scholarly journals Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

2016 ◽  
Vol 9 ◽  
pp. MBI.S38517 ◽  
Author(s):  
Jing Zhang ◽  
Guo-Chiuan Hung ◽  
Kenjiro Nagamine ◽  
Bingjie Li ◽  
Shien Tsai ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Candida Species ◽  
Rapid Detection ◽  
Corneal Transplantation ◽  
High Sensitivity ◽  
Turnaround Time ◽  
Pcr Assays ◽  
Primer Sets ◽  
Pcr Primer

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Taqman Real-Time PCR Assays for Rapid Detection of Avian PathogenicEscherichia coliIsolates

2014 ◽  
Vol 58 (4) ◽  
pp. 628-631 ◽  
Author(s):  
Nilo Ikuta ◽  
Fabiana de Oliveira Solla Sobral ◽  
Fernanda Kieling Moreira Lehmann ◽  
Vinicius Proença da Silveira ◽  
Silvia de Carli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Pcr Assays

scholarly journals Rapid detection ofYersinia pestiswith multiplex real-time PCR assays using fluorescent hybridisation probes

2003 ◽  
Vol 38 (2) ◽  
pp. 117-126 ◽  
Author(s):  
Herbert Tomaso ◽  
Emil C Reisinger ◽  
Sascha Dahouk ◽  
Dimitrios Frangoulidis ◽  
Alexander Rakin ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Pcr Assays

scholarly journals Effective Light-Upon-Extension Real-Time PCR Primer Systems for Rapid Detection of Human Viruses

2010 ◽  
Vol 41 (3) ◽  
pp. 150-155
Author(s):  
Zlatko N. Kalvatchev ◽  
Iliya D. Tsekov ◽  
Svetoslav N. Slavov ◽  
Pavel I. Draganov
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Human Viruses ◽  
Pcr Primer

scholarly journals Real-time evaluation of an optimized real-time PCR assay versus Brilliance chromogenic MRSA agar for the detection of meticillin-resistant Staphylococcus aureus from clinical specimens

2011 ◽  
Vol 60 (3) ◽  
pp. 323-328 ◽  
Author(s):  
J. Danial ◽  
M. Noel ◽  
K. E. Templeton ◽  
F. Cameron ◽  
F. Mathewson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Test Performance ◽  
High Sensitivity ◽  
Turnaround Time ◽  
Homology Search ◽  
Pcr Assay ◽  
Indirect Measure ◽  
The Mean

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

scholarly journals Rapid Diagnosis of Intestinal Parasitic Protozoa, with a Focus onEntamoeba histolytica

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Anjana Singh ◽  
Eric Houpt ◽  
William A. Petri
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Rapid Diagnosis ◽  
Amoebic Liver Abscess ◽  
Human Gut ◽  
Entamoeba Dispar ◽  
Highly Sensitive ◽  
Pcr Assays ◽  
Economic Constraints

Entamoeba histolyticais an invasive intestinal pathogenic parasitic protozoan that causes amebiasis. It must be distinguished fromEntamoeba disparandE. moshkovskii, nonpathogenic commensal parasites of the human gut lumen that are morphologically identical toE. histolytica. Detection of specificE. histolyticaantigens in stools is a fast, sensitive technique that should be considered as the method of choice. Stool real-time PCR is a highly sensitive and specific technique but its high cost make it unsuitable for use in endemic areas where there are economic constraints. Serology is an important component of the diagnosis of intestinal and especially extraintestinal amebiasis as it is a sensitive test that complements the detection of the parasite antigens or DNA. Circulating Gal/GalNac lectin antigens can be detected in the serum of patients with untreated amoebic liver abscess. On the horizon are multiplex real-time PCR assays which permit the identification of multiple enteropathogens with high sensitivity and specificity.

Real-time PCR assays for rapid detection ofZeugodacus cucumisandBactrocera jarvisi(Diptera: Tephritidae) for quarantine application

2018 ◽  
Vol 143 (1-2) ◽  
pp. 155-163 ◽  
Author(s):  
Dongmei Li ◽  
Sreelakshmi Nair ◽  
Diane Anderson ◽  
Prasad Doddala ◽  
Disna N. Gunawardana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Pcr Assays

scholarly journals Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

2000 ◽  
Vol 46 (3) ◽  
pp. 324-331 ◽  
Author(s):  
Danbing Ke ◽  
Christian Ménard ◽  
François J Picard ◽  
Maurice Boissinot ◽  
Marc Ouellette ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Culture Method ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Group B Streptococci ◽  
Standard Culture ◽  
Pcr Assays ◽  
Group B

Abstract Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance

2006 ◽  
Vol 54 (1) ◽  
pp. 91-99 ◽  
Author(s):  
T. Limpiyakorn ◽  
F. Kurisu ◽  
O. Yagi
Keyword(s):  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Full Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Ammonia Oxidizing Bacteria ◽  
Primer Sets ◽  
Activated Sludge Systems ◽  
Pcr Primer

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 102 copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea–Nitrosococcus. mobilis solely existed in one A2O system of which the influent contained twice the chloride concentrations than those of other systems.

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