scholarly journals Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica

2000 ◽  
Vol 38 (9) ◽  
pp. 3429-3435 ◽  
Author(s):  
J. Hoorfar ◽  
P. Ahrens ◽  
P. Rådström
Keyword(s):  
Salmonella Enterica ◽  
Relative Sensitivity ◽  
Positive Control ◽  
Taqman Assay ◽  
Diagnostic Specificity ◽  
Pcr Assay ◽  
Sample Tube ◽  
Simple Alternative ◽  
Taqman Pcr ◽  
Internal Positive Control

A simple and ready-to-go test based on a 5′ nuclease (TaqMan) PCR technique was developed for identification of presumptiveSalmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic “rough” isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonellastrains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [ΔRn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (ΔRn, ≤0.5). All 100 roughSalmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at −20°C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification ofSalmonella, particularly in large reference laboratories.

scholarly journals Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control

2017 ◽  
Vol 29 (3) ◽  
pp. 351-356
Author(s):  
Amaresh Das ◽  
Ming Y. Deng ◽  
Shawn Babiuk ◽  
Michael T. McIntosh
Keyword(s):  
Real Time ◽  
False Negative ◽  
Positive Control ◽  
Lumpy Skin Disease ◽  
Outbreak Management ◽  
Negative Results ◽  
Beta Actin ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Analytical Sensitivities

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Claudin-5-positive angioleiomyoma in the uterus of a degu ( Octodon degus )

2010 ◽  
Vol 58 (3) ◽  
pp. 331-340 ◽  
Author(s):  
Csaba Jakab ◽  
Miklós Rusvai ◽  
Nóra Biró ◽  
Zoltán Szabó ◽  
Péter Gálfi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Immunohistochemical Analysis ◽  
Positive Control ◽  
Histopathological Diagnosis ◽  
Mesenchymal Tumour ◽  
Tissue Mass ◽  
Octodon Degus ◽  
Internal Positive Control ◽  
The Right ◽  
Endothelial Marker

A 5-year-old female degu ( Octodon degus ) showed the clinical sign of metrorrhagia. During ovariohysterectomy a circumscribed tumoural lesion was found in the right uterine horn. The histopathological diagnosis of this soft tissue mass was primary benign cavernous angioleiomyoma of the uterus. During immunohistochemical analysis the neoplastic endothelial cells of this mixed mesenchymal tumour showed strong membrane positivity for the endothelial marker claudin-5 but were negative for CD31 (another endothelial marker). The endothelial cells of the internal positive control tissues such as intact peritumoural vessels were positive for claudin-5 but negative for the CD31 endothelial marker. As it has been described also in other species, it seems that claudin-5 is a better endothelial marker than CD31 for the detection of normal and neoplastic endothelial cells in different tissues of degus.

scholarly journals Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive control

2020 ◽  
Vol 127 (6) ◽  
pp. 763-767
Author(s):  
Luitgardis Seigner ◽  
Marion Liebrecht ◽  
Linda Keckel ◽  
Katharina Einberger ◽  
Carolin Absmeier
Keyword(s):  
Real Time ◽  
Detection Method ◽  
Plant Protection ◽  
False Negative ◽  
Positive Control ◽  
Pcr Detection ◽  
Economic Losses ◽  
Rt Pcr ◽  
Validation Data ◽  
Internal Positive Control

Abstract Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.

scholarly journals Multiplex PCR for Detection of Genes forStaphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin Resistance

2000 ◽  
Vol 38 (3) ◽  
pp. 1032-1035 ◽  
Author(s):  
Manisha Mehrotra ◽  
Gehua Wang ◽  
Wendy M. Johnson
Keyword(s):  
Multiplex Pcr ◽  
Toxic Shock Syndrome ◽  
Methicillin Resistance ◽  
Positive Control ◽  
Toxic Shock ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Internal Positive Control ◽  
Toxic Shock Syndrome Toxin

A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB,entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection offemA was used as an internal positive control. The multiplex PCR assay combined the primers for sea tosee and femA in one set and those foreta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.

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