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Amplified-fragment length polymorphism fingerprinting of Mycoplasma species.

ABSTRACT: Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneumoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5). AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and MfeI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp. The method was able to discriminate the analyzed strains at species and intraspecies levels as well. Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis. Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma species analyzed. The extent of polymorphism varied markedly between the analyzed mycoplasmas, comprising pattern similarity levels from 61.7% detected among M. dispar strains to 95.9% detected among M. genitalium strains. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of mycoplasmas.

SUBMITTER: Kokotovic B

PROVIDER: S-EPMC85551 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing.

Project description:We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

| S-EPMC120682 | biostudies-literature

Epidemiology of human sporotrichosis investigated by amplified fragment length polymorphism.

Project description:Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of Peruvian strains of Sporothrix schenckii and to compare them to a panel of non-Peruvian strains. AFLP analysis suggests that the Peruvian strains can be divided into two homogeneous clusters with no reference to geographical origin or the clinical form of sporotrichosis. The strains from abroad present heterogeneous profiles, with the Bolivian strain and the Colombian strains related to one of the Peruvian population. Sequencing of internal transcribed spacer 2, used to examine the relationships over a longer distance, confirmed the division of Peruvian strains into two populations that can be identified on the basis of a single but specific sequence divergence. This paper introduces automated AFLP analysis as a valuable tool for further investigation of the epidemiology and ecology of S. schenckii.

| S-EPMC1081277 | biostudies-literature

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments.

Project description:A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

| S-EPMC262260 | biostudies-literature

Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis.

Project description:Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media. Several pigmented variants of B. subtilis have been reclassified as B. atrophaeus, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B. subtilis var. niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the B. atrophaeus type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B. atrophaeus than to B. subtilis and should be reclassified as B. atrophaeus. A very closely related cluster of B. atrophaeus strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B. subtilis subspecies, subtilis and spizizenii. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B. atrophaeus subsp. globigii.

| S-EPMC404429 | biostudies-literature

A single domestication for potato based on multilocus amplified fragment length polymorphism genotyping.

Project description:The cultivated potato, Solanum tuberosum, ultimately traces its origin to Andean and Chilean landraces developed by pre-Colombian cultivators. These Andean landraces exhibit tremendous morphological and genetic diversity, and are distributed throughout the Andes, from western Venezuela to northern Argentina, and in southern Chile. The wild species progenitors of these landraces have long been in dispute, but all hypotheses center on a group of approximately 20 morphologically very similar tuber-bearing (Solanum section Petota) wild taxa referred to as the S. brevicaule complex, distributed from central Peru to northern Argentina. We present phylogenetic analyses based on the representative cladistic diversity of 362 individual wild (261) and landrace (98) members of potato (all tuber-bearing) and three outgroup non-tuber-bearing members of Solanum section Etuberosum, genotyped with 438 robust amplified fragment length polymorphisms. Our analyses are consistent with a hypothesis of a "northern" (Peru) and "southern" (Bolivia and Argentina) cladistic split for members of the S. brevicaule complex, and with the need for considerable reduction of species in the complex. In contrast to all prior hypotheses, our data support a monophyletic origin of the landrace cultivars from the northern component of this complex in Peru, rather than from multiple independent origins from various northern and southern members.

| S-EPMC1253605 | biostudies-literature

Amplified fragment length polymorphism analysis supports the valid separate species status of Lucilia caesar and L. illustris (Diptera: Calliphoridae).

Project description:Common DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy. This is a practical problem, and it has also been interpreted as casting doubt on the validity of some morphologically defined species. An example is Lucilia illustris and L. caesar, which co-occur in Europe whilst only L. illustris has been collected in North America. Reports that these species shared both mitochondrial and nuclear gene sequences, along with claims that diagnostic morphological characters are difficult to interpret, were used to question their separate species status. We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis, and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.

| S-EPMC6197086 | biostudies-literature

Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified-fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis.

Project description:One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.

| S-EPMC87873 | biostudies-literature

Whole Genome-Based Amplified Fragment Length Polymorphism Analysis Reveals Genetic Diversity in Candida africana.

Project description:This study aimed at investigating the genetic diversity of a panel of Candida africana strains recovered from vaginal samples in different countries. All fungal strains were heterozygous at the mating-type-like locus and belonged to the genotype A of Candida albicans. Moreover, all examined C. africana strains lack N-acetylglucosamine assimilation and sequence analysis of the HXK1 gene showed a distinctive polymorphism that impair the utilization of this amino sugar in this yeast. Multi-locus sequencing of seven housekeeping genes revealed a substantial genetic homogeneity among the strains, except for the CaMPIb, SYA1 and VPS13 loci which contributed significantly to the classification of our set of C. africana strains into six existing diploid sequence types. Amplified fragment length polymorphism fingerprint analysis yielded greater genotypic heterogeneity among the C. africana strains. Overall the data reported here show that in C. africana genetic diversity occurs and the existence of this intriguing group of C. albicans strains with specific phenotypes associated could be useful for future comparative studies in order to better understand the genetics and evolution of this important human pathogen.

| S-EPMC5377543 | biostudies-literature

Development of amplified fragment length polymorphism (AFLP) markers for the identification of Cholistani cattle.

Project description:The identification issue of livestock can be resolved by using molecular identification tools that are acceptable to preserve and maintain pure breeds worldwide. The application of a molecular identification methodology is more important for developing nations, e.g., Pakistan, where uncontrolled crossbreeding has become a common practice and the import of exotic animals and germplasm is ever increasing. This presents a risk to local breeds as also stated by the FAO. Therefore, the current study was designed to develop standard molecular markers for Cholistani cattle to ascertain their purity for breeding purpose. In this study 50 and 48 unrelated males were sampled for Cholistani and each crossbred cattle, respectively. Candidate molecular markers present in Cholistani but absent in crossbred cattle and vice versa were detected using the amplified fragment length polymorphism (AFLP) method. Eleven markers were developed and were converted to single nucleotide polymorphism (SNP) markers for genotyping. The allele frequencies in both breeds were determined for discrimination ability using polymerase-chain-reaction-restriction-fragment-polymorphism (PCR-AFLP). The probability of identifying the Cholistani breed was 0.905 and the probability of misjudgment was 0.073 using a panel of markers. The identified markers can ascertain the breed purity and are likely to extend the facility for breed purity testing before entering into a genetic improvement program in the country.

| S-EPMC7065403 | biostudies-literature

Amplified fragment length homoplasy: in silico analysis for model and non-model species.

Project description:BACKGROUND: AFLP markers are widely used in evolutionary genetics and ecology. However the frequent occurrence of non-homologous co-migrating fragments (homoplasy) both at the intra- and inter-individual levels in AFLP data sets is known to skew key parameters in population genetics. Geneticists can take advantage of the growing number of full genome sequences available for model species to study AFLP homoplasy and to predict it in non-model species. RESULTS: In this study we performed in silico AFLPs on the complete genome of three model species to predict intra-individual homoplasy in a prokaryote (Bacillus thuringiensis ser. konkukian), a plant (Arabidopsis thaliana) and an animal (Aedes aegypti). In addition, we compared in silico AFLPs to empirical data obtained from three related non-model species (Bacillus thuringiensis ser. israelensis, Arabis alpina and Aedes rusticus). Our results show that homoplasy rate sharply increases with the number of peaks per profile. However, for a given number of peaks per profile, genome size or taxonomical range had no effect on homoplasy. Furthermore, the number of co-migrating fragments in a single peak was dependent on the genome richness in repetitive sequences: we found up to 582 co-migrating fragments in Ae. aegypti. Finally, we show that in silico AFLPs can help to accurately predict AFLP profiles in related non-model species. CONCLUSIONS: These predictions can be used to tackle current issues in the planning of AFLP studies by limiting homoplasy rate and population genetic estimation bias. ISIF (In SIlico Fingerprinting) program is freely available at http://www-leca.ujf-grenoble.fr/logiciels.htm.

| S-EPMC2875239 | biostudies-literature

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