An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

Karakousis, A.; Tan, Lorwai; Ellis, David; Alexiou, H.; Wormald, Peter‐John

doi:10.1016/j.mimet.2005.06.008

Cited by 116 publications

(87 citation statements)

References 38 publications

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“…A series of comparisons was required to determine the optimal extraction technique for fungal biofilm community DNA. Based on methods described by Karakousis et al (2006), four factors were compared: (i) lyticase (Sigma-Aldrich, St. Louis, MO, USA) concentration: 200 U (Campbell et al 2009) and 400 U (Karakousis et al 2006); (ii) digestion buffer type: sorbitol buffer (Griffiths et al 2006) and MoBio-supplied extraction kit buffer; (iii) initial digestion time: 4 h (Campbell et al 2009) and overnight (Karakousis et al 2006); and (iv) extraction kit: UltraClean Microbial DNA Extraction kit and PowerBiofilm DNA Extraction kit (MoBio Laboratories, Inc., Carlsbad, CA, USA). For this study, these comparisons revealed that digestion with 200 U lyticase in MoBio PowerBiofilm buffers BF1 and BF2 for 4 h at 30°C in a dry bath incubator, then physical disruption with a tissue homogenizer and micropestle, followed by extraction with MoBio PowerBiofilm DNA Extraction kit produced the greatest yield of DNA for downstream reactions.…”

Section: Optimization Of Dna Extraction From Biofilms In Preparation mentioning

confidence: 99%

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

2016

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Abstract:We present the first characterization of fungal community diversity of natural mixed-species biofilms on artificial marine reefs. Four artificial reefs in the Mississippi (MS) Sound, USA, representing low-profile (underwater) and high-profile (periodically air-exposed) conditions were sampled every 3 months over a 23-month period to investigate changes in fungal diversity within reef biofilms. Fungal presence was assessed via PCR amplification of the internal transcribed spacer (ITS) region of fungal ribosomal DNA, and by terminal restriction fragment length polymorphism (T-RFLP) analysis of fungal ITS regions -the latter being used to track variation in fungal community structure with respect to season, location, and reef profile type. Fungal communities were also characterized taxonomically through both morphological identification and phylogenetic comparisons of ITS gene sequences, with 36 fungal genera cultured from reef biofilms. Using a multivariate statistical approach, significant temporal and spatial differences in fungal biofilm communities were detected. High-profile reefs differed significantly in biofilm fungal community composition across the 10 sampling periods. This assessment of marine fungal biofilm communities over time provides novel insights into the fungal diversity present on artificial reefs in an understudied region, the north-central Gulf of Mexico.

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Section: Optimization Of Dna Extraction From Biofilms In Preparation mentioning

confidence: 99%

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

2016

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“…The dried mycelia were then ground into a fine powder with liquid nitrogen using mortar and pestle (Karakousis et al, 2006). DNA extraction was done using DNeasy Mini Plant kit (Qiagen) and the eluted DNA was stored at -20°C until further use.…”

Section: Molecular Analysismentioning

confidence: 99%

<i>Ganoderma</i> Species Associated with Basal Stem Rot Disease of Oil Palm

Wong¹

2012

American Journal of Applied Sciences

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Problem statement: Basal Stem Rot disease (BSR) is one of the most serious diseases that have been causing major losses in the oil palm industry in Southeast Asia, especially in Malaysia and Indonesia. Several species of Ganoderma have been reported pathogenic to oil palm, however, the diversity and differentiation of the Ganoderma species were not widely studied and the identity of these species are still unclear which may lead to inaccurate and inefficient decision-making in disease management. Approach: In this study, several isolates of Ganoderma were collected in Sarawak, Malaysia and the Multiplex Polymerase Chain Reaction was carried out to differentiate the isolates into species level. This was followed by morphological studies of basidiocarp of the Ganoderma isolates cultivated via artificial cultivation whereby parameters, such as basidiocarp and spore size, color and physical morphology were recorded. Results: Multiplex PCR could be used to differentiate the Ganoderma isolates, however, optimization had to be done to obtain convincing results.

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“…A. flavus mycelia grows in parallel for 48, 72, 96 and 120 h in Cove's minimal salt medium (CMSM)supplemented with ten mM nitrate (nitrate medium) [5], collected on Miracloth, blotted dry, quickly frozen in liquid nitrogen, and stored at 28˚C until use [11]. Mycelia is grind to a fine powder in a mortar and pestle in the presence of liquid nitrogen [16].…”

Section: Fungal Strains and Isolation Of Total Rnamentioning

confidence: 99%

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2017

JBMOA

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The aflR gene is a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. There is a positive regulatory gene, aflR which required for transcriptional activation of most, if not all, of the structural genes by binding to the palindromic sequence 5'-TCGN5CGA-3' in the promoter region of the structural genes in A. parasitic, A. flavus and in A. nidulans. Adjacent to the aflR gene, a gene, aflS aflJ, is also involved in the regulation of transcription. Finally, the laeA gene, for loss of aflR expression, was shown to be involved in the global regulation of secondary metabolites, aflatoxins, sterigmatocystin (ST), penicillin and gliotoxin, in several fungal species. Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius and a few other species. The toxic effects of aflatoxins have opposing consequences for the health of human and agricultural economics. This study will examine to understand the mechanisms of aflR gene regulation, documentation of transcriptional activation.

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An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

Cited by 116 publications

References 38 publications

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

<i>Ganoderma</i> Species Associated with Basal Stem Rot Disease of Oil Palm

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