After extracting dna from a sample and amplifying it with the technique known as polymerase chain. Restriction fragment length polymorphism rflp is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their dna. Isolates represented by lanes 3, 5, 6, 9, and 10 have the same pattern and were epidemiologically linked. Full text get a printable copy pdf file of the complete article 1. Restriction fragment length polymorphism analysis of.
Is6110 based restriction fragment length polymorphism rflp is6110 based rflp genotyping detects variations generated by the insertion element is6110. Research article open access first insights into the. The role of is6110 in the evolution of mycobacterium. Author summary since the pioneering discovery of transposition by barbara mcclintock in eukaryotes and later in prokaryotes by robert w. Is6110 is the element most widely used as a probe for rflp. Rflp analysis rflp analysis of all mtb strains was performed using is6110 as probe. Commonwealth of virginia division of consolidated laboratory services introduction microbial identification and. Aimsto evaluate the usefulness of two is6110 based typing methods, an amplityping assay and restriction fragment length polymorphism rflp analysis, for fingerprinting respiratory isolates of mycobacterium tuberculosis. Is6110 rflp uses the variability of the numbers and positions of is6110 elements in mtbc genomes to generate strainassociated molecular fingerprints van embden et al. Evaluation of semiautomated is6110based restriction. Because a better understanding of the dynamics of tb transmission is necessary, the present study. By studying 2,236 clinical isolates typed by is6110rflp and covering the mtbc, we remarked. Several studies indicated that vntr typing is as discriminative as rflp typing and more suitable to type strains with few copies of is6110 3,4. Miruvntr, a very powerful technique, provides adequate discrimination between mtb clinical isolates and is comparable to is6110rflp in terms of its accuracy for estimating tb outbreaks and for use in phylogenetic investigations 5, 8.
The most commonly used typing system for tuberculosis is is6110 based restriction fragment length polymorphism rflp. The numbers and chromosomal positions of is6110 elements have been shown to be highly variable corresponding author and reprint requests. Interpretation of restriction fragment length polymorphism. If two organisms differ in the distance between sites of cleavage of a particular restriction. Despite many advantages, the is6110 rflp method has certain disadvantages and limitations. From the division of infectious diseases, department of medicine drs barnes and jones and ms.
Molecular methods for mycobacterium tuberculosis strain. Is6110 restriction fragment length polymorphism typing of. Pdf the rate of change of is6110 restriction fragment length polymorphism rflp patterns of mycobacterium tuberculosis was determined in serial. Insertion elements are capable of making copies of themselves and then inserting the copy anywhere in the genome in a process known as transposition. Restriction fragment length polymorphism rflp restriction fragment length polymorphism rflp is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their dna. Genotyping guide is6110based rflp analysis laboratory. Overall, the diversity of rflp among 95 tuberculosis patients were 48, 50 and 45 on the basis of is6110, pgrs and dr patterns, respectively. Restriction fragment length polymorphism rflp analysis of is6110 insertion sequence relies on the determination of the number of copies and location of insertion on the chromosome of the is6110 element 3. Epidemiological analysis of mycobacterium tuberculosis. Is6110rflp analysis was performed as previously described method. Computerassisted analysis of is6110 rflp patterns was done with gelcompar software, version 4.
Theanalysis of the band patterns of rflpis6110 requires the use of expensive software and specialized professionals, and are quite dif. This genome mapping lecture explains the process of restriction fragment length polymorphism. Arnold, afgu, centre for infections, hpa, 61 colindale avenue, london. Tb detection using molecular methods denise toney, ph. Methods eight cases of tuberculosis were diagnosed in residents of the hostel over a period of 28 months. Restriction fragment length polymorphisms rflp analysis. Restriction fragmentlength polymorphism typing ofmycobacterium tuberculosis achayward restriction fragment length polymorphism rflp typing ofmycobacterium tuberculosis is a recently developed technique which allows strains oftuberculosis to bedifferentiatedwith a high degree ofconfidence.
Dna fingerprinting by is6110rflp has shown a high incidence of mycobacterium tuberculosis isolates having no and low copies of the insertion sequence in kerala, south india. Trflp terminalrflp analysistrflp is a cultureindependent rflp method only terminal fragments are labeled and detected used to study highcomplexity microbial populations based on variations in 16s rrna ribosomal rna. Restriction fragment length polymorphism rflp technique. The is6110 fingerprint patterns generated were highly variable. Pdf is6110 based amplityping assay and rflp fingerprinting. The cluster sizes ranged from 2 to 6 isolates figure 1. The extracted genomic dna was restricted with pvu ii 20 in a reaction mix final volume 30 ml consisting of 3. Restriction fragment length polymorphism rflp, a molecular biology technique 16, is used to type different virus strains 1718192021. Since 1990, characterization of mycobacterium tuberculosis isolates by molecular methods has been useful in confirming suspected laboratory contamination and as an adjunct to epidemiologybased contact investigation.
Usefulness of is6110based restriction fragment length. However, some studies have found microepidemics that were not supported by genotyping data. Molecular analysis of mycobacterium tuberculosis strains. Rflp patterns denote endogenous reactivation of the previously infecting bacteria. In an effort to improve is6110based rflp to make it a faster format, dupont molecular diagnostics recently introduced the is6110pvuii kit for semiautomated typing of.
In molecular biology, restriction fragment length polymorphism rflp is a technique that exploits variations in homologous dna sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence. Miruvntr analysis also generates readily comparable. For large scale epidemiological studies the portability and standardization of is6110 restriction fragment length polymorphism rflp means that this remains the gold standard technique. Is6110 restriction fragment length polymorphism rflp genotyping is the most widely used genotyping method to study the epidemiology of mycobacterium tuberculosis. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites. The is6110 rflp patterns obtained from this region were compared to those, available from previous studies in various parts of the country. Evolution of is6110 rflp patterns sequencing of the is6110 element has revealed that the sequence is conserved within the m. New insights into the transposition mechanisms of is6110. The insertion sequence is6110 is widely recognized for its utility in tb diagnosis and epidemiology. Using restriction endonuclease enzymes fragments of dna is obtained and the desired fragment is detected by using restriction probes. Applying efficient and suitable methods for genotyping mycobacterium tuberculosis mtb isolate.
Tb programs can request the genotyping laboratories to perform is6110based rflp analysis on isolates that match by both spoligotyping and miru analysis. Due to high polymorphism of the is6110 rflp element, methods based on this sequence are characterized by a considerable discriminative potential, and are commonly used in molecular epidemiology. Trflp analysis is a technique used to study complex microbial communities based on variation in the 16s rrna gene 1. The semiautomated rflp system is technically simple and significantly faster than the manual rflp method 8 h versus 5 days. However, due to the complexity of the is6110 rflp genotyping technique, and the interpretation of rflp data, mycobacterial interspersed repetitiveunitvariablenumber tandemrepeat miruvntr genotyping. Discordance between mycobacterial interspersed repetitive. Restriction fragment length polymorphism an overview. The high variability of the number of copies detected by the rflpis6110 of m. Research article open access molecular diversity of.
However, the epidemiological relationship was greater when the clusters were detected by rflp is6110 as compared to miruvntr p 0. They support the use of miruvntr typing as a more reliable and faster method for transmission analysis. Amplified fragment length polymorphism aflp would scan the entire genome rather than a few repetitive elements, we thought that this technique would help us in differentiating the large reservoir of isolates. Between june 2008 and june 2009, all smear positive pulmonary specimens were collected prospectively at the national tuberculosis program ntp center of duhok province.
This technique is thus useful in distinguishing between recent epidemiological events transmission and distant epidemiological events reactivation 12. However, the epidemiological relationship was greater when the clusters were detected by rflpis6110 as compared to miruvntr p 0. However, some studies have found microepidemics that. Restriction fragment length polymorphism rflp introduction restriction fragment length polymorphism rflp is a difference in homologous dna sequences that can be detected by the presence of fragments of different lengths after digestion of the dna samples in question with specific restriction endonucleases. Aim to investigate a possible outbreak of tuberculosis in a hostel for homeless men using is6110 profiling, a polymerase chain reaction pcr based fingerprinting technique. The range of is6110 copies among isolates studied varied from 016. It also states the role of rflp in genome mapping studies.
Restriction fragment length polymorphism wikipedia. The number of is6110 copies per strain varied from 0 to 14. The insertion sequence is6110, only present in the pathogens of the mycobacterium tuberculosis complex mtbc, has been the goldstandard epidemiological marker for tb for more than 25 years, but biological implications of is6110 transposition during mtbc adaptation to humans remain elusive. To provide epidemiological data, a heminested inverse pcr hip assay targeting the insertion sequence. Molecular epidemiology of multidrugresistant strains of. Usefulness of 3 5 is6110rflp genotyping and spoligotyping. Molecular typing of mycobacterium tuberculosis by mycobacterial. Combined use of amplified fragment length polymorphism and is.
Is6110 restriction fragment length polymorphism rflp. Southern hybridization using restriction endonuclease enzymes for isolation of desired length of dna fragments is an. L of the prescribed restriction buffer amersham biosciences, greece. This technique is generally recognized as a reference standard for strain differentiation. Epidemiology and molecular typing of an outbreak of. Transmission of tuberculosis among the urban homeless. Restriction fragment length polymorphism rflp is a technique invented in 1984 by the english scientist alec jeffreys during research into hereditary diseases. The request for rflp analysis must list the cluster. Rflp technique uses the 6110 insertion sequence is6110 to analyze polymorphism. Is6110 based amplityping assay and rflp fingerprinting of. The aim of this study was to evaluate the genotypic diversity of mycobacterium tuberculosis strains using is6110 rflp, spoligotyping and miruvntr typing. The role of is6110 in the evolution of mycobacterium tuberculosis.
New insights into the transposition mechanisms of is6110 and. In an effort to improve is 6110 based rflp to make it a faster format, dupont molecular diagnostics recently introduced the is 6110 pvuii kit for semiautomated typing of mycobacterium. First insight into the genetic diversity of mycobacterium. Chromosomal dna fingerprint patterns produced with is6110. The high discriminatory power and the agreement demonstrate that both techniques can be used. Aims tuberculosis tb is still an important disease in the world, especially in developing countries. Is6110,6 and identical rflp patterns were shown by means of secondary genotyping methods to be genetically distinct 1012. Europe pmc is an archive of life sciences journal literature. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the rflp and drepcr methods.
In the next few years the internationally standard mycobacterial interspersed repetitive unit miru may come to challenge this primacy. Molecular typing of mycobacterium tuberculosis isolates. Flip and flap, and hybridization technique, is6110rflp. Such dna fingerprints, or restriction fragment length polymorphism rflp patterns, of clinical isolates are remarkably polymorphous, even in. Comparison of molecular epidemiological study on mycobacterium tuberculosis using is6110 rflp and is6110 pcr typing. Genotyping guide description of genotyping methods. It is an insertion sequence belonging to the enterobacterial is3 family mcadam et al. Tuberculosis microepidemics are considered as such when a proven epidemiological link is identified between the cases. Rflp restriction fragment length polymorphism rflp analysis measures fragments of dna containing short sequences that vary from person to person, called vntrs.
However, doubt remained whether vntr typing is as good as rflp. In a crosssectional study, 44 linked pairs from 33 microepidemics identified during a 5year period in madrid, spain were analysed to evaluate whether the epidemiological findings were. The dna fragments produced by the digest are then separated by length through a process known as agarose gel. Is6110 based amplityping assay and rflp fingerprinting of clinical isolates of mycobacterium tuberculosis article pdf available in journal of clinical pathology 4810. Rflp, as a molecular marker, is specific to a single clonerestriction enzyme. Analysis of rate of change of is6110 rflp patterns of. Tb programs can request the genotyping laboratories to perform is6110 based rflp analysis on isolates that match by both spoligotyping and miru analysis.
Restriction fragment length polymorphism rflp analysis and tuberculosis epidemiology1 jorge enrique gmez marn, leen rigouts, luz emilia villegas londoo, c franoise portaels in order to study polymorphisms of the dna insertion sequence 6110 is6120 in myco. By studying 2,236 clinical isolates typed by is6110 rflp and covering the mtbc, we remarked a. Another molecular fingerprinting method is spoligotyping, a robust polymerase chain reaction pcr based technique which relies on the detection of. See chapter 5, developing a tuberculosis genotyping program, for a description of when to request rflp analysis. This sequence hybridized with a plasmid isolated from m. Apr 10, 2014 this genome mapping lecture explains the process of restriction fragment length polymorphism. However, the major drawbacks of is6110 rflp have been reported widely. The basic technique for the detection of rflps involves fragmenting a sample of dna with the application of a restriction enzyme, which can selectively cleave a dna molecule wherever a short, specific sequence is recognized in a process known as a restriction digest. In molecular biology, restriction fragment length polymorphism, or rflp commonly pronounced riflip, is a technique that exploits variations in homologous. Sc department of biotechnology central mizoram university 2. An is6110targeting fluorescent amplified fragment length. However, the technique may be efficient in interspecies differentiation, and hence potentially useful in identifying and developing species specific markers. Occasional restriction fragmentlength polymorphism. Pdf hosted at the radboud repository of the radboud.
Insertion sequence is6110 is the genetic marker most frequently used to distinguish clinical mycobacterium tuberculosis isolates 1, 2. Most studies used the restriction fragment length polymorphism rflp technique, s based on is6110 and specific to the m. The isolated strains were divided in terms of location 3 towns of podkarpackie voivodeship differing in population size and relationship 8 members of 4 families, each represented by 2 patients. Molecular epidemiology of tuberculosis in a sentinel. Chromosomal dna fingerprint patterns produced with is6110 are excellent stainspecific markers for the epidemiologic study of tuberculosis. Reanalysis of epidemiologically linked tuberculosis cases not supported by is6110rflpbased genotyping a. Jacob, it has become clear the key role of mobile genetics elements in chromosome remodelling, microbial evolution and host adaptation. In molecular biology, restriction fragment length polymorphism, or rflp commonly pronounced riflip, is a.
Rflp was performed using the standardized is6110 technique as described previously. On the other hand, the rflp is6110, despite being more laborious than the miruvntr, a pcr based method using 12 or 24 interspaced markers, or spoligotyping, requiring higher amounts of dna, has the advantage of a strong genetic stability, which makes this technique appropriate to identify true clusters in old population strains 9. The manual is6110based restriction fragment length polymorphism rflp typing method is highly discriminatory. Consequently, studies involving lowcopynumber isolates commonly use additional genotyping techniques in conjunction with is6110 rflp typing to better describe relatedness among clinical isolates, 14. Restriction fragment length polymorphism rflp targeting the insertion sequence is 6110 was the first standardised molecular typing method for mtbc strains. Combined use of amplified fragment length polymorphism and. Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in havana, cuba, were characterized by is 6110 restriction fragment length polymorphism rflp analysis and the doublerepetitiveelement pcr drepcr method. The manual is 6110 based restriction fragment length polymorphism rflp typing method is highly discriminatory. Pdf analysis of rate of change of is6110 rflp patterns of. Restriction fragment length polymorphism rflp analysis and. Reanalysis of epidemiologically linked tuberculosis cases. Evaluation of semiautomated is6110based restriction fragment. The rflp is the genotyping method most widely used to differentiate among mtb strains and has been useful in carrying out various types of epidemiologic studies6,8. Comparison of the genetic convergence between mycobacterium.
This technique allows you to examine complex communities without the need for any genomic sequence information. In brief, the procedure involve mtb dna digestion pvuii restriction enzyme. Genetic diversity and molecular epidemiology of multidrug. Is6110based rflp requires much more isolate material than do the pcrbased tests to y01ield sufficient dna for analysis. Rflp is an enzymatic procedure for separation and identification of desired fragments of dna. Molecular analysis of mycobacterium tuberculosis strains from. Is6110rflp and spoligotyping of mycobacterium tuberculosis isolates in iran article pdf available in infectious diseases 326. Therefore, this technique has become the new international typing method for m. However, some studies have found microepidemics that were not supported by genot.
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