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Detection of mutation in rpoB gene of Multi Drug Resistant strains of Mycobacterium tuberculosis from the isolates of Nepal by Amplification

Refractory Mutation System (ARMS) A pilot study


A thesis report presentation in partial fulfillment of the requirement for The Bachelor of science degree in Biotechnology

BY Mr. Krishna Sundar Twayana Mr. Nipin Shrestha Supervisor: Mr. Prajwal Bhandari Mr. Bal Hari Poudel
Department of Biotechnology Tribhuwan University
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Introduction:
TB is an infectious disease caused by Mycobacteria species namely Mycobacterium tuberculosis, along with M. bovis, M. africanum, and M. microti; tuberculosis species complex. M tuberculosis was discovered by Robert Koch in 1882. Primary infection takes place in respiratory system called pulmonary TB, secondary infections may develop in other organs called extra pulmonary TB. Mycobacterium tuberculosis- a slow growing acid fast bacilli and an obligate aerobe.
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Current Scenario of TB cases:


1/3rd of global population infected. Nepal 49 thousands population was reported to bear new cases in TB. 6.2 thousand were dead. China 1 million new cases . India 2.3 million new cases.

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Drugs against TB
First drug Streptomycin (SM)1944, in the same year paraaminosalicylic (PAS) introduced and first combinatorial regimen trialed in the year 1950. In 1952, INH + SM +PAS treatment regimen for 18-24 months. 1960 EMB replaced PAS, regimen for 18 months 1970 Rif, regimen 9 months 1980 Pyrazinamide, regimen reduced to 6 months. Directly Observed treatment short course (DOTS) introduced by WHO in 1991 with combinatorial regimen of INH+RIF+PZA+EMB. Drugs adminstered in two phases, Initial - INH+RIF+PZA+EMB, for two months, Continuation phase - INH+RIF, for 4-7 months These are the first line drugs.
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Drug resistance:
Soon after development of drugs its resistant strains have been reported, particular genomic mutation in specific gene is traced as molecular cause. Isoniazid (INH): A prodrug, activated by peroxidase catalase, coded by katG to form reactive oxygen species i.e superoxides, peroxides, hydroxyl radicals, nitric oxides and reactive organic species like isonicotinic-acyl radical. Primary target product of InhA ;enoyl-acyl carrier protein reductase and DfrA ; NADPH dependent dihydrofolate reductase required for DNA synthesis. Resistant strains found to be mutated in - katG, most frequent point mutation S315T - InhA, six point mutations reported Ile16Thr, Ile21Thr, Ile21Val,Ile47Thr, Val78Ala and Ile95Pro
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Rifampicin
Introduced in1972, helped to reduce the treatment regimen to 6 months in combination with PZA. Rifampicin inhibits the transcription by binding to -subunit of DNA dependent RNA polymerase, coded by rpob. rpoB is a 3534 bp ORF which codes for 1178 amino acids. Resistant development due to mutation in rpoB gene. Mutation reported to be concentrated to 81bp region from codon 507 -533 in more than 90% cases, so known as Rifampicin resistance determinant region (RRDR) Mutation involves point mutations at codon 531,526, 522,516, 513 and 511and some deletions 513-514. The resistant MTB against firstline drugs is countered with the secondline drug regimen involving aminoglycosides kanamycin, amikacins, capreomycins, cycloserines, ethionamide and some fluroquinones.
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Pyrazinamide (PZA): A prodrug activated to pyrazinoic acid by enzyme pyrazinamidase coded by pncA. Ethambutol (EMB): Used in combination with INH, RIF and PZA EMB is a bacteriostatic agent that is active for growing bacilli and has no effect on non-replicating bacilli. EMB arabinogalactan(cell wall component) synthesis Arabinogalactan is synthesized by arabinosyl transferase coded by embB

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MultiDrug resistance(MDR) and diagnostic practices


MTB resistant to at least rif and inh is refered as MDR Rif resistant taken as surrogate marker.

A. Conventional phenotypic methods for diagnosis Assessing inhibition of MTB growth in presence of antibiotics ; distinguishing susceptible and resistance strains Proportion method Resistance ratio method Absolute concentration method BACTEC radiometric method

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B. Genotypic method
Nucleic acid amplification , part of genome known to be altered Assessing the product for mutation analysis by, DNA sequencing Solid phase hybridisaton Restriction fragment length polymorphism (RFLP) Real time PCR high resolution melt analysis Micro array Line probe assay Genotype MTBDR Single stranded conformation polymorphism analysis (SSCP) Heteroduplex analysis (HA) Amplification refractory mutation system (ARMS) or allele specific PCR
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Amplification Refractory Mutation System(ARMS)


Simple PCR technique used in detection of point mutation and small deletions. Exploits the absence of 3 5 exonuclease proofreading activity of Taq DNA polymerase. Mismatch of primer at 3 OH renders taq polymerase unable to extend the chain. Used in diagnosis of various genetic diseases like CFTR, 1- anti trypsin deficiency, - thalessemia and other SNPs. Can be an efficient and economic diagonostic tool in MDR TB diagonosis.
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General mechanism
ARMS assay can be directed either against wild type or mutant allele. Deliberate mismatch in the primer at 3OH, resulting two mismatches in mutant allele and a single mismatch in wild type allele or vice versa.
Mutant strands

High probability that polymerase extends allele with single mismatch but double mismatch is definitely refracted.
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Materials and methods


Sample collection- 50 random samples From Germen Nepal Tuberculosis Project (GENTUP), Kalimati. DNA Isolation: Performed by using ACCU prep Purification Kit standard protocols. PCR amplification: 1)Amplification of 537 bp of Rpob gene involving RRDR , for confirmation. - Primers used- control forward 5-CGAATATCTGGTCCGCTTGC-3 - common reverse 5-GTCGACCACCTTGCGGTACG-3 2) Amplification using ARMS primer, -Primers used - ARMS 516 -5CAGCTGAGCCAATTCACGGA 3 - ARMS 526 5CGCTGTCGGGGTTGTCCC 3 - ARMS 531 5 ACCCACAAGCGCCGACAGGTC 3 Gel electrophoresis in 2% agarose gel.

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PCR cocktail
Sn
1
2

Reagent
DNTPs
Taq polymerase

Amount
0.5
0.2U

Sn
1
2 3

Reagent
DNTPs
Taq polymerase Taq buffer Forward Primer Reverse primer ARMS primer Template

Amount
0.5
0.2U 5 1 3 1.5 1.5

3 4 5 6 7

Taqbuffer

5 4

Forward Primer 1 5 Reverse primer 1 Template NFW 1.5 15.8 6 7

NFW
Total

12.3
25

Total

25

Master mix for PCR of 537bp rpoB


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Master mix for ARMS PCR


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THERMOCYCLER PROGRAM
Sn 1 Step Initial Denaturation Temperature 94C Time 10 min

Cycle - 35
i) Denaturation ii)Annealing 94C 56C 35s 35s

iii)Extension
3 Final Extension

72C
72C

35s
10 min

Thermocycler program for both normal PCR and ARMS


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Result:
The initial amplification of 537 bp fragment of rpoB containing RRDR confirmed the presence of desired segment in our template used. 33 out of 50 samples showed the amplification. Out of 33 phenotypic resistant samples analyzed by ARMS assay, 30 samples showed mutation in any of the 3 codons we selected on the basis of high prevalence. Highest frequency of mutation was in codon 531, which accounts for 48% of total samples analyzed, followed by 36% in codon 526 and 24% in codon 516.

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Result of ARMS PCR representing Frequency of mutation in respective codons


Sn 1 2 3 Mutated codon 531 526 516 Number of sample 16 12 8 Frequency 48.4% 36.3% 24.2%

537 bp control band


Control fwd

GAC

CAC

TCG
Common reverse

216bp ARMS 531WT 246bp ARMS 526WT 261bp ARMS 516WT Schematic representation of rpoB gene targeted by ARMS PCR
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Discussion:
Due to lack of 3 exonucleolytic proofreading activity in Taq DNA polymerase, oligonucleotides with any mismatch at 3 OH end will not function as primer. ARMS assay can be successfully developed as a diagnostic tool for detection of mutation in rpoB gene, which is partly justified by this study though further verification is required. Codon 531, 526 and 516 are the most frequently mutated region that has been reported in case of rif resistance. Some samples analyzed showed no mutation in the respective regions, though the samples were phenotypically rif resistant, it can either be due to presence of mutation some where other than the selected codon or might not have mutation in RRDR at all.

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The assay described may be used for rapid detection of mutation in rpoB as it is convenient sensitive and fast. Under optimized condition the whole process can be completed in a day. Integration of the assay to microarray technique and other probing, huge number of samples can be analyzed at a time. The assay can be developed as a multiplex PCR shortening the time and making cost effective. Limitations: Can not be used for detection of all the mutations as only RRDR covers 87 types of mutation. The assay can detect only existence of mutation but not the nature of mutation.
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Conclusion:
Rif resistance serve as a surrogate marker for MDR , so detection of mutation in rpob can help early diagnosis and treatment stopping rapid transmission. ARMS can be developed as a prominent tool for mutation detection in resource limited nation like Nepal. The study is just a pilot scale thesis research, if it could be upgraded to higher level could be helpful in discovery of new diagnosis technique and even Novel drug discovery is possible.
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References:
The rpoB gene of Mycobacterium. L P Miller, J T Crawford and T M Shinnick, 1994, Antimicrob. Agents Chemother. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence Issar Smith*,TB Center, Public Health Research Institute, International Center for Public Health, Newark, New Jersey 07103-3535 Drug Resistance in Mycobacterium tuberculosis, Rabia Johnson, Elizabeth M. Streicher, Gail E. Louw, Robin M. Warren, Paul D. van Helden,and Thomas C. Victor* Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients In North India, Noman Siddiqi, Mohammed Shamim, Seema Hussain, Rakesh Kumar Choudhary, Niyaz Ahmed, Prachee, Sharmistha Banerjee, G. R. Savithri, Mahfooz Alam, Niteen Pathak, Amol Amin, Mohammed Hanief, V. M. Katoch, S. K. Sharma and Seyed E. Hasnain. Antimicrob, Agents Chemother. 2002 Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs Pedro Eduardo Almeida Da Silva1 and Juan Carlos Palomino2* Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection T. Prammananan1,2, W. Cheunoy3, D. Taechamahapun3, J. Yorsangsukkamol3, S. Phunpruch4, P. Phdarat2, M. Leechawengwong2,5 and A. Chaiprasert2,3 Gene Mutations in rpoB Rapid Detection of Mycobacterium Rifampin-Resistant Isolates in Shanghai by Using tuberculosis the Amplification Refractory Mutation System

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Analysis of any point mutation in DNA. The ampliflcation refractory mutation system (ARMS) C.R.Newton, A.Graham, L.E.Heptinstall, S.J.Powell, C.Summers, N.Kalshekerl, J.C.Smith and A.F.Markham Evaluation of a new efficient procedure for single-nucleotide polymorphism genotyping: tetra-primer amplification refractory mutation system-polymerase chain reaction. Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance Xing Tanga,*, Sheldon L. Morrisb, John J. Langonea, Larry E. Bockstahlera Analysis for a Limited Number of Gene Codons Can Predict Drug Resistance of a Mycobacterium tuberculosis in High-Incidence Community. Annelies Van Rie, Robin Warren, Idris Mshanga, AnnemarieM Jordaan, Gian D. van der Spuy, Madalene RichardsonJohn Simpson, Robert P. Gie, Donald A. Enarson, NuldaBeyers, Paul D. van Helden and Thomas C. Victor,2001, J. Clin. Microbiol.

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