Rapid Detection of DNA Methylation with a Novel Real-Time Fluorescence Recombinase-Aided Amplification Assay

2021 ◽  
Vol 17 (7) ◽  
pp. 1364-1370
Author(s):  
Ziyu He ◽  
Zengrui Tong ◽  
Boyu Tan ◽  
Xuliang He ◽  
Tao Zhang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Quantitative Detection ◽  
Amplification Efficiency ◽  
Clinical Settings ◽  
Specific Sequence ◽  
Fluorogenic Probe ◽  
Detection Assay ◽  
Amplification Assay ◽  
Entire Experiment

Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent (R2 > 95%) when the concentration range was 4.64×108 copies/μL˜4.64×104 copies/μL. The test assay can also detect positive samples when the template concentration is below 4.64×104 copies/μL. Remarkably, the entire experiment process only takes 15–20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.

scholarly journals Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System

2020 ◽  
Vol 8 (1) ◽  
pp. 14
Author(s):  
Shengwen Calvin Li ◽  
Kara J. Sparks ◽  
Leonard S. Sender
Keyword(s):  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Range ◽  
Measurement Range ◽  
Quantitative Detection ◽  
Clinical Settings ◽  
Stem Cell Therapies ◽  
Cell Therapies ◽  
Copy Numbers

Allogenic stem-cell therapies benefit patients in the treatment of multiple diseases; however, the side effects of stem-cell therapies (SCT) derived from the concomitant use of immune suppression agents often include triggering infection diseases. Thus, analysis is required to improve the detection of pathogen infections in SCT. We develop a polymerase chain reaction (PCR)-based methodology for the qualitative real-time DNA detection of cytomegalovirus (CMV), with reference to herpes simplex virus types 1 (HSVI), Epstein–Barr virus (EBV), and varicella-zoster virus (VZV) in blood, urine, solid tissues, and cerebrospinal fluid. This real-time PCR of 96-well plate format provides a rapid framework as required by the Food and Drug Administration (FDA) for clinical settings, including the processing of specimens, reagent handling, special safety precautions, quality control criteria and analytical accuracy, precisely reportable range (analyst measurement range), reference range, limit of detection (LOD), analytical specificity established by interference study, and analyte stability. Specifically, we determined the reportable range (analyst measurement range) with the following criteria: CMV copies ≥200 copies/mL; report copy/mL value; CMV copies ≤199 copies/mL; report detected but below quantitative range; CMV copies = 0 with report <200 copies/mL. That is, with reference range, copy numbers (CN) per milliliter (mL) of the LOD were determined by standard curves that correlated Ct value and calibrated standard DNA panels. The three repeats determined that the measuring range was 1E2~1E6 copies/mL. The standard curves show the slopes were within the range −2.99 to −3.65 with R2 ≥ 0.98. High copy (HC) controls were within 0.17–0.18 log differences of DNA copy numbers; (2) low copy (LC) controls were within 0.17–0.18 log differences; (3) LOD was within 0.14–0.15 log differences. As such, we set up a fast, simple, inexpensive, sensitive, and reliable molecular approach for the qualitative detection of CMV pathogens. Conclusion: This real-time PCR of the 96-well plate format provides a rapid framework as required by the FDA for clinical settings.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

scholarly journals Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment

2010 ◽  
Vol 76 (15) ◽  
pp. 5097-5104 ◽  
Author(s):  
M. H. Josefsen ◽  
C. L�fstr�m ◽  
T. B. Hansen ◽  
L. S. Christensen ◽  
J. E. Olsen ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Real Time ◽  
Control Strategies ◽  
Quantitative Risk Assessment ◽  
Quantitative Detection ◽  
Amplification Efficiency ◽  
Sample Treatment ◽  
Propidium Monoazide ◽  
Real Time Quantitative Pcr ◽  
Chicken Carcass

ABSTRACT A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT ) values (R 2 = 0.993), with a quantification range of 1 � 102 to 1 � 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R 2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.

scholarly journals Direct Quantitative Detection and Identification of Lactococcal Bacteriophages from Milk and Whey by Real-Time PCR: Application for the Detection of Lactococcal Bacteriophages in Goat's Raw Milk Whey in France

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Mai Huong Ly-Chatain ◽  
Loïc Durand ◽  
Véronique Rigobello ◽  
Annabelle Vera ◽  
Yann Demarigny
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Raw Milk ◽  
Quantitative Detection ◽  
Amplification Efficiency ◽  
The Real ◽  
Milk Whey ◽  
Detection And Identification ◽  
Milk Fermentation

The presence ofLactococcusbacteriophages in milk can partly or completely inhibit milk fermentation. To prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups ofLactococcusbacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 102PFU/mL. The curve slopes were −3.49, −3.69, and −3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient () of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. This method was then used to detect the bacteriophages in whey and goat's raw milk coming from three farms located in the Rhône-Alpes region (France).

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