Treatment of cytomegalovirus retinitis: intravitreal injection of antiviral drugs under the control of the real time polymerase chain reaction of anterior chamber fluid

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription–PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

Download Full-text

Molecular diagnostics in clinical parasitology and mycology: limits of the current polymerase chain reaction (PCR) assays and interest of the real-time PCR assays

Clinical Microbiology and Infection ◽

10.1046/j.1469-0691.2003.00677.x ◽

2003 ◽

Vol 9 (6) ◽

pp. 505-511 ◽

Cited By ~ 55

Author(s):

S. Bretagne

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Molecular Diagnostics ◽

Real Time Pcr ◽

Chain Reaction ◽

The Real ◽

Polymerase Chain ◽

Pcr Assays

Download Full-text

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2014000100005 ◽

2014 ◽

Vol 34 (1) ◽

pp. 29-33 ◽

Cited By ~ 4

Author(s):

Gisele M. Bacanelli ◽

Carlos A. N. Ramos ◽

Flábio R. Araújo

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Anaplasma Marginale ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Chain Reaction ◽

The Real ◽

End Point ◽

Polymerase Chain

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

Download Full-text

Protein gene candidates for the qualitative molecular detection of Theileria parva in African buffalo (Syncerus caffer) using the real-time SYBR Green polymerase chain reaction

10.5176/2382-5685_vetsci13.20 ◽

2013 ◽

Cited By ~ 1

Author(s):

Ronel Pienaar ◽

Abdalla A. Latif ◽

Ben J. Mans ◽

Oriel M. M. Thekisoe

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Molecular Detection ◽

Protein Gene ◽

Theileria Parva ◽

African Buffalo ◽

Syncerus Caffer ◽

Chain Reaction ◽

The Real ◽

Polymerase Chain

Download Full-text

Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses

Phytopathology ◽

10.1094/phyto.2002.92.8.870 ◽

2002 ◽

Vol 92 (8) ◽

pp. 870-876 ◽

Cited By ~ 88

Author(s):

Min Qi ◽

Yinong Yang

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Northern Blot ◽

Host Resistance ◽

Real Time Pcr ◽

Magnaporthe Grisea ◽

Chain Reaction ◽

Fungal Pathogenicity ◽

The Real ◽

Polymerase Chain

Rice blast, caused by Magnaporthe grisea, is a serious fungal disease of rice worldwide. Currently, evaluation of the fungal pathogenicity and host resistance is mainly based on a disease rating or measurement of blast lesion number and size. However, these methods only provide visual estimation rather than accurate measurement of fungal growth in rice plants. In this study, DNA-based real-time polymerase chain reaction (PCR) and RNA-based northern blot/phosphoimaging analyses were evaluated to quantify M. grisea. Both methods were sensitive, specific, and reproducible and could accurately measure the relative growth and absolute biomass of M. grisea. The real-time PCR analysis showed that the growth of M. grisea in seedling leaves of susceptible cultivars (M201 and Wells) was ≈46 to 80 times higher than that of a resistant cultivar (Drew) at 4 and 6 days after inoculation. The data obtained from the real-time PCR assays also were consistent with that from northern blot/ phosphoimaging analysis. However, the real-time PCR approach was much faster and more convenient in most cases. Therefore, it is an excellent tool for in planta quantification of M. grisea and can be used for reliable assessment of fungal pathogenicity and host resistance

Download Full-text