Kit Qiagen Research Articles

Comparison of six methods for Loa loa genomic DNA extraction.

Good-quality and sufficient DNA is essential for diagnostics and vaccine development. We aimed to compare six DNA extraction techniques applied to Loa loa microfilariae in order to evaluate the purity and integrity of extracts in terms of quality and quantity. The microfilariae were purified via a Percoll gradient procedure with blood from hyper-microfilaremic individuals (> 30,000 microfilaria [mf]/ml). DNA extraction was carried out in duplicate at a rate of 350,000 mf/tube for each technique: phenol/chloroform, commercial Qiagen kit, salting out, Tris-EDTA, methanol, and cetyltrimethylammonium bromide (CTAB). The integrity, purity, concentration, and quality of the DNA extracts were successively verified by agarose gel electrophoresis, spectrophotometry (A260/A280 and A260/A230 wavelength ratio), Qubit fluorometry, and endonuclease and polymerase activity. The six techniques were compared on the basis of the following parameters: concentration, purity, efficiency, effectiveness, integrity, safety of the technique, as well as cost and duration of the protocol. The ratios of the optical densities of the extracts A260/A280 and A260/A230 were, respectively: phenol/chloroform (1.82; 1.11), Qiagen (1.93; 1.36), salting-out (1.9; 2.04), Tris-EDTA (1.99; 1.183), methanol (2.126; 1.343), and CTAB (2.01; 2.426). The DNA yield was: phenol/chloroform (3.920 μg), Qiagen (10.280 μg), salting-out (10.390 μg), Tris-EDTA (0.5528 μg), methanol (0.1036 μg), and CTAB (1.115 μg). Endonuclease and polymerase activity was demonstrated by digestion of DNA and through amplicons obtained via polymerase chain reaction assays with phenol/chloroform, Qiagen, and salting-out extracts. The phenol/chloroform, Qiagen, and salting-out DNA extracts were all of good quality. Salting out had the best yield followed by Qiagen and then phenol/chloroform. Endonuclease and polymerase activity was effective in all three extracts despite the presence of some contaminants. These methods are therefore suitable for the extraction of DNA from Loa loa microfilariae. Tris-EDTA and methanol did not show adequate sensitivity, while CTAB was found to be unsuitable.

PPI ADMINISTRATION CAUSES COLONIC TIGHT JUNCTION BARRIER DYSFUNCTION VIA INCREASE INENETROCOCCUS FECALIS

Abstract BACKGROUND Proton pump inhibitors (PPIs) are highly effective antagonists of gastric acid secretion and are widely used to treat a number of gastroesophageal disorders, including peptic ulcer disease, GERD, Barrett’s esophagus. PPI-induced elevation in intra-gastric pH and subsequent alterations of gastrointestinal physiology are known to cause undesired effects on the entire GI tract. Gastric acid suppression can change the composition of the intestinal microbiota causing intestinal dysbiosis and increased risk for intestinal inflammation. The intrinsic intestinal barrier is composed of a single layer of columnar epithelial cells and inter-epithelial tight junctions (TJ) residing at the apical-most region of the intercellular space. Defective intestinal TJ barrier is an important pathogenic factor for intestinal inflammation. The role of PPI on Intestinal TJ barrier is not known. AIM The aim of the present study was to study the effect of PPI on intestinal dysbiosis and mouse colonic TJ permeability. METHODS 8 to 12-week-old male C57BL/6N mice were divided into two groups (Controls and PPI treatments (Omeprazole 20mg/kg B.W.) and were fed a nutritionally adequate feed for 30 days. Fecal samples were collected from both treated and untreated mice after 30 days. Fecal DNA was isolated using the Qiagen kit according to the manufactures Instructions. 16s amplicon DNA was sequenced by Genewiz (South Plainfield, NJ). Degenerate primers that amplify the V3-V4 region of bacterial rDNA genes were used (F341-806R pair).The amplicon pool sequenced on Illumina MiSeq generated paired-end 301 bp reads was multiplexed using Illumina’s fastq. At the end of the experiments, the colonic permeability was measured on Ussing chambers. Western blot (WB) and Immunofluorescence (IF) was used to study myosin light chain kinase (MLCK, a key regulator of TJ barrier) protein expression in mice colon. RESULTS The microbiome changes identified by 16S rRNA sequencing showed a significant increased proportion of Firmucutis in the fecal samples of PPI treated mice compared with untreated mice. Also, PPI group showed prominent increase in enterococcus spp which was identified as Enterococcus fecalis (EF). In Ussing chambers studies, PPI administration caused a decrease in transepithelial resistance (TER), and increase in urea and inulin flux when compared to untreated colonic tissues. Immunofluorescence and Western blot analysis showed that PPI administration also caused an increase in MLCK and phospho- MLC protein expression in mice colonic tissues. Also, PPI treatment caused increased focal sloughing of colonic surface epithelium with focal to diffuse lymphocytic infiltration in the colonic lamina propria in mice colon. CONCLUSION Our results suggest that the PPI causes overgrowth of a pathobiont EF associated increase in mouse colonic TJ barrier permeability.

Comparison of COVID-19 laboratory diagnosis by commercial kits: Effectivity of RT-PCR to the RT-LAMP.

Coronavirus disease 2019 or COVID‐19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 or 2019‐nCoV) is an ongoing pandemic that has emerging global effects and requires rapid and reliable diagnostic testing. Quantitative reverse transcription‐polymerase chain reaction (q‐RT‐PCR) is the gold standard method for SARS‐CoV‐2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as one of these novel approaches may also contribute to faster and cheaper field‐based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30–45 min and to compare the effectiveness of LAMP to the q‐RT‐PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS‐CoV‐2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT‐LAMP as well as by conventional q‐RT‐PCR. The patient samples were tested with four different kits (SENSObiz COVID‐19 [SARS‐CoV‐2] LAMP Assay, the QIAseq DIRECT SARS‐CoV‐2 kit, Biospeedy SARS‐CoV‐2 Variant Plus kit, and CoVirion‐CV19‐2 SARS‐CoV‐2 OneStep RT‐PCR kit) and two different PCR devices (GDS Rotor‐Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor‐Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor‐Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS‐CoV‐2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.

Determination of critical cut-off values of PCA3 index in patients with prostate gland pathology

Introduction. Prostate cancer antigen-3 (PCA3) is a genetic biomarker, which got widespread and demonstrated high diagnostic potential. This gene expresses only in prostate gland tissue, furthermore, malignant prostate neoplastic transformation leads to 70 – 100 time overexpression of it. Polyadenylated matrix RNA (mRNA) is a functional product of this gene, which do not translating into protein. Gene contains of four introns and three exon. Product of PCA’s gene detecting in post massage urine using polymerase chain reaction (PCR) in combination with reverse transcription. It could use as prognostic biomarker in patients with first negative biopsy or before it performing. Moreover, index of PCA3 can be useful in detecting tumor aggressiveness and decisions about further treatment options. Taking into account these facts, patients with high initial PSA level can avoid unnecessary biopsies. Objective: to determine critical cut-off value of PCA3 index as prognostic biomarker of PCa development. Materials and methods. Research based on routine and special test’s data of 243 men in Institute of Urology, National Academy of Medical Sciences of Ukraine in period of 2015–2020 year. RNA extraction from postmassage urine samples performed using RNeasy (Qiagen) kit. Statistical analysis performed using SPSS Statistics 19.0 (IBM SPSS Statistics 19.0) та SAS 12.0 (IBM Statistical Analysis System 12.0). Critical cut-off values determined using three methods: calculating of average value and (Xave) and standard deviation (SD) of it, Heiner index, and Youden’s index. Evaluation of optimality of determined critical cut-off values performed by comparative analysis using index of positive prognosis (IPP), index of negative prognosis (INP), diagnostic accuracy of method (DAM), Sp and Se for each of used methods. Results and discussion. First method demonstrated than 97,5 % of all results, which means absent of disease, was below critical cut-off values, due to statistical deviations and accurate demonstrate than in case of asymmetry or multimodal distribution this method was unsatisfied. Wherein IPP was 95 %, INP – 72 %, DAM – 78 %, Sp – 97,5 % and Se – 52 %. Calculation of PCA3 index area under ROC-curve was 0,810 (0,771–0,849). Using Heiner’s method critical cut-off value of PCA3 index was 14,0 с. u., where in IPP was 75 %, INP – 82 %, DAM – 78 %, Sp – 78 % and Se – 78 %. Critical cut-off values of PCA3 index 33,4 c. u. which calculated by Youden’s index demonstrated IPP – 98 %, INP – 73 %, DAM – 80 %, Sp – 99 % and Se – 57 %. Conclusions. The most optimal critical cut-off value for PCA3 index was determined using Youden’s index and was 33,4 c. u. Keywords: prostate cancer, PCA , prostate cancer antigen-3, PCA3, prostate cancer antigen-3 index, PCA3 index, critical cut-off values of PCA3 index.

Alternaria alternata Causing Disease on Sugar Beet (Beta vulgaris) Steckling in Arizona, USA

Sugar beet (Beta vulgaris. L) is an economically important crop which is contributing 55% of the total sugar in the USA. In April 2018, irregular dark brown symptoms were observed in sugar beet stecklings in Arizona where different sugar beet cultivars were grown for seed production (Figure 1). The symptoms covered approximately 5% on the sugar beet root surface. Symptomatic beet root tissue were excised from the junction of diseased and healthy tissue. Small pieces (5mm²) were surface sterilized with 10% sodium hypochlorite for 1min, rinsed thrice with sterile distilled water, air dried and transferred to clarified V8 (CV8), and Potato Dextrose Agar (PDA), and incubated at 24°C with a 12h photoperiod for 5 days. White-dark green velvety colony appeared on both media (Figure 2). Isolates were developed by the single spore isolation technique. Conidia were obclavate or oviod, two to four transverse septa, and pale brown, often in chains (4 to 8 conidia) and or solitary (Figure 3). The dimension of conidia varied from 20.21-40.15 x 7.50-14.12 μm [1- 3]. Based on the morphological characters, the fungus was tentatively identified as Alternaria species. Genomic DNA were extracted via Qiagen kit. A total of 40ng genomic DNA was used to generate a library using NEBNext® Fast DNA Fragmentation & Library Prep Set for Ion TorrentTM for sequencing in Ion TorrentTM Personal Genome MachineTM System.

Dysregulated Neutrophil Iron Homeostasis in β-Thalassemia Impairs Phagocytosis and Reactive Oxygen Species Production

IntroductionBeta thalassemia is an inherited disorder characterised by ineffective erythropoiesis leading to anemia and secondary iron overload. Neutrophils are the first line of innate immune defence against infection is dysfunctional in thalassemia patients. Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, the role of iron contributing to the neutrophil dysfunction is unclear. This is the first study to characterise the neutrophil iron metabolism in β-thalassemia and its association with oxidative burst capacity, phagocytosis, and systemic iron homeostasis.MethodSixteen thalassemia patients and fourteen healthy individuals were recruited in the department of Haematology, Christian Medical College, Vellore, India. Neutrophils were purified from human whole blood collected in EDTA tube, using neutrophil magnetic isolation kit (Miltenyi Biotec). Purified population (>95%) was confirmed by the presence of the surface marker CD62L evaluated using flow cytometry. Haematological parameters were analysed according to standard methods. Serum ferritin, iron, soluble transferrin receptor were measured using immunoassay. Serum hepcidin was measured using ELISA. Neutrophil RNA was isolated using trizol method and was reverse transcribed into complementary DNA using QIAGEN kit. The relative quantification of iron related genes were measured using real-time PCR. In oxidative burst assay, neutrophils were incubated with dihydrorhodamine 123 (DHR) and stimulated with Phorbol 12-Myristate 13-Acetate (PMA). Respiratory burst of the cell was analysed by flow cytometry. Phagocytosis and acidification capacity of human neutrophils were quantified using the pHrodo Green Staphylococcus aureus BioParticles kit (Thermo Fisher). Acquisition was performed using the Beckman Coulter(Navios) flow cytometer and analysed using kaluza software. Statistical analysis was performed using SPSS software.ResultsWe investigated a cohort of β thalassemia Major (n=5), intermedia (n=6) and sickle beta thalassemia (n=5) patients who were on regular iron chelation therapy. The demographic and biochemical parameters are tabulated in Table 1. Serum iron, ferritin levels and transferrin saturation were significantly increased in thalassemia cohort as compared to healthy donors (Fig1a). There was no significant association between ferritin and hepcidin levels. The percentage of neutrophils in thalassemia was significantly reduced incomparision to healthy donors (p=0.032). Oxidative burst capacity of neutrophils from thalassemia major and intermedia patients were significantly decreased (p=0.002) compared to healthy donors upon stimulation with PMA (Fig1b). Neutrophil phagocytosis capacity indicated by the relative amounts of phagocytized fluorescein S.aureus particles were significantly lower in thalassemia patients compared with controls, after 15min/30min incubation (Fig1c). The recognition capacity of neutrophils towards bioparticles significantly decreased at 45-minute incubation in patients (p=0.017) (Fig1d). Serum iron overload and transferrin saturation had negative association with neutrophil phagocytosis capacity (r=-0.714; p=0.045 & r=-0.857; p=0.014), respectively. Neutrophil iron related gene expression was analysed and found significantly lower expression of FPN 1A (FPN1 containing iron regulatory element (IRE)), DMT1B without IRE region and IRP2 respectively (p=0.029, p=0.029 & p=0.016) (Fig1e). FPN1B without IRE region was upregulated in thalassemia patients (p=0.016). Serum ferritin had positive correlation with FPN1B (r=0.786; p=0.036). Soluble transferrin receptor had negative association with DMT1A (containing IRE region) and IRP2 respectively (r=-0.900; p=0.037 and r=-0.750; p=0.052).Aberrant neutrophil function was found in all thalassemia patients. Although oxidative burst activity was decreased in thalassemia major and intermedia patients, sickle β-thalassemia patients had normal burst activity. Systemic iron status had inverse correlation with phagocytosis capacity of neutrophils. Dysregulation of iron transporters in neutrophils was indicated by decreased expression of DMT1 and augmented FPN1B expression, despite systemic iron overload. These findings have to be explored in a larger cohort to elucidate the clinical significance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparative Analyses of Liquid-Biopsy MicroRNA371a-3p Isolation Protocols for Serum and Plasma.

Simple SummaryThe active disease status of patients with a malignant germ cell tumor can be evaluated using detection of specific body-circulating microRNAs. However, various methods are reported to isolate and detect microRNAs from blood, possibly influencing the score as positive or negative. Here, we investigated two frequently used techniques for microRNA isolation from blood, either serum or plasma, to evaluate possible differences. These data are required to compare published studies and to select the best methods in the future. No effect of either starting with plasma or serum was found, indicating that both blood products can be used. The bead-based method was more stable and applicable on small blood volumes, whereas the total RNA method exhibited a higher sensitivity due to a larger starting volume. These results are important to develop the optimal method for the detection of microRNAs in blood to monitor malignant germ cell tumor patients in clinic practice.MicroRNAs (miRNAs) are short, non-coding RNAs involved in translation regulation. Dysregulation has been identified in cancer cells. miRNAs can be secreted and detectable in body fluids; therefore, they are potential liquid-biopsy biomarkers. The miR-371a-3 cluster members are an example, monitoring the presence of malignant germ cell tumors based on patient serum/plasma analyses. However, a large variety of isolation techniques on sample types (serum vs. plasma) are reported, hampering interstudy comparisons. Therefore, we analyzed the impact of using the miRNeasy Serum/Plasma Kit (cell-free total RNA purification) Qiagen extraction kit and the TaqMan anti-miRNA bead-capture procedure of ThermoFisher for miRNA isolation. Ten normal male matched serum and plasma samples and seventeen testicular germ cell tumor patient serum samples were investigated. The Qiagen kit requires a higher input volume (200 µL vs. 50 µL), resulting in higher sensitivity. Serum and plasma comparison demonstrated high similarity in miRNA levels. Titration experiments showed that the bead-capture procedure is superior in cases of lower starting volumes (<100 µL). This study highlights the strengths and limitations of two different isolation protocols, relevant for in vivo analysis with small starting volumes. In summary, miRNA detection levels results varied little between plasma and serum, whereas for low volumes the bead capture isolation method is preferable.

Optimization of DNA Extraction from Field-Collected Mammalian Whole Blood on Filter Paper for Trypanosoma cruzi (Chagas Disease) Detection.

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen’s DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of β-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

Abstracts

Abstracts

ANALISIS PENGARUH PAPARAN FISIK PADA SAMPEL GIGI TERHADAP HASIL KUANTIFIKASI DNA FORENSIK MENGGUNAKAN METODE KIT PURIFIKASI DNA KOMERSIAL

Physical exposure to biological samples has an enormous influence on the results of forensic DNA analysis. The lack of molecular research on the effect of physical exposure on dental samples is the reason for the need for further research. This study aims to determine the effect of physical exposure on dental samples on the results of forensic DNA quantification. The parameter used is the concentration value of isolated DNA obtained from real time PCR analysis. The use of real time PCR allows the detection and quantification of specific sequences of DNA samples at the same time to be analyzed. The dental samples used were obtained from different individuals. Teeth are used as identification media because teeth are the hardest part of the body and are chemically the most stable and most resistant to degradation and decomposition. The method in this study is to give three types of treatment to the tested samples in the form of sea water immersion, river water immersion and exposure to free air at room temperature with each treatment consisting of three test samples. All samples were extracted using a Commercial DNA purification KIT with a reagent in the form of a Qiagen KIT (QIAamp® DNA Investigator) then a quantification process was carried out to see the value of the DNA concentration of each sample using real time PCR. The results of DNA quantification of dental samples from each treatment showed that the highest sample concentration value was based on the average of each treatment, namely samples with treatments exposed to free air at room temperature with a concentration value of 1.34 ng/µl, followed by samples soaked using river water with a concentration value of 0.15 ng/µl, while the sample with the lowest concentration is shown by a sample treated with seawater immersion with a concentration value of 0.10 ng/µl. Physical exposure in the form of exposure to free air, exposure to river water and exposure to sea water on dental samples, gave a not too significant effect on the results of DNA quantification produced.

Buccal swabs as non-invasive specimens for detection of severe acute respiratory syndrome coronavirus-2.

IntroductionThe current gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA involves subjecting nasopharyngeal or oropharyngeal swabs to reverse transcription quantitative PCR (RT-qPCR). However, both sample types need to be collected by trained professionals. Using self-collected buccal swabs as an alternative could simplify and accelerate diagnosis of coronavirus disease 2019 (COVID-19).ObjectiveTo assess self-collected buccal swab samples as an alternative method for SARS-CoV-2 detection in patients with COVID-19.MethodsBuccal swab samples were self-collected by 73 patients with COVID-19. Total RNA was extracted using Qiagen kits. RNA encoding the SARS-CoV-2 Env protein and human RNase P as an internal control was amplified using the TRUPCR® SARS-CoV-2 RT-qPCR kit version 2.1 and a Bio-Rad CFX96 Real-Time Detection System.ResultThe sensitivity of RT-qPCR from buccal swabs was 58.9% (43/73; 95% confidence interval [CI] 46.77%–70.27%) and that of RT-qPCR from saliva was 62.90% (39/62; 95% CI 49.69%–74.84%) taking positive SARS-CoV-2 RT-qPCR from nasopharyngeal swabs as the gold standard.ConclusionSelf-collected buccal swabs are promising alternatives to nasopharyngeal or oropharyngeal swabs for SARS CoV-2 detection.

Significant compartment-specific impact of different RNA extraction methods and PCR assays on the sensitivity of hepatitis E virus detection.

RNA detection in plasma/stool is the gold-standard for diagnosis of hepatitis E virus (HEV) infection. The impact of viral extraction methods on HEV RNA detection is poorly investigated. We determined the limit of detection of the RealStar HEV RT-PCR V2.0 Kit (altona Diagnostics, RS) utilizing 3 RNA extraction methods (COBAS® AmpliPrep Total Nucleic Acid Isolation Kit, TNAi Roche; MagNA Pure 96 DNA, Viral NA SV Kit, MgP; QIAamp Viral RNA mini Kit Qiagen; VRK) in plasma and stool. The most sensitive method was evaluated in a total of 307 longitudinal samples of patients with HEV infection (acute=18/chronic=36) and compared to results with the former diagnostic standard of our centre (TNAi/FastTrack Diagnostic; FTD). The plasma-LOD was 49, 94 and 329IU/mL for extraction with MgP, VRK and TNAi respectively. In stool, the LOD was 21IU/mL, 528IU/mL and indefinable for extraction with TNAi, VRK and MgP respectively. Utilizing longitudinal patient plasma samples, MgP/RS revealed 56 HEV RNA-positive samples in 158 negative samples as determined by TNAi/FTD. In stool, from 37 HEV negative samples (TNAi/FTD), 15 were positive with TNAi/RS. At end of treatment, 8 out of 27 chronically infected patients were RNA positive with MgP/RS, while classified negative with TNAi/FTD. A relapse occurred in 3 of these patients. Different methods for RNA extraction and quantification have a significant, compartment-specific impact on the sensitivity of HEV detection. Knowledge about the favourable combinations of extraction and quantification has important implications for diagnosis and patients receiving antiviral therapy.

DNA barcode of Enggano hill myna, Gracula religiosa enganensis (Aves: Sturnidae) based on mitochondrial DNA cytochrome oxidase subunit I

Abstract. Jarulis, Muslim C, Kamilah SN, Utama AF, Permana D, Sari MM, Prayitno AH, Jannah IM. 2021. DNA barcode of Enggano hill myna, Gracula religiosa enganensis (Aves: Sturnidae) based on mitochondrial DNA cytochrome oxidase subunit I. Biodiversitas 22: 1635-1643. The sharp decline of the Enggano hill myna population due to illegal trading and habitat degradation needs to be our concern to prevent this bird from extinction. Taxonomically, Enggano hill myna is referred to as a sub-species, but this has not been confirmed by genetic data. We have sequenced seventeen Enggano hill myna mitochondrial DNA COI genes to describe their genetic identity (barcode), genetic distances, and phylogeny. DNA genome from seventeen blood samples was isolated with DNeasy® Blood and Tissue Kit Qiagen, while PCR amplification was performed using a pair primers, namely COIGRF (5'-TTCTGATTCTTTGGCCATCC-3') and COIGRR (5'-GTTGGAAGGCTTTGCGTTTA-3'). We used Clustal W alignment in MEGA 10.2.2 software to search single nucleotide polymorphisms. Genetic distance was analyzed by using the Kimura 2-parameter, and the phylogenetic tree was reconstructed with Neighbor-Joining models. We found 98.60% conservative sites, 0.69% parsimony sites, and 0.83% singleton sites from the 716 bp sequence. The highest nucleotide composition was cytosine (32.20%), and the lowest was guanine (16.80%), followed by 49% GC content. Seven SNP sites were found in 716 bp COI gene sequences of seventeen individuals. The genetic distance between Enggano hill myna individuals was ranged from 0.0-0.8%, and all Enggano hill myna individuals separated from Chinese and Indian populations in the phylogenetic tree with a genetic distance of 0.9% and 1.1%. Our data suggest that the Enggano hill myna population is still classified as a sub-species. The COI gene sequences that we found can be used to quickly identify this species and are also important to prevent illegal trading in Indonesia.

First Report of Alternaria alternata Causing Leaf Spot on Beta vulgaris in North Dakota, USA

Sugar beet is an economically important crop which is contributing 55% of the total sugar in the USA. In June 2018, irregular dark brown somewhat circular spots were observed on sugar beet leaves in Hickson, North Dakota. The symptoms covered approximately 5% on the lower leaves. Symptomatic leaf tissue were excised from the junction of diseased and healthy tissue. Small pieces (5 mm²) were surface sterilized with 10% sodium hypochlorite for 1 min, rinsed thrice with sterile distilled water, air dried and transferred to Potato Dextrose Agar (PDA), and incubated at 24°C with a 12-h photoperiod for 5 days. Dark-green velvety colony appeared in two weeks. Three isolates were developed by the single spore isolation technique. Conidia were club-shaped, two to four transverse septa, and pale brown, without any beak, often in chains (4 to 8 conidia) and or solitary. The dimension of conidia varied from 25-40x7-14 μm [1,2]. Based on the morphological characters, the fungus was tentatively identified as Alternaria species. Genomic Deoxyribonucleic Acids (gDNAs) were extracted from the culture generated from a single spore using Qiagen kit. ITS4/ITS5 were used to amplify the fragments of the Internal Transcribed Spacer (ITS) region. The amplified PCR products were cleaned and sent for Sanger sequencing by GenScript (GenScript, Piscataway, NJ). The sequences from GenScript were congruence to the reference sequence ID MT126620.1. The entire sequences were deposited at NCBI (GenBank accession nos. MK441717). Koch postulates were followed by spraying conidia suspension (5×105 conidia/ml) to 8-week age of 20 plants of and kept in humidity chamber at 28-30 °C, 80-85 % RH. Mock-inoculated seeds were also sown as a control. Three weeks of post inoculation, the similar irregular dark brown symptoms observed in twelve plants. No symptoms were found in the mock. The experiment was conducted twice. The fungus was reisolated from the diseased leaf tissue, as described above. Macroscopic and microscopic analysis indicated the similar dark-green colony and morphology, respectively. Molecular detection performed using the same ITS primers and sent for Sanger sequencing by GenScript, this study further confirmed that the isolate was similar to A. alternata [3]. Another close species of Alternaria was recently reported in sugar beet to cause leaf spot in Minnesota [4,5]. To our best knowledge, this is the first report of A. alternata causing leaf spot on sugar beet in North Dakota.

DNA Barcode Authentication and Improvement of Andrographolide Yield in Andrographis paniculata Plant

Background: Andrographolide, the major bioactive compound responsible for most pharmacological activities such as anticancer, antimicrobial activity exhibited by the Andrographis paniculata plant is present in small quantities. In addition, the genus Andrographis has about 28 species most of which possess no medicinal value. The deoxyribonucleic acid (DNA) barcode is utilized in species identification and plant authentication.Objectives: This study aimed at authenticating Andrographis paniculata using DNA barcodes and improving the yield of andrographolide via enzymatic treatment.Materials and Method: The DNA of Andrographis plant was obtained using the Qiagen kit. The psbA-trnH and rbcL DNA barcode regions were amplified using polymerase chain reaction (PCR). Presence of amplified regions was confirmed using gel electrophoresis and the amplicons were sequenced. A blast N search was performed on the sequenced DNA. The constituents of A. paniculata dried leaves was extracted using methanol, followed by treatment with and without β glucosidase. The extract obtained was dried and partitioned using ethyl acetate. The ethyl acetate fraction was concentrated and dissolved in methanol. Andrographolide content was determined using high performance liquid chromatography (HPLC).Results: The psbA-trnH and rbcL DNA regions were successfully amplified having 358 and 604 bp respectively. The DNA barcode sequences obtained were identical to the psbA-trnH (97%) and rbcL (99%) genes of A. paniculata voucher MICET P00101. The mean andrographolide yield was 9.4±0.11mg/g and 8.9±0.13mg/g dry weight for the treatment and control groups respectively; statistical analysis at p = 0.05 shows a significant difference.Conclusion: The Andrographis plant used in this study was confirmed to be Andrographis paniculata, enzymatic treatment increased andrographolide yield from the plant.&#x0D; Keywords: Andrographis paniculata, andrographolide, authentication, DNA barcodes, β-glucosidase.

Evaluation of a New Rapid Antigen Detection Test for the Diagnosis of Influenza Virus Infection in a Tertiary Care Hospital of Southern India

Introduction: Rapid antigen testing can help in early diagnosis of Influenza in resource limited settings in patients presenting with Influenza Like Illness (ILI). Aim: To evaluate the usefulness of Rapid Influenza Direct antigen Testing (RIDT) in comparison to Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for diagnosis of Influenza virus infection in clinically suspected patients with ILI in a tertiary care hospital. Material and Methods: A prospective observational independent study was done to evaluate RIDT for Influenza virus infection, considering RT-PCR as gold standard. Throat swabs and nasal swabs from patients meeting Category 3 definition of ILI by Ministry Of Health and Family Welfare (MOHFW) were collected from July 20th, 2019 to October 20th, 2019 from hospitals in and around the study area. Onsite Rapid test (Biogenix, China) was used as per manufactures instructions for rapid detection of Influenza antigen. RNA extraction was done using Qiagen kit followed by RT-PCR using primer, probes recommended by National Institute of Virology (NIV, Pune) as per their protocols. Data was recorded on a predesigned proforma and managed using Microsoft Excel 2018 (Microsoft Corp, Redmond, WA). The Statistical software MedCalc version 9.1 for Windows was used for data analysis. Results: The overall sensitivity of the RIDT was 37.9% and specificity was 94.4%. The Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 73.3% and 78.8%, respectively. Kappa was calculated to be 0.37 that suggested a fair agreement between RIDT and RT-PCR. When the Cycle threshold (Ct) values were compared to the results of RIDT, there were more false positives at low Ct value and more false negatives at high Ct values. Conclusion: The RIDT is not useful in screening during an outbreak in acute care settings. The negative result of RIDT should be followed up with RT-PCR. With a strong clinical suspicion, it is better to start antivirals than wait for laboratory report in severely ill patients with ILI.

Plasmodium falciparum Allelic Diversity: A Comparison of DNA Extraction from Isolates Collected on Rapid Diagnostic Tests (Rdts) and Filter Paper

Background:To perform molecular epidemiologic studies based on large cohorts, material such as RDTs or filter papers are useful for biological sample collection and extraction of RNA or DNA of good quality. Thus, we aimed to assess the quality of DNA extracted from malaria rapid diagnostic tests (RDTs) stored at various temperatures for the analysis of Plasmodium falciparum genetic diversity.Methods:Febrile patients benefitted from free malaria diagnosis using microscopy in a malaria sentinel site, at the Regional Hospital Estuaire-Melen, in Gabon, in 2015. P. falciparum isolates were collected onto one filter paper and 2 similar RDTs devices (Acon®) per patient. Nucleic acids were extracted with QiAmp Qiagen kit from paper and RDTs and the quality of the DNA was analyzed by msp1 gene amplification.Results:Msp1gene amplification was achieved in nucleic acids extracted from all filter papers and RDTs devices (n = 45, 100%). K1 alleles were detected in 93.3% (n = 42/45) of the samples and Mad20 alleles in 73.3% (n = 33/45). The number and the intensity of K1 and/or Mad20 fragments were comparable according to the sample collection material and the storage conditions (room temperature vs −20°C) of the samples. The size of the fragments indicating allelic diversity was comparable in 80% (n=36) of the samples.Conclusion:These data show that RDTs are a valuable source of DNA for malaria parasite genetic polymorphism analysis. Storage conditions of the devices did not influence the quality of DNA extracted from RDTs device, although some alleles may be missed.

Comparison of performances between three commercial ELISA kits for detection of antibodies against Porcine Reproductive and Respiratory Syndrome Virus (PPRSV) in swine sera samples

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases in pigs, worldwide. Just in the US, the total costs to the swine industry have been estimated at $664 million per year. Therefore, the continuous and reliable monitoring of the PRRS status of a pig herd is required in order to prevent and reduce costs due to this infection. Mostly used methods for diagnosis of PRRS infection nowadays are serological (ELISA) and molecular (PCR) ones. This study aimed to assess the sensitivity and specificity of three different commercially available ELISA kits for detection of antibodies against PRRSV (IDEXX PRRS X3 Ab Test (IDEXX, USA), INgezim PRRS Universal (Ingenasa, Spain), Pigtype PRRSV Ab (Qiagen, Germany)) using 91 blood serum samples collected from pigs in Serbia. Our study showed no significant differences in specificity and sensitivity between three commercially available ELISA kits. However, IDEXX ELISA proved to be more reliable kit for detecting antibodies against PRRSV with sensitivity of 97,4% and specificity of 98,1%, considering INgezim and Qiagen kits specificity of 92,5% and 83%, respectively, and sensitivity of 94,7 % for both kits. In order to achieve maximal reliability of obtained results, ELISA diagnostic protocol for diagnosis of PRRS infection should be complemented with additional tests such as PCR and virus neutralization test.

Whole genome sequencing and genome annotation of Dermacoccus abyssi strain HZAU 226 isolated from spoiled eggs

Dermacoccus abyssi strain HZAU 226 is a spoilage bacterium isolated from eggs. So far, there are still few genomic resources available on the Dermacoccus abyssi. Here, we reported the complete genome sequence of Dermacoccus abyssi strain HZAU 226. High-quality DNA was extracted using the Qiagen kit, then single-molecule sequencing was performed by GridION sequencer. The raw data was quality-controlled and assembled to obtain the final genome, which consisted of a complete genome of 2,992,060 bp circular chromosome and a 64,524 bp plasmid. The structural and functional annotations of the genome were achieved through the analysis of different available databases, including antibiotic resistance genes, secondary metabolite synthesis genes and stress-related genes. Meanwhile, comparative genomic analyses of the strains were also performed. This is the first report on the complete genome of Dermacoccus abyssi, which will provide genomic resources for the study of spoilage bacteria in eggs

Epidemiology and immuno-molecular status of Nepal dengue outbreak

Background: The emergence and circulation of dengue infection have become one of the major public health concerns in the world with its high prevalence in the sub-tropical and tropical countries. Hundreds of Dengue cases have been reported annually and the shift of serotypes in every year has raised the concern of public health in Nepal. The detection of dengue virus infections and circulation of different serotypes in different areas has a great role for the clinical management of patients, surveillance, and clinical trial assessments in the days to come. Methods and materials: Clinically suspected dengue cases (n = 240) were subjected to sero analysis for anti-dengue IgM, IgG antibody and NS1 antigen followed by IgG end-point ELISA. Viral RNA extraction was performed for all samples using the Qiagen kit. cDNA was prepared, and RT-PCR was performed using D1 and DencomR2 primers. Dengue Serotyping was done for PCR positive samples using gene-specific primers. Results: Out of 240 dengue suspected cases, male (n = 153/63.75%), female (n = 87/36.25%) and children below 10 years (6.25%) were diagnosed as positive by ELISA. NS1 (59.16%), IgM (33%) and IgG (7.9%) were detected by ELISA, however, only 14 samples were positive alone to each test. Most of the cases were found to be of primary infection. Antibody level for each serum was calculated and categorized as high (43228.70), medium and low (953.53). Only 35.83% of the suspected cases were confirmed positive for dengue virus by PCR and were of Dengue Serotype-1 in the year 2016 in Nepal. Conclusion: The single test alone could not be confirmatory as only 35.83% of the clinically suspected cases were confirmed for Dengue virus infection by PCR. This implies that confirmatory tests like PCR should be coupled with the ELISA results for the proper diagnosis and management of the disease.