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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
Subject(s)
Candida albicansABSTRACT
Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supple mented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m2s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.
Vitex trifolia es una especie arbustiva de uso popular como planta medicinal, sus hojas, raíces y flores se han reportado para la cura de diferentes aflicciones. El aumento de la explotación de estas plantas ha puesto en peligro su conservación y ha justificado el uso de herramientas biotecnológicas para su propagación. El objetivo de esta investigación fue presentar un protocolo eficiente para la regeneración de estas plantas a través de la organogénesis, y analizar la homogeneidad genética de las líneas clonales establecidas por ADN polimórfico amplificado aleatoriamente (RAPD) mediante la repetición de marcadores de inter secuencia simple (ISSR). La regeneración de plántulas se logró en cultivos de callos derivados de explantes de tallo, hoja y pecíolo de V. trifolia en un medio diferenciado Murashige & Skoog, que se incubaron a 25±2ºC bajo una intensidad de luz de 61μmol/m2s con lámparas fluorescentes blancas y un fotoperíodo de 16h. La tasa de regeneración de brotes se correlacionó positivamente con la concentración de las hormonas en el medio nutritivo. Los brotes se regeneraron más rápidamente a partir de explantes de tallo y pecíolos en comparación con explantes de hoja. La mayor tasa de regeneración de brotes se obtuvo en los explantes de tallo utilizando 11.10μM BAP en combinación con 0.54μM NAA, 271.50μM Ads y 1.44μM GA3. Este protocolo puede ayudar a la propagación masiva y conservación de esta importante planta medicinal de gran potencial terapéutico.
Subject(s)
Plants, Medicinal/physiology , Regeneration/physiology , Vitex/physiology , Microsatellite Repeats , Plant Growth Regulators/pharmacology , Plant Shoots/drug effects , Plant Shoots/growth & development , Plants, Medicinal/classification , Plants, Medicinal/drug effects , Random Amplified Polymorphic DNA Technique , Regeneration/drug effects , Vitex/classification , Vitex/drug effectsABSTRACT
Objective To test the antimicrobial susceptibility of Staphylococcus aureus from children with impetigo,and to assess the differences in randomly amplified polymorphic DNA profiles between sensitive and resistant Staphylococcus aureus strains.Methods Secretion specimens were obtained from the impetiginous lesions of 178 children,and subjected to bacterial culture.The susceptibility of 162 Staphylococcus aureus isolates against 21 antibiotics was tested.Randomly amplified polymorphic DNA PCR(RAPD-PCR)was performed to characterize the genotype of Staphylococcus aureus.Results Totally,180 bacterial strains were isolated from 178 children with impetigo in Chengdu,including 162(90.00％)Staphylococcus aureus strains.Of the 162 Staphylococcus aureus strains,148 were methicillin sensitive Staphylococcus aureus(MSSA),14 methicillin resistant Staphylococcus aureus(MRSA).The most active antibiotic was minocycline,followed by teicoplanin,quinupristin,vancomycin and nitrofurantoin,while the resistance rate to penicillin was highest,followed by that to erythromycin,clindamycin,compound sulfamethoxazole and tetracycline.All the Staphylococcus aureus isolates were sensitive to fusidic acid,nitrofurantoin,vancomycin,minocycline and teicoplanin.According to RAPD-PCR,the 162 Staphylococcus aureus strains were divided into 8 genotypes,with the three most prevalent genotypes being Ⅲ(31.48％),Ⅱ(26.54％)and Ⅵ(25.93％),which accounted for 65.43％(106/162)in all the strains.The 148 MSSA strains fell into 8 genotypes,with genotype Ⅲ(50 strains,33.78％),Ⅵ(39 strains,26.35％)and Ⅱ(33 strains,22.30％)being the most prevalent genotypes;the 14 MRSA strains fell into 3 genotypes,i.e.,genotype Ⅱ(10 strains,71.43％),Ⅵ(3 strains,21.43％),and Ⅲ(1 strain,7.14％).Conclusions Staphylococcus aureus is the most prevalent pathogenic bacteria in children with impetigo in Chengdu area,which is highly sensitive to minocycline,teicoplanin and quinupristin,and falls into 8 genotypes according to RAPD-PCR with genotype Ⅲ being the most common genotype.
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OBJECTIVE To study the homology of the pandrug-resistant Acinetobactor baumannii(PRABA) by randomly amplificed polymorphic DNA(CRPD) to guide the control of the nosocomial infection and epidemology investigation.METHODS Eighteen strains of PRABA were collected from Jul 2008 to Mar 2009 in our hospital.The bacteria were examined by DADE BEHRING Microscan WalkAway 96SI;DNA extracted from all isolates was amplified by polymerase chain reaction using random primers.In addition epidemology investigation and antimicrobial susceptibility testing were also under taken.RESULTS Fifteen strains were divided into 6 RAPD profiles.There were two clonal strains derived,respectively,from two intensive care units.One strain was isolated from a patient in surgical ward who was transferred from ICU1,showing same homology with that in the ICU1.The homology was different from sensitive strains and drug-resistant strains obviously.CONCLUSIONS An outbreak of PRABA happens in intensive care unit.
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OBJECTIVE To survey the hospital infection of Acinetobacter baumannii in an ICU during one week's time,and study its antibiotic resistance pattern and homogeneity.METHODS The antibiotic resistance patterns of 17 A.baumannii strains were obtained by K-B method,and their homogeneity information was determined using random amplified polymorphic DNA(RAPD).RESULTS Twelve strains of A.baumannii were isolated from the ICU,among which 3 were isolated from patients,and 9 were isolated from the surface of facilities in the ICU.Eleven strains were found to be multi-antibiotic resistant,and their resistance patterns were the same with the 5 strains from patients.RAPD results showed that the 3 strains from patients and 8 strains from the environment had the same genotype.CONCLUSIONS A small outbreak of a genotype of A.baumannii infection happens in the ICU.
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OBJECTIVE To establish a fingerprinting method by randomly amplified polymorphic DNA.Epidemiological study was carried out on Pseudomonas aeruginosa in Ruijin Hospital. METHODS To obtain optimum scheme on reaction system for randomly amplified polymorphic DNA(RAPD) of P.aeruginosa. RESULTS P.aeruginosa strains isolated from the same ward shared the same RAPD fingerprint type,except for pulmonary ward.Different ward was with different fingerprint type. CONCLUSIONS Prevalent strain was not found in the whole hospital,but within ward exists hospital-acquired infection phenomenon.
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Objective To evaluate the indeterminate growth mutant of tomato derived by space mutagenesis to provide the basis for selecting and cultivating the molecular markers of tomato growth habit. Methods Fifty 10-mer randomly amplified polymorphic DNA(RAPD) primers were used to examine the polymorphism of M1 and 10-3-2. Their polymorphic fragments were cloned, and then were transferred to SCAR markers. Results Of all the 50 10-mer RAPD primers, 44 primers amplified polymerase chain reaction(PCR) products and 2 primers (S165 and S168) amplified stable reproducible polymorphic products. The molecular weight of the specific amplified products were 300 bp and 1 500 bp respectively, therefore they were named as TRS165300 and TRS1681500 temporarily. And TRS1681500 was transferred into stable sequence characterized amplified region(SCAR) marker and this marker could be a specific genetic marker of this indeterminate growth habit mutant. Conclusion Space mutation can produce mutants at DNA level from the loaded materials. The indeterminate growth mutant of tomato is derived by space mutagenesis which can provide a valuable material for studying the regulation and control of tomato growth habit.
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BACKGROUND: Recently, we noticed an increased isolation rate for Candida tropicalis from urine of the patients in the intensive care unit (ICU) of the neurosurgery department in our hospital. We inves-tigated the risk factors for funguria and performed randomly amplified polymorphic DNA (RAPD) analysis for the isolates. METHODS: A total of 27 strains including 12 strains of C. tropicalis from the urine of ICU patients collected during a one-month period, 13 strains from other wards than ICU, and 2 control strains were analyzed. RAPD analysis was performed using 2 primers (UBC78 and CD16S). Medical re-cords of the patients were reviewed to determine the risk factors for funguria by C. tropicalis. RESULTS: RAPD analysis showed an identical pattern for all strains of C. tropicalis from ICU patients and a heterogeneous pattern for the isolates from other wards. C. tropicalis funguria of these ICU patients was significantly associated with the use of an urinary catheter (100%, P < 0.001), previous surgery (44%, P < 0.05) and trachostomy (40%, P < 0.05), when compared with those of non-ICU patients. CONCLUSIONS: The identical RAPD pattern of all C. tropicalis strains from ICU patients indicates that they possibly originated from one clone. Through the investigation of risk factors, we can postulate that an urinary catheter might be a source for the outbreaks of urinary tract infections in the ICU. In addition, RAPD analysis might be a very useful test as one of the molecular epidemiologic tools for C. tropicalis funguria.
Subject(s)
Humans , Candida tropicalis , Candida , Clone Cells , Disease Outbreaks , DNA , Epidemiologic Studies , Intensive Care Units , Neurosurgery , Risk Factors , Urinary Catheters , Urinary Tract Infections , Urinary TractABSTRACT
To investigate the influential factors of different concentrations of some reagents and the comparability among the laboratories of RAPD. RAPD was carried out with different concentrations of reagents and three different amplified cyclers to amplify 11 strains of pathogenic vibrios. 11 strains of pathogenic vibvios were genotyped into 4 types, and could be easily repeated.The different concentrations of primer and 4 dNTP could influence the result of RAPD,but the fingerprints of RAPD produced respectively with 3 amplified cyclers were consistent. It is suggested that RAPD can be compared among the laboratories with the standard reagents.