scholarly journals Temperature-controlled Primer Limit for Multiplexing of Rapid, Quantitative Reverse Transcription-PCR Assays: Application to Intraoperative Cancer Diagnostics

2002 ◽  
Vol 48 (8) ◽  
pp. 1329-1337 ◽  
Author(s):  
Siva Raja ◽  
Talal El-Hefnawy ◽  
Lori A Kelly ◽  
Melissa L Chestney ◽  
James D Luketich ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Positive Control ◽  
Cancer Diagnostics ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Temperature Controlled ◽  
Pcr Assays

Abstract Background: Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate. Methods: We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples. Results: The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1–2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples. Conclusion: Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.

scholarly journals Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis

2005 ◽  
Vol 109 (4) ◽  
pp. 365-379 ◽  
Author(s):  
Stephen A. Bustin ◽  
Reinhold Mueller
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Therapy Monitoring ◽  
Clinical Diagnostics ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Diagnostic ◽  
Pcr Assays

qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

scholarly journals Comparison of three TaqMan Real-Time Reverse Transcription-PCR assays in detecting SARS-CoV-2

2020 ◽  
Author(s):  
Yan Xiao ◽  
Zhen Li ◽  
Xinming Wang ◽  
Yingying Wang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Detection Rate ◽  
N Gene ◽  
Detection Rates ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Linear Detection ◽  
Gene Assay ◽  
Pcr Assays

AbstractQuick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LOD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LOD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92% and 100%, respectively) than that of the WHO assays (with a detection rate of 60%), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64%) than those of the WHO assays and the CCDC assays (with detection rates of 48% and 20%, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.

scholarly journals Validation of Reference Genes in Cervical Cell Samples from Human Papillomavirus-Infected and -Uninfected Women for Quantitative Reverse Transcription-PCR Assays

2008 ◽  
Vol 15 (9) ◽  
pp. 1369-1373 ◽  
Author(s):  
Ibrahim I. Daud ◽  
Mark E. Scott
Keyword(s):  
Human Papillomavirus ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Single Gene ◽  
Amplification Efficiency ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Cervical Cell ◽  
Overall Stability

ABSTRACT Reference genes for quantitative reverse transcription-PCR (qRT-PCR) studies must be validated for the cell type studied and should be stable between the groups that represent the independent variable in an experimental design. We sought to identify the reference genes in cervical cell specimens showing the most stable expression between human papillomavirus (HPV)-infected and -uninfected women without high-grade cervical intraepithelial neoplasia. Using endocervical cells collected by cytology brush and Sybr green-based qRT-PCR, eight candidate genes were screened for amplification efficiency, specificity, and overall stability (by use of geNorm software). The five most stable genes were then further evaluated both for overall stability (geNorm) and intergroup stability (by use of NormFinder software) in specimens from HPV-negative and HPV-positive women. The combination of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and RPLP0 was the most stable overall, with a geNorm stability measure of 0.603. The intergroup analysis showed GAPDH to be the most stable single gene and RPLP0 to be second most stable and also showed that these genes represent the most stable two-gene combination, with a NormFinder stability value of 0.130. The fact that these two distinct approaches identified the same pair of genes provides added confidence that, when the focus is on HPV infection, a normalization factor derived from these two genes is likely to be appropriate.

scholarly journals Application of a Receptor-Binding Capture Quantitative Reverse Transcription-PCR Assay To Concentrate Human Norovirus from Sewage and To Study the Distribution and Stability of the Virus

2011 ◽  
Vol 78 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Peng Tian ◽  
David Yang ◽  
Liangwen Pan ◽  
Robert Mandrell
Keyword(s):  
Receptor Binding ◽  
Reverse Transcription ◽  
Magnetic Beads ◽  
Sample Volume ◽  
Sewage Effluent ◽  
Human Norovirus ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Charged Membrane

ABSTRACTWater is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method and compared it to the existing negatively charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (CT) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV were measured as averageCTvalues of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE-concentrated HuNoV was not protected from RNase I and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 103genomic copies per liter (gc/liter) and 4.3 × 104gc/liter, respectively. No GI and <2% GII HuNoV were detected in sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage.

scholarly journals Inability To Catabolize Galactose Leads to Increased Ability To Compete for Nodule Occupancy in Sinorhizobium meliloti

2012 ◽  
Vol 194 (18) ◽  
pp. 5044-5053 ◽  
Author(s):  
Barney A. Geddes ◽  
Ivan J. Oresnik
Keyword(s):  
Genetic Analysis ◽  
Reverse Transcription ◽  
Sinorhizobium Meliloti ◽  
Nodule Occupancy ◽  
Content Type ◽  
Qrt Pcr ◽  
Genetic Region ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr

ABSTRACTA mutant unable to utilize galactose was isolated inSinorhizobium melilotistrain Rm1021. The mutation was found to be in a gene annotateddgoK1, a putative 2-keto-3-deoxygalactonokinase. The genetic region was isolated on a complementing cosmid and subsequently characterized. Based on genetic and bioinformatic evidence, the locus encodes all five enzymes (galD,dgoK,dgoA,SMc00883, andilvD1) involved in the De Ley-Doudoroff pathway for galactose catabolism. Although all five genes are present, genetic analysis suggests that the galactonase (SMc00883) and the dehydratase (ilvD1) are dispensable with respect to the ability to catabolize galactose. In addition, we show that the transport of galactose is partially facilitated by the arabinose transporter (AraABC) and that both glucose and galactose compete with arabinose for transport. Quantitative reverse transcription-PCR (qRT-PCR) data show that in adgoKbackground, the galactose locus is constitutively expressed, and the induction of thearalocus seems to be enhanced. Assays of competition for nodule occupancy show that the inability to catabolize galactose is correlated with an increased ability to compete for nodule occupancy.

scholarly journals Development and Assessment of a Quantitative Reverse Transcription-PCR Assay for Simultaneous Measurement of Four Amplicons

2003 ◽  
Vol 49 (9) ◽  
pp. 1467-1475 ◽  
Author(s):  
Marcy B Grace ◽  
Christopher B McLeland ◽  
Steven J Gagliardi ◽  
Jeffrey M Smith ◽  
William E Jackson ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Whole Blood ◽  
18S Rrna ◽  
Ex Vivo ◽  
Detection System ◽  
Simultaneous Detection ◽  
Positive Control ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Pcr Assays

Abstract Background: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. Methods: We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl2, and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. Results: The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log10 cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of −3.322. Conclusions: This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.

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