Gene Restriction Fragment Length Polymorphism Research Articles

Molecular Analysis of the Microbial Guild Fixing Nitrogen in Ricefield Soils in Missouri

Non-symbiotic diazotrophic microbes are important contributors to global N budgets in cereal crops. Knowledge of the biogeography of the organisms in this functional guild increases our understanding of biological N fixation in diverse locations and climates. Here, we describe the diazotrophic community in the previously unstudied, extensive ricefields of southeast Missouri, using restriction fragment length polymorphism (RFLP) analysis and sequencing of nifH gene clones. While nine RFLP patterns were observed in random nifH clones, these groups were not all supported by gene sequencing, suggesting that the RFLP of nifH genes alone is not suitable for describing diazotrophic guilds. Dozens of nifH clones from Missouri ricefield soils were sequenced and analyzed phylogenetically. The nifH genes detected were predominantly from Geobacteraceae, most closely related to Geobacter and Geomonas species. There were substantial clusters of nifH clones most closely related to Desulfovibrionales and other Proteobacteria. Many of the clones did not closely cluster with nifH sequences from known isolates or clades. No cyanobacterial or archaeal sequences were detected in the Missouri ricefield soils. The microbial guild fixing N appeared to be rich in anaerobes and lithotrophs. Organisms in Geobacter and Geomonas seem to be cosmopolitan, but endemism was evident, since nifH clones were recovered that formed clusters not previously reported from ricefields in other locations.

PAEP gene restriction fragment length polymorphism and its effects on milk composition in cross-bred Hamdani sheep

β-lactoglobulin (βLG) stands as the primary whey protein in ruminant milk, synthesized by mammary gland cells during lactation and encoded by the βLG gene. This study aimed to assess the impact of βLG gene exon II polymorphism on milk composition traits in crossbreed Hamdani sheep. Sheep were examined for clinical diseases and mastitis. Milk and blood samples were only collected from healthy ewes. The composition and physical properties of milk were analyzed using milk autoanalyzer. The βLG gene exon II region's 452 bp PCR products were subjected to RFLP analysis using the RsaI restriction enzyme. Two genotypes, AA and AB, were identified for the βLG gene exon II region, with A and B allele frequencies of 0.7 and 0.3, respectively. Statistical analysis, conducted with Minitab® (Version: 19.2020.2.0), revealed that the AA genotype is associated with a higher milk fat percentage (p

Molecular typing of canine parvovirus type 2 by VP2 gene sequencing and restriction fragment length polymorphism in affected dogs from Egypt.

Canine parvovirus-2 (CPV-2) is one of the most common infectious diseases in dogs characterized by severe gastroenteritis, vomiting, and bloody diarrhea. Little information is available about this topic in Egypt, particularly in the Delta region. This study reports the prevalence and molecular analysis of CPV-2 variants collected from El-Gharbia and Kafrelsheikh governorates in the Delta of Egypt. In this study, 320 rectal swabs were collected from infected domestic dogs from two districts in delta Egypt. The samples were investigated by rapid immunochromatographic test and polymerase chain reaction for detection the prevalence of CPV-2 variants. The genetic characterization was performed using restriction fragment length polymorphism (RFLP) analysis and partial VP2 gene sequence. The viral antigen was detected in (264/320, 82.5%) of samples by IC test, while PCR was found more sensitive by detecting (272/320, 85%) positive samples. The RFLP technique using MboII restriction enzyme was successfully used for the differentiation of CPV-2c antigenic variants from CPV-2a/2b strains. Interestingly, the molecular and phylogenetic analysis revealed that both CPV-2a and CPV-2c are circulating in the study area. Deduced amino acid sequence analysis showed changes at residue (N426E) and residue (T440A).: Our results indicated that CPV-2 is prevalent among dogs in Egypt, and therefore further molecular and epidemiological studies of CPV-2 are warranted.

Campylobacter jejuni and other emerging Campylobacteraceae in retail beef liver - an underestimated potential source?

Bovine by-products, such as liver, could be an underestimated source of Campylobacter jejuni. Therefore, our aims were to evaluate the occurrence of C. jejuni and other Campylobacteraceae in retail beef liver and characterize their antibiotic resistance (ciprofloxacin, tetracycline, erythromycin and gentamicin) and potential genetic relationship by flagellin gene restriction fragment length polymorphism (flaA-RFLP) and multilocus sequence typing with clinical strains. Seventy-six out of 206 samples (36·9%) were positive for Campylobacter and related organisms. Arcobacter butzleri was the most frequently isolated species (21·8%), followed by C. jejuni (9·7%), C. fetus (7·8%) and C. coli (1%). The C. jejuni strains showed resistance to tetracycline (17·2%) or ciprofloxacin (6·9%), with only one strain resistant to both antibiotics. Meanwhile, 8·3% of ciprofloxacin resistance was observed in C. fetus. The other species showed no resistance. Most of the clonal complexes (CC) in which the C. jejuni genotypes were grouped (CC-21, 42, 48 and 52), coincided with genotypes of clinical strains previously reported in Chile. As such, this study provides evidence that beef liver could be an underestimated route for resistant C. jejuni to humans. Further studies should assess whether this food could play a role in the transmission of other emerging Campylobacteraceae such as those reported here.

Effects of the commercial biostimulant BC204 on the rhizosphere microbial community of Solanum lycopersicum L.

The biostimulant BC204 is a commercially available biostimulant based on an extract from Citrus aurantium, comprising a mixture of bioflavonoids, other plant extracts and organic acids. In this study, the potential of BC204 to cause positive effects on plant growth and overall plant health by altering the rhizosphere microbial community of Solanum lycopersicum L. was investigated. Mycorrhizal colonization of plant roots was analysed by trypan blue-staining and light microscopy, followed by polymerase chain reaction (PCR) amplification of a region of the 18S rRNA gene conserved among arbuscular mycorrhizal fungi. No difference in mycorrhizal colonization of roots was observed between control and treated plants. The structural and functional diversity of rhizosphere bacteria between different treatment groups was assessed using 16S rRNA gene restriction fragment length polymorphism (RFLP) analysis and community-level physiological profiling (CLPP), respectively. Nearest sequence matches for three isolates from the rhizosphere of plants treated with BC204 as a soil drench in soil supplemented with Mycoroot™ revealed bacterial genera which play a role in promoting plant growth. Furthermore, the microbial community from this treatment group showed increased richness of substrate utilization on Biolog EcoPlates™. These results indicate some form of interaction between BC204 and Mycoroot™ which causes a shift in the structural and functional diversity of the rhizosphere bacterial community.

A critical appraisal on the recurrence of sandalwood spike disease and its management practices

AbstractSandal (Santalum album) is an important forest tree that has been utilized for centuries, valued for its wood and scented essential oil derived from the heartwood. Sandalwood spike disease (SSD) is one of the most destructive problems that affect Indian sandalwood. In 1969, three research groups confirmed that the causal agent of SSD was a phytoplasma. Although research literature on sandalwood spike disease has been published for over a century, many earlier publications reported only primarily descriptive macro‐morphological data. Identification of the causal agent was confirmed only recently, using nested PCR of 16S rRNA‐encoding gene sequences and restriction fragment length polymorphism (RFLP) analysis. The identified phytoplasma exhibited high sequence similarity to the aster yellows subgroup 16SrI‐B in NCBI BLAST search. The use of tissue culture in transgenic and disease‐free cloning strategies to produce disease‐resistant sandalwood tress might be the most realistic solution for SSD. The aim of this review is to increase understanding of SSD among phytopathologists in order to encourage research on SSD to discover best management practices. The increased cultivation of sandalwood plantations has significant importance, and it is essential to review the status of SSD.

Evaluating the antibiotic resistance and frequency of adhesion markers among Escherichia coli isolated from type 2 diabetes patients with urinary tract infection and its association with common polymorphism of mannose-binding lectin gene

The present paper aims to determine the frequency and antibiotic resistance patterns of pathogenic bacteria, the virulence factor profile of Escherichia coli and mannose-binding lectin (MBL) gene polymorphism in individuals with diabetes mellitus (DM) and urinary tract infection (UTI). The population under study was 130 individuals with type 2 diabetes mellitus (T2DM) and UTI. The patients' clinical characteristics and urine and blood samples (5 mL) were collected. Antibiotic resistance was determined using a disc diffusion method, and the results were interpreted according to CLSI. The presence of virulence genes was detected by multiplex PCR. To detect the MBL gene polymorphism, PCR and restriction fragment length polymorphism methods were applied. The predominant Gram-negative and Gram-positive bacteria included E. coli and Streptococcus spp.viridans group, respectively. Women were more susceptible to the incidence of UTI than men. The E. coli isolates showed a high level of resistance to amoxicillin-clavulanic acid (87.35%), and nitrofurantoin and ceftizoxime were the most effective antimicrobial agents for E. coli. Cefotaxime and ceftizoxime were the most effective antimicrobial agents for Enterobacter spp., norfloxacin and ciprofloxacin were the most effective antimicrobial agents for Staphylococcus epidermidis and Staphylococcus saprophyticus. papGII (52.87%) and papEF (1.14%) had the highest and lowest frequency among examined genes in E. coli isolates, respectively. The GG genotype had the highest frequency among patients with T2DM and UTI. Results showed that the detection of E. coli in individuals with an AA genotype, codon 54 of the MBL gene, can play an important role in the molecular diagnosis and timely treatment of bacterial infections in individuals with diabetes.

Molecular and restriction fragment length polymorphism analysis of canine parvovirus 2 (CPV-2) in dogs in southeast Anatolia, Turkey.

Canine parvovirus-2 (CPV-2) is the aetiological agent of an infectious viral disease of dogs, characterised by diarrhoea and vomiting. Mutations of the CPV-2 genome have generated new variants circulating worldwide. This article reports the molecular analysis of CPV-2 variants collected in the dog population in southeast Anatolia, Turkey. Twenty blood samples previously taken for the laboratory diagnosis of dogs with suspected parvovirus were screened for CPV-2 by polymerase chain reaction (PCR). Of the 20 samples, 18 tested positive for CPV-2. Partial VP2 gene sequencing and restriction fragment length polymorphism (RFLP) analysis revealed CPV-2a (n = 1), CPV-2b (n = 16) and CPV-2c (n = 1) variants. Phylogenetic analysis based on the partial length VP2 gene showed that CPV-2b (n = 15) variants showed sequences clustering separately in the phylogenetic tree. The CPV-2c sample was phylogenetically related to Chinese strains and Indonesia strain, whereas the CPV-2a sample was phylogenetically related to the Portuguese strain. These results, which are the first to demonstrate the presence of CPV-2c in the dog population of southeast Anatolia, Turkey, indicate that CPV-2a/2b/2c variants co-exist in Turkey’s dog population.

Differential identification of Anaplasma in cattle and potential of cattle to serve as reservoirs of Anaplasma capra, an emerging tick-borne zoonotic pathogen

Bovine anaplasmosis is a tick-borne, infectious, non-contagious disease caused by Anaplasma marginale, A. centrale, A. bovis, and zoonotic A. phagocytophilum. Recently, Anaplasma capra detected in goats was identified as a novel zoonotic pathogen. To determine whether A. capra can infect bovines, we used PCR to differentially diagnose Anaplasma spp. in 1219 South Korean cattle by performing multilocus gene typing and restriction fragment length polymorphism (RFLP). Nucleotide sequencing and phylogenetic analysis detected the 16S rRNA gene of A. bovis and four genes from A. capra in 12 (1.0%) and five (0.4%) cattle, respectively. Supplementary discrimination between A. bovis and A. capra was accomplished by RFLP. The 16S rRNA, msp4, groEL, and gltA genes of A. capra identified in this study had much lower degrees of identity to those in A. centrale and other Anaplasma spp. A. phagocytophilum was not detected in any of the tested cattle. Although the prevalence was low, this study suggests the potential of cattle to serve as reservoirs of A. capra. Thus, further studies are needed to clarify the pathogenesis of A. capra in cattle and its possible involvement in transmission to humans.

Genotyping Points to Divergent Evolution of 'Candidatus Phytoplasma asteris' Strains Causing North American Grapevine Yellows and Strains Causing Aster Yellows.

Grapevine yellows diseases occur in cultivated grapevine (Vitis vinifera L.) on several continents, where the diseases are known by different names depending upon the identities of the causal phytoplasmas. In this study, phytoplasma strains associated with grapevine yellows disease (North American grapevine yellows [NAGY]) in vineyards of Pennsylvania were characterized as belonging to 16S ribosomal RNA (rRNA) gene restriction fragment length polymorphism group 16SrI (aster yellows phytoplasma group), subgroup 16SrI-B (I-B), and variant subgroup I-B*. The strains (NAGYI strains) were subjected to genotyping based on analyses of 16S rRNA and secY genes, and to in silico three-dimensional modeling of the SecY protein. Although the NAGYI strains are closely related to aster yellows (AY) phytoplasma strains and are classified like AY strains in subgroup I-B or in variant subgroup I-B*, the results from genotyping and protein modeling may signal ongoing evolutionary divergence of NAGYI strains from related strains in subgroup 16SrI-B.

Antibiograms, toxin profiling and molecular typing of Staphylococcus aureus isolates from two tertiary hospitals in Jordan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infections. This study was undertaken to investigate toxin profiles as well as antibiotic resistance patterns of S. aureus isolates form two tertiary hospitals in Jordan. A total of 250 S. aureus isolates from clinical samples of two tertiary hospitals were analyzed for the presence of the mecA, vanA, vanB, and 16 Staphylococcus toxin encoding genes using PCR. The isolates were further tested for antimicrobial sensitivities using the disc diffusion method. DNA from all the isolates were fingerprinted by coa gene Restriction Fragment Length Polymorphism (RFLP) to study relationships between isolates from the two hospitals. 73.2% of the isolates contained the mecA gene and thus were designated MRSA. All MRSA isolates showed high levels of resistance to many of the antibiotics compared to those of MSSA. All MRSA isolates were susceptible to vancomycin and teicoplanin while all MSSA isolates were susceptible to nitrofurantoin, teicoplanin, vancomycin, cefoxitin, clindamycin, erythromycin and gentamycin. The isolates exhibited high prevalence of the toxin genes and none of the isolates contained less than 4 genes with one isolate contained 14 genes with no apparent differences in gene profiles among MRSA and MSSA. About 60% of the isolates contained 12 to 13 toxin genes and were isolated either from pus or blood. Antibiograms of the MRSA isolates were significantly different from MSSA antibiograms while there were no apparent differences in the toxin genes profiles. Further, coagulase gene RFLP of the isolates showed that the isolates are very heterogenic.

Detection of newly defined superantigenic toxin genes and coagulase gene polymorphism in Staphylococcus aureus isolates.

The current study aims to use coagulase (coa) polymorphism gene to identify Staphylococcus aureus isolated from stool samples, evaluate the efficiency of these methods in discriminating variable strains, and compare these subtypes with antibiotypes. A total of 100 specimens were collected from patients in Babylon province, Iraq, between July 2016 and September 2016. Twenty S. aureus strains were isolated and identified using standard laboratory microbiological tests. The bacterial isolates were then examined by coa gene restriction fragment length polymorphism genotyping. Out of 20 isolates, coa gene types were classified, and the amplification products showed multiple size bands (500, 600, 700, 800, and 900-bp bands). Coa gene PCR restriction fragment length polymorphisms exhibited seven patterns that ranged from one to four fragments with AluI digestion. The results have demonstrated that many variants of the coa gene are present. At least one type of S. aureus newly described enterotoxin gene (staphylococcal enterotoxins) was harboring in all 20 (100%) of the isolates. The most frequently encountered gene were sei (100.%), seh (5%), seg (65%). Many S. aureus isolates carry at least one of the enterotoxin genes, and (95%) strains harbored more than one toxin gene coding.

Identification of Fasciola spp. in the East of Iran, Based on the Spermatogenesis and Nuclear Ribosomal DNA (ITS1) and Mitochondrial (ND1) Genes

Background: Fascioliasis is a neglected zoonotic disease with worldwide distribution. Phylogenetic analysis of Fasciola species is critical because of their different epidemiology. Objectives: The current study aimed at identifying Fasciola spp. based on spermatogenesis and phylogenetic analysis of mitochondrial (ND1) gene. Methods: One hundred and fifty adults with fascioliasis were selected from the Eastern provinces of Iran, based on spermatogenetic ability, measurement criteria, and ITS (nuclear ribosomal DNA internal transcribed spacers) 1 gene restriction fragment length polymorphism (RFLP) pattern. Then, their genetic diversity indices and phylogenetic relationships were analyzed using mitochondrial DNA marker of Fasciola population spread in the East of Iran. Results: Aspermic F. gigantica was observed in the eastern part of Iran. Partial sequences of mtDNA showed new haplotypes in both species. Pairwise fixation index between different F. gigantica populations calculated from the nucleotide data set of ND1 gene were statistically significant and showed the genetic differences in pairwise population. Conclusions: The results of the study showed that F. hepatica, lives in this region of Iran, had different genetic structures compared with the other Fasciola populations in the world. Fasciola gigantica present in the East of Iran had different genetic structures, compared with the other Fasciola population, based on genetic index.

Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran

Aim:Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker.Materials and Methods:In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed.Results:Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference.Conclusion:F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world.

Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001-2006.

Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004–2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004–2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.

Transforming Growth Factor-β1 T869C Gene Polymorphism Is Associated with Acquired Sick Sinus Syndrome via Linking a Higher Serum Protein Level

BackgroundFamilial sick sinus syndrome is associated with gene mutations and dysfunction of ion channels. In contrast, degenerative fibrosis of the sinus node tissue plays an important role in the pathogenesis of acquired sick sinus syndrome. There is a close relationship between transforming growth factor-β1 mediated cardiac fibrosis and acquired arrhythmia. It is of interest to examine whether transforming growth factor-β1 is involved in the pathogenesis of acquired sick sinus syndrome.MethodsOverall, 110 patients with acquired SSS and 137 age/gender-matched controls were screened for transforming growth factor-β1 and cardiac sodium channel gene polymorphisms using gene sequencing or restriction fragment length polymorphism methods. An enzyme-linked immunosorbent assay was used to determine the serum level of transforming growth factor-β1.ResultsTwo transforming growth factor-β1 gene polymorphisms (C-509T and T+869C) and one cardiac sodium channel gene polymorphism (H588R) have been identified. The C-dominant CC/CT genotype frequency of T869C was significantly higher in acquired sick sinus syndrome patients than in controls (OR 2.09, 95% CI 1.16–3.75, P = 0.01). Consistently, the level of serum transforming growth factor-β1 was also significantly greater in acquired sick sinus syndrome group than in controls (5.3±3.4 ng/ml vs. 3.7±2.4 ng/ml, P = 0.01). In addition, the CC/CT genotypes showed a higher transforming growth factor-β1 serum level than the TT genotype (4.25 ± 2.50 ng/ml vs. 2.71± 1.76 ng/ml, P = 0.028) in controls.ConclusionTransforming growth factor-β1 T869C polymorphism, correlated with high serum transforming growth factor-β1 levels, is associated with susceptibility to acquired sick sinus syndrome.

Molecular types of Cryptococcus gattii/Cryptococcus neoformans species complex from clinical and environmental sources in Nairobi, Kenya.

Cryptococcal meningitis infections cause high mortality rates among HIV-infected patients in Sub-Saharan Africa. The high incidences of cryptococcal infections may be attributed to common environmental sources which, if identified, could lead to institution of appropriate control strategies. To determine the genotypes of Cryptococcus gattii/C. neoformans- species complex from Nairobi, Kenya, 123 clinical and environmental isolates were characterised. Typing was done using orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism (URA5-RFLP). The majority of the isolates [105/123; 85.4%] were C. neoformans genotype (AFLPI/VNI) and 1.6% AFLP1A/VNB/VNII, whereas (13%) were C. gattii (AFLP4/VGI). This is the first report on the genotypes of C. gattii/C. neoformans species complex from clinical and environmental sources in Nairobi, Kenya and the isolation of C. gattii genotype AFLP4/VGI from the environment in Kenya.

Peritoneal Pigmentation in Purebred and Hybrid River Herring

AbstractRiver herring (Alewives Alosa pseudoharengus and Blueback Herring A. aestivalis [bluebacks]) are congeneric, anadromous clupeid fishes that hybridize in areas of sympatry. Peritoneal pigmentation is the most diagnostic characteristic used to distinguish the species, but pigmentation has not been examined in hybrids. We developed a molecular‐based assay to distinguish purebred river herring species from F1 hybrids and compared the peritoneal pigmentation among captive‐raised purebred and hybrid individuals. Both wild‐caught Alewife and blueback females tank‐spawned with conspecific and congeneric males, but Alewives required exogenous hormone administration. Larvae of both species and hybrids were raised for ∼290 d posthatch, and peritoneal pigmentation was quantified. A nuclear gene (rag2) restriction fragment length polymorphism assay was used for species and hybrid identification. The peritoneal pigmentation was significantly darker in hatchery‐spawned bluebacks than in Alewives, and hybrids exhibited consistent, intermediate expression. Adult individuals collected from the wild, however, exhibited darker (Alewives) or more variable (bluebacks) peritoneal coloration than their respective, captive‐reared juveniles. These results indicated that peritoneal pigmentation alone is insufficient for diagnostic river herring species identification and should be coupled with genetic assays.

Study on genotype and virulence of Cryptococcus neoformans and Cryptococcus gattii clinical isolates in Guigang, Guangxi Zhuang Autonomous Region

To understand the species, genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii isolated from clinical samples in Guigang, Guangxi Zhuang Autonomous Region. A total of 20 Cryptococcus strains were isolated from clinical samples in Guigang from 2009 to 2012. The biological identification was conducted by polymerase chain reaction (PCR) to amplify internal transcribed spacer (ITS) sequences. The serotypes and mating types of C. neoformans and C. gattii were identified by PCR with serotype-specific and mating type-specific primers. The genotype was characterized by PCR fingerprinting and URA5 gene restriction fragment length polymorphism (URA5-RFLP). Phenotype study included growth test at 37 °C, melanin production test and urease test. Among the 20 strains, 19 (95%) were identified as C. neoformans varieties (var.) grubii (serotype A, mating type α, genotype VN I), and only 1 was identified as C. gattii (mating type α, genotype VG I). The results of virulence test showed that all the strains grew well at 37 °C and positive in both urease test and melanin production test. C. neoformans var. grubii (serotype A, genotype VN I and mating type α) was the predominant pathogen causing cryptococcosis in Guigang, and C. gattii strain was also detected.

Genetic Characteristics of Actinomycetes Isolates (GST, KP, KP11, Kp16, T24, T37 Code) by RFLP of NRPS Genes

Actinomycetes is the one of antibiotic producing microorganisms. Actinomycetes isolates with GST, KP, KP11, KP16, T24, T37 have been isolated from soil. This study aims to determine the diversity of actinomycetes isolates by RFLP (Restriction Fragment Length Polymorphism) of NRPS (Non Ribosomal Peptide Synthetase) genes from GST, KP, KP11, KP16, T24, and T37 isolates. This study is divided into several steps of the DNA isolation from isolate GST, KP16, T24, T37, KP, KP11, PCR of the 16S rRNA and NRPS genes and cutting the NRPS genes fragmens with HaeIII enzyme to determine the diversity of isolates. Results of DNA isolate, PCR of 16S rRNA genes and NRPS genes, and RFLP of NRPS genes performed by agarose gel electrophoresis. The diversity of the isolates were analyzed by multivariate analysis UPGMA with MVSP 2.0 applications. The results showed the diversity of 6 actinomycetes isolates are T37 and GST have 64 % similarity, KP11 and KP16 have 96 % similarity, KP has 76 % similarity with KP11 and KP16, while T24 has 60 % similarity with KP and 12 % similarity with T37 and GST. Based on RFLP analysis of the NRPS genes, among of 6 isolates can divided into 4 groups are T37 and GST isolates as group 1, KP11 and KP16 isolates as group 2, KP isolate as group 3, and T24 isolate as group 4. Keyword : Actinomycetes, diversity, 16S rRNA genes, NRPS genes, RFLP