COMPARATIVE MIRU-VNTR PROFILE of the LCP TB ISOLATES and

the WILD TYPE STRAIN H37Rv

Crist John M. Pastor1, Ursela G. Bigol1, Jay T. Dalet1, Maria Teresa A. Barzaga2,
Nelia S.Tan-Liu2 and Francisco M. Heralde III1
1Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines-Manila and
2 Lung Center of the Philippines, Quezon City, Philippines
 
Corresponding author: FM Heralde III, [email protected]

ABSTRACT
The Mycobacterium Interspersed Repeating Units-Variable Number Tandem Repeats (MIRU-VNTR) profiling of heat killed
Mycobacterium tuberculosis isolates from the Lung Center of the Philippines was done through optimization of the genotyping procedure using 12
MIRU-VNTR primers to amplify the MIRU-VNTRs of the wild type Mycobacterium tuberculosis strain, H37Rv. The PCR products were sequenced and verified
through online NCBI GenBank BLAST tool. The optimized protocol was then used to amplify MIRU-VNTRs from the Lung Center of the Philippines and
further calculate the number of MIRU-VNTR repeats which served as identification code for each isolate. Results show that some MIRU-VNTRs are
highly polymorphic while others are moderately variable across strains. Comparison of the profile of the samples and with the wild type H37Rv
show that different strains of Mycobacterium tuberculosis thrive in our country which calls for further studies on correlation of these observed
differences on bacterial characteristics.

Methodology
Optimization of MIRU - VNTR Amplification Process

INTRODUCTION
Tuberculosis
is one of the most deadly and widespread
infectious diseases accounting for 2 to 3 million deaths worldwide1.
According to the World Health Organization Global TB Report in
Mtb ( H37Rv) DNA Lysate PCR Separation by Visualization Sequencing of PCR
2006, one-third of the entire world’s population is infected with the Bacteria Grown in Preparation Amplification Gel thru BioRad Products and NCBI
Culture Medium
disease2 and incidence is highest in Africa and Sub-Saharan Electrophoresis GelDoc System BLAST Analysis

regions. Among the South East Asian nations, the Philippines is

considered as the ninth highest burden in terms of mortality as of
20083. Extensive efforts are being exerted by different countries to
address this problem through drug development and improvement
DNA Lysates OPTIMIZED
Calculation of
of diagnostic procedures, processes which are made complicated MIRU - VNTR Repeats
from Lung MIRU - VNTR
by emergence of drug-resistant strains that are less susceptible or Center of the and Designation
even highly resistant to anti-tuberculosis antibiotics. Studies have PROTOCOL of Bacterial
Philippines Identification
shown that strains of Mycobacterium tuberculosis undergo Codes
mutation and there are existing predominant types of
Mycobacterial strain that have developed stable association with
their target human host population4. This emphasizes the point RESULTS AND DISCUSSION
that specific strain of tuberculosis must first be identified and its The PCR results (Fig 1) show that ten out of twelve
characteristics are determined before administering treatment to MIRUs were successfully amplified and visualized.
prevent drug resistance. It can be done through genotyping using Using linear regression, the formula for the number
Variable Number of Tandem Repeats (VNTRs). This method relies of base pairs for any band with a given distance
on the length variation of twelve independent mini-satellite loci travelled was calculated. The number of repeats of
the following MIRUs was then computed through
scattered through the Mycobacterium tuberculosis genome.7 MIRU linear regression (see Table 1).
or mycobacterial interspersed repeating units are VNTRs specific to  
mycobacteria and can be considered as the most practical and  
The sequences of the The number of repeats exhibited by the LCP isolates was
efficient method owing to its portability compared with other different from the wild type H37Rv. Of interest is the
amplicons were determined
typing methods like IS6110, the “gold standard” in TB genotyping from submitted PCR apparent differential polymorphism of the MIRUs. Only
that requires highly trained personnel, sub-culturing of the bacteria products to Macrogen, MIRU 26 remained the same for the four isolates
and a turn around time of 30 days. MIRU-VNTR typing can also Korea. The results were including the wild type strain. Majority of the MIRUs
verified through an online exhibited moderate polymorphism (MIRU 2, 4, 10, 16, 20,
address the problem of low copy number of insertions which is a 24, 27, 31, 39 and 40). One MIRU exhibited high
feature of highly infectious TB strain such as the MTb Beijing BLAST tool of NCBI GenBank.
The ten MTB amplicons polymorphism (MIRU 23), suggestive of its strong
strain5. Since this technology is PCR-based, typing procedure can visualized in the gel potential in differentiating the local Tb isolates.
be done in less than 5 hours. The gel profile of the PCR products  provided a correct match Eleven out of 12 MIRUs were able to differentiate the
can be used to calculate the number of repeats for each VNTR  on
  the MIRU sequence of wild type H37Rv from the LCP isolates. The same set of
 
the H37Rv gene. These MIRUs was able to rule out the three LCP isolates to be
locus and these set of numbers can be used as code for a different from each other. Notable is the difference
findings suggest that the
particular isolate. This study aims to compare the MIRU profiles primers used as well as obtained in MIRU 2, 4 and 26 in Table 1 and Table 2 for
of TB isolates from the Lung Center of the Philippines (LCP) and the PCR conditions H37Rv. This difference in the computation of number of
compare them with the wild type tuberculosis strain, H37Rv. utilized correctly repeats will be further validated.
determine the number of
MIRUs of a given TB
isolate .
The optimized protocol was then used to determine
the number of repeats for the other isolates
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DOI: 10.1128/JCM.42.5.1986–1993.2004
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