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

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.

2006 ◽  
Vol 72 (9) ◽  
pp. 5750-5756 ◽  
Author(s):  
Melanie W. Kuiper ◽  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Harry Boonstra ◽  
Hauke Smidt ◽  
...  

ABSTRACT A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 � 10−1 and 1.14 � 104 cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 � 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (≥98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.


2014 ◽  
Vol 62 (S 01) ◽  
Author(s):  
M. Arar ◽  
A. Rotärmel ◽  
A.-K. Knoefel ◽  
H. Baraki ◽  
I. Kutschka ◽  
...  

2019 ◽  
Vol 14 (6) ◽  
pp. 454-459
Author(s):  
Xuejing Hou ◽  
Ying Liu ◽  
Isabelle Streuli ◽  
Patrick Dällenbach ◽  
Jean Dubuisson ◽  
...  

Asherman’s Syndrome or Intrauterine adhesions is an acquired uterine condition where fibrous scarring forms within the uterine cavity, resulting in reduced menstrual flow, pelvic pain and infertility. Until recently, the molecular mechanisms leading to the formation of fibrosis were poorly understood, and the treatment of Asherman’s syndrome has largely focused on hysteroscopic resection of adhesions, hormonal therapy, and physical barriers. Numerous studies have begun exploring the molecular mechanisms behind the fibrotic process underlying Asherman’s Syndrome as well as the role of stem cells in the regeneration of the endometrium as a treatment modality. The present review offers a summary of available stem cell-based regeneration studies, as well as highlighting current gaps in research.


2016 ◽  
Vol 5 (2) ◽  
pp. 145-151
Author(s):  
Ana Muñoz ◽  
Víctor Galvez ◽  
María Camarasa

Author(s):  
Richard N. Puzzitiello ◽  
Jeremy Dubin ◽  
Mariano E. Menendez ◽  
Michael A. Moverman ◽  
Nicholas R. Pagani ◽  
...  

2017 ◽  
Vol 25 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Aimée C. Colbath ◽  
David D. Frisbie ◽  
Steven W. Dow ◽  
John D. Kisiday ◽  
C. Wayne McIlwraith ◽  
...  

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.


2014 ◽  
Vol 168 (3) ◽  
pp. 328-337 ◽  
Author(s):  
Paola Cipriani ◽  
Piero Ruscitti ◽  
Roberto Giacomelli

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