WOLLO UNIVERSITY COLLEGE OF MEDICINE AND HEALTH

SCIENCES

DEPARTMENT OF MEDICAL LABORATORY SCIENCES

Assignment Paper on Spoligotyping

Molecular Biology; Submitted to Getachew Gugsa (DVM,MSc, Associate Professor)

By; Amanuel Mengesha ID; SGSR/0022/17

Saturday, December 21, 2024 Dessie, Ethiopia

i
Acknowledgment

I would like to express my sincere gratitude to my instructor, Getachew Gugsa (DVM, MSc,
Associate Professor) for your unwavering support, guidance, and dedication to nurturing my
academic and personal growth. Your passion for teaching and commitment to excellence has
truly inspired me to strive for success and pursue knowledge with enthusiasm. I am immensely
grateful for your invaluable mentorship and the profound impact you have had on my
educational journey.

ii
Contents

Acknowledgment.........................................................................................................................................ii

Acronym and abbreviation.........................................................................................................................iv

List of figures...............................................................................................................................................v

Objectives...................................................................................................................................................vi

Methodology.............................................................................................................................................vii

1. Introduction.........................................................................................................................................1

2. Principle of the Technique...................................................................................................................1

3. Materials and Reagents.......................................................................................................................2

[Link]-by-Step Methodology.......................................................................................................................3

5. Applications.............................................................................................................................................9

6. Advantages and Limitations....................................................................................................................9

7. Troubleshooting....................................................................................................................................10

8. Comparison with Similar Techniques.....................................................................................................11

9. Future Directions...................................................................................................................................12

9.1 Innovations or recent advancements in the technique...................................................................12

9.2 Potential improvements or new applications............................................................................13

10. Conclusion.....................................................................................................................................14

10.1 Summary of key points..................................................................................................................14

10.2 Emphasis on the significance of the technique in advancing molecular biology.........................15

11. References...........................................................................................................................................16

iii
Acronym and abbreviation

DR; Direct repeat

SNP; Single nucleotide polymorphism

MTBC; Micobacterium Tuberculosis complex

PCR; polymerase chain reaction

RFLP; Restriction fragment length polymorphism

MTB; mycobacterium tuberculosis

MIRU; mycobacterial interspersed repetitive units

DNA; Deoxyribonucleic acid

SSPE; saline sodium phosphate EDTA

ECL; Enhanced chemiluminescence

SDS; sodium dodecyl sulphate

NGS; next generation sequencing

iv
List of figures

Fig 1; Basis of Spoligotyping methodology

Fig 2; Conversion of a raw Spoligotype results to octal code representation

Fig 3; Comparisons of Spoligotyping with other techniques

v
 Objectives

General Objectives

Generally, this assignment aimed to discuss about Spoligotyping technique which is currently in use
for the Molecular identification of Mycobacterium species strains

Specific objectives

This assignment specifically aimed to;

 Clarify how Spoligotyping technique is performed in the laboratory

 To compare Spoligotyping with others techniques
 To look at current advancements of Spoligotyping technique
 To look at the application of Spoligotyping technique

vi
Methodology

To get materials and information for this assignment, I used systematic searching on different
search databases particularly Google Scholar and PubMed using key words like Spoligotyping,
Mycobacterium Tuberculosis, Molecular Techniques, RFLP, and the like. Different articles
published to Describe the effectiveness and accessibility of Spoligotyping Techniques as well as
the tendency to Increase the specificity of Mycobacterium strain differentiation were assessed
using Google Scholar and related references were used. I have included scientific reviews and
published articles about Spoligotyping technique

vii
1. Introduction

Spacer oligonucleotide typing, or Spoligotyping, is a rapid, polymerase chain reaction

(PCR)-based method for genotyping strains of the Mycobacterium tuberculosis complex
(MTB). Spoligotyping data can be represented in absolute terms (digitally), and the
results can be readily shared among laboratories, thereby enabling the creation of large
international databases.

Spoligotyping is a method for typing the DR (direct repeat) region, which is structurally
composed of 43 spacers, each of which is surrounded by direct repeats. Identification is
based on the absence or presence of a particular spacer sequence(1). Several PCR-based
methods have been developed for MTBC strain typing that require little DNA like
Spoligotyping.

Since the Spoligotypes assay was standard sized more than 10 yr ago, tens of thousands
of isolates have been analyzed, giving a global picture of MTB strain diversity. The
method is highly reproducible and has been developed into a high-throughput assay for
large molecular epidemiology projects. In the United States, Spoligotyping is employed
on nearly all newly identified culture-positive cases of tuberculosis as part of a national
genotyping program. (2)

2. Principle of the Technique

The technique involves the amplification of the spacers using primers targeting the
extremes of the DR region, and hybridization of the product in a membrane with
immobilized complementary probes. 43 spacers were selected for the assay, to
discriminate between M. Bovis and the most frequently isolated M. tuberculosis strains.
Spoligotypes mainly evolve through the loss of these spacers and provide a binary

1
 barcode of 43 digits for each isolate, indicating the presence or absence of the
corresponding spacer. This technique was able to discriminate samples from different
locations, constituting an interesting first-line approach to explore the likely transmission
events. It also helped identify relatedness between samples, leading to the definition of
Spoligotypes families (such as East-African-Indian, African, Beijing, etc.) due to its
relatively slow evolutionary rate. These families are highly congruent with single
nucleotide polymorphism (SNP)-based lineages(3)

3. Materials and Reagents

a. Stock Buffers

1. 20X SSPE: 0.2M Na2HPO4, 3.6M NaCl2, 20 mM ethylenediaminetetraacetic

acid (EDTA), final pH should be 7.4–7.6, autoclaved and stable for 1 yr.

2. 0.5M EDTA, pH 8.0, autoclaved and stable for 1 yr.

3. 10% (w/v) sodium dodecyl sulfate (SDS), made fresh as required.

b. Polymerase Chain Reaction

 Primers for DR region amplification: DRa (GGTTTTGGGTCT- GACGAC,

5” biotinylated) and DRb (CCGAGAGGGGACG- GAAAC). Store
reconstituted DRa and DRb and post-PCR products at 4°C

c. Hybridization

1. Spoligotyping membrane (Ocimum Biosolutions Inc., for- merly Isogen

Biosciences B.V., Hyderabad, India).

2. MN45 Mini blotter and support cushions (Immunetics, Inc., Boston, MA).

3. Rotating hybridization oven.

2
 d. Detection

1. 500 U streptavidin-peroxidase conjugate (Roche Diagnostics, Indianapolis,

IN), resuspended in 1 mL H2O.

2. Enhanced chemiluminescence (ECL) detection reagents 1 and 2 (GE

Healthcare Life Sciences, Piscataway, NJ).

3. X-ray film.

4. X-ray film developer.

[Link]-by-Step Methodology

The Spoligotyping assay is currently performed by one of two methods. The most
commonly employed method utilizes a nylon membrane to which 43 different
oligonucleotides, corresponding to the 43 unique spacer sequences, have been
individually bound(4). A second method utilizes a high-throughput, multianalyte
flow system (Luminex)(5), permitting analysis of high numbers of strains without
the need for membranes.
Detailed instructions on how to manufacture Spoligotyping membranes have been
previously published(6). Commercially prepared Spoligotyping membranes are
commonly used A wide variety of samples is suitable for the PCR reaction. Extracted
DNA, heat-killed cell suspensions from growth medium, and even primary specimens
have been successfully used as templates in PCR reactions(4).

 PCR Amplification of the DR Region

1. Prepare a 25-µL PCR reaction using 1–5 µL of cell suspension or 0.5–1 µL of

extracted genomic DNA. A wide range of template concentrations seem suitable
for DR region amplification.

2. Use the following PCR conditions: 3 min at 96°C, followed by 20 (extracted

3
 DNA) to 30 (cell suspension) cycles of 1 min at 96°C, 1 min at 55°C, and 30 s
at 72°C, final extension of 5 min at 72°C.

 Hybridization of PCR Samples to Spoligotyping Membrane

1. Add 150 µL of 2X SSPE/0.1% SDS to each tube containing the 20–25 µL
post-PCR products.

2. Heat denatures the diluted PCR products for 10 min at 100°C and cool on ice
water for 2 min.

3. Wash the Spoligotyping membrane for 5 min at 60°C in 2X SSPE/0.1% SDS.

4. Place the membrane and a support cushion into the Mini blotter in such a way
that the slots are oriented perpendicular to the line pattern of the applied
oligonucleotides.

5. Fill the slots of the diluted PCR product (avoid air bubbles) and hybridize for 1 h
at 60°C.
 Post hybridization Steps
1. Following hybridization, remove the samples from the mini- blotter by
aspiration.

2. Wash the membrane twice in 2X SSPE/0.5% SDS for 10 min per wash at
60°C.

3. Place the membrane in a rolling bottle and allow it to cool to prevent

inactivation of the peroxidase in the next step.

4. Add 2.5 µL of 500 U/mL streptavidin-peroxidase to 10 mL of 2X

SSPE/0.5% SDS and add to roller bottle. Incubate the membrane in this
solution for 45–60 min at 42°C with rotation.

5. Following this incubation, wash the membrane twice in 2X SSPE/0.5% SDS

for 10 min per wash at 42°C.

6. Rinse the membrane twice with 2X SSPE for 5 min per wash at room
temperature.

4
 Chemiluminescent Detection

1. For chemiluminescent detection of hybridizing DNA, incubate the membrane for

1 min in 10 mL ECL detection reagent 1 mixed with 10 mL ECL detection
reagent 2 at room temperature.

2. Briefly blot off excess ECL liquid, cover the membrane with plastic wrap, and
expose to X-ray film for 2 min or longer.

 Regeneration of the Membrane

The hybridized PCR product is dissociated from the membrane to regenerate the membrane for
the next hybridization. A membrane can typically be regenerated for reuse at least 20 times.
1. Wash the membrane twice in 1% SDS at 80°C for 30 min.

2. Wash the membrane in 20 mM EDTA at room temperature for 15 min.

3. Store the membrane at 4°C sealed in a plastic bag containing 10 mL of 20 mM

EDTA.
 Data Analysis
 Octal Code Nomenclature

The Spoligotype patterns from X-ray film can either be read manually or scanned into a
software package. For manual reads, it is recommended to have two people independently
score the results for maximum accuracy. illustrates the process of assigning a 15-digit
octal code(7) to a Spoligotype result based on the pattern of hybridization. Spacers are
grouped into triplets except for spacer 43. There is a number designation for each of the
eight possible hybridization combinations for a group of three spacers as shown. The
15th digit of the octal code is either a 1 or a 0 based on the hybridization of spacer 43
alone.

 Spoligotype Family Assignments

Once the octal code for a strain has been determined, the strain can then be assigned to a

5
 global strain family. Visual rules established as part of SpolDB(8) and an online software
tool, SpotClust ([Link] are available for aiding in the
assignment of a Spoligotype to one of the global strain families. The family assignment is
useful for producing an overall picture of the strain diversity in a given population, for
tracking changes in the TB population over time, and for comparing TB diversity between
populations or areas. The criteria used to define Spoligotype global families have been vali-
dated through comparison with MIRU data(10).

The online version of the most recent international Spoligotype database, SpolDB4, is called SIT
VIT ([Link] [Link]/SITVITDemo/). The user can enter a spoligo-
type octal or binary code to search whether that Spoligotype has been previously reported.

If it has, a shared type (ST) number is returned. That number can then be used to produce a
map showing laboratories that have previously submitted that pattern. This information has the
potential to be useful in deciphering the global origin or spread of a particular Spoligotype.

 Expansion of the Spoligotype Assay: Examination of Additional Spacer Sequences

Since the initial Spoligotyping assay was based on a Mini blotter with 43 usable sample
chambers, the assay was limited to 43 different spacer sequences. Researchers have explored
the potential of additional spacer sequences for “expanded” or “extended”
Spoligotyping(11).

The hope was that screening for Additional spacer sequences would improve the
differentiability of the commonly observed Spoligotypes. These expanded assays have not
been standardized and are not commercially available, and they have unfortunately shown
limited success in improving differentiation within the commonly observed patterns.

6
7
8
5. Applications

Spoligotyping has been very successful in providing a tool for the rapid acquisition of MTB
genotyping information and for the establishment of a global picture of MTB diversity.
(12), The method is highly reproducible and has been developed into a high-throughput assay
for large molecular epidemiology projects.

In the United States, Spoligotyping is employed on nearly all newly identified culture-positive
cases of tuberculosis as part of a national genotyping program.(2)Alone or in combination with
other techniques, Spoligotyping has been also extensively used for M. bovis during the last
decade. It has been useful for the study of the epidemiology of infection in livestock and
wildlife, allowing detection of transmission between herds, the source of new detected cases,
and the transmission of infection between domestic animals and wildlife.(13)

6. Advantages and Limitations

The strengths of this method include its low cost, its digital data results, the good correlation of
its results with other genetic markers, its fair level of overall differentiation of strains, its high-
throughput capacity, and its ability to provide species information.

However, the method’s weaknesses include the inability of Spoligotyping to differentiate well
within large strain families such as the Beijing family, the potential for convergent evolution of
patterns, the limited success in improving the assay through expansion, and the difficulty in
obtaining the specialized membranes and instrumentation.(2)

This technique has a significant advantage over the other typing methods by being easy to
perform and the results are easy to interpret. However, despite its extensive use and impact [a
recent search in the PubMed website of the National Center for Biotechnology Information

9
([Link] entrez) retrieved 328 papers on this topic since 1996], it has
received very little attention regarding improvement in terms of discrimination of isolates. (13)

7. Troubleshooting

1. To minimize handling of PCR products, use a 25-µL total volume PCR reaction in a 0.5-mL
tube. The 150 µL of hybridization buffer may be directly added to the tube following
amplification.

2. To confirm PCR amplification of DR region, run a 5-µL aliquot from the reaction on a 1%
Mini agarose gel. A successful PCR reaction should appear as a ladder or smear of faint
bands. If no PCR reactions can be observed, check oligonucleotide stocks for
degradation/incorrect concentration
3. Never store biotinylated primers or biotinylated PCR products below 4°C.

4. Validate the proper manufacture of a new Spoligotyping membrane on the first use by
including a series of previously characterized strains.

5. Do not reuse plastic support cushions in Mini blotter.

6. Leakage into adjoining wells usually results from a dry mem- brane “wicking” sample into
the adjoining well. Improper placement of the support cushion or membrane can also lead
to this problem. Avoid wrinkling the membrane in the Mini blotter. Ensure that the Mini
blotter is evenly tightened. Do not completely fill or overfill wells. Hybridization fluid may
transfer to adjacent wells.

7. Discard stocks of streptavidin alkaline phosphatase 6 Mo after rehydration.

8. Check hybridization temperature and the temperatures of the post hybridization wash
buffers. Lowering the hybridization temperature and stringent washes from 60 to 55°C may
help and does not add to any background problems or nonspecific hybridization.

9. With proper handling and storage, Spoligotyping membranes can be reused 30 or more
times

10
8. Comparison with Similar Techniques

In understanding strain diversity of M. tuberculosis, whole genome sequencing is believed to

offer many advantages compared to the classical methods like RFLP, Spoligotyping and MIRU-
VNTR studies. Nevertheless, whole genome sequencing remains relatively costly and requires
advanced capacity ranging from wet lab to big data analysis(14)

Spoligotyping has lower discriminatory power compared to MIRU-VNTR (Mycobacterial

Interspersed Repetitive Units-Variable Number of Tandem Repeats), which analyzes
multiple loci for higher resolution. RFLP (Restriction Fragment Length Polymorphism)
offers high discriminatory power but requires more time and resources. SNP Typing (Single
Nucleotide Polymorphism) provides detailed phylogenetic information but demands advanced
sequencing technologies(15)

Fig; 3 comparisons of Spoligotyping with other techniques

11
9. Future Directions

9.1 Innovations or recent advancements in the technique.

Recent advancements in the Spoligotyping technique, which is crucial for the identification and
characterization of Mycobacterium tuberculosis complex (MTBC) strains, include several
innovative approaches and technologies:

 In Silico Spoligotyping: The development of software like SpoTyping allows for

fast and accurate in silico Spoligotyping from next-generation sequencing (NGS)
reads. This method is significantly faster than traditional techniques and integrates
epidemiological data from global databases, enhancing the utility of Spoligotyping
in public health surveillance (15).
 High-Throughput Methods: Innovations in high-throughput Spoligotyping have
improved the efficiency of strain typing. Automated systems enable the
simultaneous processing of multiple samples, which is particularly beneficial in
clinical and epidemiological settings.
 Integration with NGS: The combination of Spoligotyping with NGS technologies
provides a more comprehensive understanding of the genetic diversity of MTBC
strains. This integration allows for detailed phylogenetic analysis and better tracking
of transmission dynamics(15) .
 Enhanced Sensitivity and Specificity: Recent advancements have focused on
improving the sensitivity and specificity of Spoligotyping through optimized primer
design and amplification techniques. These improvements facilitate the detection of
a wider range of MTBC strains, including those that may be less prevalent(16).
 Use of Digital Platforms: The application of digital tools for data analysis has
streamlined the interpretation of Spoligotyping results. Automated pattern
recognition and comparison against extensive global databases enhance the
accuracy of strain classification (15).

These innovations not only enhance the accuracy and efficiency of Spoligotyping but also
broaden its application in public health and epidemiological research.

12
9.2 Potential improvements and new applications.

Application in Environmental Studies: Spoligotyping is increasingly being utilized in

environmental microbiology to monitor MTBC in wildlife and assess zoonotic transmission
risks, expanding its relevance beyond clinical settings (17).

 Potential Improvements in Spoligotyping

Enhanced Discriminatory Power: Recent studies have introduced new spacer oligonucleotides
that improve the discriminatory power of Spoligotyping. For instance, the addition of 51 new
spacer oligonucleotides allowed for the identification of 14 additional Spoligotypes, which
helped to differentiate clusters of isolates that were previously indistinguishable using traditional
methods (11).

Improved Readability and Reproducibility: Redesigning existing oligonucleotides has led to

better readability of Spoligotype patterns. This enhancement is crucial for accurate interpretation
and comparison of results across different laboratories (11).

 New Applications of Spoligotyping

 Molecular Epidemiology: Spoligotyping is increasingly used in molecular
epidemiology to study the transmission dynamics of tuberculosis. It helps in
identifying outbreaks and understanding the spread of specific strains within
populations.
 Environmental Monitoring: The application of Spoligotyping in environmental
studies can help track MTBC in wildlife and assess zoonotic transmission risks. This
is particularly relevant for understanding the ecology of tuberculosis and its
reservoirs.
 Vaccine Development: Spoligotyping can aid in identifying and characterizing
strains for vaccine research, potentially leading to the development of more effective
vaccines tailored to specific strains prevalent in certain regions.

13
  Drug Resistance Studies: Investigating the relationship between Spoligotyping
patterns and drug resistance profiles can enhance our understanding of resistance
mechanisms and inform treatment strategies for tuberculosis (18).
 Clinical Diagnostics: Implementing Spoligotyping as part of routine diagnostic
protocols in clinical laboratories can improve the identification of TB strains,
facilitating timely and appropriate treatment decisions(16).

[Link]

Spoligotyping is a molecular technique used for genotyping strains of Mycobacterium

tuberculosis complex. It targets the Direct Repeat (DR) region of the genome, using PCR
amplification followed by hybridization with specific oligonucleotide probes. The resulting
binary pattern of spacer sequences provides a unique fingerprint for each strain, making it useful
for epidemiological studies and tracking the spread of tuberculosis. While it is less
discriminatory than some other methods, such as MIRU-VNTR, it is particularly effective for
strains with a low number of IS6110 elements.

One of the key advantages of Spoligotyping is its simplicity and speed, which makes it a
valuable tool in settings where rapid results are needed. It can be easily performed with standard
laboratory equipment and provides reproducible results. However, its lower discriminatory
power compared to other techniques means that it is often used in conjunction with additional
methods for more comprehensive strain differentiation.

Spoligotyping has contributed significantly to our understanding of the genetic diversity and
evolutionary history of Mycobacterium tuberculosis. By identifying specific strain types and
tracking their distribution, researchers can better understand the dynamics of tuberculosis
outbreaks and transmission patterns. This information is crucial for informing public health
interventions and developing strategies to control and prevent the spread of this serious
infectious disease.

14
10.1 Summary of key points.
Spoligotyping: A molecular technique used for genotyping Mycobacterium tuberculosis
complex strains. It targets the Direct Repeat (DR) region of the genome, producing a binary
pattern of spacer sequences.

Procedure: Involves PCR amplification of the DR region and hybridization with specific
oligonucleotide probes to generate a unique fingerprint for each strain.

Advantages: Simple, rapid, and effective for strains with a low number of IS6110 elements. It
can be performed with standard laboratory equipment and provides reproducible results.

Limitations: Lower discriminatory power compared to other methods, such as MIRU-VNTR,

which means it is often used in conjunction with additional techniques for comprehensive strain
differentiation.

Applications: Useful for epidemiological studies, tracking tuberculosis spread, understanding

genetic diversity, and informing public health interventions and strategies.

10.2 Emphasis on the significance of the technique in advancing molecular

biology

Spoligotyping has been instrumental in advancing our understanding of the genetic diversity and
epidemiology of Mycobacterium tuberculosis. This technique has enabled researchers to quickly
and effectively genotype bacterial strains, which is crucial for tracking disease outbreaks and
understanding the dynamics of tuberculosis transmission. By providing insights into the genetic
variations and evolutionary patterns of these bacteria, spoligotyping has significantly contributed
to the field of molecular epidemiology.

Moreover, the simplicity and rapidity of Spoligotyping have made it an accessible tool for
laboratories worldwide, facilitating extensive studies and collaborations. Its application has
extended beyond tuberculosis, inspiring the development of similar molecular typing methods
for other pathogens. This has broadened the horizons of molecular biology, enabled detailed
genetic analysis and fostering advancements in disease control and prevention strategies.
15
11. References

1. Coscolla M, Gagneux S. Consequences of genomic diversity in Mycobacterium tuberculosis.

Semin Immunol. 2014;26(6):431-44.
2. Driscoll JR. Spoligotyping for molecular epidemiology of the Mycobacterium tuberculosis
complex. Molecular Epidemiology of Microorganisms: Methods and Protocols. 2009:117-28.
3. Kato-Maeda M, Gagneux S, Flores LL, Kim EY, Small PM, Desmond EP, et al. Strain classification
of Mycobacterium tuberculosis: congruence between large sequence polymorphisms and spoligotypes.
Int J Tuberc Lung Dis. 2011;15(1):131-3.
4. Kamerbeek J, Schouls L, Kolk A, Van Agterveld M, Van Soolingen D, Kuijper S, et al. Simultaneous
detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.
Journal of clinical microbiology. 1997;35(4):907-14.
5. Cowan LS, Diem L, Brake MC, Crawford JT. Transfer of a Mycobacterium tuberculosis genotyping
method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex
multianalyte profiling system. Journal of Clinical Microbiology. 2004;42(1):474-7.
6. Molhuizen HO, Bunschoten AE, Schouls LM, van Embden JD. Rapid detection and simultaneous
strain differentiation of Mycobacterium tuberculosis complex bacteria by spoligotyping. Mycobacteria
Protocols. 1998:381-94.
7. Dale J, Brittain D, Cataldi A, Cousins D, Crawford J, Driscoll J, et al. Spacer oligonucleotide typing
of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardised
nomenclature [The Language of Our Science]. The International Journal of Tuberculosis and Lung
Disease. 2001;5(3):216-9.
8. Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, et al. Mycobacterium
tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4)
for classification, population genetics and epidemiology. BMC microbiology. 2006;6:1-17.

16
9. Vitol I, Driscoll J, Kreiswirth B, Kurepina N, Bennett KP. Identifying Mycobacterium tuberculosis
complex strain families using spoligotypes. Infection, Genetics and Evolution. 2006;6(6):491-504.
10. Ferdinand S, Valétudie G, Sola C, Rastogi N. Data mining of Mycobacterium tuberculosis complex
genotyping results using mycobacterial interspersed repetitive units validates the clonal structure of
spoligotyping-defined families. Research in Microbiology. 2004;155(8):647-54.
11. Van der Zanden A, Kremer K, Schouls LM, Caimi K, Cataldi A, Hulleman A, et al. Improvement of
differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by
introduction of new spacer oligonucleotides. Journal of clinical microbiology. 2002;40(12):4628-39.
12. Brudey K, Driscoll J, Rigouts L, Prodinger W, Gori A, Al-Hajoj S, et al. An Appraisal of the
geographic prevalence of Major Genotypic Families of Mycobacterium tuberculosis complex through the
updated SpolDB4 database. BMC Microbiol. 2006;6:23.
13. Javed MT, Aranaz A, de Juan L, Bezos J, Romero B, Álvarez J, et al. Improvement of spoligotyping
with additional spacer sequences for characterization of Mycobacterium bovis and M. caprae isolates
from Spain. Tuberculosis. 2007;87(5):437-45.
14. Gagneux S. Genetic diversity in Mycobacterium tuberculosis. Pathogenesis of Mycobacterium
tuberculosis and its Interaction with the Host Organism: Springer; 2013. p. 1-25.
15. Xia E, Teo Y-Y, Ong RT-H. SpoTyping: fast and accurate in silico Mycobacterium spoligotyping
from sequence reads. Genome Medicine. 2016;8(1):19.
16. Gori A, Bandera A, Marchetti G, Degli Esposti A, Catozzi L, Nardi GP, et al. Spoligotyping and
Mycobacterium tuberculosis. Emerging infectious diseases. 2005;11(8):1242.
17. Hussien B, Zewude A, Wondale B, Hailu A, Ameni G. Spoligotyping of clinical isolates of
Mycobacterium tuberculosis complex species in the oromia region of Ethiopia. Frontiers in Public Health.
2022;10:808626.
18. Sola C, Filliol I, Legrand E, Rastogi N. Recent developments of spoligotyping as applied to the
study of epidemiology, biodiversity and molecular phylogeny of the Mycobacterium tuberculosis
complex. Pathologie-biologie. 2000;48(10):921-32.

17