Reverse Transcription-Quantitative real-time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

2021 ◽  
Author(s):  
Bryanna Lexus Freitas ◽  
Lynn Leach ◽  
Vishnu Chaturvedi ◽  
Sudha Chaturvedi
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Ribosomal Gene ◽  
Qpcr Assay ◽  
Molecular Diagnostic ◽  
Quantitative Real Time Pcr ◽  
Candida Auris ◽  
Healthcare Environments

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted the development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcription (RT)-quantitative real-time PCR (RT-qPCR) assay to detect live C. auris in healthcare environments rapidly. This assay targeted the internal transcribed spacer 2 (ITS2) ribosomal gene by obtaining pure RNA followed by reverse transcription (ITS2 cDNA) and qPCR. ITS2 cDNA was not detectable in bleach-killed cells but detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT-qPCR reaction. Validation studies yielded positive Ct values from sponge matrix samples spiked with 10 2 to 10 5 CFU of live C. auris while dead (bleach-killed) C. auris (10 5 /ml) or other live Candida species (10 5 /ml) had no cycle threshold (Ct) values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT-qPCR assay, confirming the concordance between culture and the PCR assay. The RT-qPCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples. Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

scholarly journals Reverse Transcriptase Real-Time PCR Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

2021 ◽  
Author(s):  
Bryanna Lexus Freitas ◽  
Lynn Leach ◽  
Vishnu Chaturvedi ◽  
Sudha Chaturvedi
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Multidrug Resistant ◽  
Molecular Diagnostic ◽  
Pcr Assay ◽  
Candida Auris ◽  
Healthcare Environments ◽  
Rapid Enumeration

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcriptase (RT) real-time PCR assay for rapid detection of live C. auris in healthcare environments. The assay targets the internal transcribed spacer 2 (ITS2) cDNA of C. auris, amplified by obtaining pure RNA followed by reverse transcription and real-time PCR assay. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT real-time PCR reaction. In C. auris viability studies, ITS2 cDNA was detectable in heat- and ethanol-killed C. auris cells, but not bleach-killed cells. Validation studies showed no cycle threshold (Ct) values were obtained from samples on a sponge matrix spiked with either dead C. auris (105/ml) or other Candida species (105/ml), while most environmental samples spiked with 102 to 105 CFU of live C. auris yielded positive Ct values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT real-time PCR assay, confirming concordance between culture and the assay. The RT real-time PCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples, and could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

scholarly journals Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ana Érika Inácio Gomes ◽  
Leonardo Prado Stuchi ◽  
Nathália Maria Gonçalves Siqueira ◽  
João Batista Henrique ◽  
Renato Vicentini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

scholarly journals A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽  
2020 ◽  
Vol 10 (12) ◽  
Author(s):  
Siti Suriawati Badai ◽  
Omar Abd Rasid ◽  
Ghulam Kadir Ahmad Parveez ◽  
Mat Yunus Abdul Masani
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Oil Palm ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests

2006 ◽  
Vol 53 (8) ◽  
pp. 195-202 ◽  
Author(s):  
G. Garcés ◽  
M. Effenberger ◽  
M. Najdrowski ◽  
C. Wackwitz ◽  
A. Gronauer ◽  
...  
Keyword(s):  
Anaerobic Digestion ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Combined Treatment ◽  
Quantitative Real Time Pcr ◽  
Anaerobic Digesters ◽  
Hsp70 Mrna ◽  
Cryptosporidium Parvum Oocysts

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic–thermophilic–mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of ≥2 and ≥5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

scholarly journals Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

2014 ◽  
Vol 80 (10) ◽  
pp. 3086-3094 ◽  
Author(s):  
Hyatt C. Green ◽  
Richard A. Haugland ◽  
Manju Varma ◽  
Hana T. Millen ◽  
Mark A. Borchardt ◽  
...  
Keyword(s):  
Real Time ◽  
Surface Waters ◽  
Real Time Pcr ◽  
Fecal Pollution ◽  
Qpcr Assay ◽  
Taqman Assay ◽  
Reference Database ◽  
Quantitative Real Time Pcr ◽  
Ambient Surface

ABSTRACTQuantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genusBacteroidesare among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

scholarly journals Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

ISRN Oncology ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-6
Author(s):  
Anders Ståhlberg ◽  
Pierre Åman ◽  
Linda Strömbom ◽  
Neven Zoric ◽  
Alfredo Diez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Real Time ◽  
B Lymphocytes ◽  
Light Chain ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Light Chains ◽  
Quantitative Real Time Pcr ◽  
Healthy Humans

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

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