Polymerase Chain Reaction Amplification Of DNA Research Articles

New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales)

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Prevalence of viral DNA in high-grade serous epithelial ovarian cancer and correlation with clinical outcomes.

Currently 11 infectious agents are classified as carcinogenic but the role of infectious agents on outcomes of epithelial ovarian cancer is largely unknown. To explore the association between infectious agents and ovarian cancer, we investigated the prevalence of viral DNA in primary ovarian cancer tumors and its association with clinical outcomes. Archived tumors from 98 patients diagnosed with high-grade serous epithelial ovarian cancer were collected between 1/1/1994 and 12/31/2010. After DNA extraction, Luminex technology was utilized to identify polymerase chain reaction-amplified viral DNA for 113 specific viruses. Demographic data and disease characteristics were summarized using descriptive statistics. We used logistic regression and Cox proportional hazards model to assess associations between tumor viral status and disease outcome and between tumor viral presence and overall survival (OS), respectively. Forty-six cases (45.9%) contained at least one virus. Six highly prevalent viruses were associated with clinical outcomes and considered viruses of interest (VOI; Epstein-Barr virus 1, Merkel cell polyomavirus, human herpes virus 6b, and human papillomaviruses 4, 16, and 23). Factors independently associated with OS were presence of VOI (HR 4.11, P = 0.0001) and platinum sensitivity (HR 0.21, P<0.0001). Median OS was significantly decreased when tumors showed VOI versus not having these viruses (22 vs 44 months, P<0.0001). Women <70 year old with VOI in tumors had significantly lower median OS versus age-matched women without VOI (20 vs 57 months, P = 0.0006); however, among women ≥70 years old, there was no difference in OS by tumor virus status. The presence of a VOI was significantly associated with a lower OS. These findings may have implications for clinical management of ovarian cancer but require additional studies.

THE EFFECT OF HELICOBACTER PYLORI ON INTERLEUKIN-6 IN PATIENTS WITH ISCHEMIC HEART DISEASE

Several studies have shown that the presence of Helicobacter pylori (H. pylori) may be a risk factor for Ischemic Heart Diseases (IHD). Therefore, it is needed to look into the relation between IHD and H. pylori infection. One hundred Iraqi patients with IHD and 60 healthy Iraqis as a control group participated in the current study. The ELISA assay was used for the measurement of the sera levels for anti- H. pylori IgG, IgM, and Interleukin-6 (IL-6). Also, H. pylori DNA in the human venous blood was investigated by the polymerase chain reaction (PCR) using 16S rDNA primers. Between IHD patients and the control group, H. pylori-IgG and IL-6 mean ± SD were (Patient =1.814±0.08, Control = 0.823±0.09, Patient = 49.73±6.60, Control = 0.21±0.02, respectively), and this results showed a highly significant variation (p &lt; 0.05) in anti-H. pylori IgG and IL-6, while no significant variations (p &gt; 0.05) were found in anti- H. pylori IgM. It was also found that PCR amplification of Helicobacter pylori DNA using 16S rDNA primers was positive in the venous blood of 81 (81%) IHD patients. The current study results proposed that there was a relation between H. pylori and Ischemic Heart Disease.

Isolation and some basic characteristics of lactic acid bacteria from beetroot (Beta vulgaris L.) —A preliminary study

The carbohydrate content of fruit and vegetable juices has made them a suitable environment for the growth of lactic acid bacteria. Various studies have shown that beetroot can be a good substrate for the production of probiotic drinks for consumers. Therefore, this study was performed to isolate and identify lactic acid bacteria from beetroot juice. Beetroot juice was prepared from two kilograms of washed beetroot with a cold press. Sterile samples of beetroot juice were first enriched in MRS broth and then cultured on MRS agar. Gram and catalase tests were used for the initial detection of lactic acid bacteria. Then, to identify the species of lactic acid bacteria, biochemical confirmation tests such as the melting of gelatin, and the growth of bacteria has been studied in the ideal condition that is 15-45° C, sugars fermentation, gas production from glucose, and growth in at different concentrations of salt were performed. The final verification of species was done using polymerase chain reaction-amplified 16S rRNA-specific DNA. All isolates formed creamy white round colonies on MRS agar medium, were gram-positive, were able to grow in anaerobic conditions, and were also rod-shaped and catalase-negative. Based on biochemical tests, four types of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus salivarius, and Lactobacillus acidophilus) were identified. The findings of this study indicated the presence and diversity of lactic acid bacteria in beet juice, which can be a potential platform for the production of probiotic beetroot juice as a non-dairy product.

Optimized method for dna extraction and PCR amplification in aroeira tree

Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of β-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

P455 Identification of Cryptic species of Aspergillus using Beta-tubulin gene in a tertiary care center in South India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesTo identify cryptic species of the genus Aspergillus using Beta-tubulin gene by sequence typing.Methods Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud's Dextrose Agar/Oatmeal Agar.Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape, and arrangement of the conidia.DNA Extraction using phenol-chloroform method: DNA was extracted and purified directly from 4-day-old culture and used as a template for polymerase chain reaction (PCR) amplification.DNA Amplification: A fragment of the target gene were amplified using PCR.The primer pairs used for Beta-tubulin gene was Bt2a (Forward primer) and Bt2b (Reverse primer).Beta-tubulin.Bt2a FGGTAACCAAATCGGTGCTGCTTTCBt2b RACCCTCAGTGTAGTGACCCTTGGCDNA Purification: The PCR product was purified with multi-screen filter plates.Gel electrophoresis: The amplified DNA product was subjected to agarose gel electrophoresis and specific band formation was to be observed for the species of Aspergillus.DNA Sequencing: The purified product will be used as a template for sequencing. An applied biosystem 3730 sequencer will be used to obtain DNA sequences.Phylogenetic Analysis: Parsimony analysis of individual and combined matrices will be conducted using PAUP version 4.0b10 software/free software at phylogeny.fr by the maximum parsimony (MP) and Neighbor-joining methods.The results will be represented using a dendrogram.ResultsA total of 30 Aspergillus isolates were collected and identified using Phenotypic methods.The DNA extraction and PCR amplification were done and sent for Sequencing. The results of Sequence typing are awaited.ConclusionCryptic species are morphologically indistinguishable forms of Aspergillus and their identifications can be confirmed exclusively by using molecular techniques which have led to the description of previously unknown or rare species among different Aspergillus species complex. According to studies, the frequency of such species was found to be 10% in TRANSNET study U.S and 12% in FILPOP study Spain. Most of these species are less susceptible and certain species are multidrug-resistant (azoles and amphotericin B) and the emergence of antifungal-resistance among the Aspergillus species forms the importance in identifying Cryptic species.

P219 Fungal keratitis caused by Pseudallescheria boydii: Clinical and mycological characteristics

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Pseudallescheria boydii keratitis is rare but important type of fungal keratitis because of the inherently resistance of the organism to many existing antifungal agents. We present the clinical characteristics, risk factors, treatment, and prognosis of patients with P. boydii keratitis, and also present the antifungal sensitivities of the isolated strain.MethodsSlit-lamp and confocal microscopy were used for clinical examinations. Fungal isolates were identified based on morphological characteristics and DNA sequence of the internal transcribed spacer region (ITS). In vitro antifungal susceptibility testing for fungal isolates was performed according to the Clinical and Laboratory Standards Institute (CLSI).Results Pseudallescheria boydii was identified in four patients. All patients had a history of ocular trauma. In clinical examination hypopyon was seen in three patients. The main antifungal medications were oral and topical voriconazole. After treatment the visual activity of all patients improved in 2-3 weeks.ConclusionThese patients bold the importance of determining causative organism of fungal keratitis and their antibiotic susceptibility. Culture findings are limited in identifying organisms. Sequencing of polymerase chain reaction-amplified DNA is good for accurate and rapid identification of species that can be helpful for optimizing treatment.

(Invited) DNA Sequencing with Carbon Nanotube Electronics

Nanoscale materials and devices provide new opportunities to monitor and understand biomolecules. This presentation will review novel devices in which single molecules of DNA polymerase have been joined to single-walled carbon nanotube (SWNT) transistors.DNA polymerases are the enzymes that convert single-stranded DNA into double-stranded helices. This biochemical process is the primary step in DNA replication, amplification, and most sequencing technologies. When one of these polymerases is attached to a SWNT transistor, the enzyme’s native activity is simultaneously transduced into a high-resolution electrical signal [1]. Each time the enzyme works its way down a DNA strand, it generates a nucleotide-by-nucleotide signal that encodes the DNA sequence.This hybrid, biofunctional device is the key element for developing a new, electronic DNA sequencing technology. As a solid-state, manufacturable element, the SWNT transistors provide exciting possibilities for high-density, high-throughput, and low cost sequencing solutions. In addition, the technique is easily generalized to DNA polymerases with specialized properties. Proof-of-principle measurements have been accomplished with DNA polymerases from five different organisms, including the highly processive polymerase of φ29 phage and the high-temperature polymerase Thermus aquaticus (Taq), which is the foundation of the polymerase chain reaction (PCR) and commercial DNA amplification. The SWNT transistor technique operates equally well at room temperature and the elevated PCR temperature of 72 °C, which opens opportunities for novel, high-temperature sequencing strategies with Taq or similar thermostable polymerases.[1] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

A Genotype-Phenotype Study of Multiple Hereditary Exostoses in Forty-Three Patients.

Multiple hereditary exostoses (MHE) is a rare autosomal dominant skeletal disorder with a variety of clinical manifestations. We aimed to evaluate the general clinical phenotypic severity of MHE using our own scoring system and analyzed the risk factors associated with severe clinical phenotypes. In this study, 43 patients from 30 families were analyzed. The mutations were identified by direct sequencing of polymerase chain reaction-amplified genomic DNA or by multiplex ligation-dependent probe amplification. According to a new scoring system devised by the authors, the severity of the phenotype was assessed as mild, moderate, or severe based on the deformity of each segment, number of exostoses, leg length discrepancy, and functional limitations. Of 43 patients from 30 families, 39 patients (90.7%) and 24 families (80%) presented with EXT1 or EXT2 mutations. Patients with EXT1 mutations had a significantly worse phenotype than that of patients with EXT2 mutations or without any detectable mutation. The mean clinical score of patients with an EXT1 mutation (5.76; range, 2.0–8.0; SD = 1.60) was higher than that of patients with an EXT2 mutation (4.06; range, 2.0–7.0; SD = 1.47) or of those without any detectable mutation (4.63; range, 3.0–6.0; SD = 1.44; p = 0.005). According to our classification system, more patients with EXT1 mutations had ‘severe disease’ than those with EXT2 mutations. Deformity scores were also higher in patients with EXT1 mutations (p = 0.018). In the multivariate analysis, the deformity score was found to be associated with the ‘severe’ class (p = 0.031). In conclusion, 90.7% of patients with MHE showed EXT mutations. Our scoring system showed reliable results. We suggest that the extent of deformity is an important factor in determining the phenotype of MHE and close monitoring for the development of severe disease is recommended in patients with high deformity scores.

Molecular Analysis of HBV in Histologically Confirmed Hepatocellular Carcinoma in a Tertiary Hospital in Ghana.

Hepatocellular carcinoma (HCC) is a leading cause of death in Africa. Viral hepatitis B is a leading cause of hepatocellular cancer in Ghana and most African countries except Egypt where hepatitis C virus is more prevalent. This study aims at reviewing the histopathological patterns of HCC and its association with hepatitis B virus in our environment. Demographics and histological diagnosis were retrieved from the surgical daybook and archival FFPE tissue samples with histopathologically confirmed HCC were used for this study. Sections (10μm) were taken from the tissues and digested to obtain DNA lysates. The DNA lysates were used in polymerase chain reaction (PCR) to determine the prevalence of HBV in the biopsies. Of the 24 confirmed cases of HCC seen in the 5-year period, there were 17 males and 7 females with M:F ratio of 2.4:1. The mean age of our patients was 39.92 ± 1.98 years with age range 13-85 years. 50% of the cases were moderately differentiated while 25% each were well and poorly differentiated. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 (45.83%) after the restriction fragment length polymorphism PCR reaction. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 (45.83%) after the restriction fragment length polymorphism PCR reaction. Eight of the 11 cases were found in the male and 3 in females. Of the 11 (45.83%) samples that were positive for HBV DNA, 3 were above 40 years and 8 were 40 years and younger. The overall prevalence of HBV DNA in our study was 45.83% and a greater proportion seen in ≤ 40 years. This suggests that most of our patients are infected with HBV early in life in our environment.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

BARKODE DNA DAN KEKERABATAN IKAN LAIS TIMAH (Kryptopterus apogon) BERDASARKAN GEN SITOKROM C OXIDASE SUBUNIT I (SOI)

Lais (Krypyoterus apogon) is highly economic important catfish in South Sumatera. Investigating fish diversity of catfish is of importance for species conservation. Many species or subspecies extincted due to environment decreasing caused by habitat loss, pollution and over fishing. Cytochrome C Oxidase subunit I (COI) is one of mitochondrial DNA markers used for species barcoding in freshwater, brackishwater and marine fish. This research aim to explore the use of COI gene for species barcoding in investigating the diversity of COI nucleotide, constructing phylogenetic tree of lais, and knowing physical and chemistry characteristic of habitat at Musi and Kelekar River, South Sumatera. The methods used in the research consisted of DNA extraction, PCR (Polymerase Chain Reaction) amplification and sequencing mtDNA COI gene of Kryptopterus apogon obtained from Musi and Kelekar River (South Sumatera). A 615 bp (sample code CM1) and 693 bp sequences (sample code CK1 and CK2) of parsial coding were sequenced from this fish. Nucleotide BLAST analyzes showed COI gen can distinguish lais at Musi and Kelekar River from others fish at GenBank database with high identity (92.29% and 92.21%) to Phalacronotus bleekeri (Thailand) and Phalacronotus apogon (Australia).Phylogenetic analyzes indicated that obviously, lais was at the same cluster with others genus in Siluridae family, but different cluster from Hemibagrus nemurus (Bagridae), Chanidae and Oreochomis niloticus (Cichlidae). Water quality criteria (water tranparancy, temperature, ammonia, pH and alkalinity) at Musi and Kelekar River were still in tolerance for maintainning survival rate of lais. Further study using more species in Siluridae family and various habitat are needed to investigate the diversity of DNA of Siluridae at South Sumatera water resources.Keywords: Catfish, DNA barcode, COI gene, Musi River, Kelekar River

DNA extraction and pattern of crab and macrobentos from North Sumatran mangrove forest, Indonesia

Mangrove forest ecosystem is one of the most productive and unique ecosystems that serves to protect coastal areas from various disturbances, as well as provide habitat for various animal species. The large number of crab species and macrobentos in mangrove ecosystems results in frequent errors in the naming of species that have similarities in morphological features. This problem can be solved through a comprehensive approach by combining morphological analysis with genetic analysi.This study aims to report a DNA extraction and PCR amplification prior was used for the identification of crab and macrozoobentos from mangrove forest, North Sumatra. Primer 16S has a conserved area so it is appropriately used in Polymerase Chain Reaction (PCR) and sequencing analysis to determine taxonomy, phylogeny and diversity between specie. Visualization of PCR amplification results with primer16S from crab samples and macrobentos resulting a low DNA band with a size of 206 bp and a high of 678bp

Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study

The present study aimed to carry out DNA extraction and polymerase chain reaction (PCR) amplification to isolate the physiological and phenotypic traits of Candida albicans and Candida dubliniensis in denture wearer patients with and without denture-induced stomatitis. A total sample size of 160 participants were divided into two equal groups (80 each), patients in the study group having 40 males and 40 females with Newton type II denture stomatitis, and in the control group, healthy 40 males and 40 females those who wear complete denture were selected. All the samples were collected from the hard palate with a sterile swab and inoculated on CHROM agar plate; samples that displayed dark green colored colonies were selected for DNA extraction. DNA isolation was done on agarose gel using electrophoresis. Biorad gene identification was used. Strands depicting the presence of DNA in particular samples were identified, and further standardization of the procedure was done. PCR amplification was done using Candida species-specific primer, preset to the hyphal wall of the protein 1 gene with the CRR forward and reverse primers, under strict standard conditions with reverse transcriptase technique. Results showed that prevalence of C. albicans was more in females with denture stomatitis which was 67.50% than in males, i.e., 52.50%, and prevalence of C. dubliniensis was found in one female and in one male who were having denture stomatitis and it was not isolated from patients without denture stomatitis. Statistical analyses were performed using the Chi-square test. Denture stomatitis is the most common problem faced by long-term denture wearers, with C. albicans as one of the causative organisms. However, recent findings show an emerging pathogenic yeast species, C. dubliniensis, which was isolated from denture-induced stomatitis candidates in the present study, which is closely related to the C. albicans species. The identification of candidal strains causing denture stomatitis with DNA extraction and PCR amplification and its management by determination of its susceptibility to antifungals may improve the treatment outcome of the same. Candidiasis is the most frequently seen mucocutaneous infection of the oral cavity especially in denture wearers. It is caused mainly by the genus Candida. C. dubliniensis is phenotypically similar but genotypically different from C. albicans. This affects the treatment outcome drastically as there is enough literature suggesting resistance to the common antifungal drugs. Hence, drugs like fungus-specific calcineurin inhibitors should also be considered in resistant patients. Therefore, DNA identification of Candida genus plays a major role in deciding the treatment outcome. How to cite this article: Patil A, Boparai NK, Shankargouda SB, et al. Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study. J Contemp Dent Pract 2021;22(5):517-521.

Detection of toxoplasmosis in aborted women in Alexandria, Egypt using ELISA and PCR.

Toxoplasmosis is a worldwide infection that can be acquired through the ingestion of tissue cysts in poorly cooked meat, and/or oblivious intake of sporulated oocysts in cat faeces, and transplacental. The infection in pregnant women is mainly asymptomatic. It produces abortion or congenital infection. The present study aimed to test the utility of polymerase chain reaction (PCR) on placental tissues in comparison to enzyme-linked immunosorbent assay (ELISA) to detect infections with Toxoplasma gondii in aborted women presented to Al-Shatby Maternity Hospital, Alexandria University, Egypt. Specific Toxoplasma gondii IgG and IgM were detected in serum by ELISA. Placental tissues from each participant were subjected to DNA extraction and PCR amplification. It was found that overall seroprevalence was 73%, DNA was detected in placenta tissues by using PCR analysis in 46% of cases. {× 2 (p) 18.124(< 0.001)}. Toxoplasma IgG/IgM by ELISA was positive in 23% of the cases, 20% showed amplified DNA by PCR. Positive IgG without IgM was seen in 27% cases, only 2% of them were positive by PCR. Moreover, positive PCR among positive ELISA IgM aborted women was 21 of the 23 cases. Positive PCR was obtained in three seronegative women. Our results showed that PCR sensitivity was 58.90 specificity 88.89, positive predictive value was 93.48%, and negative predictive value 44.44%. Although ELISA assay is still the gold standard of diagnosis of Toxoplasmosis, other diagnostic modalities are highly required particularly in those ELISA seronegative cases. PCR can be used as a sensitive and precise modality.

First Record of Fungi Isolated from Thrips tabaci Lindeman on Garlic (Ilocos White) in Batac, Philippines

The study aimed to isolate, and identify fungi associated with live adult female Thrips tabaci Lindeman (Thysanoptera: Thripidae) from garlic (Ilocos White) in Tubug Experimental Area of Mariano Marcos State University in Batac, Ilocos Norte, Philippines. Thrips were collected randomly from garlic (Ilocos White) plants within 200 m2 area in one garlic field of about 400 m2 using the tapping/beating method. Thrips were sorted and examined under the microscope. For the isolation of fungi, 20 live adult female thrips were selected from the collection consisting of females and larvae. Standard isolation of fungi from thrips was conducted and pure cultures of fungal isolates were maintained on PDA (potato dextrose agar) slants. Fungal isolates were processed for DNA extraction, PCR (polymerase chain reaction) amplification, and gel electrophoresis. Forward and reverse reads were paired to generate a consensus sequence, which was used in the Basic Local Alignment Search Tool (BLAST) queries to determine taxonomic identity. Out of the 10 fungal isolates, nine were identified as Fusarium proliferatum while one is Aspergillus candidus based on ITS (internal transcribed spacer) gene sequence. They both belong to Class Ascomycota. F. proliferatum on PDA shows varying characteristics of its mycelial growth from being scanty to fluffy, off-white to light yellow, brown, or purple while A. candidatus exhibits the entire margin and bluish-green pigmentation with a fertile layer composed of catenate conidia formed from the vesicle of the conidiophore. F. proliferatum was dominant among the isolates. The manner of isolating them from Thrips tabaci proved that they are endophytes living within them, and these are new records in the Philippines.

Molecular Characterization and Pathogenicity of an Indian Isolate of Duck Enteritis Virus Recovered From a Natural Outbreak

Background: Duck plague is a highly contagious viral disease reported in our country very often with significant economic loss. There are some bottlenecks with the currently used ‘Holland strain’ vaccine that involves cumbersome process of vaccine production in embryonated chicken eggs. With the future goal of development of an indigenous cell culture vaccine for duck plague, the present study is aimed at isolation of an Indian strain of DEV from a natural outbreak and its characterization for the seed virus purpose. Methods: Liver samples were collected from the suspected ducks died during a natural outbreak in Kerala and subjected to polymerase chain reaction (PCR) to confirm presence of viral DNA. The duck enteritis virus (DEV) was isolated by inoculation of PCR positive samples in embryonated duck eggs/ducklings and its pathogenicity was studied. Further, the DEV recovered from the infected duck embryo and duckling liver was confirmed by PCR amplification of the viral DNA polymerase gene and its sequence analysis. Result: Out of 12 liver samples tested eight (8) were found to be positive for duck plague by PCR. The DEV infected duck embryos and ducklings died showing typical signs and characteristic gross and microscopic lesions. PCR amplification of viral DNA targeting the DNA polymerase gene yielded amplicon of expected size of 446bp. The amplicon sequence showed 99-100% homology with other DEV isolates, thus confirming the new isolate as DEV, named as DEV/India/IVRI-2016 and the gene sequence has NCBI acc. no. KX511893.

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Battery-Powered Portable Rotary Real-Time Fluorescent qPCR with Low Energy Consumption, Low Cost, and High Throughput.

The traditional qPCR instrument is bulky, expensive, and inconvenient to carry, so we report a portable rotary real-time fluorescent PCR (polymerase chain reaction) that completes the PCR amplification of DNA in the field, and the reaction can be observed in real-time. Through the analysis of a target gene, namely pGEM-3Zf (+), the gradient amplification and melting curves are compared to commercial devices. The results confirm the stability of our device. This is the first use of a mechanical rotary structure to achieve gradient amplification curves and melting curves comparable to commercial instruments. The average power consumption of our system is about 7.6 W, which is the lowest energy consumption for real-time fluorescence quantification in shunting PCR and enables the use of our device in the field thanks to its self-contained power supply based on a lithium battery. In addition, all of the equipment costs only about 710 dollars, which is far lower than the cost of a commercial PCR instrument because the control system through mechanical displacement replaces the traditional TEC (thermoelectric cooler) temperature control. Moreover, the equipment has a low technical barrier, which can suit the needs of non-professional settings, with strong repeatability.

Isolation, Identification and Antimicrobial Evaluation of Bactericides Secreting Bacillus subtilis Natto as a Biocontrol Agent

Herein, a bactericide-secreting Bacillus strain, potentially useful as a biocontrol agent, was isolated from the commercial Yanjing Natto food. Following the biochemical and physiological evaluation, the molecular identification was performed using 16S rDNA sequencing of polymerase chain reaction-amplified DNA that confirmed the natto isolate as Bacillus subtilis natto (B. subtilis natto). The biocontrol (microbial inhibitory) capability of B. subtilis natto was investigated against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and yeast (Yarrowia lipolytica) and recorded. The antimicrobial activity of B. subtilis natto was further enhanced by optimizing the growth medium for optimal bactericides secretion. Under optimized conditions, B. subtilis natto exhibited much higher inhibitory activity against S. aureus with a zone of inhibition diameter up to 27 mm. After 48 h incubation, the optimally yielded B. subtilis natto broth was used to extract and purify the responsible bactericides by silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. Structural identification of purified bactericides (designated as NT-5, NT-6, and NT-7) from B. subtilis natto was performed by 13C-nuclear magnetic resonance (NMR) and mass spectral analyses. The NMR comparison also revealed that NT-5, NT-6, and NT-7 had identical structures, except for the fatty chain. In summary, the present study suggests the improved biocontrol and/or microbial inhibitory potential of newly isolated bactericides secreting B. subtilis natto.