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BackgroundEnvironmental water samples are increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and next generation sequencing (NGS) offer a potentially inclusive surveillance platform for a wide range of AMR genes. However, molecular methods are dependent upon the extraction of DNA of high yield and quality. Current options for DNA extraction from complex environmental matrices for downstream molecular applications are either expensive or low yielding. We present here a novel magnetic bead-based DNA extraction method, for the detection of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract. MethodsMagnaExtract involves initial filtration of 250ml freshwater, followed by an overnight incubation of the filter in 15ml buffered peptone water (BPW), common procedure in microbiology laboratories. 200{micro}l is then taken for a boil (95{degrees}C) and spin step and mixed with magnetic beads to bind DNA. Following washes with ethanol, the DNA is eluted in nuclease-free water. To determine the effectiveness of this method, 98 freshwater samples were collected from two rivers in Southern Malawi, and DNA was isolated using the MagnaExtract method, two commercial Qiagen (Germany) kits; PowerWater and DNeasy Blood and tissue, alongside a boil and spin of BPW, and a boil and spin from bacterial isolate grown on agar media. All samples were screened with a high-resolution melt (HRM) PCR panel previously validated for the detection of third generation cephalosporin and carbapenem ARGs. We compared the DNA yield obtained using all extraction methods, as well as the identification of each ARG. ResultsDNA yield using MagnaExtract was statistically greater than both boil and spin methods and DNeasy Blood & Tissue (Qiagen, Germany). DNA yield was slightly lower than using PowerWater (Qiagen) but the difference was not statistically significant. MagnaExtract was the only method to identify ARGs in all 98 water samples compared with PowerWater (n=82), DNeasy (n=95) boilate of BPW (n=75) and boilate of bacterial isolate (n=87). The most commonly detected ARG was OXA-48 (n=93). In addition, we found overnight incubation in non-selective enrichment broth (BPW) to promote the growth of bacteria harbouring extended spectrum beta lactamase (ESBL) genes and reduction in the detection of carbapenemase genes. ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding DNA extraction method for the detection of ARGs isolated from river water samples.

Iridoviruses are DNA virus and have caused huge economic losses in the aquaculture industry. The aim of this study was to establish a colorimetric loop-mediated isothermal amplification (LAMP) protocol for the on-site detection of Singapore grouper iridovirus (SGIV). The SGIV-VP61 gene was chosen as the target gene to develop a colorimetric LAMP assay. The optimized condition of the colorimetric LAMP assay was incubation at 63 °C for 1 h. Samples infected with SGIV could be detected with the color change from yellow into pink. The sensitivity of the developed assay is 5.66 copies/μL of the viral DNA template. This sensitivity was about 1000 times higher than that of conventional PCR while it was slightly lower than the one-step semi-nested PCR assay. A total of 60 DNA samples extracted from the fin tissue of the SGIV-infected Asian seabass were examined for SGIV by colorimetric LAMP, semi-nested PCR and conventional PCR. The results of the colorimetric LAMP assay showed 94.87 % agreement with the semi-nested PCR. In addition, the DNA extraction method using NaOH showed a better performance in the colorimetric LAMP assay. Taken together, the colorimetric LAMP established was a sensitive, rapid and specific method for the detection of SGIV. SGIV was not detected in samples randomly taken from a genetically improved line of the Asian seabass. However, some seabass obtained from the local markets were found to contain SGIV. Thus, the LAMP assay has the potential application in the diagnosis of iridovirus diseases in the aquaculture industry.

Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 105 to 2.7 × 102 cfu/mL, with a limit of detection of 2.7 × 102 cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.

Backgrounds PCR is a proper technique that significantly improves toxoplasmosis diagnosis. However, a more sensitive technique is required. This study compared real-time PCR with nested PCR using B1, SAG-4, and MAG-1 bradyzoite genes to diagnose toxoplasmosis in toxoplasmic retinochoroiditis patients. Methods Blood samples were collected from 10 patients with active toxoplasmic chorioretinal lesions and 10 healthy individuals. Blood samples including peripheral blood mononuclear cells (PBMCs), serum and whole blood samples were used for DNA extraction. Serum was also used to detect anti-toxoplasma IgG and IgM antibodies. Nested PCR and real-time PCR were performed using B1, SAG-4, and MAG-1 target genes. Results Five (50%) out of the 10 patients were tested positive for toxoplasmosis with nested PCR using the PBMC samples. All the five patients tested positive with nested PCR were also tested positive for toxoplasmosis with real-time PCR using the PBMC samples. The real-time PCR results demonstrated that 9(90%) out of the 10 patients were positive based on B1 and the remaining one (10%) was positive only based on MAG-1. In general, of the patients, five (50%) were positive using SAG-4 and three (30%) were positive in term of MAG-1 using PBMCs with real-time PCR. Conclusion It appears that PBMC samples have the best performance as the PCR extraction method and are a good source for toxoplasmosis diagnosis. The use of B22 and B23 target genes due to their high sensitivity and specificity along with bradyzoite genes are recommended for toxoplasmosis diagnosis using PBMC samples with real-time PCR.

Extraction of high quantity and quality DNAs from marine sponges, which contain diverse and abundant microbial communities, is important to molecular biology techniques for the analysis of nucleic acids. Several marine sponges and their associated microorganisms have been known to produce cytotoxic natural products on several cancer cell lines via DNA damage mechanisms. These marine cytotoxic substances might be one of the factors that cause the low quantity and quality of DNAs during the DNA extraction from its living origin. Therefore, the extraction of DNA of a Thai blue marine sponge Xestospongia sp. with sufficient purity and quantity for molecular study can be challenging. In this study, we developed an efficient extraction method to prepare DNAs from a Thai blue marine sponge Xestospongia sp. which accumulated a highly potent cytotoxic alkaloid with DNA-damaging activity, named Renieramycin M (RM), as a major constituent in high quantity. We demonstrated that removal of RM from the sponge samples by a simple methanolic extraction before DNA extraction dramatically increased the yield and purity of DNAs compared to the RM-unremoved sponge samples. High molecular weight (HMW) genomic DNA was obtained from sponge samples with 8 times of RM elimination by using modified NaOAc salting-out extraction method. The quantity and quality of the prepared DNAs were comparatively determined via spectrophotometry, electrophoresis, and 16S rRNA gene amplification. Our result suggests that the removal of DNA-damaging constituents from the samples is a crucial step and must be seriously taken as the necessary consideration for the practical protocol of DNA extraction.

Determining the diet of flying-foxes can increase understanding of how they function as pollinators and seed dispersers, as well as managing any negative impacts of large roosts. Traditional methods for diet analysis are time consuming, and not feasible to conduct for hundreds of animals. In this study, we optimised a method for diet analysis, based on DNA metabarcoding of environmental DNA (eDNA) from pollen and other plant parts in the faeces. We found that existing eDNA metabarcoding protocols are suitable, with the most useful results being obtained using a commercial food DNA extraction kit, and sequencing 350–450 base pairs of a DNA barcode from the internally transcribed spacer region (ITS2), with ~550 base pairs of the chloroplast rubisco large subunit (rbcL) as a secondary DNA barcode. A list of forage plants was generated for the little red flying-fox (Pteropus scapulatus), the black flying-fox (Pteropus alecto) and the spectacled flying-fox (Pteropus conspicillatus) from our collection sites across Queensland. The diets were determined to comprise predominantly Myrtaceae species, particularly those in the genera Eucalyptus, Melaleuca and Corymbia. With more plant genomes becoming publicly available in the future, there are likely to be further applications of eDNA methods in understanding the role of flying-foxes as pollinators and seed dispersers.

Purpose Morphological and molecular description of a new species of Haematoloechus dehradunensis sp. nov. collected from the lungs of Euphlyctis cyanophlyctis (Schneider 1799) from Dehradun (Uttarakhand), India and reporting first record of H. singaporensis from India. Methods Digeneans were fixed in AFA (alcohol-formalin-acetic acid), stained with Borax's carmine, studied and photomicrographed with a BX53 DIC/BF Olympus research microscope. Molecular studies were done by DNA isolation using Qiagen, DNeasy® Blood and Tissue Kit and PCR amplification using r-DNA ITS-1 marker situated between 18S and 1.58S gene. Results and conclusion The new species is differentiated from other known species of Haematoloechus in having larger oral sucker, kidney-shaped ovary and oval-lobed testes. H. singaporensis collected from E. cyanophlyctis represents a first record for India and a new host record. ITS sequences submitted and compared at NCBI GenBank support the uniqueness of the new species.

Toxoplasma gondii is a parasite of significant public health importance. We attempted to detect T. gondii contamination and assess advantages and disadvantages of contamination indicators through surveilling soil, wildlife, cats (Felis catus), and cows (Bos taurus) on a farm in Tennessee, U.S. in 2016 and 2017. Twenty-two soil samples were collected from the farm and subjected to oocyst flotation, DNA extraction, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting 18S ribosomal RNA (18S rRNA) gene to detect and identify T. gondii. Three samples (13.6%) were positive for the parasite; however, T. gondii DNA was not consistently detected from repeated tests. Blood samples were collected from small mammals, cats, and mesopredators live-trapped on the farm, and serum from 30 of the farm's cows were obtained. Serological testing by the modified agglutination test (MAT; cutoff 1:50) found 2.5% (1/40) of small mammals, 52.9% (9/17) of raccoons (Procyon lotor), and 50% (1/2) of domestic cats were seropositive for T. gondii antibodies. No antibodies were found in 16 opossums (Didelphis virginiana), two skunks (Mephitis mephitis), and 30 cows. Small mammal tissue samples were subjected to PCR-RFLP detection. Four out of 29 (13.7%) tissue samples were positive for T. gondii; however, T. gondii DNA was not consistently detected during repeated PCR-RFLP testing. Our results indicate the ability to detect T. gondii varies greatly by contamination indicator. We found detection of soil oocysts to be challenging, and results suggest limited utility of the method performed. The ability to detect T. gondii in animals was highly variable among species. Our research emphasizes the importance of a holistic approach when surveilling for T. gondii to compensate for shortcomings of each contamination indicator. Future research should be conducted to further investigate the most effective T. gondii surveillance methods and species with increased sample sizes at other agricultural facilities., Graphical abstract Image 1, Highlights • We investigated T. gondii contamination at a farm and the efficacy of T. gondii detection indicators through examining soil, small mammals, mesopredators, domestic cats, and agricultural animals • Soil oocyst detection was low suggesting limited utility of the method performed in the study region • Small rodents and agricultural animals had low detection of T. gondii • Mesopredators (i.e., raccoons and domestic cats) had the greatest affinity for T. gondii detection out of all tested animals and fomites • Multiple indicators of potential contamination should be examined when trying to detect T. gondii

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens’ mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Bovine Trichomoniasis (BT) is an infectious disease caused by Tritrichomonas foetus that can be transmitted either sexually or by fomites. In males, the disease is asymptomatic and permanent. T. foetus has been detected in semen samples where it is able to remain viable even when frozen. The objective of this study was to investigate the presence of T. foetus in 27 samples of commercial frozen bovine semen by culture and Polymerase Chain Reaction (PCR). Samples were thawed in water at 37°C. Part of the samples was inoculated in a test tube containing Diamond’s medium and incubated at 35°C. Growth was evaluated every 24 hours via direct examination under a microscope. The other part was placed in an Eppendorf tube and frozen for later molecular analysis. After 10 days of culture, all samples were negative for T. foetus. The Quick-DNA Miniprep Kit (Zymo Research) without proteinase K was used for DNA extraction. The primers used in PCR were TRF3 and TRF4. PCR results were negative for all samples. In conclusion, bovine semen samples were negative for T. foetus in both diagnostic methods, according to the adopted methodology. RESUMO A tricomonose genital bovina (TGB), uma doença infectocontagiosa causada pelo Tritrichomonas foetus, é transmitida por via venérea e fômites contaminados. Em machos a doença é assintomática e permanente. O agente já foi encontrado em amostras de sêmen e é capaz de permanecer viável quando congelado. Este trabalho teve por objetivo investigar a presença de T. foetus em 27 amostras de sêmen bovino comercial congelado, por meio de cultivo e reação em cadeia da polimerase (PCR). As amostras foram descongeladas em água a 37ºC; parte foi inoculada em tubo de ensaio contendo meio Diamond, incubada a 35ºC com consequente avaliação de crescimento e avaliada a cada 24 horas, via exame direto em microscópio, e a outra parte foi diluída em PBS para análise molecular. Após 10 dias de cultivo, todas as amostras foram negativas. Para a detecção molecular foi utilizado o kit Quick-DNA Miniprep (Zymo Research) sem proteinase K para extração do DNA. Os iniciadores utilizados na PCR foram TRF3 e TRF4. O resultado da PCR foi negativo para todas as amostras. Conclui-se que as amostras utilizadas foram realmente negativas para a presença do patógeno em ambos os métodos diagnósticos, o que comprovou a inocuidade do sêmen testado.

DNA metabarcoding is an emerging tool used to quantify diet in environments and consumer groups where traditional approaches are unviable, including small‐bodied invertebrate taxa. However, metabarcoding of small taxa often requires DNA extraction from full body parts (without dissection), and it is unclear whether surface contamination from body parts alters presumed diet presence or diversity.We examined four different measures of diet (presence, rarefied read abundance, richness, and species composition) for a terrestrial invertebrate consumer (the spider Heteropoda venatoria) both collected in its natural environment and fed an offered diet item in contained feeding trials using DNA metabarcoding of full body parts (opisthosomas). We compared diet from consumer individuals surface sterilized to remove contaminants in 10% commercial bleach solution followed by deionized water with a set of unsterilized individuals.We found that surface sterilization did not significantly alter any measure of diet for consumers in either a natural environment or feeding trials. The best‐fitting model predicting diet detection in feeding trial consumers included surface sterilization, but this term was not statistically significant (β = −2.3, p‐value = .07).Our results suggest that surface contamination does not seem to be a significant concern in this DNA diet metabarcoding study for consumers in either a natural terrestrial environment or feeding trials. As the field of diet DNA metabarcoding continues to progress into new environmental contexts with various molecular approaches, we suggest ongoing context‐specific consideration of the possibility of surface contamination., In this study, we present results of a study examining the effects of a surface sterilization treatment of invertebrate consumers prior to diet DNA metabarcoding on various metrics of diet. We find that no measures of diet (detection, abundance, richness, or composition) are altered by surface sterilization treatments, suggesting a validation of the use of DNA metabarcoding of small‐bodied consumers to identify diet interactions.

Objective This study aimed to detect the presence of Wolbachia and Spiroplasma endosymbionts in Musca flies through molecular approaches. Methods In total, 40 Musca spp. (20 female and 20 male) were used. Before DNA extraction, the flies were dissected and their heads, wings and legs were detached from their bodies under a stereomicroscope. Genomic DNA was analysed by standard polymerase chain reaction (PCR) using primers against Musca beta-tubulin. Afterward, the samples were examined for the presence of Wolbachia spp. using primers against Wolbachia wsp and GroEL. Furthermore, the DNA samples were analysed by PCR to detect the presence of Spiroplasma using primers against the 16S rRNA. Results No Wolbachia positivity was detected in Musca flies, as shown by the negative PCR results for wsp and GroEL. Spiroplasma positivity was detected in 5% (1/20) of the female Musca flies but not in the male flies (0/20). Conclusion Wolbachia spp. were not detected in Musca flies. Of the total Musca flies, only one was positive for Spiroplasma spp. To our knowledge, this is the first study to detect the presence of Spiroplasma in Musca flies.

Foodborne illnesses are a major threat to public health also leading to significant mortality and financial and reputational damage to industry. It is very important to detect pathogen presence in food products early, rapidly, and accurately to avoid potential outbreaks and economic loss. However, "gold standard" culture methods, including enrichment of pathogens, can take up to several days. Moreover, the food matrix often interferes with nucleic acid amplification methods of detection, requiring DNA extraction from the sample for successful molecular detection of pathogens. Here, we introduce a "biphasic" amplification method that can achieve high sensitivity detection with background noise from ground beef food samples without culture or other extraction methods in 2.5 h. Homogenized ground beef is dried resulting in an increase in porosity of the dried food matrix to allowing amplification enzymes and primers to access the target DNA and initiate the reaction within the dried food matrix. Using Loop Mediated Isothermal Amplification, we demonstrate the detection of 1-3 cfu of Escherichia coli bacteria in 30 mg of dried food matrix. Our approach significantly lowers the time to result to less than a few hours and have a pronounced impact on reduction of instrumentation complexity and costs.

Objective Klebsiella pneumoniae is one of most opportunistic pathogens that can be related to nosocomial infections. Increased acquisitions of multidrug resistance in this bacterium as well as the transfer of genes to other strains have caused concern. Integrons play key role in the acquisition and the spread of resistance genes. The aim of this study was evaluated the frequency of resistance genes sulI, sulII, tetA, tetB, class I (intI gene), class II integrons (intII gene) and the association between multidrug resistance and the presence of integrons in K. pneumoniae. Results Antibiotics susceptibility test was performed on 126 of K. pneumoniae isolates. Also, DNA extraction was done and genes were detected using PCR method. In this study, 67 isolates (53%), carrying both the sulI and sulII genes. Forty-five percent tetracycline-resistant isolates were tetA or tetB positive. The prevalence of intI gene was 96%, while only sixteen isolate harboring intII gene (12.5%). Our results showed the high prevalence of integrons in MDR K. pneumoniae, indicating the important role of these genes in the transmission of antibiotic resistance.

Introduction: Toxigenic aspergilli are storage contaminants of poultry feeds and feed ingredients which produce secondary metabolites known as mycotoxins in these products. This research is aimed at detecting and characterizing mycotoxin producing aspergillus species present in feeds given to poultry birds and in the ingredients used in the production of these feeds. Method: A total of 102 samples of feed (n=30) and feed ingredients (n=72) were collected across 5 agro-ecological zones of Nigeria and analyzed for toxigenic Aspergilli using morphometric and molecular genomic characterization techniques. Different mycological media was used for isolation, by deploying pour plate method. The Aspergilli isolates morphometric identity were characterized using macroscopic and microscopic observed features. The isolated Aspergillus species were further characterized molecularly using genomic characterization protocols. Results: The results of the morphometric characterization indicated that nine (9) Aspergillus species were detected in the feed and feed ingredients, with Aspergillus flavus having the highest rate of occurrence among the isolates. Furthermore, the genomic typing using internal transcribe spacer (ITS) as a barcode, as well as comparison of the fungal isolates with other Aspergillus species in the genebank showed 99-100% similarity. Also, toxigenic gene typing indicated that some of the A. flavus were toxigenic with the detection of the following genes Nor-1, ver-1, OmtB and AflR. Conclusion: The findings of this study showed that toxigenic Aspergilli isolates are present in both poultry feed and feed ingredients from 5 agro-ecological zones in Nigeria and poses great public health issues.

Recently, metagenomic DNA based analysis gained importance in fields such as environmental sciences and bioenergy, where mixed microbial communities embedded in complex matrixes, play a crucial role. Therefore, financially economical, and high quality metagenomic DNA extraction protocols are needed. In this work, a rapid and inexpensive method for high quality DNA extraction from a variety of complex samples was performed; the average DNA yield was 71.65 ng µL−1, with an average purity of 1.68 (A260/A280). The cost of extraction per sample was about 76 % less in comparison with commercial kits and the time needed to obtain the DNA pellets was about 4 hours. The DNA was suitable for 16S rRNA gene amplification by PCR and for next generation sequencing analysis, employing a MiSeq Ilumina platform. A high microbial diversity was detected in this study, and three main groups of bacteria were observed, which were developed according to the effect of the activated carbon had on them. The analysis performed showed a great difference between the samples, highlighting the differences between the microbial communities developed in the activated carbon biofilm and the bacteria detected in the reactor without activated carbon.

The flagship animal species of Sundarbans, the Royal Bengal tiger (Panthera tigris tigris) is under threat of extinction. Its natural population is declining day by day. So, to avoid killing and harming the animal, the use of non-invasive samples such as scat, hair, or scent is preferred for DNA extraction and subsequent genotyping of tiger species. DNA has been extracted from scat samples of the Bengal tiger in the present study, and a fragment of the cytochrome b gene has been sequenced after PCR with species-specific primers. DNA has been extracted manually using a previously described methodology with slight modifications. The size of the PCR product and sequence of cytochrome b gene indicates that tiger DNA is successfully extracted from scat samples using tigerspecific primers. Thus, presence of tiger DNA can be detected by using this method just by the PCR product size in the gel. This is the first report of a partial sequence of mitochondrial cytochrome b gene of P. t. tigris from Bangladesh. Dhaka Univ. J. Biol. Sci. 30(2): 325-330, 2021 (July)

Aim 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. Materials and methods 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 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. Conclusion 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. Clinical significance 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.

Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop‐mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross‐sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens’ parasitological examination was performed by using the direct wet mount and formalin‐ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica‐positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51–0.72 and p, The liver flukes F. hepatica and F. gigantica are agents for fasciolosis that is a zoonotic parasitic disease. Bennett and Ijpelaar (Journal of Agriculture Economy, 2005, 56, 135) estimated that F. hepatica causes annual economic loss by£40.4 million in the UK cattle industry. Fasciolosis not only infects the liver and biliary ducts, ectopic fasciolosis, but it may also infect the peritoneal cavity, lungs, subcutaneous tissue, lymph nodes, eye, and other locations. Fasciolosis can induce mortality and morbidity in the sheep and cattle industry. Many advances are observed in vaccination against F. hepatica but validated methods for the diagnosis of fascioliasis are still unknown. Fascioliasis is commonly diagnosed by fecal testing and finding parasitic eggs, bile, or fluid in the duodenum through examining direct stool and/or condensation techniques such as formalin‐ether and telman or Kato‐kats assays. Some serologic techniques such as Fas2‐ELISA, IFA, and IHA are applicable at all stages of the disease. These techniques have disadvantages such as the expertise of the test person and the number of parasite eggs in the stool sample. This study aimed to compare the loop‐mediated isothermal amplification (LAMP), one of the relatively new molecular techniques with PCR assay in the diagnosis of F. hepatica in sheep.

Identification of insect species using molecular approach is one of the first steps in managing Ceratocystis. Proper species identification of the vectorsof Ceratocystiswill provide an effective way in limiting the distribution of this pathogenic fungi. The isolation of insect DNA is a crucial step in species identification using molecular characters. The aim of this research was to obtain the most effective method for isolating the COI gene and to confirm the insect DNA using amplification of the DNA through polymerase chain reaction (PCR). Twelve unidentified Coleoptera specimen collected from Acacia spp. plantation in Pelalawan Riau were randomly selected for this study. The insect DNA were isolated using the CTAB buffer with four different pre-incubation treatments, involved a non destructive method, i.e. soaking (R), destructive methods, i.e. crushing (H), soaking-crushing (RH) and freezing-crushing (FH). Two sets of primer, LCO1490/HCO2196 and LCO1490/HCO2198 were used to amplify the DNA of COI gene. The results shows, the COI gene was isolated from all pre-incubation treatments, except in the non-destructive treatment. The isolated DNA of COI gene was successfully amplified using both primer sets used in this study

Objective — to develop a method of modern molecular genetic diagnosis of microsporia in children based on polymerase chain reaction (PCR), which will allow identification of the pathogen of Microsporum canis at the DNA level. Materials and methods. The study included 40 patients with microsporia of smooth skin, scalp, scalp and smooth skin. The biological materials for the research were scales from the smooth skin and scalp, hair from the scalp of patients with microsporia. A study of 40 samples of biological material was carried out in patients with microsporia of smooth skin, microsporia of the scalp, microsporia of the scalp and smooth skin. At the first stage, DNA isolation of Microsporum canis was carried out. Then PCR was carried out to increase the copies of the DNA region using specific primers. The final step was typing 40 samples of clinical material of patients. Results and discussion. PCR diagnostics made it possible to identify the DNA of Microsporum canis in all 40 samples of biological material of patients with microsporia. In our study, we developed a PCR-based method for diagnosing microsporia, which uses a set of two MC primers (regions of the beta tubulin gene of Microsporum canis). For internal control of the course of amplification and the quality of biomaterial sampling, specific primers of APOE (a region of the human apolipoprotein E gene) were also used. Conclusions. In order to improve the precise specific diagnosis of microsporia in children, a method of modern molecular genetic diagnostics based on polymerase chain reaction (PCR) has been developed, which allows identification of the Microsporum canis pathogen at the DNA level. Analysis of the molecular structure of the genome of Microsporum canis proved that the most objective diagnosis of microorganisms is the PCR method. The developed method of DNA diagnostics based on PCR using specific primers can be included in the algorithm for detecting Microsporum canis in humans.

Objectives The purpose of this study is to use the second internal transcribed spacer (ITS-2) to determine the molecular characteristics of Sarcoptes scabiei in rabbits from several areas of East Java. Methods Collecting S. scabiei mites from rabbits with clinical signs of scabies; DNA extraction with minikit QIAamp DNA; polymerase chain reaction amplification; nucleotide sequence analysis; homology and phylogenetic tree using the Neighbor-Joining method in the program molecular evolutionary genetics analysis-7 (MEGA-7). Results Sequence analysis of ITS-2 S. scabiei from five regions in East Java showed an identity >91.23% with isolates from China (KX695125.1). The phylogenetic analysis of ITS-2 S. scabiei from Mojokerto rabbits has a close relationship with AB82977.1; Surabaya and Nganjuk rabbits are closely related to KX695125.1; while Sidoarjo and Pasuruan rabbits are closely related to EF514469.2. and AB369384.1. Conclusions The homology analysis of all samples showed identity of more than 91.23% with isolate China (KX695125.1). The sequences of ITS-2 gen of S. scabiei from rabbits in several areas were relatively close to S. scabiei obtain various hosts from National Centre for Biotechnology Information (NCBI) data.

DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water. HIGHLIGHTS DNA extraction methods were evaluated to quantify H. pylori in water.; The tissue kit was suitable for DNA extraction from treated drinking water.; The stool kit was efficient in analyzing samples from the river after heavy rain.; The soil kit could extract DNA effectively from the river and stream with no rain.; The characteristics of the water sample were important in detecting H. pylori.

We report the first barcode identification of water flea species Moina macrocopa (strain JSK1) from the Indian subcontinent. The strain isolated from a freshwater pond near Mumbai by collective efforts of CUBE (Collaborative Undergraduate Biology Education), was morphologically characterized as Moina species. Keeping up with the growing demand of affordable science in colleges and to avoid hazardous technologies, we established a home grown porcelain-abrasion DNA extraction strategy for crustaceans, and compared it against routinely used protocols employing liquid nitrogen or insect-DNA isolation kits. Our method successfully yielded high quality genomic DNA from Moina, comparable to published protocols as further analysed by restriction digestion, ligation and PCR efficiency. We also sequenced two barcoding loci namely, COI and ITS sequences, duly submitted and curated by Genbank (accession numbers MH734122.1 and MH745035.1). Molecular phylogenetic analyses identified the Indian species as Moina macrocopa and related it closely to the Russian clade. Protein prediction analyses of the variations in COI revealed that the missense mutations lie in the aliphatic membrane helices and should not affect the catalytic capacity of cytochrome oxidase. The strain JSK1 is currently disseminated and maintained collectively by CUBE students across India, towards developing it as a model system.

Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

Microalgal contamination in algal culture is a serious problem hampering the cultivation process, which can result in considerable economic and time losses. With the field of microalgal biotechnology on the rise, development of new tools for monitoring the cultures is of high importance. Here we present a case study of the detection of fast-growing green algae Chlorella vulgaris (as contaminant) in a diatom Phaeodactylum tricornutum culture using various approaches. We prepared mixed cultures of C. vulgaris and P. tricornutum in different cell-to-cell ratios in the range from 1:103 to 1:107. We compared the sensitivity among microscopy, cultivation-based technique, PCR, and qPCR. The detection of C. vulgaris contamination using light microscopy failed in samples containing cell ratios

Aims We aimed to elucidate whether the DNA extraction kit and bacteria therein affect the characterization of bacterial communities associated with butterfly samples harbouring different bacterial abundancies. Methods and Results We analysed bacteria associated with eggs of Pieris brassicae and with adults of this butterfly, which were either untreated or treated with antibiotics (ABs). Three DNA extraction kits were used. Regardless of the extraction kit used, PCR amplification of the bacterial 16S rRNA gene detected very low bacterial presence in eggs and AB-treated butterflies. In untreated butterflies, bacterial signal intensity varied according to the kit and primers used. Sequencing (MiSeq) of the bacterial communities in untreated and AB-treated butterflies revealed a low alpha diversity in untreated butterflies because of the dominance of few bacteria genera, which were detectable regardless of the kit. However, a significantly greater alpha diversity was found in AB-treated butterflies, evidencing a true bias of the results due to bacterial contaminants in the kit. Conclusions The so-called ‘kitome’ can impact the profiling of Lepidoptera-associated bacteria in samples with low bacterial biomass. Significance and Impact of the Study Our study highlights the necessity of method testing and analysis of negative controls when investigating Lepidoptera-associated bacterial communities.

Among the methods of detecting food adulteration, the PCR amplification techniques of the endogenous reference gene are increasingly significant. In this article, the improved SDS-CTAB is chosen for the DNA extraction of Flammulina velutipes, the tre6P Phosphatase gene (Tps2) is selected an endogenous reference gene suitable to identify agricultural important fungus F. velutipes and by-products via PCR methods. The Tps2 gene was assayed in 2 varieties of F. velutipes and 9 non-F. velutipes species by both qualitative and quantitative PCR, no amplicon was captured in the non-F. velutipes species. By SYBR Green quantitative PCR, the melting curve showed that the target Tps2 sequence displayed high homogeneity in different F. velutipes varieties. In Taqman probe quantitative PCR analysis, the detection limit was as low as 3 pg/uL of DNA template. From what is said in the foregoing, we can conclude that the Tps2 gene is a reliable, convenient, and accurate endogenous reference gene for detecting F. velutipes component, even in the processed food products, such as sauce and biscuits.

High-throughput phylogenetic 16S rRNA gene analysis has permitted to thoroughly delve into microbial community complexity and to understand host-microbiota interactions in health and disease. The analysis comprises sample collection and storage, genomic DNA extraction, 16S rRNA gene amplification, high-throughput amplicon sequencing and bioinformatic analysis. Low biomass microbiota samples (e.g. biopsies, tissue swabs and lavages) are receiving increasing attention, but optimal standardization for analysis of low biomass samples has yet to be developed. Here we tested the lower bacterial concentration required to perform 16S rRNA gene analysis using three different DNA extraction protocols, three different mechanical lysing series and two different PCR protocols. A mock microbiota community standard and low biomass samples (108, 107, 106, 105 and 104 microbes) from two healthy donor stools were employed to assess optimal sample processing for 16S rRNA gene analysis using paired-end Illumina MiSeq technology. Three DNA extraction protocols tested in our study performed similar with regards to representing microbiota composition, but extraction yield was better for silica columns compared to bead absorption and chemical precipitation. Furthermore, increasing mechanical lysing time and repetition did ameliorate the representation of bacterial composition. The most influential factor enabling appropriate representation of microbiota composition remains sample biomass. Indeed, bacterial densities below 106 cells resulted in loss of sample identity based on cluster analysis for all tested protocols. Finally, we excluded DNA extraction bias using a genomic DNA standard, which revealed that a semi-nested PCR protocol represented microbiota composition better than classical PCR. Based on our results, starting material concentration is an important limiting factor, highlighting the need to adapt protocols for dealing with low biomass samples. Our study suggests that the use of prolonged mechanical lysing, silica membrane DNA isolation and a semi-nested PCR protocol improve the analysis of low biomass samples. Using the improved protocol we report a lower limit of 106 bacteria per sample for robust and reproducible microbiota analysis.

Xpert MTB/RIF testing has improved tuberculosis (TB) diagnostics and rifampicin (Rif) resistance testing worldwide. However, it has weaknesses, such as its restriction to Rif resistance testing and the inability to use extracted DNA for further testing. Herein, a holistic diagnostic workflow, including TB detection and resistance testing toward Rif, isoniazid, and important second-line drugs (SLDs), based on a novel microfluidic DNA extraction cartridge (TB-Disk), is presented. DNA from 73 precharacterized sputum samples was extracted with TB-Disk, including 45 clinical and bacteriologically confirmed TB samples, nine TB-negative samples, and 19 sputum samples spiked with twofold dilutions of TB bacteria. The extracted DNA was subjected to further testing with FluoroType MTB (FT-MTB), GenoType MTBDRplus (GT-plus), and GenoType MTBDRsl. A total of 100% (20/20) and 72% (18/25) of smear-positive and smear-negative TB samples were identified as Mycobacterium tuberculosis complex positive. A total of 79% (33/42) of subsequently GT-plus tested samples yielded a valid result. Eight samples were identified as multidrug-resistant TB by GT-plus and further tested for resistance toward SLDs using GenoType MTBDRsl, yielding 75% (6/8) valid results. FT-MTB with cartridge-based DNA extraction (Disk-DNA) and DNA extracted with FluoroLyse yielded similar analytical sensitivities. FT-MTB with Disk-DNA was 100% specific. TB-Disk in combination with FT-MTB enables sensitive TB detection. The Disk-DNA can be further used for screening resistance toward first-line drugs and SLDs.

BackgroundAs an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet.MethodsIn this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild.ResultsBead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity.ConclusionsDNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

Background New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important. Results Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution. Conclusions Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.

Summary Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species‐level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB‐based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.

Aims This study evaluated the microbial viability of fish gut microbiota in both digesta (faecal) and mucosal samples using a modified propidium monoazide (PMA) protocol, followed by 16S ribosomal RNA (rRNA) gene sequencing. Methods and results Digesta and gut mucosal samples from farmed yellowtail kingfish (Seriola lalandi) were collected and a modified PMA treatment was applied prior to DNA extraction to differentiate both active and nonviable microbial cells in the samples. All samples were then sequenced using a standard 16S rRNA approach. The digesta and mucosal samples contained significantly different bacterial communities, with a higher diversity observed in digesta samples. In addition, PMA treatment significantly reduced the microbial diversity and richness of digesta and mucosal samples and depleted bacterial constituents typically considered to be important within fish, such as Lactobacillales and Clostridales taxa. Conclusions These findings suggest that important bacterial members may not be active in the fish gut microbiota. In particular, several beneficial lactic acid bacteria (LAB) were identified as nonviable bacterial cells, potentially influencing the functional potential of the fish microbiota. Significance and impacts of the study Standardizing the methods for characterizing the fish microbiota are paramount in order to compare studies. In this study, we showed that both sample type and PMA treatment influence the bacterial communities found in the fish gut microbiota. Our findings also suggest that several microbes previously described in the fish gut may not be active constituents. As a result, these factors should be considered in future studies to better evaluate the active bacterial communities associated with the host.

Background The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. Results 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45–11.5 ng/μL, and on Qubit™ ranged between undetectable – 0.962 ng/μL. Median A260/280 ranged between 0.505–3.925, and median A260/230 ranged − 0.005 – 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. Conclusions A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Background: Tuberculosis is a critical infectious disease primarily affecting the lungs and is more common in developing countries. In the 21st century, it forms a significant problem for world public health especially with the emergence and rising of drug resistant TB. Microbiological methods are the clue for the laboratory diagnosis. The ordinary methods for TB identification showed either weak sensitivity as in microscopy or lateness for many weeks as in culture. The evolution in molecular biology gives a chance for fast diagnosis of Mycobacterium tuberculosis helping start proper treatment early and holding its spread. The initial critical step in PCR is DNA extraction. Objective: The aim to evaluate different extraction methods of Mycobacterium tuberculosis retrieved directly from sputum samples and from LJ isolates from same patients and comparing DNA yield using conventional PCR. Methodology: DNA from 32 sputum samples from TB patients extracted by solid, digestion and phenol extraction methods, DNA from 40 LJ isolates extracted by solid, boiling and Cetyl trimethylammonium bromide methods. Extracted DNA was evaluated by conventional PCR. Results: Among 32 sputum samples, the extracted DNA by phenol method was 21/32 (65.62%) with highest DNA yield, digestion method 14/32 (43.75%) and solid phase method 1/32 (2.5%) with least DNA yield. From 40 MTB LJ culture isolates, the extracted DNA by boiling method was 28/40 (70%), CTAB method 18/40 (45%) and solid phase method 2/40 (5%). Conclusion: Phenol method was the best method (mean rank 2.34) for DNA extraction from sputum samples, while the easy and economic boiling method was the best method (mean rank 2.45) for DNA extraction from LJ culture isolates. The worst method of DNA extraction from both sputum and culture was phase solid method. A greater and easier yield of DNA was obtained from MTB LJ Culture than sputum.

Vertical transmission of Trypanosomacruzi is the cause of congenital Chagas disease, a re-emerging infectious disease that affects endemic and nonendemic regions alike. An early diagnosis is crucial because prompt treatment achieves a high cure rate, precluding evolution to symptomatic chronic Chagas disease. However, early diagnosis involves low-sensitive parasitologic assays, making necessary serologic confirmation after 9 months of life. With the aim of implementing early diagnostic strategies suitable for minimally equipped laboratories, a T. cruzi-loop-mediated isothermal amplification (LAMP) prototype was coupled with an automated DNA-extraction device repurposed from a three-dimensional printer (PrintrLab). The whole process takes3 hours to yield a result, with an analytical sensitivity of 0.1 to 2 parasite equivalents per milliliter, depending on the T. cruzi strain. Twenty-five blood samples from neonates born to seropositive mothers were tested blindly. In comparison to quantitative real-time PCR, the PrintrLab-LAMP dual strategy showed high agreement, while both molecular-based methodologies yielded optimal sensitivity and specificity with respect to microscopy-based diagnosis of congenital Chagas disease. PrintrLab-LAMP detected all 10 congenitally transmitted T. cruzi infections, showing promise for point-of-care early diagnosis of congenital Chagas disease.

Identifying the newly isolated species is crucial to establishing a reliable algal database with successful commercial applications for different biotechnological applications. Morphological identification does not give sufficient description, especially for tiny unicellular microalgae. The rbcL gene encodes the large unit of ribulose-1, 5-bisphosphate carboxylase /oxygenase (Rubisco) has been widely known for barcoding in plants and developed for microalgae molecular identification. In this study, we examined the local strains of green microalgae from Indonesia using the rbcL partial gene sequence to identify the strains. Green microalgae isolates originated from Yogyakarta, Serayu, Gondol, Ancol, Cilegon, and Teluk Jakarta were cultured in f/2 media and harvested for DNA extraction. The DNA extracted was proceeded to PCR using 1AB_rbcL primer pair to amplify the sequences of rbcL gene with target band located at 582 bp, followed by the sequencing of the PCR product was conducted. Molecular identification of local green microalgae isolates was successfully carried out using primers 1AB_rbcL with a genetic similarity of 99% toward identified species in the NCBI database. Among six isolates, TJ, G, S, C, and A isolates were identified as C. pyrenoidosa . Only CP isolate from Yogyakarta identified as C. sorokiniana . Nannochloropsis gaditana rbcL sequence was selected as an outgroup. The phylogenetic analysis indicated that the five isolates of Chlorella belong to one clade and clearly distinguished from C. sorokiniana isolate from Yogyakarta.

Avocado (Persea americana Mill.) is grown for its nutritious fruit. However, the quantity and quality of these fruits are threatened by some fungal organisms which can cause health complications when it is consumed by man. DNA extraction provides a unique tool for identification of organisms. This study was conducted to isolate and identify fungal species associated with avocado fruit using both morphological and molecular techniques. Fungal species were isolated from Persea americana purchased from Choba market, Port Harcourt, Rivers State, Nigeria using Potato Dextrose Agar (PDA) as a growth medium. The morphology of isolated fungi on PDA were cotton-like blackish grey spots, white villous colonies, greyish powdery spores and black spores for isolates 1 to 4 respectively. Extraction of DNA from fungal isolates was carried out using Zymo Fungal/Bacteria DNA Miniprep Kit. PCR amplification of the ITS1-2 regions of isolates was carried out using fungal universal primer pair; ITS4 and ITS5.PCR amplification of the ITS1-2 gene sequences yielded amplicons between 537-580 base pairs. PCR products were sequenced and the sequencing result after BLAST search revealed the identity of the four fungal species as follows: Lasiodiplodia theobromae, Fusarium proliferatum, Penicillium sp. and Aspergillus niger. This study will promote the knowledge of specific fungal species associated with Persea americanna and help plant pathologists to proffer preventive and control measures to enhance fruit protection and yield quality.

Background Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database. Results Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009). Conclusions Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.

A study was carried out to compare the efficacy of different DNA extraction procedures for Eimerian oocysts. A total of 753 litter and fecal samples and intestines were collected from desi poultry farms and slaughter houses in Bengaluru, Karnataka. Out of five different methods of DNA extraction procedures the glass bead method was found to be the best method. Since the above method involves breaking of the oocysts and sporocysts walls and dissolving the sporozoites membrane. This greatly simplifies the currently used DNA extraction procedures for Eimeria species. The liquid nitrogen method was also found to be good but was more critical and time consuming. Other methods such as sodium hypochlorite, direct and sonication has failed to yield DNA from Eimeria oocysts. The present study indicates that glass beads method yielded better quality of DNA for molecular analysis and it is suitable for both large and small number (˂1x103) of oocysts in a sample. Hence, can be used as a routine procedure for DNA extraction for Eimerian oocysts.

Accurate diagnosis of the differentially aggressive fungi Leptosphaeria maculans ‘brassicae’ and L. biglobosa ‘brassicae’ causing blackleg disease in Brassica crops is crucial. Prior to the availability of fungal whole‐genome sequences, gene‐specific markers were designed on specific genes. But, the availability of the whole‐genome sequences of several isolates now offers a greater opportunity to develop more precise and novel markers. The draft genomes of L. maculans and L. biglobosa that are available in the repository were aligned, and species‐specific primers were designed from the variable sequences that encode for effector proteins. Three primer sets namely, Lm1, Lm2 and Lm5 and two primer sets namely Lb1’ and Lb3 specifically detected the isolates of target species in PCR assay. The primers Lm5 and Lb1’ were multiplexed for detection of L. maculans ‘brassicae’ and L. biglobosa ‘brassicae’, respectively, from a single PCR reaction. The markers were highly sensitive in amplifying target species from crude ‘pseudothecia and ascospore suspension’ without requiring DNA extraction. These markers, solitarily or in combination, could be used for precise, sensitive and rapid detection of L. maculans ‘brassicae’ and L. biglobosa ‘brassicae’ species and will be helpful for surveillance, management and transboundary quarantine of the devastating blackleg disease.

Hydatid cyst, the larval stage of Echinococcus granulosus, and Cysticercus tenuicollis, the larval stage of Taenia hydatigena, are prevalent in domestic, livestock, and wild ruminants. The main goal of this research was to identify the isolates of E. granulosus and C. tenuicollis by partial sequencing with PCR amplification of the cytochrome C oxidase 1 (COX1) gene. During a routine veterinary inspection at a Chabahar city slaughterhouse, two samples of hydatid cysts from sheep's liver and cattle's lung and two samples of C. tenuicollis from sheep's liver were collected. After DNA extraction, the fragment of the COX1 gene was amplified by the PCR method. Sample sequences were modified and synchronized by Chromas and CLC genomic workbench 11 software. Sequence analysis was carried out by BLAST algorithms and GenBank databases. Phylogenetic trees were performed using MEGA 7 software and the neighbor-joining and maximum likelihood method for T. hydatigena and E. granulousus. The result indicated that the main genotype of parasites and the amplified fragment size were G1 and approximately 455 bp, respectively. The analysis of phylogenetic trees based on nucleic acid for four samples showed that there was a common ancestor. However, the shift in nucleotides in the two isolates in E. granulosus and the two isolates of T. hydatigena were non-synonymous type and synonymous type, respectively. The present study showed that the dominant genotype in all isolates was G1 and this report was similar to other studies in Iran and the world. Also, the partial COX1 gene sequence was matched with T. hydatigena.

As the incidence of fungicide resistance in plant pathogens continues to increase, control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele-specific PCR (ASqPCR) is an ideal technique for the in-field analysis of fungicide resistance as it can quantify the frequency of mutations in fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b (cytb) gene in the cytochrome bc1 enzyme complex, resulting in a substitution of alanine for glycine at position 143 (G143A). We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures, within 90 min of sample collection. The in situ analysis of samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9–100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.

The aphrodisiac property of Eurycoma longifolia has led to an increase in the demand for its Herbal Medicinal Products (HMPs). However, the efficiency of such HMPs depends on the usage of their genuine raw materials. The conventional methods cannot identify species in processed form. The authentication of HMPs can be achieved effectively using DNA barcoding as the method species-specific. However, the use of this method solely relied on the extraction of high-quality DNA from the HMPs. Therefore, it is necessary to establish a satisfactory method for extracting high-quality DNA from the HMPs. Here, four DNA extraction methods were compared to evaluate the best protocol in yield, purity, polymerase chain reaction (PCR) amplification, sequencing, and species identification. The spectrophotometer analysis showed that the Nucleospin Plant II extraction kit has the best purity as this can be severely affected by the presence of various contaminants in the HMPs. Our findings reveal that DNA purity was more important as a predictor for PCR amplification than yield. Therefore, the present study results demonstrate that the Nucleospin Plant II extraction kit is the best because it produces the purest, amplifiable, and sequenceable DNA for identification and authentication of E. Longifolia HMPs.