Simple DNA Extraction Research Articles

Cell-free DNA extraction from urine of lung cancer patients and healthy individuals: Evaluation of a simple method using sample volume up-scaling.

Urine holds promise as a source for cell-free DNA (cfDNA) analysis of cancer genetics due to its nature as a self-collectable biospecimen available in large quantities. However, pre-analytical variables such as preservation of cfDNA or efficiency of up-scaling specimen volume need to be better explored to increase analysis sensitivity. Initially effects of pH levels on cfDNA stability of urine preserved with EDTA were investigated in three healthy probands. Furthermore, the efficiency of urine volume up-scaling was examined using a simple DNA extraction method and cfDNA in urine of 32 individuals. Quantification was performed by PCR detection of three relevant targets used for EGFR and KRAS gene mutational analysis. Only samples preserved with EDTA at alkaline pH levels showed cfDNA stability of up to 10 days at room temperature. Moreover, it was found that increasing the volume up to 100 mL was highly efficient. A similar amount of copies was detected in three different gene sites in all specimens indicating both a good availability and a non-random distribution pattern across genes. Since the median cfDNA copy number was 1642 copies/mL, abundance of cfDNA in this type of liquid biopsy is low. Predictable sensitivities with different urine volumes on the ground of detectable cfDNA in our study population revealed that up-scaling (>5 mL) is mandatory if the mutation allele frequency is less than 1%.

Quick preculture method for determining low concentration Vibrio spp. in contaminated seafood and its verification

This study aimed to present a new approach for detecting pathogenic Vibrio spp., including V. parahaemolyticus, V. vulnificus, and V. cholerae in contaminated seafood. This method combined polymerase chain reaction (PCR) with a concise preculturing step and a simplified DNA extraction procedure. The effectiveness of the method was confirmed using contaminated mussel samples. To validate this proposed method, 15 different shellfish samples were analyzed and the results were compared with the results obtained using a conventional method, most-probable-number-PCR (MPN-PCR). These results confirm the efficiency of this new approach in detecting pathogenic Vibrio spp. This method takes approximately 6 h for results and offers significant time saving with a reduction of 75% compared with MPN-PCR. Additionally, it demonstrated high sensitivity for the efficient Vibrio spp. detection, and the simplification of the DNA extraction process using boiling makes it accessible to non-experts and contributes to overall time savings in the sample preparation stage. The proposed method in this study suggests a time-efficient and simplified alternative approach for detecting Vibrio spp. in seafood products, providing an in-situ application for improving safety measures in the food industry.

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

Development of rapid on-site detection of Salmonella Enteritidis, S. Typhimurium, and S. Thompson in food samples using an ultrafast PCR system

The conventional Salmonella serotyping methods are labor intensive and time-consuming; therefore, rapid identification method is needed to detect outbreaks early and prevent their spread. In this study, we developed for the first time a detection method for the rapid and accurate on-site serotyping of Salmonella enterica subsp. enterica serovar Enteritidis, Thompson, and Typhimurium, all of which have epidemiological importance worldwide. This method, which is based on specific marker genes, was constructed with portable equipment and a simple DNA extraction step. For the specific detection of the three serovars, genetic markers obtained via a bioinformatics analysis were selected and showed high specificity for 97 strains. The ultrafast polymerase chain reaction (PCR) showed high linearity (≥0.997) in both pure culture and a spiked food sample, with a detection specificity of 102 colony forming unit (CFU)/ml; these results are consistent with real-time PCR. Moreover, ultrafast PCR was further improved for developing direct ultrafast PCR method for field detection, the entire procedure of which could be completed within 25 min. The test results of an artificially contaminated food sample showed that the three serovars could be detected using the direct ultrafast PCR with a very short (3 h) enrichment step when inoculated at 1 CFU/g. This method exhibited various advantages, such as its ultrafast and labor-saving characteristics, which can be applied to the diagnosis of Salmonella serovar in the field.

Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia.

Colorectal cancer (CRC) is a serious public health problem known to have a multifactorial etiology. The association between gut microbiota and CRC has been widely studied; however, the link between archaea and CRC has not been sufficiently studied. To investigate the involvement of archaea in colorectal carcinogenesis, we performed a metagenomic analysis of 68 formalin-embedded paraffin fixed tissues from tumoral (n = 33) and healthy mucosa (n = 35) collected from 35 CRC Tunisian patients. We used two DNA extraction methods: Generead DNA FFPE kit (Qiagen, Germantown, MD, USA) and Chelex. We then sequenced the samples using Illumina Miseq. Interestingly, DNA extraction exclusively using Chelex generated enough DNA for sequencing of all samples. After data filtering and processing, we reported the presence of archaeal sequences, which represented 0.33% of all the reads generated. In terms of abundance, we highlighted a depletion in methanogens and an enrichment in Halobacteria in the tumor tissues, while the correlation analysis revealed a significant association between the Halobacteria and the tumor mucosa (p < 0.05). We reported a strong correlation between Natrialba magadii, Sulfolobus acidocaldarius, and tumor tissues, and a weak correlation between Methanococcus voltae and healthy adjacent mucosa. Here, we demonstrated the feasibility of archaeome analysis from formol fixed paraffin-embedded (FFPE) tissues using simple protocols ranging from sampling to data analysis, and reported a significant association between Halobacteria and tumor tissues in Tunisian patients with CRC. The importance of our study is that it represents the first metagenomic analysis of Tunisian CRC patients' gut microbiome, which consists of sequencing DNA extracted from paired tumor-adjacent FFPE tissues collected from CRC patients. The detection of archaeal sequences in our samples confirms the feasibility of carrying out an archaeome analysis from FFPE tissues using a simple DNA extraction protocol. Our analysis revealed the enrichment of Halobacteria, especially Natrialba magadii, in tumor mucosa compared to the normal mucosa in CRC Tunisian patients. Other species were also associated with CRC, including Sulfolobus acidocaldarius and Methanococcus voltae, which is a methanogenic archaea; both species were found to be correlated with adjacent healthy tissues.

A simple and rapid DNA extraction method from meat by microneedle patch for detection of adulterated components

ABSTRACT The simple and rapid identification of meat via DNA-based methods is still subject to the conditions of complex and time-consuming preprocessing. It usually takes 3–4 h to obtain DNA sample using conventionally available DNA extraction kits. The existing approaches are cumbersome, require large equipment, and are not suitable for field operation. This study developed a swelling microneedle patch, constituted by micron-sized copolymers of methyl vinyl ether and maleic acid (PMVE/MA). The microneedle patch was applied to the meat sample for 1 min before being pulled out and rinsed with Tris-EDTA (TE) buffer to obtain DNA, which could be used for polymerase chain reaction (PCR) without purification. The DNA extracted by the microneedle patch could meet the needs for amplification and identification of meat samples. The developed approach which combines the patch-based extraction method and conventional PCR amplification is quick, simple, and reliable for DNA extraction and identification of meat samples.

Simplified detection of the species of origin of antler velvets using single-stranded tag hybridization chromatographic printed-array strip.

In this study, we developed a convenient and easy-to-use origin identification method for antler velvets based on a simple DNA extraction technique and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS). The primer sets used to detect Cervus elaphus, Rangifer tarandus, and 12S rRNA did not engage in non-specific reactions such as primer dimer formation. In both the triplex and singleplex assays, the sensitivity was < 1ng DNA. Moreover, Cervus elaphus DNA could be detected in OTC crude drug products. Although the detection sensitivity resulting from the simplified extraction was slightly lower than that obtained with extraction by conventional methods, the amount of DNA was sufficient even from a small sample. The choice of a triplex or singleplex assay will depend on the purpose of the test. For example, if it is important to determine whether the antler velvet is derived from Cervus elaphus or Rangifer tarandus, a triplex assay is appropriate. If it is necessary to explore whether antler velvet from Cervus elaphus is included in an OTC crude drug product, a singleplex assay using the Cervus elaphus primer set is informative. If it is necessary to explore whether powdered antler velvet includes counterfeit products (from Rangifer tarandus), a singleplex assay employing the Rangifer tarandus primer is appropriate. The singleplex assay detects minor components even at a 1,000:1 ratio. Our study thus demonstrated the utility of a method combining simple DNA extraction with STH-PAS for efficient identification of the origin of antler velvets.

Vertebrate environmental DNA from leaf swabs

Terrestrial vertebrates are threatened by anthropogenic activities around the world. The rapid biodiversity loss that ensues is most intense in the tropics and affects ecosystem functions, such as seed dispersal, or may facilitate pathogen transmission1. Monitoring vertebrate distributions is essential for understanding changes in biodiversity and ecosystems and also for adaptive management strategies. Environmental DNA (eDNA) approaches have the potential to play a key role in such efforts. Here, we explore whether eDNA swabbed from terrestrial vegetation in a tropical biodiversity hotspot is a useful tool for vertebrate biomonitoring. By swabbing leaves, we collected eDNA from 24 swabs at three locations in Kibale National Park, Uganda and used two metabarcoding systems to catalog the vertebrate taxa in the samples. We detected 52 wild vertebrate genera, including 26 avian and 24 mammalian genera; 30 of these assignments could be refined to the species level. We detected an average of 7.6 genera per swab. This approach, with its inexpensive and simple collection and DNA extraction, opens the door for inexpensive large-scale vertebrate biomonitoring.

Evaluation of A Simple DNA Extraction Method and Its Combination with Loop-Mediated Isothermal Amplification Assays for Rapid Plasmodium knowlesi Diagnosis.

The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in commercial DNA extraction kits. We attempted to improve an existing loop-mediated isothermal amplification (LAMP) for the detection of Plasmodium knowlesi by using a simple DNA extraction approach, making it a feasible option for mass screening. We utilized a simple nucleic acid extraction method directly from whole blood for the detection of P. knowlesi, taking only 5 min to complete. The extracted DNA was evaluated by two fluorescent-based LAMP and one colorimetric-based LAMP assay. The detection limit for both SYTO-LAMP and SYBR green-LAMP was 0.00001% and 0.0001% parasitemia, respectively. Meanwhile, neutral red-LAMP had a detection limit of 0.01% parasitemia. Combining this simple and inexpensive DNA extraction method, SYTO-LAMP could serve as an alternative molecular diagnosis for the detection of P. knowlesi and other human Plasmodium spp.

On-site detection system of Candidatus Liberibacter asiaticus by using TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR).

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus plants caused by the obligate and phloem-limiting bacterium Candidatus Liberibacter asiaticus (Las). Reliable detection methods are important for successful control of the disease. This study was aimed to develop a rapid and user-friendly on-site detection system for Las using the TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay. The Las-specific on-site detection system could be completed within one hour by simple DNA extraction coupled with a portable POCKIT device, which can perform PCR amplification and automatically provide qualitative results derived from fluorescence signals. The sensitivity of the TaqMan probe-iiPCR assay could be as low as single copy of Las, comparable to a real-time PCR method. Further testing of the field citrus samples showed 100% agreement between the TaqMan probe-iiPCR assay and the real-time PCR method, and the on-site detection system also demonstrated a great performance of Las detection. With high specificity and sensitivity, the on-site detection system developed in this study becomes a simple, rapid and powerful tool for detecting Las in fields.

Development of a portable DNA extraction and cross-priming amplification (CPA) tool for rapid in-situ visual diagnosis of plant diseases

Plant pathogens cause severe losses to crop yields and economic returns in agriculture. Despite plant tissue DNA extraction of typically constituting a preliminary step in nucleic acid-based molecular diagnostics, such lab-based methods can be time-consuming and arduous to complete many samples. To mitigate these challenges, we developed an inexpensive portable DNA extraction technique that is lightweight and suitable for deployment in sampling locations, such as fields. It includes a DNA extraction device fabricated with a Steel Microneedle Array (SMA) and a simple high-efficiency DNA extraction buffer. As a result, DNA extraction times can be reduced to within ~ 1 min, and the eluted DNA is demonstrated to be suitable for subsequent molecular biological analyses without requiring additional purification. Cross-priming amplification (CPA) technology was first established to detect Phytophthora infestans, which achieves sensitivity attainment of 10–7 ng/µL. The detection result can be conveniently estimated with naked-eye visual inspection using fluorescent dsDNA binding dye. CPA was demonstrated to be more feasible than PCR-based approaches and performed well in species-specific and practicability tests. This study elucidates a novel integrated pathogen detection technique coupled with SMA-Device extraction and a modified visual CPA assay to establish and verify various field-based samples infected with multiple pathogens. Altogether, the total sample-to-answer time for pathogen detection was reduced to ~ 1.5 h, making field-based analysis affordable and achievable for farmers or extension workers inside and outside the laboratory.

Field ready: Development of a rapid LAMP‐based colorimetric assay for the causal agent of white pine blister rust, Cronartium ribicola

AbstractThe invasive fungal pathogen Cronartium ribicola causes white pine blister rust which is considered one of the most destructive diseases of five‐needle (white) pines in North America. The disease has a life cycle that requires two hosts: white pines and Ribes spp., although other non‐Ribes species, including Castilleja and Pedicularis, have been demonstrated as alternate hosts as well. Detection of this disease can be difficult because of the ephemeral nature of sporulation on pine hosts with ambiguity in other symptoms, and the alternate hosts for C. ribicola can also be an alternate host for other pine rust species. We used the previously published C. ribicola genome and species‐specific real‐time PCR assay to develop a field‐ready loop‐mediated isothermal amplification (LAMP) specific colorimetric assay for this pathogen. Specificity results across regionally identified pine rust pathogens showed the assay is highly specific to C. ribicola and can detect as little as 40 pg of pathogen DNA. We also developed a simple DNA extraction method that works with several tissue types (bark/phloem, aeciospores, and urediniospores/telia) to prepare the DNA samples for the LAMP assay. The DNA extraction and LAMP assay take ~70 min to complete and require a relatively small investment in equipment. This tool enables quick and efficient detection of white pine blister rust.

A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection

Background: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. Methods: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. Results: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 102 CFU/mL Salmonella Typhimurium was over 2000 RFU, while 104 CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). Conclusions: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.

Point-of-Care Diagnosis of Malaria Using a Simple, Purification-Free DNA Extraction Method Coupled with Loop-Mediated Isothermal Amplification-Lateral Flow.

We propose a protocol suitable for point-of-care diagnosis of malaria utilizing a simple and purification-free DNA extraction method with the combination of loop-mediated isothermal amplification assay and lateral flow (LAMP-LF). The multiplex LAMP-LF platform developed here can simultaneously detect Plasmodium knowlesi, P. vivax, P. falciparum, and Plasmodium genus (for P. malariae and P. ovale). Through the capillary effect, the results can be observed by the red band signal on the test and control lines within 5 min. The developed multiplex LAMP-LF was tested with 86 clinical blood samples on-site at Hospital Kapit, Sarawak, Malaysia. By using microscopy as the reference method, the multiplex LAMP-LF showed 100% sensitivity (95% confidence interval (CI): 91.4 to 100.00%) and 97.8% specificity (95% CI: 88.2% to 99.9%). The high sensitivity and specificity of multiplex LAMP-LF make it ideal for use as a point-of-care diagnostic tool. The simple and purification-free DNA extraction protocol can be employed as an alternative DNA extraction method for malaria diagnosis in resource-limited settings. By combining the simple DNA extraction protocol and multiplex LAMP-LF approach, we aim to develop a simple-to-handle and easy-to-read molecular diagnostic tool for malaria in both laboratory and on-site settings.

One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.

Rapid and simultaneous detection of viable S. aureus and its penicillin susceptibility by phage amplification techniques in different food matrices

Staphylococcus aureus, a zoonotic pathogen causes diseases in humans and animals, establishing a robust analytical method is crucial to minimize its adverse impacts. Phage amplification assay (PAA) is a method for indirect detection of target bacteria by detecting the progeny phage. In this study, we proposed a phage amplification assay based on Twortvirinae phage LSA2311 with a broad host range to indirectly detect S. aureus and penicillin-resistant S. aureus. PAA-based on the plaque counting could quantitatively detect viable S. aureus by forming progeny phage plaques on the culture medium within 6.5 h, with the detection limit of 10 CFU/mL. Progeny phages produced by PAA could be quantified by qPCR for the detection of viable S. aureus while reducing the detection time by 3 h (detection limit of 10 CFU/mL), and simple DNA extraction (thermolysis) process. It can be successfully applied to quantify viable S. aureus in spiked milk and lettuce. Moreover, this assay could rapidly detect its penicillin susceptibility within 13 h, and the results were in line with the standard paper disc diffusion method (DDM). These phage amplification techniques enable the rapid and accurate detection of viable S. aureus in food matrices, as well as drug-resistant S. aureus.

Integrating microneedle DNA extraction to hand-held microfluidic colorimetric LAMP chip system for meat adulteration detection

Most microfluidic-based “sample-in-result-out” systems suffer sophisticated microfluidic production processes, high-cost chips, and expensive instruments. They cannot be used in the meat market as well as farmer's markets in rural areas. Here, we developed a hand-held microfluidic chip system for on-site meat species qualitative authentication detection which integrated a simple microneedle DNA extraction and a visual loop-mediated isothermal amplification (LAMP). The chip can be used by easily pricking meat samples, simply hand-shaking the chip, and readily available isothermal heating instead of a complicated DNA extraction process and microfluidic control device. The system demonstrates high specificity and sensitivity for selected six species of meat samples and low to 1% simulated adulteration could be detected within 60 min. Besides, the whole cost was less than 1 dollar. The integrated hand-held microfluidic detection system offers a simple, fast, low-cost “sample-in-result-out” point-of-care device which could be extended to medical diagnosis and animal/plant disease identification.

A simplified protocol for DNA extraction from FTA cards for faecal microbiome studies

As metagenomic studies continue to increase in size and complexity, they are often required to incorporate data from geographically isolated locations or longitudinal time samples. This represents a technical challenge, given that many of the commonly used methods used for sample collection, storage, and DNA extraction are sensitive to differences related to the time, storage and chemistry involved. FTA cards have been previously proposed as a simple, reliable and cost-efficient method for the preservation of animal faecal microbiomes. In this study, we report a simplified extraction methodology for recovering microbiome DNA from faeces stored on FTA cards and compare its performance to a common alternative means of characterising such microbiomes; namely, immediate freezing of the faeces followed by DNA extraction using the Qiagen PowerSoil DNA isolation kit. Our results show that overall the application of our simplified DNA extraction methodology yields microbial community results that have higher diversity and an expanded core microbiome than that found using the PowerSoil methodology. This suggests that the FTA card extraction method presented here is a viable alternative for metagenomic studies using faecal material when traditional freeze-based storage methods are not feasible.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Studies on the Rapid and Simple DNA Extraction Method, Antibacterial Activity and Enzyme Activity Involved in Plant Biomass Conversion by Cookeina sulcipes and C. tricholoma (Cup Fungi)

Cookeina sulcipes and C. tricholoma are a cup fungi (Ascomycota) collected in Saraburi, Thailand. The fungi have been isolated, cultured and confirmed as respective species. For morphology, both Cookeina sp. are white mycelium and the growth rate on potato dextrose agar (PDA) result present C. sulcipes is faster than C. tricholoma. The molecular characterization from a rapid and simple DNA extraction method that is modified based on thermolysis method, The DNA extraction is finish in thirty minutes and efficiency to continuous with polymerase chain reaction (PCR) amplification to fungi species level identification. The DNA sequence from internal transcribed spacer (ITS) gene regions by universal primer pairs ITS5/ITS4 is effective to confirm Cookeina species level that C. sulcipes has 616 bp and C. tricholoma has 570 bp. Including, DNA sequence of large subunit (LSU) gene regions by universal primer pairs LROR/LR5 is generate that C. sulcipes has 912 bp and C. tricholoma has 906 bp. The cultures are screened for antibacterial activity by agar plug diffusion method and found that both isolates have been no activity against test strains (Bacillus subtilis, Escherichia coli, Kocuria rhizophila (Micrococcus luteus), Pseudomonas aeruginosa, Staphylococcus aureus and S. epidermidis). In a preliminary screening test of enzymes involved in plant biomass breakdown by agar plate method, both Cookeina sp. show cellulolytic and hemicellulolytic enzymatic activity, and manganese peroxidase (MnP) productivity. In contrast, only C. sulcipes had additional laccase activity. Neither isolate generate pectinolytic and lignin peroxidase (LiP) activities. Thus, Cookeina spp. proved the potentiality to break down lignocelluloses.