Diversidad genética de aedes aegypti en el eje transfronterizo Central-Alto Paraná en Paraguay / Genetic diversity of Aedes aegypti in the central-upper Paraná Cross-Border axis in Paraguay

RESUMEN Objetivos: Conocer la diversidad genética de Aedes aegypti en el corredor vial transfronterizo Central-Alto Paraná de Paraguay, con registros de casos de dengue. Materiales y métodos: Se seleccionaron veinte hembras adultas de la eclosión de huevos de Ae. aegypti procedentes de casas geolocalizadas en los departamentos de Alto Paraná, Caaguazú, Cordillera y Central, entre el 2018 y 2019. Se extrajo ADN del tejido de las hembras para amplificación aleatoria de sus patrones polimórficos mediante amplificación aleatoria del ADN polimórfico por PCR (RAPD-PCR), usando cebadores H3 y B03 a fin de conocer parámetros genéticos de diversidad poblacional. Las relaciones entre las poblaciones de mosquitos según la localidad fueron visualizadas mediante análisis no apareado de la media aritmética. Las áreas idóneas de distribución geográfica real y potencial de estas poblaciones de Ae. aegypti fueron analizadas mediante DIVA-GIS 7.3.0 y MAXENT. Resultados: Se identificaron 40 loci mediante perfiles RAPD-PCR, con diferenciación génica moderada (Gst = 0,12). El corredor transfronterizo presentó condiciones bioclimáticas para la presencia de poblaciones variantes de Ae. aegypti, siendo determinantes en la distribución la precipitación del trimestre más cálido y la temperatura media del trimestre más seco. Conclusiones: Se evidencia que existe diversidad genética moderada en las poblaciones de Ae. aegypti procedentes de zonas con registros de casos de dengue ubicadas en el corredor vial transfronterizo que une los departamentos Central y Alto Paraná de Paraguay. El estudio de variabilidad genética de Ae. aegypti es de gran utilidad para la vigilancia entomoepidemiológica y evaluación de posibles eventos de resistencia al control químico.

ABSTRACT Objective: To determine the genetic diversity of Aedes aegypti in the Central-Alto Paraná cross-border road corridor of Paraguay, an area that has reports of dengue cases. Materials and methods: Twenty adult females were selected from hatching Ae. aegypti eggs from households geolocated in the departments of Alto Paraná, Caaguazú, Cordillera and Central, between 2018 and 2019. DNA was extracted from the tissue of females for amplifying their polymorphic patterns by random amplification of polymorphic DNA by PCR (RAPD-PCR), using primers H3 and B03 in order to identify genetic parameters of population diversity. The relationships between mosquito populations according to locality were observed by unpaired arithmetic mean analysis. We used DIVA-GIS 7.3.0 and MAXENT to analyze the suitable areas of actual and potential geographic distribution of these Ae. aegypti populations. Results: Forty loci were identified by RAPD-PCR profiling, with moderate gene differentiation (Gst = 0.12). The cross-border corridor presented bioclimatic conditions for the presence of variant populations of Ae. aegypti, with precipitation in the warmest quarter and mean temperature in the driest quarter being determinant in the distribution. Conclusions: There is evidence of moderate genetic diversity in Ae. aegypti populations from areas that have reported dengue cases in the cross-border road corridor linking the Central and Alto Paraná departments of Paraguay. The study of genetic variability of Ae. aegypti is very useful for entomo-epidemiological surveillance and evaluation of possible resistance to chemical control.

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

Introduction: For the rapid and accurate genetic identification and authentication of living organisms, improved random amplified polymorphic DNA (RAPD) fragment based development of sequence-characterized amplified region (SCAR) markers is an important genetic technique. Objective: This study aimed to develop SCAR markers for perennial herb Eclipta prostrate (E. prostrate). Methods: Here the RAPD fragments by improved RAPD amplification with primers A11 and N-7 for E. prostrate were cloned into pGEX-T vector, and PCR amplification identified the positive clones. After the enzymatic digestion, they were sequenced with Sanger sequencing. Results: Two SCAR markers were developed, which were very specific to E. prostrate, not found in Penthorum chinense Pursh(P. chinense). The nucleotide sequence search by BLAST GenBank database showed that they are novel in E. prostrate, therefore they were deposited in Genbank with accession number KX671034, KX671035. The markers did not show any identity to other species. Conclusions: Thus, in this study two specific SCAR markers were developed for genetically distinguishing and identifying the plant species E. prostrate from herb P. chinense and others.

Introducción: Verificación genética del arbusto Eclipta prostrate (Asteraceae) (Para la identificación y verificación genética rápida y precisa de organismos vivos, el uso de fragmentos de ADN polimórfico amplificado aleatoriamente (RAPD) mejorado de marcadores de región amplificada caracterizada por secuencia (SCAR) es una técnica genética importante. Objetivo: Este estudio tuvo como objetivo desarrollar marcadores SCAR para la hierba perenne Eclipta postrate (E. postrate). Métodos: En este estudio os fragmentos RAPD mediante amplificación RAPD mejorada con los cebadores A11 y N-7 para E. postrate se clonaron en el vector pGEX-T, y la amplificación por PCR identificó los clones positivos. Después de la digestión enzimática, se realizó una secuenciación Sanger. Resultados: Se desarrollaron dos marcadores SCAR, muy específicos para E. postrate, que no se encuentran en Penthorum chinense Pursh (P. chinense). La búsqueda de las secuencias de nucleótidos con BLAST en GenBank mostró que son nuevos en E. postrate, por lo que fueron depositados en Genbank con los números de acceso: KX671034 y KX671035. Los marcadores no mostraron ninguna identidad a otras especies. Conclusiones: En este estudio se desarrollaron dos marcadores SCAR específicos para distinguir e identificar genéticamente la especie de planta E. postrate de la hierba P. chinense y otras.

Population Structure Of Kaempferia Galanga L. From Eastern India

Objective: India has been a producer of a large number of aromatic medicinal plants which serves as a valuable genetic resource for future quality improvement to meet the ever-growing demand of human essential products. Thus, an urgent need arises for germplasm conservation of these high yielding varieties to help the pharmaceutical and other industries. For this understanding, the population structure is essential in order to explore their genetic identification by fingerprinting and molecular characterization. Methods: In the present study DNA was isolated using modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Polymerase Chain Reaction (PCR) was performed according to standardized method along with its data analysis. This study was undertaken to characterize the highly medicinal Kaempferia galanga collected from 4 different populations of Odisha using the molecular markers as Random Amplified Polymorphic DNA and Inter-Simple Sequence Repeats for the first time. Results: A dendrogram constructed through Sequential Agglomerative Hierarchical and Nested (SAHN) clustering and Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis showed an average similarity of 0.993 ranging between 0.967 to 1.000. Jaccard’s similarity coefficient of combined markers segregated the genotypes into two main clusters, 1 with six samples and the others at 0.98 similarity coefficient. Conclusion: Hence, the molecular analysis could be further used for the identification of important novel gene present in Kaempferia galanga which can be utilized for future crop improvement as well as pharmacological activities.

In vitro propagation of chia (Salvia hispanica L.) and assessment of genetic fidelity using random amplified polymorphic DNA and intersimple sequence repeat molecular markers

A well-organized micropropagation protocol has been designed for Salvia hispanica L., which bears high nutritional and medicinal value. Seeds of S. hispanica L. were germinated aseptically on half strength MS medium. Nodal explants obtained from in vitro germinated seedling were cultured on MS medium fortified with 6-benzyladenine (BAP) (1–5 mg/l) or Kinetin (Kin) (1–5 mg/l) individually or with α-naphthalene acetic acid (0.1–1 mg/l) and indole3-acetic acid (IAA) (0.1–1 mg/l) for clonal propagation. It was observed that maximum amount of shoots per explant (9.02 ± 2.65) was achieved on culture medium fortified with 3 mg/l BAP which was also optimum for subculturing of the regenerated shoots. Rooting was achieved on medium supplemented with 1 mg/l IBA. The rooted plantlets were acclimatized and transferred to field conditions, with 75% survival rate. Genetic fidelity studies were carried out on regenerated plantlets by 30 random amplified polymorphic DNA and 10 intersimple sequence repeat (ISSR) as molecular markers

Genetic analysis of Penthorum chinense Pursh by improved RAPD and ISSR in China

Background: Penthorum chinense Pursh (P. chinense) is a well-known traditional Chinese medicine (TCM) plant, which has long been used for the prevention and treatment of hepatic diseases. This study aimed to genetically characterize the varieties of P. chinense from different geographic localities of China by random amplification of polymorphic DNA (RAPD)-PCR technique and verified with inter-simple sequence repeat (ISSR) markers. Results: The P. chinense samples were collected from nine different geographic localities. Previously improved RAPD and ISSR markers were utilized for genetic analysis using DNA amplification. The genetic relationship dendrogram was obtained by conducting cluster analysis to the similarity coefficient of improved RAPD and ISSR markers. Improved RAPD yielded 185 scorable amplified products, of which 68.6% of the bands were polymorphic, with an average amplification of 9.25 bands per primer. The ISSR markers revealed 156 alleles with 7.8 bands per primers, where 59.7% bands were polymorphic. Furthermore, the similarity coefficient ranges of RAPD and ISSR markers were 0.71­0.91 and 0.66­0.89, respectively. Conclusions: This study indicated that improved RAPD and ISSR methods are useful tools for evaluating the genetic diversity and characterizing P. chinense. Our findings can provide the theoretical basis for cultivar identification, standardization, and molecular-assisted breeding of P. chinense for medicinal use.

Prevalence of Complement-Mediated Cell Lysis-like Gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis Isolates From Japan (2014–2016)

BACKGROUND: Streptococcus dysgalactiae subsp. equisimilis (SDSE; a β-hemolytic streptococcus of human or animal origin) infections are emerging worldwide. We evaluated the clonal distribution of complement-mediated cell lysis-like gene (sicG) among SDSE isolates from three central prefectures of Japan. METHODS: Group G/C β-hemolytic streptococci were collected from three institutions from April 2014 to March 2016. Fifty-five strains (52 from humans and three from animals) were identified as SDSE on the basis of 16S rRNA sequencing data.; they were obtained from 25 sterile (blood, joint fluid, and cerebrospinal fluid) and 30 non-sterile (skin-, respiratory tract-, and genitourinary tract-origin) samples. emm genotyping, multilocus sequence typing, sicG amplification/sequencing, and random amplified polymorphic DNA (RAPD) analysis of sicG-positive strains were performed. RESULTS: sicG was detected in 30.9% of the isolates (16 human and one canine) and the genes from the 16 human samples (blood, 10; open pus, 3; sputum, 2; throat swab, 1) and one canine sample (open pus) showed the same sequence pattern. All sicG-harboring isolates belonged to clonal complex (CC) 17, and the most prevalent emm type was stG6792 (82.4%). There was a significant association between sicG presence and the development of skin/soft tissue infections. CC17 isolates with sicG could be divided into three subtypes by RAPD analysis. CONCLUSIONS: CC17 SDSE harboring sicG might have spread into three closely-related prefectures in central Japan during 2014–2016. Clonal analysis of isolates from other areas might be needed to monitor potentially virulent strains in humans and animals.

Genetic relationship between Escherichia coli strains isolated from dairy mastitis and from the stable fly Stomoxys calcitrans / Caracterização genotípica de Escherichia coli isolados de leite com mastite e da mosca dos estábulos Stomoxys calcitrans

The stable fly Stomoxys calcitrans (Linnaeus, 1758) has been described as a potential spreader of infectious agents to cattle herds. Among the agents transmitted by this fly, Escherichia coli has attracted attention due to its potential to cause gastrointestinal disorders as well as environmental mastitis in dairy cows. Therefore, the aim of this study was to isolate and to assess the genetic diversity and the clonal relatedness among E. coli isolates from the milk of dairy mastitis and from stable flies anatomical sites by the Random Amplification of Polymorphic DNA (RAPD-PCR) technique. The molecular typing revealed a high degree of genetic polymorphism suggesting that these microorganisms have a non-clonal origin. Identical electrophoretic profiles were observed between E. coli isolates from different flies, different mammary quarters of the same cow and from cows on a single farm. These results reveal the circulation of the same bacterial lineages and suggest the role of the stable fly in bacterial dispersion. Considering the high pathogenic potential of this bacterial species, our findings alert to a more effective health surveillance.(AU)

A mosca dos estábulos Stomoxys calcitrans é descrita como um importante dispersor de agentes infecciosos aos bovinos. Dentre os agentes veiculados por esta mosca a bactéria Escherichia coli ganha relevância devido ao seu potencial em desenvolver alterações gastroentéricas, bem como mastite bovina ambiental. Desta forma, objetiva-se com este estudo isolar e acessar a diversidade genética e relação de clonalidade entre isolados de E. coli provenientes de casos de mastite e de moscas dos estábulos utilizando a técnica da Amplificação Randômica do DNA Polimórfico (RAPD). A tipagem molecular revelou elevado polimorfismo genético sugerindo que esses microrganismos têm origem não clonal. Perfis eletroforéticos idênticos entre si foram observados entre amostras isoladas de diferentes moscas, quartos mamários de uma mesma vaca, bem como de diferentes vacas dentro de uma mesma propriedade. Esses resultados revelam a circulação de uma mesma linhagem bacteriana e sugerem o papel da Stomoxys calcitrans na dispersão bacteriana. Considerando o elevado potencial patogênico dessa espécie bacteriana, nossos achados alertam para uma vigilância sanitária mais efetiva.(AU)

Community structures of total bacterial DNA, cultivable bacteria and prototrophs in bulk soil and rhizospheres

Aims: It has been hypothesized that root exudates can be a nutritional factor influencing the bacterial community structure as well as the occurrence of prototrophs and auxotrophs in rhizospheres. The present study was performed to examine the community structures of total bacterial DNA, cultivable bacteria and prototrophs in 3 soil samples with different levels of abundance of root exudates. Methodology and results: Denaturing gradient gel electrophoresis (DGGE) was performed to examine the community structures of total bacterial DNA, cultivable bacteria and prototrophs in 3 soil samples including bulk soil, rhizosphere of a single plant species and rhizosphere of multiple plant species. For clustering analysis, a dendrogram generated from the DGGE patterns revealed the different bacterial community structures in these soil samples. Both rhizospheres claded together, separating from bulk soil. The DGGE patterns of cultivable bacteria showed particular fingerprints corresponding to kinds of media and soil samples. Nutrient agar (NA) medium, isolation medium for prototroph (IMP) and IMP supplemented with soil extracts were used for bacterial cultivations. Prototrophs were isolated and examined by random amplified polymorphic DNA (RAPD) and 16S rRNA gene sequence analysis. The genetic diversity of prototrophs in 3 soil samples was similar (approximately 5% to 10% similarities) and most of them (13 of 28 strains) were members of Pseudomonas with 97% to 100% identities. Conclusion, significance, and impact of study: The present study provides a strong evidence of the influence of root exudates and plant species on bacterial community structures.

Modulation of mitochondrial membrane integrity and ROS formation by high temperature in Saccharomyces cerevisiae

Background Yeast strains are exposed to numerous environmental stresses during industrial alcoholic fermentation. High temperature accumulated acetic acid, enhanced the growth inhibition and decreased ethanol production. Results In this study the influence of high temperature on the cellular and mitochondrial membrane integrity of Saccharomyces cerevisiae as well as reactive oxygen species (ROS) formation was investigated to understand the mechanisms of the high temperature fermentation process. However, increasing the temperature to 42°C resulted in a clear decrease in the cytoplasmic and mitochondrial membrane potential and an increase in intracellular ROS formation. It was also determined that the different thermostability between YZ1 and YF31 strains had a clear correlation with the yeast's intracellular trehalose content of the cell. Finally, random amplified polymorphic DNA (RAPD) was used to explore the genome differences between the YZ1 and YF31 strains. Conclusions Thus, the stability of the mitochondrial membrane and subsequently, the clearance ROS ability could be important factors for the viability of S. cerevisiae at high temperatures.

Genetic characterization and authentication of Gardenia jasminoides in different regions of China by using improved RAPD analysis.

Gardenia jasminoides is a common garden medicinal plant known for its anticancer, anti-inflammatory, anti-thrombic, anti-fibrotic, antiviral, hepatoprotective, lung-protective, renal-protective, retina-protective and neuroprotective activities. It is found in several regions of the world, including China, but information about its genetic characteristics is limited. Here, we employed an improved method of random amplified polymorphic DNA (RAPD) analysis (with increased RAMP time) to investigate the genetic link between G. jasminoides samples collected from six different regions of Southern China. Total 26 RAPD primers were selected randomly, among which 23 primers generated reproducible polymorphic amplification bands. A total of 174 bands were obtained, where each primer had amplified 5-13 bands with an average of 7.56 bands per primer. The band size ranged approximately 150-2200 bp. Cluster dendrogram was obtained based on the improved RAPD amplification profiles, which showed that the similarity coefficients among six varieties of G. jasminoides ranged 0.67-0.88. To our knowledge, this is the first report of genetic characterization of G. jasminoides using improved RAPD analysis, which may be useful for the preservation of genetic diversity and identification of Gardenia population.

Efficiency of improved RAPD and ISSR markers in assessing genetic diversity and relationships in Angelica sinensis (Oliv. ) Diels varieties of China

Background Angelica sinensis is a well-known traditional Chinese medicinal plant. We aimed to assess the genetic diversity and relationships in A. sinensis cultivars collected from different locations of China and also some other Angelica species. Results We employed an improved random amplified polymorphic DNA (RAPD) technique for the amplification of DNA materials from ten Angelica cultivars, and the results were verified by inter-simple sequence repeat (ISSR) analysis. Twenty six RAPD primers were used for RAPD, and the amplified bands were found highly polymorphic (96%). Each primer amplified 8-14 bands with an average of 10.25. The cluster dendrogram showed that the similarity coefficients ranged from 0.41 to 0.92. The similarity coefficients were higher among different cultivars of A. sinensis, and lower among different species. Twenty ISSR primers were used for the amplification, and each primer generated 6-10 bands with an average of 7.2 bands per primer. The cluster dendrogram showed that the similarity coefficients ranged from 0.35 to 0.89. Conclusions This study genetically characterized the Angelica species, which might have a significant contribution to the genetic and ecological conservation of this important medicinal plant. Also, this study indicates that the improved RAPD and ISSR analyses are important and potent molecular tools for the study of genetic diversity and authentication of organisms.

Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn: by improved RAPD amplification and molecular cloning

Background Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.

Genetic relatedness of Candida albicans bloodstream infection clinical isolates in Malaysia

Aims: The aim of this study was to investigate the genetic relatedness of the most prevalent Candida bloodstream infection (BSI) species in in a Malaysian population via Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) fingerprinting. Methodology and results: The genomic DNA of 43 Candida BSI blood culture samples obtained from Universiti Malaya Medical Centre (UMMC) was isolated, after which species identification was carried out using PCR with ITS-1 and ITS-4 pan-fungal primers in conjunction with CHROMagar™ Candida. The predominant Candida species in the BSI samples is Candida albicans (14 out of 43 isolates). RAPD-PCR on these 14 C. albicans clinical isolates was performed using PST as the arbitrary primer. Data analysis using MEGA found an overall non-relatedness of these 14 clinical isolates [average similarity coefficient (SAB) value 0.733±0.172]. Following in-depth analysis, five of the 14 isolates were observed to be identical (SAB values of 1.00 each), four isolates had SAB values of 0.80-0.99, indicating that they are highly similar, but are non-identical, while five isolates are unrelated (SAB lower than 0.80). This suggests that microevolution might have occurred and that these clinical isolates may possibly belong to different strains. Conclusion, significance and impact of study: A fair degree of genetic heterogeneity was found among the 14 C. albicans isolates from UMMC. To our knowledge, this is the first report on the genetic profiles of C. albicans bloodstream infection isolates from Malaysia, warranting further studies in the possible evolutionary trends within this Candida species in Malaysia. Keywords: Randomly Amplified Polymorphic DNA PCR (RAPD-PCR), Candida albicans, Candida bloodstream infections, Genetic relatedness, DNA fingerprinting

Analysis of zoonotic dermatophytoses in 64 family-based groups / 中华皮肤科杂志

Objective To investigate the distribution and epidemiology of fungal pathogens in zoonotic dermatophytoses.Methods Seventy-four patients with dermatophytoses and 72 pets from 64 families,who were all culture positive for dermatophytes,were included in this study and classified into 64 family-based groups.Fungal culture and direct microscopic examination were carried out for species identification of fungal isolates,internal transcribed spacer (ITS) sequence analysis and random amplified polymorphic DNA (RAPD) analysis were performed for molecular identification and homology analysis.Results Dermatophyte species were consistent among the patients and pets from the same families for all the 64 family-based groups.A total of 146 fungal strains were isolated,including 93 Microsporum canis (M.canis) strains and 53 Trichophyton interdigitale (T.interdigitale) strains.M.canis was isolated from 42 (65.7％) family-based groups including 34 groups keeping cats and 8 groups keeping dogs,while T.interdigitale from 22 (34.3％) groups,including 14 groups keeping rabbits,6 groups keeping cats and 2 groups keeping dogs.There were 54 (75.0％) pets with obvious clinical symptoms (erythema,desquamation,depilation,etc),and 18 (25.0％) asymptomatic pets which were all cats.Among the 18 asymptomatic cats,14 carried M.canis,and 4 T.interdigitale.ITS sequencing and RAPD analysis revealed a high homology between the fungal pathogens in the same family-based groups.Conclusions M.canis and T.interdigitale are common species of dermatophytes in zoonotic dermatophytoses,and both of them have host specificity.Zoonotic dermatophytes can be transmitted between human and domestic animals,and attention should be paid to asymptomatic animals (carriers).

Comparative Study of Genetic Diversity of Spatolobi caulis from Guangxi by RAPD and ISSR Method / 中国药房

OBJECTIVE:To compare genetic diversity of Spatolobi caulis from different areas of Guangxi by random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR). METHODS:Through using POPGENE 32 software,Ntsys software and SPSS 17.0 software,RAPD and ISSR methods were used to study genetic diversity of 9 samples of S. caulis from dif-ferent areas of Guangxi. RESULTS:After amplification of screened 3 RAPD primers and 4 ISSR primers,and there were 198 and 315 locus,and 37 and 80 polymorphism locus. Rates of polymorphism locus were 18.7% and 25.4%;the number of effective al-leles were 1.416 8 and 1.584 0;genetic diversity index were 0.269 4 and 0.351 3;Shannon diversity index were 0.431 6 and 0.529 9. All the values of ISSR marker were higher than RAPD marker. The average genetic similarity coefficient of ISSR and RAPD were 0.757 64 and 0.683 80,indicating ISSR was more sensitive for the detection of genetic diversity. The clustering result of them was close to each other. The correlation coefficient of them were 0.847,indicating very significant positive correlation at the level of 0.001. CONCLUSIONS:ISSR could reflect more information of genetic diversity than RAPD,and is more suitable for research of genetic diversity of S. caulis from different areas of Guangxi.

Analysis of the Genetic Diversity of Germplasm Resources of Duchesnea indica from Hunan by Random Amplified Polymorphic DNA / 中国药房

OBJECTIVE:To study the genetic diversity of germplasm resources of Duchesnea indica from Hunan. METHODS:The random amplified polymorphic DNA(RAPD)reaction system of germplasm resources was established and RAPD-PCR was ad-opted to detect the expressions of 24 groups of D. indica from Hunan(including 21 wild and 3 cultivated varietos in different geo-graphical distribution). Agarose gel electrophoresis was used to analyze the results and calculate allele number (Na),effective al-lele number (Ne) the polymorphism points percentage (PPB),Nei’s gene diversity index (H) and Shannon’s information index (I). RESULTS:Totally 14 polymorphic primers were screened and 90 electrophoresis bands were amplified,including 81 polymor-phic bands with Na of 1.900 0,Ne of 1.540 1,PPB of 90.0%,H of 0.312 8 and I of 0.467 5. CONCLUSIONS:The genetic diver-sity of D. indica from Hunan is rich,and there were differences in genetic background between the cultivated species and wild spe-cies;clustering results show significant correlation with geographic distribution.

Development of RAPD-SCAR markers for Lonicera japonica (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning / Desarrollo de marcadores RAPD-SCAR para Lonicera japonica Thunb. (Caprifolicaceae) para la autenticación de variedades mediante RAPD mejorados y clonación de ADN

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with differ- ent species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR mark- ers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.

La diversidad genética dentro de una especie es una característica común, que juega un papel vital en su supervivencia y adaptabilidad, y es importante para la identificación y la autenticación de una especie. Lonicera japonica es una planta medicinal utilizada tradicionalmente, que han sido recientemente caracterizada genéticamente por amplificación aleatoria mejorada de ADN polimórfico (RAPD). En este estudio, los marcadores moleculares basados en estos fragmentos de RAPD se han desarrollado para identificar una variedad específica de L. japonica. Los ADN se extrajeron de las hojas jóvenes frescas de diferentes muestras de L. japonica recogidas de Shenzhen, Yichang, Leshan, Emei y Loudi, China. Los materiales de ADN fueron amplificados utilizando el RAPD PCR mejorado. Diferentes bandas RAPD fueron extraídas, clonadas y desarrolladas para las regiones amplificadas de secuencia conocida (SCAR) con marcado- res de diferentes especies. Dos marcadores SCAR, JYH3-3 y JYH4-3, se clonaron con éxito de los RAPD mejorados. El marcador SCAR JYH3-3 se encontró específico para todas las muestras de L. japonica recolectadas en las diferentes regiones, mientras que el otro marcador JYH4-3 era estrictamente específico para la muestra de Shenzhen de la provincia de Guangdong, que está geográficamente distante de Hubei, Sichuan y Provincias Hunan (fuente de otras muestras de L. japonica). Se encontró que JYH3-3 es un marcador molecular específico para la identificación de L. japonica, mientras que JYH4-3 se encontró como marcador molecular estrictamente específico para la muestra de Shenzhen. Los marcadores SCAR desarrollados podrían servir como marcadores moleculares más específicos para la autenticación de la variedad L. japonica. La combi- nación de RAPD mejorado y el desarrollo del marcador SCAR han dado como resultado herramientas útiles para el estudio de la variedad genética de cualquier organismo, que hemos aplicado con éxito en L. japonica.

Using molecular markers to assess Streptococcus mutans variability and the biological risk for caries

AIM: To characterize the genetic variability of Streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. METHODS: S. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16S-23S intergenic spacer region. The genetic variability of the isolates was first assessed using Random Amplified Polymorphic DNA (RAPD) markers. Next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase B (gtfB). RESULTS: Characterization using RAPD markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. By sequencing the 16S-23S intergenic region, it was found that all of the isolates belonged to the species S. mutans. Based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfB gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. CONCLUSIONS: The partial sequencing of the gtfB gene can be a useful tool for elucidating the colonization patterns of S. mutans. As amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children...

Susceptibilidad antimicrobiana y genotipificación de Pseudomonas aeruginosa de pacientes con fibrosis quística y otras patologías en Cartagena (Colombia) / Antimicrobial susceptibility and genotypification of Pseudomonas aeruginosa from cystic fibrosis patients and other diseases in Cartagena (Colombia)

Objetivo: el objetivo de este estudio fue analizar el genotipo y susceptibilidad antimicrobiana de Pseudomonas aeruginosa de pacientes con fibrosis quística y otras patologías. Materiales y métodos: se analizaron 20 aislados de pacientes con fibrosis quística y 20 de pacientes con otras enfermedades por medio de la prueba de susceptibilidad antimicrobiana por microdilución en caldo y técnica del ADN polimorfo amplificado aleatorio. Resultados: se observó que los aislados de pacientes con fibrosis quística presentaron mayor resistencia (56 %) en comparación con aislados de pacientes sin fibrosis quística (25 %). Los antimicrobianos más efectivos en ambos grupos fueron cefepima, ceftriaxona y meropenem. Desde el punto de vista genotípico, se observa heterogeneidad entre las cepas de pacientes con fibrosis quística y dos grupos con cepas idénticas de origen hospitalario, lo que sugiere una posible transmisión cruzada. Conclusión: Concluimos que los porcentajes de resistencia de Pseudomonas aeruginosa en este estudio son altas, y este hallazgo se acentúa en el caso de pacientes con fibrosis quística, lo cual deja muy pocas opciones de tratamiento. La tipificación por técnica del ADN polimórfico amplificado aleatorio permitió conocer la variabilidad de genotipos para tener control sobre la transmisión de cepas, lo cual constituye un tópico de importancia en el sistema de salud y el mejoramiento de la calidad de vida de los pacientes.

Objective: Our aim was to analyze genotype and antimicrobial susceptibility of Pseudo-monas aeruginosa from cystic fibrosis patients and other diseases. Materials and methods: We analyzed 20 isolates from cystic fibrosis patients and 20 from patients with other diseases by dilution antimicrobial susceptibility test and random amplified polymorphic DNA technique. Results: We found that isolates from cystic fibrosis patients had higher resistance (56 %) than isolates from patients without cystic fibrosis (26 %). The most effective antimicrobi-als in both groups were cefepime, ceftriaxone and meropenem. With regard to the geno-type, we observed heterogeneity between strains from cystic fibrosis patients and two clus-ters with identical strains from hospital origin, suggesting a possible cross transmission. Conclusion: We concluded that the resistance rate of Pseudomonas aeruginosa in this study was high and this finding is accentuated in patients with cystic fibrosis, leaving few treatment options. Typification by random amplified polymorphic DNA technique allowed us to know the variability of genotypes to control strain transmission; this is an important topic to optimize health services and the quality of life of our patients.

Random amplified polymeric DNA fingerprinting of antler by high performance capillary electrophoresis / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis-random amplified polymorphic DNA (HPCE-RAPD) fingerprinting method of antler.