian. 2002. Effect of Primary and Secondary
Structure of Oligodeoxyribonucleotides on The
Fluorescent Properties of Conjugated Dyes. Nucleic
Acids Research. Volume 30 (9): 2089-2195.
[46] Mackay, I. M. 2007. Real-Time PCR in Microbiology
From Diagnosis to Characterization. Norfolk, Uk:
Caister Academic Press. 1-64
Published
2018-01-25
How to Cite
RAHMARYANI, I Gusti A. A. Santhi et al.
DNA Probe Design for Detection Mutation at Codon 315 In katG Gene of Mycobacterium Tuberculosis to Real-Time Polymerase Chain Reaction.
Journal of Health Sciences and Medicine, [S.l.], v. 1, n. 2, p. 31-41, jan. 2018.
ISSN 2622-0555.
Available at: <https://ojs.unud.ac.id/index.php/jhsm/article/view/36991>. Date accessed: 26 apr. 2024.
doi: https://doi.org/10.24843/JHSM.2017.v01.i02.p08.
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