Restriction fragment length polymorphism analysis of. Using restriction endonuclease enzymes fragments of dna is obtained and the desired fragment is detected by using restriction probes. However, some studies have found microepidemics that were not supported by genot. Is6110 restriction fragment length polymorphism typing of. However, other authors have found limitations in its discriminatory power and incomplete correlation with the rflp analysis 79, indicating that miru analysis should be combined with an additional genotyping method 912. Isolates represented by lanes 3, 5, 6, 9, and 10 have the same pattern and were epidemiologically linked. 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 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. Rflp restriction fragment length polymorphism rflp analysis measures fragments of dna containing short sequences that vary from person to person, called vntrs. Aims tuberculosis tb is still an important disease in the world, especially in developing countries. 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. The semiautomated rflp system is technically simple and significantly faster than the manual rflp method 8 h versus 5 days. Is6110rflp analysis was performed as previously described method. Molecular epidemiology of multidrugresistant strains of.
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. Research article open access first insights into the. Pdf analysis of rate of change of is6110 rflp patterns of. However, the epidemiological relationship was greater when the clusters were detected by rflpis6110 as compared to miruvntr p 0. Comparison of the genetic convergence between mycobacterium. For large scale epidemiological studies the portability and standardization of is6110 restriction fragment length polymorphism rflp means that this remains the gold standard technique. Restriction fragment length polymorphism rflp is a technique invented in 1984 by the english scientist alec jeffreys during research into hereditary diseases. Is6110 restriction fragment length polymorphism rflp genotyping is the most widely used genotyping method to study the epidemiology of mycobacterium tuberculosis. 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. 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.
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 numbers and chromosomal positions of is6110 elements have been shown to be highly variable corresponding author and reprint requests. This sequence hybridized with a plasmid isolated from m. Southern hybridization using restriction endonuclease enzymes for isolation of desired length of dna fragments is an. Pdf hosted at the radboud repository of the radboud. Because a better understanding of the dynamics of tb transmission is necessary, the present study. 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. Rflp patterns denote endogenous reactivation of the previously infecting bacteria. Epidemiology and molecular typing of an outbreak of. Discordance between mycobacterial interspersed repetitive. It is an insertion sequence belonging to the enterobacterial is3 family mcadam et al. Between june 2008 and june 2009, all smear positive pulmonary specimens were collected prospectively at the national tuberculosis program ntp center of duhok province.
Comparison of molecular epidemiological study on mycobacterium tuberculosis using is6110 rflp and is6110 pcr typing. Miruvntr analysis also generates readily comparable. Rflp, as a molecular marker, is specific to a single clonerestriction enzyme. 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. In molecular biology, restriction fragment length polymorphism, or rflp commonly pronounced riflip, is a technique that exploits variations in homologous. Computerassisted analysis of is6110 rflp patterns was done with gelcompar software, version 4. Is6110rflp and spoligotyping of mycobacterium tuberculosis isolates in iran article pdf available in infectious diseases 326. To provide epidemiological data, a heminested inverse pcr hip assay targeting the insertion sequence. However, due to the complexity of the is6110 rflp genotyping technique, and the interpretation of rflp data, mycobacterial interspersed repetitiveunitvariablenumber tandemrepeat miruvntr genotyping. New insights into the transposition mechanisms of is6110 and.
Restriction fragment length polymorphism rflp targeting the insertion sequence is 6110 was the first standardised molecular typing method for mtbc strains. The number of is6110 copies per strain varied from 0 to 14. Research article open access molecular diversity of. Most studies used the restriction fragment length polymorphism rflp technique, s based on is6110 and specific to the m. 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 aim of this study was to evaluate the genotypic diversity of mycobacterium tuberculosis strains using is6110 rflp, spoligotyping and miruvntr typing. Molecular analysis of mycobacterium tuberculosis strains from. Insertion elements are capable of making copies of themselves and then inserting the copy anywhere in the genome in a process known as transposition. Pdf is6110 based amplityping assay and rflp fingerprinting. Restriction fragment length polymorphisms rflp analysis. Dna fingerprinting of mycobacterium tuberculosis isolates. This technique is generally recognized as a reference standard for strain differentiation.
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. Molecular typing of mycobacterium tuberculosis isolates. 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. The combined use of aflp and is6110rflp showed relatively better differentiation of high copy is6110 isolates, but failed to differentiate the low copy isolates. Rflp technique uses the 6110 insertion sequence is6110 to analyze polymorphism. Another molecular fingerprinting method is spoligotyping, a robust polymerase chain reaction pcr based technique which relies on the detection of.
However, some studies have found microepidemics that were not supported by genotyping data. 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 term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites. Molecular typing of mycobacterium tuberculosis by mycobacterial. The insertion sequence is6110 is widely recognized for its utility in tb diagnosis and epidemiology. Reanalysis of epidemiologically linked tuberculosis cases. Analysis of rate of change of is6110 rflp patterns of. If two organisms differ in the distance between sites of cleavage of a particular restriction. Is6110 is the element most widely used as a probe for rflp.
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. Restriction fragment length polymorphism wikipedia. 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. Europe pmc is an archive of life sciences journal literature. The manual is 6110 based restriction fragment length polymorphism rflp typing method is highly discriminatory. Pdf the rate of change of is6110 restriction fragment length polymorphism rflp patterns of mycobacterium tuberculosis was determined in serial. Rflp is an enzymatic procedure for separation and identification of desired fragments of dna. By studying 2,236 clinical isolates typed by is6110rflp and covering the mtbc, we remarked. Evaluation of semiautomated is6110based restriction. Theanalysis of the band patterns of rflpis6110 requires the use of expensive software and specialized professionals, and are quite dif. 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. Usefulness of 3 5 is6110rflp genotyping and spoligotyping. 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. Restriction fragment length polymorphism rflp technique.
Applying efficient and suitable methods for genotyping mycobacterium tuberculosis mtb isolate. The manual is6110based restriction fragment length polymorphism rflp typing method is highly discriminatory. 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. Tuberculosis microepidemics are considered as such when a proven epidemiological link is identified between the cases. The role of is6110 in the evolution of mycobacterium. Molecular analysis of mycobacterium tuberculosis strains. Restriction fragment length polymorphism rflp analysis and.
Arnold, afgu, centre for infections, hpa, 61 colindale avenue, london. However, doubt remained whether vntr typing is as good as rflp. Restriction fragment length polymorphism rflp, a molecular biology technique 16, is used to type different virus strains 1718192021. Interpretation of restriction fragment length polymorphism.
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. Insertion sequence is6110 is the genetic marker most frequently used to distinguish clinical mycobacterium tuberculosis isolates 1, 2. New insights into the transposition mechanisms of is6110. However, some studies have found microepidemics that. 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. Flip and flap, and hybridization technique, is6110rflp. 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. The is6110 fingerprint patterns generated were highly variable. 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. From the division of infectious diseases, department of medicine drs barnes and jones and ms. The cluster sizes ranged from 2 to 6 isolates figure 1. Combined use of amplified fragment length polymorphism and. Sc department of biotechnology central mizoram university 2. After extracting dna from a sample and amplifying it with the technique known as polymerase chain.
First insight into the genetic diversity of mycobacterium. Rflp was performed using the standardized is6110 technique as described previously. The high variability of the number of copies detected by the rflpis6110 of m. Trflp analysis is a technique used to study complex microbial communities based on variation in the 16s rrna gene 1. The dna fragments produced by the digest are then separated by length through a process known as agarose gel. The most commonly used typing system for tuberculosis is is6110 based restriction fragment length polymorphism rflp. This genome mapping lecture explains the process of restriction fragment length polymorphism. The role of is6110 in the evolution of mycobacterium tuberculosis. Evaluation of semiautomated is6110based restriction fragment. The is6110 rflp patterns obtained from this region were compared to those, available from previous studies in various parts of the country. The range of is6110 copies among isolates studied varied from 016. 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. Is6110 based amplityping assay and rflp fingerprinting of. 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.
Is6110 based restriction fragment length polymorphism rflp is6110 based rflp genotyping detects variations generated by the insertion element is6110. This technique allows you to examine complex communities without the need for any genomic sequence information. The request for rflp analysis must list the cluster. L of the prescribed restriction buffer amersham biosciences, greece. An is6110targeting fluorescent amplified fragment length. 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.
Tb programs can request the genotyping laboratories to perform is6110based rflp analysis on isolates that match by both spoligotyping and miru analysis. Occasional restriction fragmentlength polymorphism. Combined use of amplified fragment length polymorphism and is. Usefulness of is6110based restriction fragment length. However, the epidemiological relationship was greater when the clusters were detected by rflp is6110 as compared to miruvntr p 0. Rflp analysis rflp analysis of all mtb strains was performed using is6110 as probe. 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. Is6110 based amplityping assay and rflp fingerprinting of clinical isolates of mycobacterium tuberculosis article pdf available in journal of clinical pathology 4810. Such dna fingerprints, or restriction fragment length polymorphism rflp patterns, of clinical isolates are remarkably polymorphous, even in.
Genotyping guide is6110based rflp analysis laboratory. It also states the role of rflp in genome mapping studies. Therefore, this technique has become the new international typing method for m. This technique is thus useful in distinguishing between recent epidemiological events transmission and distant epidemiological events reactivation 12. In the next few years the internationally standard mycobacterial interspersed repetitive unit miru may come to challenge this primacy. Transmission of tuberculosis among the urban homeless.
Genetic diversity and molecular epidemiology of multidrug. 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. Molecular methods for mycobacterium tuberculosis strain. Genotyping guide description of genotyping methods. Apr 10, 2014 this genome mapping lecture explains the process of restriction fragment length polymorphism. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the. Epidemiological analysis of mycobacterium tuberculosis. However, the technique may be efficient in interspecies differentiation, and hence potentially useful in identifying and developing species specific markers. Chromosomal dna fingerprint patterns produced with is6110. Restriction fragment length polymorphism an overview. Overall, the diversity of rflp among 95 tuberculosis patients were 48, 50 and 45 on the basis of is6110, pgrs and dr patterns, respectively. By studying 2,236 clinical isolates typed by is6110 rflp and covering the mtbc, we remarked a. Molecular epidemiology of tuberculosis in a sentinel.
Reanalysis of epidemiologically linked tuberculosis cases not supported by is6110rflpbased genotyping a. Tb programs can request the genotyping laboratories to perform is6110 based rflp analysis on isolates that match by both spoligotyping and miru analysis. In molecular biology, restriction fragment length polymorphism, or rflp commonly pronounced riflip, is a. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Jacob, it has become clear the key role of mobile genetics elements in chromosome remodelling, microbial evolution and host adaptation. However, the major drawbacks of is6110 rflp have been reported widely. See chapter 5, developing a tuberculosis genotyping program, for a description of when to request rflp analysis. Consequently, studies involving lowcopynumber isolates commonly use additional genotyping techniques in conjunction with is6110 rflp typing to better describe relatedness among clinical isolates, 14. Full text get a printable copy pdf file of the complete article 1. Commonwealth of virginia division of consolidated laboratory services introduction microbial identification and. Chromosomal dna fingerprint patterns produced with is6110 are excellent stainspecific markers for the epidemiologic study of tuberculosis. Restriction fragment length polymorphism rflp is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their dna. Methods eight cases of tuberculosis were diagnosed in residents of the hostel over a period of 28 months.
Is6110 restriction fragment length polymorphism rflp. They support the use of miruvntr typing as a more reliable and faster method for transmission analysis. Is6110,6 and identical rflp patterns were shown by means of secondary genotyping methods to be genetically distinct 1012. Evolution of is6110 rflp patterns sequencing of the is6110 element has revealed that the sequence is conserved within the m.
86 74 539 1534 1025 202 433 23 238 381 104 134 878 1442 796 459 1293 230 75 844 1369 186 872 1456 1543 1269 1503 392 44 1225 62 295 145 489 465 1