scholarly journals Efficient Diagnosis of Ratoon Stunting Disease of Sugarcane by Quantitative PCR on Pooled Leaf Sheath Biopsies

Plant Disease ◽  
2016 ◽  
Vol 100 (12) ◽  
pp. 2492-2498 ◽  
Author(s):  
Anthony J. Young ◽  
Asuka Kawamata ◽  
Mark A. Ensbey ◽  
Eleanore Lambley ◽  
Catherine J. Nock
Keyword(s):  
Phase Contrast ◽  
Quantitative Polymerase Chain Reaction ◽  
Xylem Sap ◽  
Diagnostic Techniques ◽  
Leaf Sheath ◽  
Conventional Pcr ◽  
Ratoon Stunting Disease ◽  
Contrast Microscopy ◽  
Plate Counts ◽  
Polymerase Chain

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, is arguably one of the most devastating diseases of sugarcane. Four diagnostic techniques were compared for 100 fields of sugarcane (Saccharum interspecific hybrids) of unknown infection status. These were quantitative polymerase chain reaction on pooled leaf sheath biopsies (LSB-qPCR), conventional PCR on the same templates (LSB-PCR), evaporative-binding enzyme immunoassay (EB-EIA) coupled with phase contrast microscopy (PCM) on expressed xylem sap from the same fields, and conventional PCR on the same xylem sap samples. LSB-qPCR and LSB-PCR detected the causal agent in 27 and 18 fields, respectively, whereas, from samples of expressed xylem sap from the same fields, conventional PCR identified 12 infections and EB-EIA/PCM detected L. xyli subsp. xyli in 3 fields. The sensitivities of qPCR and PCR were approximately 103 and 104 CFU ml−1, respectively, determined from plate counts of a dilution series. Tests were conducted on a further 139 LSB samples from across the Australian industry, with qPCR and PCR diagnosing RSD in 31 and 25 fields, respectively. Using qPCR and PCR on LSB samples, RSD was diagnosed in a range of cultivars throughout the year, and qPCR and PCR could detect L. xyli subsp. xyli in sugarcane ranging from 3 months to greater than 1 year old.

scholarly journals Molecular Detection of Diverse Leifsonia Strains Associated With Sugarcane

Plant Disease ◽  
2017 ◽  
Vol 101 (8) ◽  
pp. 1422-1431 ◽  
Author(s):  
Anthony J. Young ◽  
Catherine J. Nock
Keyword(s):  
Interspecific Hybrids ◽  
Molecular Detection ◽  
Phylogenetic Analyses ◽  
Xylem Sap ◽  
Leaf Sheath ◽  
Ratoon Stunting Disease ◽  
Sugarcane Varieties ◽  
Multiple Samples ◽  
Broad Complex ◽  
Epidemiological Significance

Leifsonia xyli subsp. xyli, causal agent of ratoon stunting disease (RSD) of sugarcane (Saccharum interspecific hybrids), is the most well-known member of the Microbacteriaceae genus Leifsonia. However, the presence of other Leifsonia strains associated with sugarcane has not been reported. A total of 697 Australian and 40 Indonesian sugarcane fields were screened by leaf sheath biopsy (LSB) PCR using primers specific for L. xyli subsp. xyli, in addition to primers designed to amplify DNA from other members of the genus Leifsonia. While L. xyli subsp. xyli was detected in 126 fields, a total of 37 distinct and novel Leifsonia and non-Leifsonia strains were detected in 116 fields. Representatives of these strains were also detected in multiple samples of expressed xylem sap. Sequencing and phylogenetic analyses demonstrated the presence of a broad complex of novel Leifsonia strains, in addition to strains closely related to the recently erected Cnuibacter genus. Attempts to isolate Leifsonia strains were unsuccessful; however, one strain related to Cnuibacter was recovered from expressed xylem sap. Among the genetically diverse lineages discovered, identical genotypes were present in multiple sugarcane varieties growing in disparate regions in different years, strongly suggesting an ongoing association with sugarcane. The epidemiological significance of these strains is unknown, but there is evidence that they can interfere with serological and microscopic RSD diagnostics, and there is the potential that they may represent new and distinct pathologies of sugarcane.

scholarly journals Diagnosis of Schistosoma Infection in Non-Human Animal Hosts: A Systematic Review and Meta-Analysis

2021 ◽  
Author(s):  
Song Liang ◽  
Keerati Ponpetch ◽  
Yi-Biao Zhou ◽  
Jia-Gang Guo ◽  
Berhanu Erko ◽  
...  
Keyword(s):  
Systematic Review ◽  
Meta Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Techniques ◽  
Molecular Technique ◽  
Significant Heterogeneity ◽  
Animal Hosts ◽  
Polymerase Chain ◽  
Human Animal ◽  
Meta Analyses

Background Reliable and field-applicable diagnosis of schistosome infections in non-human animals is important for surveillance, control, and verification of interruption of human schistosomiasis transmission. This study aimed to summarize uses of available diagnostic techniques through a systematic review and meta-analysis. Methods and principal findings We systematically searched the literature and reports comparing two or more diagnostic tests in non-human animals for schistosome infection. Out of 4,909 articles and reports screened, 18 met our inclusion criteria, four of which were considered in the meta-analysis. A total of 14 techniques (parasitologic, immunologic, and molecular) and nine types of non-human animals were involved in the studies. Notably, four studies compared parasitologic tests (miracidium hatching test (MHT), Kato-Katz (KK), the Danish Bilharziasis Laboratory technique (DBL), and formalin-ethyl acetate sedimentation-digestion (FED-SD)) with quantitative polymerase chain reaction (qPCR), and sensitivity estimates (using qPCR as the reference) were extracted and included in the meta-analyses, showing significant heterogeneity across studies and animals hosts. The pooled estimate of sensitivity was 0.21 (95% confidence interval (CI): 0.03 – 0.48) with FED-SD showing highest sensitivity (0.89, 95% CI: 0.65 – 1.00). Conclusions and significance Our findings suggest that the parasitologic technique FEA-SD and the molecular technique, qPCR, are the most promising field-applicable techniques for schistosome diagnosis in non-human animal hosts. Future studies are needed for validation and standardization of the techniques for real-world field applications.

scholarly journals Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 833
Author(s):  
Phuc H. Pham ◽  
Ehab Misk ◽  
Fotini Papazotos ◽  
Ginny Jones ◽  
Mark P. Polinski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Quantitative Polymerase Chain Reaction ◽  
Head Kidney ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Rna Amplification ◽  
Host Tropism ◽  
Contrast Microscopy ◽  
Polymerase Chain ◽  
Kidney Liver

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

scholarly journals Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer

Biomedicines ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 447
Author(s):  
Julia Oto ◽  
Emma Plana ◽  
Álvaro Fernández-Pardo ◽  
Fernando Cana ◽  
Manuel Martínez-Sarmiento ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Diagnostic Marker ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Techniques ◽  
Expression Study ◽  
Non Invasive ◽  
Good Expression ◽  
Polymerase Chain ◽  
Early Diagnose ◽  
Independent Cohort

Bladder cancer (BC) is among the most frequent malignancies worldwide, being the most expensive cancer to treat and monitor and the most lethal urological cancer. Urine microRNAs (miRNAs) have been proposed as novel non-invasive biomarkers to early diagnose and monitor BC patients in order to avoid the performance of current aggressive diagnostic techniques. However, huge discrepancies arise among studies mainly due to the lack of standardization in the normalization, a crucial step in all miRNA studies. Our aim was to identify the best miRNA normalizer for miRNA studies in urine of BC patients. We evaluated the performance of 110 candidate miRNAs in urine of 35 BC patients and 15 healthy controls by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) followed by a stability analysis with RefFinder. In this screening stage, miR-29c-3p arose as the most stably expressed miRNA in BC and controls, with a good expression level. Stability of miR-29c-3p expression was validated in an independent cohort of 153 BC patients and 57 controls. Finally, we evaluated the robustness of miR-29c-3p as normalizer in the expression study of miR-200c-3p, a potential diagnostic marker for BC. We propose miR-29c-3p as a normalizer for miRNA studies in BC urine. This is the first study that characterizes a reliable normalizer that may allow the comparison of future urine miRNA studies as non-invasive biomarkers for BC diagnosis and monitoring.

scholarly journals RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

Diagnostics ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1646
Author(s):  
Kasturi Selvam ◽  
Mohamad Ahmad Najib ◽  
Muhammad Fazli Khalid ◽  
Suharni Mohamad ◽  
Fahreddin Palaz ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Public Attention ◽  
Laboratory Equipment ◽  
Highly Sensitive ◽  
Polymerase Chain

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has attracted public attention. The gold standard for diagnosing COVID-19 is reverse transcription–quantitative polymerase chain reaction (RT-qPCR). However, RT-qPCR can only be performed in centralized laboratories due to the requirement for advanced laboratory equipment and qualified workers. In the last decade, clustered regularly interspaced short palindromic repeats (CRISPR) technology has shown considerable promise in the development of rapid, highly sensitive, and specific molecular diagnostic methods that do not require complicated instrumentation. During the current COVID-19 pandemic, there has been growing interest in using CRISPR-based diagnostic techniques to develop rapid and accurate assays for detecting SARS-CoV-2. In this work, we review and summarize reverse-transcription loop-mediated isothermal amplification (RT-LAMP) CRISPR-based diagnostic techniques for detecting SARS-CoV-2.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

Detachment of biofilm bacteria due to variations in nutrient supply

1998 ◽  
Vol 37 (4-5) ◽  
pp. 211-214 ◽  
Author(s):  
Linda K. Sawyer ◽  
Slawomir W. Hermanowicz
Keyword(s):  
Phase Contrast ◽  
Digital Image Analysis ◽  
Specific Growth ◽  
Nutrient Supply ◽  
Surface Shear Stress ◽  
Surface Shear ◽  
Nutrient Limitations ◽  
Contrast Microscopy ◽  
Biofilm Bacteria ◽  
Observable Parameter

Growth and detachment rates of an environmental isolate of Aeromonas hydrophila attached to a surface were determined under varying nutrient supply conditions in a complex medium. Growth and detachment of cells were observed in real time using phase contrast microscopy in glass parallel plate flow chambers. Surface shear stress was controlled in all experiments at 3 N m−2. Images were taken every 15 min. Digital image analysis was used to determine specific growth and detachment rates. An observable parameter proportional to the nutrient depletion at the surface due to transfer limitations was used to indicate nutrient limitations. Specific detachment rates increased as the depletion parameter increased, indicating that nutrient limitations cause this bacterium to detach at greater rates.

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