Document Detail

Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India.
Jump to Full Text
MedLine Citation:
PMID:  16645345     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The usefulness of random amplification of polymorphic DNA (RAPD) analysis for typing Indian strains of M. tuberculosis was investigated. M. tuberculosis H37Rv, M. tuberculosis DT and 42 clinical isolates of M. tuberculosis were subjected to RAPD-PCR using 7 random decamer primers. All 7 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for M. tuberculosis. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. In addition, the results of the present study showed heterogeneity in the M. tuberculosis strains in the population studied.
Authors:
J P Singh; Rishendra Verma; P Chaudhuri
Related Documents :
18273415 - Fluorescent nanoparticle-based indirect immunofluorescence microscopy for detection of ...
21377215 - Prevalence of body dysmorphic disorder in a german psychiatric inpatient sample.
10822925 - Mycobacterium tuberculosis presenting as sternal osteomyelitis.
402095 - Persistence of mycobacterium tuberculosis in sputum without chest roentgenographic evid...
23022815 - Ear disorders in scuba divers.
22343145 - Autoimmune encephalopathies and epilepsies in children and teenagers.
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of veterinary science     Volume:  7     ISSN:  1229-845X     ISO Abbreviation:  J. Vet. Sci.     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-04-28     Completed Date:  2006-08-16     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  100964185     Medline TA:  J Vet Sci     Country:  Korea (South)    
Other Details:
Languages:  eng     Pagination:  181-7     Citation Subset:  IM    
Affiliation:
Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar-243122, U.P., India.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Animals
Cattle
Cattle Diseases / microbiology
DNA, Bacterial / analysis*,  genetics
Gene Expression Profiling
Genetic Variation
Humans
India / epidemiology
Lung / microbiology
Mycobacterium tuberculosis / genetics*,  isolation & purification*
Phylogeny
Random Amplified Polymorphic DNA Technique*
Sputum / microbiology
Swine
Swine Diseases / microbiology
Tuberculosis, Pulmonary / epidemiology*,  microbiology*,  veterinary
Chemical
Reg. No./Substance:
0/DNA, Bacterial
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): J Vet Sci
Journal ID (publisher-id): JVS
ISSN: 1229-845X
ISSN: 1976-555X
Publisher: The Korean Society of Veterinary Science
Article Information
Download PDF
Copyright © 2006 The Korean Society of Veterinary Science
Print publication date: Month: 6 Year: 2006
Electronic publication date: Day: 30 Month: 6 Year: 2006
Volume: 7 Issue: 2
First Page: 181 Last Page: 187
ID: 3242112
PubMed Id: 16645345
DOI: 10.4142/jvs.2006.7.2.181

Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India
J. P. N. Singh1
Rishendra Verma1
P. Chaudhuri2
1Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar-243122, U.P., India.
2Division Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar-243122, U.P., India.
Correspondence: Corresponding author: Tel: +91-581-2301757; Fax: +91-581-2301757, rishendra_verma@yahoo.com

Introduction

The Mycobacterium tuberculosis complex group includes: M. tuberculosis, M. bovis, M. africanum, M. microti [19] and a newly described species M. canetti [24]. Mycobacterium tuberculosis is the primary causative agent of human tuberculosis, but may also infect animals in contact with infected humans [15]. Tuberculosis has re-emerged as one of the leading causes of death worldwide, causing nearly three million deaths annually [2]. In India alone, half a million people die of TB every year, i.e. more than 1000 people every day, and one patient every minute [29]. Both M. tuberculosis and M. bovis have been isolated from humans and animals in India [26]. However, the origin and transmission of infection between animals and humans have not been investigated. Therefore, in view of the global prevalence of tuberculosis, there is an urgent need to develop techniques that not only identify and characterize tubercular bacilli, but also facilitate epidemiological studies to trace the source of infection thereby facilitating formulation of effective control strategies.

Rarely does the antibiotic susceptibility patterns including: serotyping [10], biotyping and bacteriophage typing [11,18] allow for strain differentiation. DNA based techniques are now available for molecular characterization of M. tuberculosis. Restriction fragment length polymorphisms (RFLP) using probes for insertion sequences IS986, IS1081 and IS6110 that have been extensively used to differentiate strains of M.tuberculosis [12,21]. However, the relatively complex nature of the standard methods, as well as the lack of utility of some of the probes (such as IS6110) for some of the Indian strains indicates the need for alternate rapid procedures. Random amplification of polymorphic DNA (RAPD) is a multiplex PCR-based molecular system [27,28]. This method uses short oligonucleotide primers of an arbitrary sequence, and low-stringency PCR, to amplify discrete DNA fragments that can be used as molecular markers. RAPD analysis is rapid, inexpensive, easy to perform and can be used for determination of genetic heterogeneity based on DNA sequence diversity [3,4,27,28].

This method, which requires no previous genetic knowledge of the target organism, relies on the presence of low-stringency priming sites, for a single arbitrary primer on both strands of the DNA molecule, close enough to permit PCR amplification. This DNA fingerprinting has been successfully used to type M. tuberculosis [8,13,17,22] and other bacteria including: E. coli [5], P. multocida [7] and Staphylococcus aureus [9]. The present study reports on the use of RAPD analysis of M. tuberculosis strains to identify the heterogeneity in these strains.


Materials and Methods
Mycobacterial strains

Details of the M. tuberculosis strains used in the present study are given in the Tables 1. M. tuberculosis strains used included 40 strain isolated from human patients with pulmonary tuberculosis from the Medical Hospital, IVRI, Izatnagar (U.P.), India and from the District Tuberculosis Hospital, Bareilly, India, 2 reference strains (H37Rv and MT-DT) and 1 strain each from bovine and swine samples. All of the mycobacterium strains were typed by conventional morphological (Ziehl-Neelsen staining) and biochemical tests [26] and maintained on Lowenstein-Jensen medium at the Mycobacteria Laboratory, Indian Veterinary Research Institute, Izatnagar, India.

RAPD analysis

A number of primers were used for RAPD analysis of mycobacterium isolates. Details of the primers used in the present study are given in the Table 2. All of the primers from the OPN-series were obtained from Operon Technologies (USA); the primers for the BG-series were synthesized by M/s Bangalore Genei Pvt. Ltd. India. Genomic DNA was extracted as per the method of van Soolingen et al. [23]. Amplification of mycobacterium DNA, using random primers, was performed in a total volume of 25 µl. The reaction mixture contained 1 unit of Taq DNA polymerase (Bioloine GmBH, Germany), 1.5 mM MgCl2, 200 µM of each dNTP, 30 pmol primers, and 50 ng of template DNA. Amplification was carried out in a thermal cycler (Eppendorf, Germany). The cycling conditions consisted of an initial denaturation step for 5 min at 94℃, followed by 45 cycles of 94℃ for 1 min denaturation step, an annealing step for 1 min at 36℃, and an extension step for 1 min at 72℃ and a final extension at 72℃ for 5 min. The products obtained from RAPD-PCR were analyzed on a 1.5% agarose gel stained with ethidium bromide. Subsequently, the gel was visualized and photographed using a gel documentation and analysis system (AlfaImager, Germany). The banding patterns obtained by RAPD were noted on a photograph. A data matrix composed of the numerals 1 and 0 was built on the basis of presence (1) or absence (0) of a DNA band appearing in replicates for each isolate. Only distinct and prominent bands were scored and used in assessing RAPD patterns. The molecular size of bands was calculated using software provided by AlfaImager (Germany). The size of the bands, that differed by ±5% on different gels, was considered to be the same bands. The genetic diversity of isolates was analyzed by RAPDistance version 1.04 software that operates on the basis of UPGMA clustering.


Results

RAPD-PCR revealed the presence of amplicons of a variety of sizes in M. tuberculosis strains. In this study, several fragments were amplified in each sample, and most of these fragments were observed to be common to different strains. However, there were some fragments unique to certain strains. All 44 isolates of M. tuberculosis showed a high degree of polymorphism with RAPD analysis (Fig. 1A, B, C). The number of RAPD patterns generated by each primer is shown in Table 1. All 7 primers revealed discriminating patterns. The primer OPN-01 and the primer BG-66 generated the maximum and minimum number of amplimers, respectively. Among the seven primers, OPN-01 showed maximum discrimination for the ability to type mycobacterium isolates, and produced 10 RAPD patterns (Table 2).

Upon dendrogram analysis, with primer OPN-02, four clusters were formed, the largest cluster consisted of 17 strains, the second largest cluster contained 10 strains and the remaining two smaller clusters contained 5 strains in each; genetic relatedness was closest among strains within clusters (Fig. 2). Five strains were identified in different clusters along with M. tuberculosis strain DT, while the M. tuberculosis strain H37Rv was found in one of the smaller clusters. Two strains, 5/S and 9/S, were in the same cluster but remained separated from the rest of the strains and clusters (Fig. 2). Strain SpS19 demonstrated a unique pattern with all of the primers used (Table 1). Bovine strain 1/86 and swine strain 125/92 were identified within the largest and second largest clusters respectively (Fig. 2).


Discussion

Detailed epidemiological studies of M. tuberculosis have been hampered by difficulties in differential characterization of causative strains. The ability to distinguish strains of M. tuberculosis would be useful for investigating the source of outbreaks of infection, the relatedness of strains recovered from different patient, and the identities of multiple strains recovered from the patients from similar localities. Infections caused by mycobacterium are known to be transmitted from human to human [1], animal to human [6], and animal to animal. [16] In an outbreak investigation of tuberculosis, it is often important to know whether the disease is due to a new strain or relapse of a known strain. This information has a special bearing on our understanding of the emergence of multi-drug resistant disease. In India, the status of M. tuberculosis infection in animals is poorly understood.

In the present study, RAPD showed both similarities and differences among M. tuberculosis strains. All primers amplified scorable fragments in each strain analyzed. The common or monomorphic bands among the different strains likely represent highly conserved regions in the genome. Clusters of M. tuberculosis, consisting of the largest number strains, showed a possible close genetic relationship; these were isolated at different occasions from human sputum and from two animals, one from a bovine lymph node and the other from a swine lung. Therefore, the outcome of our analysis is consolidated, in a comprehensive manner, to draw a phylogenetic relationship, which is consistent with prior reports [13,20,25].

It was interesting to note that a previous study of RFLP using IS 6110 and IS 1081, of these M. tuberculosis strains (13 overlapping M. tuberculosis stains, including H37Rv), showed no polymorphism; [21] this suggested that this RFLP could not differentiate these M. tuberculosis strains. However, in this study using the RAPD analysis, we found differentiation among the M. tuberculosis strains. Standardization of PCR mixtures and conditions are very important for reproducibility of RAPD-PCR results. We found that it was necessary to perform RAPD-PCR in duplicate to obtain valid results. Our findings show that RAPD-PCR yields reliable and reproducible results under precise assay conditions.

Isolation of M. tuberculosis from animals is not common. M. tuberculosis strains from a bovine lymph node and a swine lung were similar to the M. tuberculosis strain from human sputum; this suggests a possible transmission of infection from humans to animals. RAPD analysis may help to establish the molecular relatedness of M. tuberculosis strains, their distribution and zoonotic importance in an agrarian country like India, where there is a close association between livestock and human beings.


Acknowledgments

The authors are grateful to Director, Indian Veterinary Research Institute, Izatnagar, India, for providing necessary facilities.


References
1. Blazquez J,Espinosa de Los Monteros LE,Samper S,Martin C,Guerrero A,Cobo J,van Embden J,Baquero F,Gomez-Mampaso E. Genetic characterization of multidrug-resistant Mycobacterium bovis strains from a hospital outbreak involving human immunodeficiency virus-positive patientsJ Clin MicrobiolYear: 199735139013939163450
2. Bloom BR,Murray CJ. Tuberculosis: Commentary on a reemergent killerScienceYear: 1992257105510641509256
3. Bowditch BM,Albright DG,Williams JG,Braun MJ. Use of randomly amplified polymorphic DNA markers in comparative genome studiesMethods EnzymolYear: 19932242943098264394
4. Caetano-Anolles G. Amplifying DNA with arbitrary oligonucleotide primersPCR Methods ApplYear: 1991385948268791
5. Chansiripornchai N,Ramasoota P,Sasipreeyajan J,Svenson SB. Differentiation of avian pathogenic E. coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysisVet MicrobiolYear: 200180758311278125
6. Cosivi O,Grange JM,Daborn CJ,Raviglione MC,Fujikura T,Cousins D,Robinson RA,Huchzermeyer HF,de Kantor I,Meslin FX. Zoonotic tuberculosis due to Mycobacterium bovis in developing countriesEmerg Infect DisYear: 1998459709452399
7. Dziva F,Christensen H,Olsen JE,Mohan K. Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocidaVet MicrobiolYear: 20018236137211506929
8. Harn HJ,Shen KL,Ho LI,Yu KW,Liu GC,Yueh KC,Lee JH. Evidence of transmission of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) fingerprinting in Taipei City, TaiwanJ Clin PatholYear: 1997505055089378819
9. Hermans K,De Herdt P,Baele M,Devriese LA,Haesebrouck F. Sequence analysis or a RAPD band differentiating high and low virulence Staphylococcus aureus strains from rabbitsVet MicrobiolYear: 200182616711423196
10. Jones WD Jr,Kubica GP. Fluorescent antibody technique with mycobacteria. III. Investigation of five serologically homogenous groups of mycobacteriaZentralbl Bakteriol OrigYear: 196820758624986179
11. Jones WD Jr. Geographic distribution of phage types among cultures of Mycobacterium tuberculosisAm Rev Respir DisYear: 1990142100010032122783
12. Katoch VM,Singh D,Chauhan DS,Sharma VD,Singh HB,Das R,Srivastava K. Mahajan RC,Therwath ANewer DNA fingerprinting techniques for tuberculosis-relevance in controlMulti-drug resistance in emerging and re-emerging diseasesYear: 2000New DelhiIndian Science Academy & Narosa Publishing House8796
13. Linton CJ,Jalal H,Leeming JP,Millar MR. Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysisJ Clin MicrobiolYear: 199432216921747814542
14. Menard C,Brousseau R,Mouton C. Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalisFEMS Microbiol LettYear: 1992741631681326466
15. Michalak K,Austin C,Diesel S,Bacon JM,Zimmerman P,Maslows JN. Mycobacterium tuberculosis infection as a zoonotic disease; transmission between humans and elephantsEmerg Infect DisYear: 199842832879621200
16. Perumaalla VS,Adams LG,Payeur J,Baca D,Ficht TA. Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strainVet MicrobiolYear: 19997026927610596810
17. Richner SM,Meiring J,Kirby R. A study of the genetic diversity of Mycobacterium tuberculosis isolated from patients in the eastern province of South Africa using random amplified polymorphic DNA profilingElectrophoresisYear: 199718157015769378124
18. Snider DE Jr,Jones WD,Good RC. The usefulness of phage typing Mycobacterium tuberculosis isolatesAm Rev Respir DisYear: 1984130109510996439088
19. Rynyon EH,Karlson AG,Kubica GP,Wayne LG. Lennette EH,Balows A,Hausler Jr WJ,Truant JPMycobacteriumManual of Clinical MicrobiologyYear: 19803rd edWashington DCAmerican Society for Microbiology150179
20. Singh HB,Chauhan DS,Singh D,Das R,Srivastava K,Yadav VS,Kumar A,Katoch VM,Sharma VD. Rapid discrimination of Indian isolates of M. tuberculosis by random amplified polymorphic DNA (RAPD) analysis - A preliminary reportIndian J Med MicrobiolYear: 200220697117657034
21. Singh SK,Verma R,Shah DH. Molecular fingerprinting of clinical isolates of Mycobacterium bovis and Mycobacterium tuberculosis from India by restriction fragment length polymorphism (RFSP)J Vet SciYear: 2004533133515613817
22. Tazi L,El Baghdadi J,Lesjean S,Locht C,Supply P,Tibayrenc M,Banuls AL. Genetic Diversity and Population Structure of Mycobacterium tuberculosis in Casablanca, a Moroccan City with high incidence of tuberculosisJ Clin MicrobiolYear: 20044246146614715806
23. van Soolingen D,de Haas PEW,Kremer K. Restriction Fragment Length Polymorphism (RFLP) typing of mycobacteriaYear: 1999Bilthoven, NetherlandsNational Institute of Public Health and Enivronment
24. van Soolingen D,Hoogenboezem T,de Haas PE,Hermans PW,Koedam MA,Teppema KS,Brennan PJ,Besra GS,Portaels F,Top J,Schouls LM,van Embden JD. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, canetti: Characterization of an exceptional isolate from AfricaInt J Syst BacteriolYear: 199747123612459336935
25. Verma R,Singh HB,Sharma VD,Katoch VM. Molecular characterization of Indian Mycobacterium bovis isolates by Random Amplified Polymorphic DNA (RAPD) Analysis -A Preliminary ReportIndian J Vet ResYear: 2002113941
26. Verma R,Srivastava SK. Mycobacterium isolated from man and animals; twelve year recordIndian J Anim SciYear: 200171129132
27. Welsh J,McClelland M. Fingerprinting genomes using PCR with arbitrary primersNucleic Acids ResYear: 199018721372182259619
28. Williams JG,Kubelik AR,Livak KJ,Rafalski JA,Tingey SV. DNA polymorphism amplified by arbitrary primers are useful as genetic markersNucleic Acids ResYear: 199018653165351979162
29. World health organization (WHO)Global tuberculosis controlWHO report 2001Year: 2001GenevaWHO

Article Categories:
  • Original Article

Keywords: Mycobacterium tuberculosis, RAPD, typing.

Previous Document:  Evaluation of a Bacillus stearothermophilus tube test as a screening tool for anticoccidial residues...
Next Document:  Matricaria chamomilla CH12 decreases handling stress in Nelore calves.