scholarly journals Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples

2021 ◽  
Author(s):  
Mahboubeh Berizi ◽  
Jalal Babaie ◽  
Pezhman Fard-Esfahani ◽  
Marjan Enshaeieh ◽  
Rahmah Noordin ◽  
...  
Keyword(s):  
Detection Limit ◽  
Toxoplasma Gondii ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dynamic Range ◽  
Quantitative Real Time Pcr ◽  
Linear Dynamic Range ◽  
Standard Curve ◽  
Linear Dynamic ◽  
Highly Sensitive

Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

scholarly journals Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

2007 ◽  
Vol 73 (20) ◽  
pp. 6557-6565 ◽  
Author(s):  
Pascal E. Saikaly ◽  
Morton A. Barlaz ◽  
Francis L. de los Reyes
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Fate And Transport ◽  
Biological Warfare ◽  
Quantitative Real Time Pcr ◽  
Pcr Assays ◽  
Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

scholarly journals Quantitation of Cell-Free and Cell-Associated Kaposi's Sarcoma-Associated Herpesvirus DNA by Real-Time PCR

2000 ◽  
Vol 38 (5) ◽  
pp. 1992-1995 ◽  
Author(s):  
Irene E. White ◽  
Thomas B. Campbell
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Kaposi's Sarcoma ◽  
Dynamic Range ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Linear Dynamic Range ◽  
Pcr Assay ◽  
Herpesvirus 8 ◽  
Linear Dynamic

A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 105 copies of KSHV DNA (r 2 > 0.99). The accuracy of DNA purification and quantitation was less than ±0.4 log10copies for samples that contained from 10 to 105 copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0.1 to 10−5, and cell-free KSHV DNA in plasma was quantitated over a range of 100 to 106 copies/ml. Real-time PCR provides a convenient method for quantitation of cell-free and cell-associated KSHV DNA in laboratory and clinical specimens.

Highly Sensitive and Broadband Organic Photodetectors with Fast Speed Gain and Large Linear Dynamic Range at Low Forward Bias

Small ◽  
2017 ◽  
Vol 13 (24) ◽  
pp. 1603260 ◽  
Author(s):  
Riming Nie ◽  
Xianyu Deng ◽  
Lei Feng ◽  
Guiguang Hu ◽  
Yangyang Wang ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Forward Bias ◽  
Linear Dynamic Range ◽  
Linear Dynamic ◽  
Fast Speed ◽  
Highly Sensitive ◽  
Organic Photodetectors

Detection ofOphiocordyceps sinensisin soil by quantitative real-time PCR

2013 ◽  
Vol 59 (3) ◽  
pp. 204-209 ◽  
Author(s):  
Qingyun Peng ◽  
Xin Zhong ◽  
Wei Lei ◽  
Guren Zhang ◽  
Xin Liu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Amplification Efficiency ◽  
Horizontal Distance ◽  
Quantitative Real Time Pcr ◽  
Internal Transcribed Spacer Region ◽  
Soil Dna ◽  
Standard Curve ◽  
Pcr Targeting

Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10of the initial amount of O. sinensis genomic DNA (r2= 0.999) over 7 orders of magnitude (4 × 101to 4 × 107fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P < 0.05). Our protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

Highly sensitive all-polymer photodetectors with ultraviolet-visible to near-infrared photo-detection and its application as optical switch

2021 ◽  
Author(s):  
Zijin Zhao ◽  
Baiqiao Liu ◽  
Chunyu Xu ◽  
Ming Liu ◽  
Kaixuan Yang ◽  
...  
Keyword(s):  
External Quantum Efficiency ◽  
Near Infrared ◽  
Dynamic Range ◽  
Optical Switch ◽  
Signal To Noise Ratio ◽  
Linear Dynamic Range ◽  
Signal To Noise ◽  
Linear Dynamic ◽  
Highly Sensitive ◽  
Photo Detection

All-polymer photodetectors with photomultiplication are fabricated with PMBBDT and N2200 as photoactive layers, exhibiting external quantum efficiency of 20700% under 4 V. Signal-to-noise ratio, linear dynamic range and specific detectivity...

Convenient Fiber-Optic-Based Sample Cell for Shpol'skii and Low-Temperature Phosphorescence Spectrometry

1989 ◽  
Vol 43 (3) ◽  
pp. 422-425 ◽  
Author(s):  
Richard T. Madison ◽  
Mary K. Carroll ◽  
Gary M. Hieftje
Keyword(s):  
Low Temperature ◽  
Fiber Optic ◽  
Dynamic Range ◽  
Detection Limits ◽  
Linear Dynamic Range ◽  
Sample Cell ◽  
Linear Dynamic ◽  
Light Guides

A sample cell for observing the Shpol'skii effect at 77 K is described and analytically assessed. The cell employs fiber-optic light guides to transport excitation and emission radiation. The system is compact, inexpensive, and simple to construct from commercially available laboratory components, and it alleviates several problems inherent in conventional refrigerated-cell designs. Detection limits for anthracene, coronene, and pyrene obtained with the sample cell are 8.8 × 10−8 M, 8.4 × 10−7 M, and 3.5 × 10−7 M, respectively. The linear dynamic range for each compound is 2 to 3 orders of magnitude.

Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
Author(s):  
ANNA-CLARA RÖNNER ◽  
HANS LINDMARK
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Close Correlation ◽  
Quantitative Detection ◽  
Quantitative Real Time Pcr ◽  
Direct Plating ◽  
Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

Sign in / Sign up

Export Citation Format

Share Document