scholarly journals Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

2018 ◽  
Vol 258 ◽  
pp. 35-40 ◽  
Author(s):  
Hodon Ryu ◽  
Karen A. Schrantz ◽  
Nichole E. Brinkman ◽  
Laura A. Boczek
Keyword(s):  
Cell Culture ◽  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Simultaneous Detection

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

2021 ◽  
Author(s):  
Liyun Chang ◽  
Zhiyong Liu ◽  
Yuelan Zhao ◽  
Yan Li ◽  
Jianhua Qin
Keyword(s):  
Quantitative Pcr ◽  
Quantitative Methods ◽  
Diarrheal Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
N Gene ◽  
Established Method ◽  
Diarrhea Virus ◽  
Fluorescence Quantitative Pcr

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

scholarly journals Optimization of RNA Extraction for PCR Quantification of Aromatic Compound Degradation Genes

2009 ◽  
Vol 76 (4) ◽  
pp. 1282-1284 ◽  
Author(s):  
Weidong Kong ◽  
Cindy H. Nakatsu
Keyword(s):  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Aromatic Compound ◽  
Rna Extraction ◽  
Maximum Activity ◽  
Bacterial Strains ◽  
Aromatic Compound Degradation ◽  
Polluted Sites ◽  
Primer Sets ◽  
Mixed Community

ABSTRACT Seven different bacterial strains and primer sets and a mixed community were used to evaluate the use of reverse transcriptase quantitative PCR (Q-PCR) and Q-PCR of oxygenase genes to assess various approaches for monitoring the bioremediation of polluted sites. Differences in maximum activity were seen when different RNA extraction kits were compared.

Reverse Transcription with a Random Pentadecamer Primer Increases the Sensitivity of Quantitative PCR for BCR-ABL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2339-2339
Author(s):  
David M. Ross ◽  
Timothy P. Hughes ◽  
Susan Branford
Keyword(s):  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Negative Control ◽  
Gene Copy ◽  
Imatinib Treatment ◽  
Control Gene ◽  
Simple Modification ◽  
Number Of Patients

Abstract Real-time quantitative reverse transcriptase PCR for BCR-ABL (RQ-PCR) is used to monitor treatment response in chronic myeloid leukaemia (CML). BCR-ABL levels continue to decline over several years of imatinib treatment and increasing numbers of patients have BCR-ABL levels at or below the limit of detection. The sensitivity of current RQ-PCR assays limits our ability to identify patients who have a continuing decline in BCR-ABL levels; or to assess the effects of novel therapies to improve outcome in patients who have a good response to ABL kinase inhibitors, but harbour minimal residual disease. Improvements in therapy for these patients will be hard to assess without more sensitive monitoring of BCR-ABL. BCR-ABL levels are usually reported relative to a control gene. The control gene value gives an indication of the sensitivity achieved within each RNA sample; this varies with sample quality and efficiency of reverse transcription (RT). Control gene copy number below a defined value indicates that the analysis may be unreliable. We investigated the use of a random pentadecamer (RP; 15mer) primer in the RT reaction to improve the sensitivity of RQ-PCR. The RP primer is reported to be more efficient than the random hexamer (RH; 6mer) which is commonly used for RQ-PCR. BCR-ABL and BCR control gene transcripts were measured in 30 peripheral blood samples. After Trizol extraction 2μg RNA and 400U Superscript II reverse transcriptase were added to two RT reactions using RP or RH at a final concentration of 25μM. Each RT and quantitative PCR was performed 2–3 times and results compared using the Wilcoxon signed rank test or paired t-test. A more efficient RT is indicated by higher transcript levels. The table shows that BCR-ABL and BCR control gene values were significantly higher with RP primers. There was a proportionate increase in all transcripts so that the change in BCR-ABL/BCR% was not significant. BCR-ABL and control gene values with pentadecamer or hexamer primers No. of Samples Primer Median 25th Percentile 75th Percentile P value (RP v HP) * missing values due to inclusion of samples with undetectable BCR-ABL BCR transcripts 30 RH 438900 241800 1090000 < 0.001 RP 1203700 836500 2125000 BCR-ABL transcripts 16* RH 280 110 11340 < 0.001 RP 390 220 32910 BCR-ABL/BCR ratio 16* RH 0.10% 0.06% 5.6% =0.083 RP 0.08% 0.05% 4.2% We tested samples with undetectable BCR-ABL including 10 from CML patients on imatinib, and 10 from BCR-ABL negative control subjects. Each RNA was tested in two independent RTs and Q-PCRs. If the results were discordant a third RQ-PCR was performed and a consensus result determined. Using RP primers 6/10 patient samples were positive; 2/10 were positive with RH. None of the BCR-ABL negative control samples was positive with either primer. Sensitivity was calculated using the lower limit of detection and the standardised baseline value for untreated CML patients.The median calculated sensitivity increased from 4.5-log with RH to 5.0-log with RP. The use of pentadecamer primer made possible the detection of BCR-ABL in a small number of patients who had undetectable BCR-ABL with random hexamer RQ-PCR. This initial analysis suggests that the sensitivity of BCR-ABL detection may be improved 2–3-fold with a simple modification to RT methodology. This also has the potential to reduce the number of samples deemed unsatisfactory due to low control gene levels.

scholarly journals Importance of Micromonospora spp. as Colonizers of Cellulose in Freshwater Lakes as Demonstrated by Quantitative Reverse Transcriptase PCR of 16S rRNA

2012 ◽  
Vol 78 (9) ◽  
pp. 3495-3499 ◽  
Author(s):  
Alexandre B. de Menezes ◽  
James E. McDonald ◽  
Heather E. Allison ◽  
Alan J. McCarthy
Keyword(s):  
16S Rrna ◽  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Lake Sediment ◽  
Relative Abundance ◽  
Bacterial Communities ◽  
Bacterial Population ◽  
Freshwater Lakes ◽  
Content Type ◽  
Water Columns

ABSTRACTThe relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA.Micromonosporaspp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU.

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