Rapid identification and differentiation of Gram-negative and Gram-positive bacterial bloodstream infections by quantitative polymerase chain reaction in preterm infants*

2009 ◽  
Vol 37 (8) ◽  
pp. 2441-2447 ◽  
Author(s):  
Kathy Y. Y. Chan ◽  
Hugh S. Lam ◽  
Hon M. Cheung ◽  
Allen K. C. Chan ◽  
Karen Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Preterm Infants ◽  
Quantitative Polymerase Chain Reaction ◽  
Bloodstream Infections ◽  
Rapid Identification ◽  
Chain Reaction ◽  
Gram Positive ◽  
Gram Negative ◽  
Polymerase Chain

scholarly journals Multiplex Polymerase chain reaction to diagnose bloodstream infections in patients after cardiothoracic surgery

2018 ◽  
Author(s):  
Kevin Pilarczyk ◽  
Peter-Michael Rath ◽  
Jörg Steinmann ◽  
Matthias Thielmann ◽  
Maximillian Dürbeck ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cardiac Surgery ◽  
Blood Culture ◽  
Real Time ◽  
Multiplex Polymerase Chain Reaction ◽  
Bloodstream Infections ◽  
Chain Reaction ◽  
Blood Samples ◽  
Gram Negative ◽  
Polymerase Chain

Abstract Background: Sepsis and other infectious complications are major causes of mortality and morbidity in patients after cardiac surgery. Whereas blood culture (BC) as the current diagnostic gold standard suffers from low sensitivity as well as a reporting delay of approximately 48–72 h, polymerase chain reaction (PCR) based technologies might offer a fast and reliable alternative for detection of bloodstream infections (BSI). The aim of this study was to compare the performance of real time multiplex-PCR “SeptiFast” (SF), a real-time multiplex PCR assay, with conventional BC testing in patients after cardiac surgery. Methods: 279 blood samples from 168 individuals with suspected BSI were analyzed by SF and BC. Receiver operating characteristic (ROC) curves were generated to determine the accuracy of clinical and laboratory information for the prediction of positive SF results. Results: Excluding results attributable to contaminants, 14.7% (n = 41) of blood samples were positive using SF and 17.2% (n=49) using conventional BC (p= n.s.). In six samples, SF detected more than one pathogen. Among the 47 microorganisms identified by SF, only 11 (23.4%) could be confirmed by BC. SF identified a significantly higher number of Gram-negative bacteria than BC (28 vs. 12, χ2=7.97, p=0.005). The combination of BC and SF significantly increased the number of detected microorganism, including fungi, when compared to BC alone (86 vs. 49, χ2=13.51, p<0.001). C-reactive protein (CRP) (21.7±11.41 vs. 16.0±16.9 mg/dl, p=0.009), procalcitonin (PCT) (28.7±70.9 vs. 11.5±30.4 ng/dl, p=0.015) as well as interleukin 6 (IL 6) (932.3±1306.7 vs. 313.3±686.6 pg/ml, p=0.010) was significantly higher in patients with a positive SF result. In addition, incidence of severe acute kidney injury (AKI) was higher in SF positive than in SF negative patients (31/42 [76%] vs. 125/237 [53%], p=0.01). Using ROC analysis, IL-6 (AUC 0.836) as well as CRP (AUC 0.804), but not PCT showed the best predictive values for positive SF results. Microbiological diagnostic information gained through SF led to 8 therapy adaptations. Conclusion: The real time PCR-based SF test might represent a valuable addition to the traditional BC method for rapid etiologic diagnosis of BSI in patients after cardiothoracic surgery. This powerful method furthermore applies in particular for individuals with fungal infections, Gram-negative bacteremia, AKI and/or elevated CRP and IL-6-concentration. However, due to the low performance in detecting Gram-positive pathogens and the inability to determine antibiotic susceptibility, it should always be used in combination with BC. [1] Key words: Blood stream infection, blood culture, real time multiplex Polymerase Chain Reaction

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

2017 ◽  
Vol 69 (6) ◽  
pp. 1443-1450 ◽  
Author(s):  
M.P. Campos ◽  
M.F. Madeira ◽  
D.A. Silva ◽  
M.S. Solcà ◽  
O.M. Espíndola ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Healthy Skin ◽  
Sample Collection ◽  
Chain Reaction ◽  
Dna Recovery ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Polymerase Chain ◽  
Commercial Kits

ABSTRACT The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

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