scholarly journals Use of Nested and Real-Time PCR for the Detection of Ceratocystis fagacearum in the Sapwood of Diseased Oak Species in Minnesota

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 480-486 ◽  
Author(s):  
A. Yang ◽  
J. Juzwik
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Detection Methods ◽  
Red Oak ◽  
Eastern United States ◽  
White Oak ◽  
Oak Wilt ◽  
Ceratocystis Fagacearum ◽  
Oak Trees

Oak wilt caused by Ceratocystis fagacearum is a significant disease of Quercus spp. in the eastern United States. Early and accurate detection of the pathogen is particularly important when disease control is planned. Nested and real-time polymerase chain reaction (PCR) methods utilizing fungal DNA extracted from sapwood drill shavings of red, bur, and white oak at different stages of disease development were compared with culture-based detection from sapwood. The pathogen was detected in all (n = 3) actively wilting branches of each of nine red oak trees using all three methods. The lowest detection rate (33% of assayed branches; 6 of 8 trees) for actively wilting branches was found for white oak using isolation while nested PCR had a branch detection rate of 100% (8 of 8 trees) and real-time PCR of 87% (8 of 8 trees) for the same samples. For both bur and white oak, the pathogen was not detected by isolation in branches over 1 year after their death but was detected using both PCR methods. Only the PCR assays detected the fungus in sapwood samples underlying remnants of sporulation mats (n = 21; 90%, nested and 62%, real-time) on red oak. These PCR methods offer several significant improvements for laboratory-based detection methods of C. fagacearum.

scholarly journals Three Colopterus Beetle Species Carry the Oak Wilt Fungus to Fresh Wounds on Red Oak in Missouri

Plant Disease ◽  
2008 ◽  
Vol 92 (2) ◽  
pp. 270-275 ◽  
Author(s):  
Maya Hayslett ◽  
Jennifer Juzwik ◽  
Bruce Moltzan
Keyword(s):  
Abundant Species ◽  
Pathogen Transmission ◽  
Red Oak ◽  
Beetle Species ◽  
Vector Species ◽  
Oak Wilt ◽  
Ceratocystis Fagacearum ◽  
Oak Trees ◽  
The Family ◽  
Nitidulid Beetles

Beetles in the family Nitidulidae can transmit the oak wilt fungus, Ceratocystis fagacearum, to fresh wounds on healthy oak trees, leading to infection and disease development. Historically, nitidulid beetles have not been considered important vectors of the pathogen in Missouri. Studies were conducted in the spring of 2005 and 2006 to determine frequencies of nitidulid beetle species contaminated with C. fagacearum visiting fresh wounds on red oak trees in central Missouri. Colopterus truncatus, C. niger, and C. semitectus were the most abundant species collected from fresh wounds and the only species found to be contaminated with Ceratocystis fagacearum. Of 230 beetles assayed for C. fagacearum, 23 yielded the fungus. Contamination frequencies were higher for beetles collected in April than May; no beetles collected in June were contaminated. We hypothesize that Colopterus truncatus, C. niger, and C. semitectus are principal nitidulid beetle vector species in Missouri during spring. The risk for pathogen transmission by these beetles appears to be greatest in April and least in June.

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.

O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Panacheva ◽  
D Pochernikov ◽  
E Voroshilina
Keyword(s):  
Cluster Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Rt Pcr ◽  
Silhouette Index ◽  
Enterococcus Spp ◽  
Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

Biology and Control of Oak Wilt in Michigan Red Oak Stands

1992 ◽  
Vol 9 (2) ◽  
pp. 47-51 ◽  
Author(s):  
Johann N. Bruhn ◽  
Robert L. Heyd
Keyword(s):  
Public Relations ◽  
Control Measure ◽  
Integrated Approach ◽  
Early Summer ◽  
Red Oak ◽  
Aerial Surveillance ◽  
Oak Wilt ◽  
Oak Trees ◽  
And Control ◽  
Sanitary Treatment

Abstract This paper explains the components of an integrated approach to oak wilt control which has strong potential for adaptation to red oak forests throughout the Lake States. Oak wilt epicenter containment involves (1) separating grafted root systems with a vibratory plow line or Vapam barrier, followed by (2) removal of all living red oaks inside the barrier, and sanitary treatment of wood capable of producing fungal mats. Use of a reference table developed for proper barrier location is described. Epicenters are established by insect vectors, often as a consequence of human activity. Establishment of epicenters can be prevented by: (1) sanitary treatment of diseased trees which may yet produce mats, (2) discouraging unsupervised movement of diseased wood, (3) minimizing the wounding of oaks during the spring and early summer, and (4) immediately painting all spring and summer wounds on oak trees with a tree wound dressing. Detection of new epicenters is important. Treatment of small epicenters minimizes site disturbance and the number of fungal mats produced, as well as the number of trees lost. Systematic aerial surveillance is useful for effective detection in large acre-ages of oak type. Prevention is the best control measure. Unfortunately, public relations are often inadequate. North. J. Appl. For. 9(2):47-51

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.

Increase EGFR Mutations Detection Rate in Lung Adenocarcinoma by Real-Time PCR Screening Followed by Direct Sequencing

2015 ◽  
Vol 23 (5) ◽  
pp. 343-348 ◽  
Author(s):  
Tze-Kiong Er ◽  
Chin-Wen Lin ◽  
Ta-Chih Liu ◽  
Chih-Chieh Chen ◽  
Li-Hsuan Wang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Direct Sequencing ◽  
Egfr Mutations ◽  
Pcr Screening

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.

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