scholarly journals Detection of Root-Knot Nematode Meloidogyne luci Infestation of Potato Tubers Using Hyperspectral Remote Sensing and Real-Time PCR Molecular Methods

2021 ◽  
Vol 13 (10) ◽  
pp. 1996
Author(s):  
Uroš Žibrat ◽  
Barbara Gerič Stare ◽  
Matej Knapič ◽  
Nik Susič ◽  
Janez Lapajne ◽  
...  
Keyword(s):  
Real Time ◽  
Hyperspectral Imaging ◽  
Real Time Pcr ◽  
Detection Methods ◽  
Parasitic Nematodes ◽  
Tuber Quality ◽  
Limiting Factor ◽  
Potato Tubers ◽  
Root Knot Nematode ◽  
Efficient Detection

Root-knot nematodes (Meloidogyne spp.) are considered the most aggressive, damaging, and economically important group of plant-parasitic nematodes and represent a significant limiting factor for potato (Solanum tuberosum) production and tuber quality. Meloidogyne luci has previously been shown to be a potato pest having significant reproductive potential on the potato. In this study we showed that M. luci may develop a latent infestation without visible symptoms on the tubers. This latent infestation may pose a high risk for uncontrolled spread of the pest, especially via seed potato. We developed efficient detection methods to prevent uncontrolled spread of M. luci via infested potato tubers. Using hyperspectral imaging and a molecular approach to detection of nematode DNA with real-time PCR, it was possible to detect M. luci in both heavily infested potato tubers and tubers without visible symptoms. Detection of infested tubers with hyperspectral imaging achieved a 100% success rate, regardless of tuber preparation. The real-time PCR approach detected M. luci with high sensitivity.

scholarly journals Ability of Real-time PCR in diagnose Differentiation Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

2019 ◽  
Author(s):  
Maryam Fekri Soofi Abadi ◽  
Meisam Fekri ◽  
alireza moradabadi ◽  
Reza Vahidi ◽  
Simin Shamsi Meymandi ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Relative Number ◽  
Parasite Load ◽  
Detection Methods ◽  
Tissue Samples ◽  
Microscopic Evaluation ◽  
Histopathological Evaluation ◽  
Histopathological Studies

Abstract objective: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify leishmania tropica parasites in paraffin-embedded tissue samples. Results: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The parasite loads were quantified by qPCR assay and microscopic evaluation were highly correlated ( r =0.598; P <0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite loads than chronic CL lesions), but there was no difference in parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P<0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P=0.549), which indicates the superiority of histopathological evaluation in CL forms differentiation.

scholarly journals Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

2006 ◽  
Vol 72 (3) ◽  
pp. 2031-2042 ◽  
Author(s):  
Linda N. Ward ◽  
Asim K. Bej
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Detection Methods ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Reading Frame ◽  
Initial Inoculum ◽  
Hemolysin Gene ◽  
Pure Cultures

ABSTRACT We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

2004 ◽  
Vol 67 (4) ◽  
pp. 823-832 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Food Microbiology ◽  
Detection Methods ◽  
Target Sequence ◽  
Bacterial Enumeration

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid–based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR–based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

2008 ◽  
Vol 56 (4) ◽  
pp. 451-458 ◽  
Author(s):  
Jitu Patel ◽  
Arvind Bhagwat
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Molecular Beacon ◽  
False Negative ◽  
Detection Methods ◽  
Time Data ◽  
Pcr Assay ◽  
Serovar Typhimurium ◽  
Meat Samples

A real-time PCR assay was evaluated for the rapid detection (10 h) ofSalmonellain meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated withSalmonella entericaserovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

scholarly journals Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

2008 ◽  
Vol 98 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Xinshun Qu ◽  
Leslie A. Wanner ◽  
Barbara J. Christ
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Common Scab ◽  
Cost Effective ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Streptomyces Species ◽  
Standard Curve ◽  
Target Dna

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R2 = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 101 to 106 pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 103 to 106 per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

scholarly journals Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation

2018 ◽  
Vol 101 (2) ◽  
pp. 507-514 ◽  
Author(s):  
Junichi Mano ◽  
Shuko Hatano ◽  
Yasuaki Nagatomi ◽  
Satoshi Futo ◽  
Reona Takabatake ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sensitive Detection ◽  
Detection Methods ◽  
35S Promoter ◽  
Processed Foods ◽  
Gmo Detection ◽  
Nos Terminator ◽  
Highly Sensitive ◽  
Dna Template

Abstract Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.

scholarly journals Efficient detection of Frankliniella schultzei (Thysanoptera, Thripidae) by cytochrome oxidase I gene (mtCOI) direct sequencing and real-time PCR

2018 ◽  
Vol 60 (0) ◽  
Author(s):  
Evelynne Urzêdo Leão ◽  
David Marques de Almeida Spadotti ◽  
Kelly Cristina G. Rocha ◽  
Élison Fabricio B. Lima ◽  
Luciana Tavella ◽  
...  
Keyword(s):  
Cytochrome Oxidase ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Sequencing ◽  
Cytochrome Oxidase I ◽  
Frankliniella Schultzei ◽  
Efficient Detection ◽  
Cytochrome Oxidase I Gene ◽  
I Gene
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