Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae)

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Prevalence of mitochondrial DNA mutations in sporadic patients with nonsyndromic sensorineural hearing loss / Prevalência de mutações no DNA mitocondrial em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica

ABSTRACT INTRODUCTION: Several mitochondrial DNA mutations have been reported to be associated with nonsyndromic hearing loss in several families. However, little is known about the prevalence of these mutations in sporadic patients with nonsyndromic sensorineural hearing loss. OBJECTIVE: The purpose of our study was to investigate the incidence of these mitochondrial DNA mutations in such population. METHODS: A total of 178 sporadic patients with nonsyndromic sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral blood sample. We employed the SNaPshot(r) sequencing method to detect five mitochondrial DNA mutations, including A1555G and A827G in 12S rRNA gene and A7445G, 7472insC, and T7511C in tRNASerUCN gene. Meanwhile, we used polymerase chain reaction and sequenced the products to screen GJB2 gene mutations in patients carrying mitochondrial DNA mutations. RESULTS: We failed to detect the presence of A1555G mutation in 12S rRNA gene, and of A7445G, 7472insC, T7511C mutations in tRNASerUCN gene in our population. However, we found that 6 patients (3.37%) were carriers of a homozygous A827G mutation and one of them also carried homozygous GJB2 235delC mutation. CONCLUSION: Our findings in the present study indicate that even in sporadic patients with nonsyndromic sensorineural hearing loss, mitochondrial DNA mutations might also contribute to the clinical phenotype.

Resumo Introdução: Diversas mutações do DNA mitocondrial tem sido descritas, em diferentes famílias, associadas à deficiência auditiva não sindrômica. No entanto, pouco se sabe sobrea prevalência dessas mutações em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica. Objetivo: A finalidade do nosso estudo foi investigar a incidência dessas mutações no DNA mitocondrial nessa população. Método: No total, 178 pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica foram recrutados para participação no estudo. O DNA genômico foi extraído de amostra, de sangue periférico. Utilizamos o método de sequenciamento SNaPshot(r) para detecção de cinco mutações do DNA mitocondrial: A1555G e A827G no gene 12S rRNA e A7445G, 7472insCe T7511C no gene tRNASerUCN. Paralelamente, utilizamos a reação de polimerase em cadeia e sequenciamos os produtos para triagem das mutações no gene GJB2 nos pacientes portadores de mutações no DNA mitocondrial. Resultados: Em nossa população, não conseguimos detectar a presença da mutação A1555G no gene 12S rRNA e nem as mutações A7445G, 7472insC e T7511C no gene tRNASerUCN. Entretanto, constatamos que seis pacientes (3,37%) eram portadores da mutação homozigota A827G; e um deles também portava a mutação homozigota GJB2 235delC. Conclusão: Nossos achados no presente estudo indicam que, mesmo em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica, as mutações do DNA mitocondrial também podem contribuir para o fenótipo clínico.

Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO₂ concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 x 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

Production of polypeptide antibiotic from Streptomyces parvulus and its antibacterial activity

A highly potent secondary metabolite producing actinomycetes strain is isolated from marine soil sediments of Visakhapatnam sea coast, Bay of Bengal. Over all ten strains are isolated from the collected soil sediments. Among the ten actinomycetes strains the broad spectrum strain RSPSN2 was selected for molecular characterization, antibiotic production and its purification. The nucleotide sequence of the 1 rRNA gene (1261 base pairs) of the most potent strain evidenced a 96% similarity with Streptomyces parvulus 1044 strain, Streptomyces parvulus NBRC 13193 and Streptomyces parvulus BY-F. From the taxonomic features, the actinomycetes isolate RSPSN2 matches with Streptomyces parvulus in the morphological, physiological and biochemical characters. Thus, it was given the suggested name Streptomyces parvulus RSPSN2. The active metabolite was extracted using ethyl acetate (1:3, v/v) at pH 7.0. The separation of active ingredient and its purification was performed by using both thin layer chromatography (TLC) and column chromatography (CC) techniques. Spectrometric studies such as UV-visible, FTIR, and NMR and mass were performed. The antibacterial activity of pure compound was performed by cup plate method against some pathogenic bacteria including of streptomycin resistant bacteria like (Pseudomonas mirabilis. Pseudomonas putida and Bacillus cereus). In conclusion, the collected data emphasized the fact that a polypeptide antibiotic (Actinomycin D) was produced by Streptomyces parvulus RSPSN2.

Yeast diversity associated to sediments and water from two Colombian artificial lakes

In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them.

Antimycobacterial activity of a Brevibacillus Laterosporus strain isolated from a moroccan soil

The treatment of tuberculosis has become more difficult with the worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. Moreover, the prevalence of human disease caused by atypical mycobacteria has also increased in the past two decades and has further complicated the problem of the treatment of mycobacterial infections. It is therefore urgent to develop new highly active molecules against these bacteria. The present study reports the isolation from a Moroccan soil of a Bacillus strain that exhibits an important antimycobacterial activity. The strain was identified as Brevibacillus laterosporus using DNA sequencing of the 16S ribosomal RNA gene. The antimycobacterial activity was assigned to a substance with a protein nature. This nature was revealed using a liquid-liquid extraction with organic solvents, precipitation with ammonium sulfate and treatment with a protease. This study suggested the identification and the characterization of this active metabolite enabling therapeutic investigations further.

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

mtDNA mutations, hearing loss and aminoglycoside treatment in Mexicans / Mutações em DNA mitocondrial, hipoacusia e tratamento de mexicanos com aminoglicosídeos

Streptomycin and aminoglycoside derivatives are commonly used to treat tuberculosis and other stubborn infections; these drugs may alter auditory and/or vestibular function. Mutations in mitochondrial DNA have been associated with hypersensitivity to aminoglycosides; no studies have been conducted in Mexicans, which are very prone to such alterations because aminoglycosides have been prescribed carelessly for many years, irrespective of the ailment to be treated. AIM: We investigated "hot spot" mutations described previously as causing inner ear alterations. METHODS: Hot spot mutations at the 12S rRNA gene and the tRNA Serine (UCN) gene were screened by PCR-RFLP and sequencing in 65 subjects undergoing audiological and vestibular testing. STUDY DESIGN: Experimental. RESULTS: 32 individuals had healthy auditory and vestibular function, whereas 33 subjects had auditory affections. We found none of the previously reported mutations related to aminoglycoside hypersensitivity, or non-syndromic hearing loss. Two hearing-impaired patients that had been treated with streptomycin had the T1189C variant of the mitochondrial 12S rRNA region. CONCLUSION: Mutations related to hearing loss in other ethnic backgrounds were not found in Mexicans. However, the T1189C variant is possibly a putative mutation related to aminoglycoside hypersensitivity and was present in 2 patients.

Derivados de aminoglicosídeos e estreptomicina são comumente utilizados para tratar tuberculose e outras infecções mais resistentes; esses medicamentos podem alterar a função vestibular e/ou auditiva. Mutações no DNA mitocondrial têm sido associadas à hipersensibilidade a aminoglicosídeos; não há estudos conduzidos com mexicanos, que são muito predispostos a tais alterações, uma vez que aminoglicosídeos têm sido exageradamente prescritos há anos, sem associações à doença sendo tratada. OBJETIVO: investigamos mutações "hot spot" previamente descritas como causas de alterações no ouvido interno. MÉTODOS: Mutações hot spot no gene 12S rRNA e gene SerinatRNA (UCN) foram triados pela PCR-RFLP e sequenciados em 65 indivíduos sujeitos a exames audiométricos e vestibulares. Desenho do estudo: Experimental. RESULTADOS: 32 indivíduos com funções auditiva e vestibular normais, e 33 indivíduos com doenças auditivas. Não encontramos nenhuma das mutações previamente relatadas como associadas à hipersensibilidade aos aminoglicosídeos, ou perda auditiva não-sindrômica. Dois pacientes com hipoacusia que haviam sido tratados com estreptomicina tinham a variante T1189C na região 12S rRNA. CONCLUSÃO: Mutações associadas à hipoacusia em outras etnias não foram encontradas em mexicanos. Entretanto, a variante T1189C é possivelmente uma mutação associada à hipersensibilidade a aminoglicosídeos, e esteve presente em dois pacientes.

Giardia duodenalis: genotypic comparison between a human and a canine isolates / Giardia duodenalis: comparação genotípica entre isolados humano e canino

INTRODUCTION: Evidence suggests that giardiasis is a zoonotic disease. The present work aimed to evaluate the genetic identity of Giardia duodenalis isolated from human and dog fecal samples from Belo Horizonte. METHODS: Human and dog fecal samples were cultured for isolation of G. duodenalis. To determine the genotype of the isolates, primers that amplify a specific region in rRNA of the protozoan were used. RESULTS: Two G. duodenalis isolates were obtained, which belong to the subgroup A genotype. CONCLUSIONS: These findings suggest that the transmission of giardiasis follows a zoonotic pattern.

INTRODUÇÃO: Evidências sugerem que a giardíase é uma doença zoonótica. O presente trabalho tem como objetivo avaliar a identidade genética da Giardia duodenalis isolada de fezes humanas e de cães de Belo Horizonte. MÉTODOS: Amostras de fezes humanas e de cães foram cultivadas para isolamento de G. duodenalis. Para determinação do genótipo dos isolados, foram usados oligonuclotídeos que amplificam regiões específicas do gene para rRNA. RESULTADOS: Dois isolados de G. duodenalis foram obtidos, os quais apresentaram o genótipo do sub-grupo A. CONCLUSÕES: Estes dados sugerem que a transmissão da giardíase segue um padrão zoonótico.

Application of mtDNA polymorphism in species identification of sarcosaphagous insects / 法医学杂志

Species identification of sarcosaphagous insects is one of the important steps in forensic research based on the knowledge of entomology. Recent studies reveal that the application of molecular biology, especially the mtDNA sequences analysis, works well in the species identification of sarcosaphagous insects. The molecular biology characteristics, structures, polymorphism of mtDNA of sarcosaphagous insects, and the recent studies in species identification of sarcosaphagous insects are reviewed in this article.

First Report of Brain Abscess Associated with Pseudozyma species in a Patient with Astrocytoma / 대한진단검사의학회지

A yeast-like strain was isolated from the brain abscess of a patient diagnosed with astrocytoma. Morphological and molecular analysis on D1/D2 domain in the 26S rRNA gene and internal transcript spacer region of the strain revealed that the strain belonged to the genus Pseudozyma. To the best of our knowledge, this is the first report on the isolation of a Pseudozyma strain from brain abscess.

Mutação mitocondrial C1494T, deficiência auditiva e uso de antibióticos aminoglicosídeos / C1494T mitochondrial dna mutation, hearing loss, and aminoglycosides antibiotics

Tendo em vista a complexidade do mecanismo da audição, não é difícil compreender que a deficiência auditiva possa resultar de ampla variedade de anomalias geneticamente determinadas, bem como de diversos fatores ambientais. Mutações específicas no gene 12S rRNA em DNA mitocondrial são responsáveis por perda da audição não-sindrômica de herança materna, e pelo aumento da susceptibilidade ototóxica dos antibióticos aminoglicosídeos. Objetivo: Neste trabalho, nós avaliamos a presença da mutação C1494T entre os indivíduos ouvintes e com deficiência auditiva que utilizaram aminoglicosídeos e os que não tiveram contato com o antibiótico. Material e método: Foram estudados 20 pacientes com deficiência auditiva neurossensorial não-sindrômica sem histórico de sensibilização aos aminoglicosídeos e 40 recém-nascidos, prematuros e de alto-risco que utilizaram a droga ototóxica, dos quais 20 eram ouvintes e 20 com perda auditiva. As amostras foram analisadas por PCR-RFLP com a enzima de restrição Hph I. Forma de estudo: Experimental. Resultados: A mutação mitocondrial C1494T no gene 12S rRNA não foi detectada em nenhuma das amostras analisadas. Conclusão: Nossos dados sugerem que a deficiência auditiva dos indivíduos analisados não está relacionada com a ototoxicidade da mutação C1494T, demonstrando que esta mutação não é frequente em nossa população.

In view of the complex mechanism of hearing, it is not difficult to understand that hearing impairment may result from a wide variety of genetically determined anomalies and various environmental factors. Specific mutations in the mitochondrial DNA 12S rRNA gene are responsible for maternally inherited non-syndromic hearing loss, and for increased susceptibility to the ototoxicity of aminoglycoside antibiotics. AIM: To asses the presence of C1494T mutation among individuals with normal hearing and hearing impairment who used aminoglycosides and those who had not had contact with the antibiotic. Material and method: The study was composed of 20 patients with nonsyndromic sensorineural hearing loss without prior use of aminoglycosides and 40 premature and high-risk newborns who used ototoxic drugs, of whom 20 had good hearing and 20 had hearing loss. The samples were analyzed by PCR-RFLP with the restriction enzyme Hph I. Study design: Experimental. Results: The mitochondrial 12S rRNA C1494T mutation was not detected in any of the samples analyzed. Conclusion: Our data suggest that the hearing loss of the individuals we analyzed was not related to the ototoxicity of mutation C1494T, showing that this mutation is not frequent in our population.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil / Caracterização molecular de Cryptosporidium spp. de pacientes de área urbana do Brasil infectados por HIV

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0 percent) isolates of C. hominis, four (14.8 percent) C. parvum, five (18.5 percent) C. felis and one (3.7 percent) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Cryptosporidium spp. são importantes causas de doenças entéricas em humanos, mas podem também ser encontrados em animais. O presente estudo descreve a frequência relativa de diversas espécies de Cryptosporidium em amostras de humanos da cidade de São Paulo, Brasil, obtidas de janeiro a julho de 2007. Análises de sequências de produtos de nested PCR direcionadas ao genes codificadores da menor unidade ribosomal e da proteina de parede de oocistos revelaram 17 (63,0 por cento) isolados de C. hominis, quatro (14,8 por cento) C. parvum, cinco (18,5 por cento) C. felis, e um (3,7 por cento) C. canis. Estes resultados sugerem que, em ambientes urbanos no Brasil, o genótipo adaptado ao gato pode desempenhar potencial papel na transmissão zoonótica de criptosporidiose, enquanto a transmissão antroponótica da criptosporidiose causada pelo C. hominis parece predominar.

Role of connexin 26 (GJB2) & mitochondrial small ribosomal RNA (mt 12S rRNA) genes in sporadic & aminoglycoside-induced non syndromic hearing impairment.

Non syndromic hearing impairment is a common sensory disorder, which affects one in 600 newborns. Though more than 50 nuclear genes are involved in causing non syndromic hearing impairment, mutations in the connexin 26 (GJB2) gene explain a high proportion of congenital deafness in several populations worldwide. The diversity of genes and genetic loci implicated in hearing loss defines the complexity of the genetic basis of hearing. This review focuses on the role of connexin 26 and mitochondrial 12S rRNA genes in hearing which will be helpful for better understanding of genes in sporadic and aminoglycosideinduced non syndromic hearing impairment.

Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches.

Background & objectives: Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India. Methods: One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing. Results: Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed gene amplification - restriction analysis method could confirm the identity of 102 of 119 (85.7%) isolates and the remaining 17 isolates (14.3%) were confirmed by 16S rRNA sequencing also. These 119 environmental mycobacterial isolates, included several potentially pathogenic species such as M. fortuitum, M. chelonae, M. avium, M. marinum, M. manitobense, M. kansasii and others belonged to nonpathogenic species, M. terrae, M. smegmatis and M. flavescens. M. chelonae was isolated from water samples only whereas M. fortuitum was isolated from both water as well as soil samples. Interpretation & conclusion: The in-house developed gene amplification restriction analysis method though failed to accurately identify 14.3 per cent of isolates, facilitated rapid differentiation of most of environmental mycobacteria including potential pathogens from this area and thus would have diagnostic potential in cases with NTM infections. This combination strategy using PCR-RFLP and 16S rRNA sequencing may be useful for characterization of mycobacteria from similar environmental settings from other parts of world.

Species identification of biomaterials by amplifying 12S rRNA gene / 法医学杂志

OBJECTIVE@#To establish an accurate, simple, quick, specific and sensitive method for species identification by amplifying 12S rRNA gene with the same reaction system.@*METHODS@#Based on the downloaded 12S rRNA gene sequences of eleven species (human, chicken, duck, goose, pig, rabbit, rat, sheep, bull, dog and goat) from GenBank, a pair of universal primers to eleven species and three pairs of specific primers to human, chicken and duck were designed. The amplicons amplified with universal primers were used for internal controls, and the amplicons amplified with specific primers were used as identification of human, chicken and duck. DNA was extracted from various samples including blood stains, fresh or freezing muscles, heat-treated muscles and hairs. Both single DNA of human, chicken or duck and mixed DNA of any two kinds of them were amplified.@*RESULTS@#The lengths of universal amplicons were about 400 bp. The lengths of specific amplicons were 163 bp for human, 286 bp for chicken, and 374 bp for duck, respectively. No cross amplification was observed, indicating a high specificity of the specific primers. The identification rate was 100% for human, 99% for chicken, and 100% for duck, respectively. The detection sensitivity ranged from 2.5 pg to 200 pg of DNA concentration depending on species, even in mixtures of different species DNA without interference.@*CONCLUSION@#The method established could identify different species under the same reaction system.

Recent advances in molecular biology of leprosy.

The last three decades have witnessed rapid progress in understanding the molecular biology of Mycobacterium leprae. Following the availability of complete genome sequence of leprosy bacillus in 2001, things have drastically changed. With the information about genetic structure, several techniques have been developed for diagnosis, molecular epidemiology and also detection of drug resistance. With the decline in the prevalence of leprosy globally, there has been some reduction in interest in the molecular methods for diagnosis, yet molecular techniques for studying the transmission dynamics and detection of drug resistance continue to be relevant. Knowledge about complete genome sequence has made it possible to undertake studies that can improve our understanding of the structure and function of this enigmatic organism. Newer information emerging about biology of M. leprae would provide insight into mechanisms of its survival and persistence in host and is likely to lead to better diagnostics and also therapeutics for mycobacterial infections in general.