scholarly journals Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

2013 ◽  
Vol 79 (17) ◽  
pp. 5394-5404 ◽  
Author(s):  
Nicola Luchi ◽  
Luisa Ghelardini ◽  
Lassaâd Belbahri ◽  
Marion Quartier ◽  
Alberto Santini
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Urban Setting ◽  
Disease Spread ◽  
Pcr Assay ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Airborne Inoculum ◽  
Plane Trees ◽  
Quantitative Pcr Assay

ABSTRACTCeratocystis plataniis the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect aC. plataniairborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity forC. platani, detecting DNA concentrations as low as 1.2 × 10−2to 1.4 × 10−2pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect theC. plataniinoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of aC. plataniinoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.

scholarly journals Real-Time Quantitative PCR Assay for Monitoring of Nervous Necrosis Virus Infection in Grouper Aquaculture

2011 ◽  
Vol 49 (3) ◽  
pp. 1090-1096 ◽  
Author(s):  
H.-C. Kuo ◽  
T.-Y. Wang ◽  
P.-P. Chen ◽  
Y.-M. Chen ◽  
H.-C. Chuang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Real Time ◽  
Quantitative Pcr ◽  
Nervous Necrosis Virus ◽  
Necrosis Virus ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Quantitative Pcr Assay

scholarly journals Determination of RhD Zygosity: Comparison of a Double Amplification Refractory Mutation System Approach and a Multiplex Real-Time Quantitative PCR Approach

2001 ◽  
Vol 47 (4) ◽  
pp. 667-672 ◽  
Author(s):  
Rossa W K Chiu ◽  
Michael F Murphy ◽  
Carrie Fidler ◽  
Benny C Y Zee ◽  
James S Wainscoat ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Gene Dosage ◽  
Amplification Refractory Mutation System ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
Quantitative Pcr Assay

Abstract Background: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. Methods: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, ΔCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. Results: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the ΔCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. Conclusion: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

A novel real-time quantitative PCR assay for detection of Helicobacter pylori in the gastric mucosa

1998 ◽  
Vol 114 ◽  
pp. A114
Author(s):  
M. Endoh ◽  
T. Shimada ◽  
N. Watanabe ◽  
Y. Ohtsuka ◽  
K. Sakata ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
Real Time ◽  
Quantitative Pcr ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Quantitative Pcr Assay
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