Application of multiple-locus variable-number tandem repeat analysis(MLVA) for molecular typing of Leptospira interrogans serogroup Icterohaemorrhagiae
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Objective To establish the method of multiple loci VNTR(variable numbers tandem-repeats) analysis (MLVA) for genotyping Leptospira interrogans serogroup ieterohaemorrhagiae . Methods Seven VNTR loci were chosen for genotyping 117 strains of L. interrogans serogroup Icterohaemorrhagiae by PCR-electrophoresis-based VNTR analysis and the results were analyzed by software BioNumerics( Version 4.0). Results One hundred and seventeen isolates of L. interrogans serogroup Icterohaemorrhagiae detec-ted with 7 VNTR loci were classified into three clusters(A,B,C), twenty-eight types were found, type A 11.97% (14/117), type B 0.85% (1/1 17), type C 87.18% (102/117). Diversity Indexes for the loci varied between 0.0831 and 0.8005. Clinical strains isolated from the same geographic area and belonging to the same serogroup shared a common VNTR pattern. Conclusion MLVA could be used to classify and identify Leptospira interrogans preliminarily. With the improvement of technology, this rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.
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Leptospira interrogate; Serogroup Icterohaemorrhagiae; Genotyping; MLVAKeywords:
Leptospira interrogans
Minisatellite
Leptospirosis is a re-emerging zoonosis of the tropical and subtropical areas of the world. The causative agent, Leptospira spp., can be classified into more than 300 serovars based on a serological technique and 15 genomospecies according to DNA-DNA hybridization. To date, the Microscopic Agglutination Test (MAT) is the only major means to identify Leptospira serovars for epidemiological tracing. MAT is a sophisticated test involving a large panel of serovar-specific antisera. The laboratory personnel is endangered by infection from the requirement of a large number of living leptospires. Therefore, the development of a new simpler technique is essential. To date, whole genome sequences of four Leptospira serovars have been released and over 60 variable number of tandem repeats (VNTR) with difference in length and copy numbers have been identified. Thus, the objective of this study is to evaluate the efficacy of multiple-locus of VNTR analysis (MLVA) in Leptospira serovars identification. A total of 30 Leptospira reference serovars and 55 Leptospira spp. isolated from patients with confirmed leptospirosis in Thailand during 2002 to 2006, were analyzed for the presence of five VNTR loci namely V27, V29, V30, V36 and V50 by PCR. A total of 38 MLVA patterns were identified in this study. Twenty-three MLVA patterns were identified from 30 reference samovars. Three serovars provided similar MLVA patterns and six serovars provided no amplification products. Some of the Leptospira patient isolates yielded identical MLVA patterns to that of the reference serovars, and they provided an extra 15 MLVA patterns. The MLVA results in this study completely conformed to the serovar identification from either MAT or latex agglutination. In conclusion, our data further emphasize the discrimination power of MLVA in Leptospira serovar identification. Nevertheless, more VNTR loci and a larger panel of reference serovars need to be investigated to obtain the complete Thai MLVA pattern database for all Leptospira serovars.
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Direct agglutination test
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OBJECTIVE To understand the genotype,geography distribution and dominant genotype of leptospirosis in Si Chuan province,in order to provide the scientific evidence for determining the outbreak,prevalent trace,infection source and diabetic foot,and to prevent and control the epidemic.METHODS 7 VNTRs(variable numbers tandem repeats)of 185 leptospirosis of SiChuan were detected by multiple locus variable number of tandem repeats analysis(MLVA),and the results were analyzed by software BioNumerics.RESULTS 164 strains of 185 leptospirosis were successfully amplificated,and the positive rate was 88.6%,which were divided into 81 genotypes.144 of 156 leptospia interrogans of Sichuan were successfully amplificated,and the positive rate was 92.3%,which were divided into 65 genotype,including 11 serogroups and 34 serotypes.CONCLUSION The leptospirosis of Sichuan demonstrates good polymorphism,and genotypes are related to geography distribution.It can provide timely and exactly scientific evidence for molecular epidemiology investigation of leptospira by MLVA method.
Leptospira interrogans
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Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.
Multilocus sequence typing
Leptospira interrogans
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Subtyping
Salmonella enteritidis
Salmonella enterica
Molecular Epidemiology
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To evaluate the feasibility of the application of variable-number tandem repeat (VNTR) loci of Salmonella Enteritidis (S. enteritidis) in subtyping mutiple-locus variable-number tandem repeat analysis (MLVA).A total of 16 isolates of S.enteritidis from different place and time in China were preliminarily assessed by choosing 11 reported VNTR loci, the loci with single amplified bands were picked to subtype all 104 S. enteritidis isolates. The isolates were also analyzed by pulse field gel electrophoresis (PFGE) to compare the superiority or inferiority of MLVA method and PFGE method.Seven VNTR loci were selected from the preliminary screening to expand the analysis, and the 7 VNTR loci had grouped 104 of S.enteritidis isolates into either 16 MLVA subtypes or 22 PFGE subtypes, with the D value at 0.7222 and 0.7974, respectively. Comparing with the isolates in MLVA subtypes, the isolates in PFGE showed a stronger resolving power. Meanwhile the results in PFGE showed a more disperse frequency distribution than those in MLVA.These results indicate that some VNTR locus which have shown a good polymorphism internationally, may fail to show polymorphism in China, thereby, more VNTR loci should be included in MLVA and the wide screening may benefit the unity of global laboratorial methods.
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Leptospirosis is a zoonotic infection for which diagnosis is difficult. It has appeared as a global emerging infectious disease over recent years. Genotype determination often requires a Leptospira strain obtained by culture, which is a long and fastidious technique. A method based on multilocus variable number tandem repeat analysis (MLVA) to determine the genotype of Leptospira interrogans, performed directly on blood or urine samples, is proposed. This method was applied to a fatal case of leptospirosis for which the geographical origin of infection was unknown. This technique will allow a genotype to be obtained for L. interrogans, even when cultures remain negative.
Leptospira interrogans
Fastidious organism
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Leptospira interrogans
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Leptospira interrogans
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Abstract Background Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism. Methods In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats [1]. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland. Results The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines. Conclusion The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.
Leptospira interrogans
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钩端螺旋体(钩体)病是一种自然疫源性疾病.近年来以可变数目串联重复序列(variable number of tandemrepeats,VNTR)为基础的分型方法,逐渐被应用于分子流行病学研究中.本研究选取我国致病性钩端螺旋体15群15型参考菌株,初步探讨多位点可变数目串联重复序列分析(MLVA)在钩体病分子流行病学研究中的应用价值。
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