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.

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.

Multiple Displacement Amplification Enables Large Scale Clonal Analysis after Retroviral Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5469-5469
Author(s):  
Stephanie Bleier ◽  
Patrick Maier ◽  
Frederik Wenz ◽  
W. Jens Zeller ◽  
Stephanie Laufs ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Large Scale ◽  
Integration Site ◽  
Multiple Displacement Amplification ◽  
Quantitative Real Time Pcr ◽  
Integration Site Analysis ◽  
Reliable Quantification

Abstract Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole genome amplification named multiple displacement amplification (MDA) with respect to the even and accurate representation of retrovirally transduced genomic DNA. We were able to show that MDA is a suitable method to subsequently specify engraftment efficiencies by quantitative real-time PCR as the retroviral integrations are amplified the same way and by the same probability as all other parts of the genome. We validated the method by analyzing a dilution series containing retrovirally transduced DNA and untransduced background DNA and retroviral integrations found in primary material from a retroviral transplantation model by quantitative real-time PCR. The representation of the portion of retroviral DNA in the amplified samples was 0.9-fold (range 0.2 – 2.1-fold) of the portion determined in the original genomic DNA. Furthermore, the succession of the combination of MDA and integration site analysis by ligation-mediated PCR showed an increase in the sensitivity of the method as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show that MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of starting material.

scholarly journals Establishment of a simpler method for measuring HDL-microRNAs

2018 ◽  
Vol 56 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Hiroaki Ishikawa ◽  
Hiroya Yamada ◽  
Kanako Kondo ◽  
Takeru Ota ◽  
Mirai Yamazaki ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phosphotungstic Acid ◽  
Density Lipoprotein ◽  
Clinical Applications ◽  
Processing Capacity ◽  
Quantitative Real Time Pcr ◽  
Simple Method ◽  
Standard Curve ◽  
Purification Methods

Background MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications. Methods We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed. Results Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08–2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30–30,000 copies/ μL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×104). Conclusions We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

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 (179) Using Quantitative Real Time PCR to Predict Levels of Botrytis allii in Stored Onion

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1016D-1016
Author(s):  
Timothy Coolong ◽  
William Randle ◽  
Ronald Walcott
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dry Weight ◽  
The United States ◽  
Quantitative Real Time Pcr ◽  
Standard Curve ◽  
Ct Value ◽  
Botrytis Allii ◽  
Log Linear ◽  
A Minor

Onion (Allium cepa L.) is an economically important vegetable in the United States. Though considered a minor crop in terms of total acreage, onions have high value when compared to other crops and, nationally, their value approaches $800 million. Because harvested onions are routinely stored for long periods, disease can be a major obstacle to the industry. The primary disease reported in stored onions is botrytis neck rot caused by the fungus Botrytis allii (syn. B. aclada). Losses from neck rot can approach 35% of the stored crop. In order to accurately quantify the level of B. allii inoculum in bulbs at harvest to be able to predict potential botrytis neck rot in storage, a quantitative real-time PCR test to quantify levels of B. allii DNA present in onion bulb tissue has been developed. We have employed the TaqMan real time PCR assay and report log-linear (R2= 0.9915) relationship between B. allii DNA concentration and cycle threshold (Ct) value with a detection limit of 5 pico gram/microliter DNA. In addition, a log-linear standard curve plotting mycelial dry weight against Ct value has been developed to allow prediction of mycelial weight in onion tissue at harvest. Currently, the ability of this test to predict botrytis neck rot during storage is being tested.

scholarly journals Optimized Quantification of Unculturable Candidatus Liberibacter Spp. in Host Plants Using Real-Time PCR

Plant Disease ◽  
2008 ◽  
Vol 92 (6) ◽  
pp. 854-861 ◽  
Author(s):  
Wenbin Li ◽  
Dayan Li ◽  
Elizabeth Twieg ◽  
John S. Hartung ◽  
Laurene Levy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Disease Diagnosis ◽  
Amplification Efficiency ◽  
Citrus Species ◽  
Geographic Locations ◽  
Standard Curve ◽  
Candidatus Liberibacter ◽  
Universal Standard

Citrus huanglongbing (HLB) is caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. and is a destructive disease of citrus that is rapidly increasing in importance. The disease was reported recently in the principle citrus-producing areas of São Paulo, Brazil in 2004 and in Florida in 2005. A variety of laboratory methods have been developed to confirm a symptom-based disease diagnosis or for the detection or identification of the pathogen; however, no quantitative information has been available on the pathogen titer in either host or vector interactions because the pathogen remains unculturable in artificial media. We previously developed a quantitative polymerase chain reaction (PCR)-based assay for detection of Ca. Liberibacter spp. and, in this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan real-time PCR. Standard curves were established using cloned plasmids containing target DNA from the pathogen and with total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than the standard curve constructed from plasmids containing the amplification targets as cloned inserts. Nontarget DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in “spiked” healthy citrus DNA extracts. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various citrus tissues of different citrus species planted in different geographic locations. Sample storage at 4°C for 2 months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host–pathogen interactions and epidemiology, and in the development of control strategies for the disease.

Sign in / Sign up

Export Citation Format

Share Document