scholarly journals Clinical Significance of Rapid and Sensitive Quantification of Pseudomonas aeruginosa by Quantitative Reverse Transcription PCR Targeting of rRNA Molecules

2020 ◽  
Author(s):  
Mai Niikura ◽  
Satomi Atobe ◽  
Akira Takahashi ◽  
Yukiko Kado ◽  
Takuya Sugimoto ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inflammatory Response ◽  
Healthy Volunteers ◽  
Reverse Transcription ◽  
Antibacterial Treatment ◽  
23S Rrna ◽  
Icu Patients ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr

Abstract Background: For Pseudomonas aeruginosa, nosocomial infection control and appropriate antimicrobial treatment have become important issues. Diagnosis is critical in managing P. aeruginosa infection, but conventional methods are not highly accurate or rapid. A novel P. aeruginosa quantification system based on 23S rRNA-targeted quantitative reverse transcription PCR (qRT-PCR) is a candidate diagnostic tool for managing P. aeruginosa infection. Here, we assessed the performance and potential impact of qRT-PCR on antibiotic therapy administered to ICU patients.Methods: We first evaluated specificity and detection sensitivity of the 23S rRNA-targeted qRT-PCR system in vitro and determined whether P. aeruginosa viable counts detected by this system reflect the inflammatory response of infected cells. Next, we utilized this system on fecal samples collected from 65 septic ICU patients and 44 healthy volunteers to identify the ICU infection status. Additionally, we monitored drug-resistant P. aeruginosa in 4 ICU patients. The trend was compared with trends in fecal microbiota composition, antibiotic use, and mechanical ventilator use.Results: The 23S rRNA-targeted qRT-PCR system quantified P. aeruginosa directly from clinical samples with high sensitivity (blood, 1 cell/mL; stool, 100 cells/g) without cross-reaction (within 6 h). Additionally, P. aeruginosa numbers detected under antibacterial treatment correlated well with the inflammatory response compared to other detection methods such as culturing and qPCR. Using this system, we confirmed that the P. aeruginosa detection ratio in ICU patients was significantly higher than that in healthy volunteers (49.2% vs. 13.6%, P<0.05). While some ICU patients had high P. aeruginosa numbers in feces (108 cells/g of feces), some patients had very low P. aeruginosa numbers (102 cells/g of feces) as observed in healthy volunteers. P. aeruginosa counts in feces of ICU patients monitored by this system correlated negatively with the proportion of total obligate anaerobes and positively with facultative anaerobes and aerobes. Additionally, trends in P. aeruginosa counts accurately reflected various treatment backgrounds of ICU patients.Conclusion: Our results suggest that this new 23S rRNA-targeted qRT-PCR system is beneficial for early diagnosis and evaluation of appropriate antibacterial treatment and may be a useful tool in combating P. aeruginosa infection.

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 Effect of Soil Clay Content on RNA Isolation and on Detection and Quantification of Bacterial Gene Transcripts in Soil by Quantitative Reverse Transcription-PCR

2011 ◽  
Vol 77 (17) ◽  
pp. 6249-6252 ◽  
Author(s):  
A. Novinscak ◽  
M. Filion
Keyword(s):  
Reverse Transcription ◽  
Clay Content ◽  
Rna Isolation ◽  
Bacterial Gene ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Gene Transcripts ◽  
Soil Clay ◽  
Soil Clay Content

ABSTRACTIn this study, we evaluated the effect of soil clay content on RNA isolation and on quantitative reverse transcription-PCR (qRT-PCR) quantification of microbial gene transcripts. The amount of clay significantly altered RNA isolation yields and qRT-PCR analyses. Recommendations are made for quantifying microbial gene transcripts in soil samples varying in clay content.

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.

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