scholarly journals The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type

Plant Disease ◽  
2018 ◽  
Vol 102 (11) ◽  
pp. 2187-2193 ◽  
Author(s):  
Felicia J. Setiono ◽  
Debotri Chatterjee ◽  
Marc Fuchs ◽  
Keith L. Perry ◽  
Jeremy R. Thompson
Keyword(s):  
Polymerase Chain Reaction ◽  
Seasonal Variations ◽  
Quantitative Polymerase Chain Reaction ◽  
Virus Titer ◽  
Tissue Type ◽  
Chain Reaction ◽  
Variable Distribution ◽  
Qpcr Method ◽  
Polymerase Chain ◽  
Virus Distribution

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

scholarly journals Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

2012 ◽  
Vol 10 (4) ◽  
pp. 594-604 ◽  
Author(s):  
Maria Raynal ◽  
Eric N. Villegas ◽  
Kara L. Nelson
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Quantitative Polymerase Chain Reaction ◽  
High Heat ◽  
Chain Reaction ◽  
Direct Microscopy ◽  
Development Stages ◽  
Target Dna ◽  
Qpcr Method ◽  
Polymerase Chain

The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH3-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

scholarly journals Quantification of Botrytis cinerea in Grapevine Bunch Trash by Real-Time PCR

2019 ◽  
Vol 109 (7) ◽  
pp. 1312-1319 ◽  
Author(s):  
Melissa Si Ammour ◽  
Giorgia Fedele ◽  
Caterina Morcia ◽  
Valeria Terzi ◽  
Vittorio Rossi
Keyword(s):  
Polymerase Chain Reaction ◽  
Botrytis Cinerea ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Qpcr Method ◽  
Polymerase Chain ◽  
Bunch Rot ◽  
Reliable Quantification

Quantification of colonization of grape bunch trash by Botrytis cinerea is crucial for Botrytis bunch rot (BBR) control. A previously developed quantitative polymerase chain reaction (qPCR) method was adapted to quantify B. cinerea DNA in grape bunch trash, and a colonization coefficient (CC) was calculated as the ratio between the DNA concentrations of B. cinerea and of Vitis vinifera. CC values increased linearly with the number of conidia of B. cinerea or the quantity of mycelium of B. cinerea added to the bunch trash increased. CC values also increased linearly in bunch trash samples containing increasing percentages of B. cinerea-colonized bunch trash; in the latter samples, CC values were correlated with subsequent assessments of B. cinerea colonization of trash (as determined by plating on agar) and sporulation on the trash (as determined by spore counts after incubation in humid chambers). The qPCR assay was also validated using trash collected from bunches treated or not treated with fungicides in three vineyards in two seasons. CC values reflected the reduction in sporulation and in latent infections of mature berries caused by fungicide application. The qPCR assay enables rapid, specific, sensitive, and reliable quantification of the degree of colonization of bunch trash by B. cinerea, which makes it a useful tool for studies of the epidemiology and management of BBR.

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.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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