Molecular Detection of Bacterial Contamination in Blood Components Using Magnetic-based Enrichment

Abstract Bacterial contamination of blood products is a major problem in transfusion medicine, in terms of both morbidity and mortality. Platelets (PLTs) are stored at room temperature (under constant agitation) for more than 5 days, and bacteria can thus grow significantly from a low level to high titers. However, conventional methods like blood culture and lateral flow assay have disadvantages such as long detection time, low sensitivity, and the need for a large volume of blood components. We used real-time polymerase chain reaction (PCR) assays with antibiotic-conjugated magnetic nanobeads (MNBs) to detect enriched Gram-positive and -negative bacteria. The MNBs were coated with polyethylene glycol (PEG) to prevent aggregation by blood components. Over 80% of all bacteria were captured by the MNBs, and the levels of detection were 101 colony forming unit [CFU]/mL and 102 CFU/mL for Gram-positive and -negative bacteria, respectively. The detection time is < 3 h using only small volumes of blood components. Thus, compared to conventional methods, real-time PCR using MNBs allows for rapid detection with high sensitivity using only a small volume of blood components.

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Real-Time Polymerase Chain Reaction for Detection of Schistosoma DNA in Small-Volume Urine Samples Reflects Focal Distribution of Urogenital Schistosomiasis in Primary School Girls in KwaZulu Natal, South Africa

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.13-0406 ◽

2014 ◽

Vol 90 (3) ◽

pp. 546-552 ◽

Cited By ~ 23

Author(s):

Pavitra Pillay ◽

Eyrun F. Kjetland ◽

Eric A. T. Brienen ◽

Myra Taylor ◽

Lisette van Lieshout ◽

...

Keyword(s):

South Africa ◽

Polymerase Chain Reaction ◽

Primary School ◽

Real Time ◽

Small Volume ◽

Chain Reaction ◽

Urogenital Schistosomiasis ◽

Kwazulu Natal ◽

School Girls ◽

Polymerase Chain

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Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822011000500021 ◽

2011 ◽

Vol 44 (5) ◽

pp. 631-632

Author(s):

Vlademir Cantarelli ◽

Bianca Cavalcante ◽

Diogo André Pilger ◽

Fabiana Souza ◽

Cícero Gomes Dias ◽

...

Keyword(s):

Real Time ◽

Rapid Detection ◽

Surveillance Program ◽

Southern Brazil ◽

Rt Pcr ◽

Chain Reaction ◽

Vancomycin Resistant Enterococci ◽

Vancomycin Resistant ◽

Polymerase Chain ◽

Low Sensitivity

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

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An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products

PLoS ONE ◽

10.1371/journal.pone.0134743 ◽

2015 ◽

Vol 10 (7) ◽

pp. e0134743 ◽

Cited By ~ 1

Author(s):

Jin-ju Zhang ◽

Jing-jing Tian ◽

Shuang-shi Wei ◽

Sheng-bao Duan ◽

Hong-mei Wang ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Polymerase Chain Reaction Assay ◽

Bacterial Contamination ◽

Blood Products ◽

Chain Reaction ◽

Internal Reference ◽

Polymerase Chain

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Reliability of Cobas Amplicor PCR test in detection of Mycobacterium tuberculosis in respiratory and nonorespiratory specimens

Vojnosanitetski pregled ◽

10.2298/vsp0912992l ◽

2009 ◽

Vol 66 (12) ◽

pp. 992-997

Author(s):

Zorica Lepsanovic ◽

Dejana Savic ◽

Branka Tomanovic

Keyword(s):

Mycobacterium Tuberculosis ◽

High Sensitivity ◽

High Specificity ◽

Diagnostic Methods ◽

Nucleic Acid Amplification ◽

Culture Methods ◽

Predictive Values ◽

Polymerase Chain ◽

Low Sensitivity ◽

Sensitivity Specificity

Background/Aim. Traditional methods for detection of mycobacteria, such as microscopic examination for the presence of acid-fast bacilli and isolation of the organism by culture, have either a low sensitivity and/or specificity, or take weeks before a definite result is available. Molecular methods, especially those based on nucleic acid amplification, are rapid diagnostic methods which combine high sensitivity and high specificity. The aim of this study was to determine the usefulness of the Cobas Amplicor Mycobacterium tuberculosis polymerase chain reaction (CAPCR) assay in detecting the tuberculosis cause in respiratory and nonrespiratory specimens (compared to culture). Methods. Specimens were decontaminated by the N-acetyl-L-cystein- NaOH method. A 500 ?L aliquot of the processed specimen were used for inoculation of L?wenstein-Jensen (L-J) slants, a drop for acid-fast staining, and 100 ?L for PCR. The Cobas Amplicor PCR was performed according to the manufacturer's instructions. Results. A total of 110 respiratory and 355 nonrespiratory specimens were investigated. After resolving discrepancies by reviewing medical history, overall sensitivity, specificity, and positive and negative predictive values for CA-PCR assay compared to culture, were 83%, 100%, 100%, and 96.8%, respectively. In comparison, they were 50%, 99.7%, 87.5%, and 98%, respectively, for the nonrespiratory specimens. The inhibition rate was 2.8% for respiratory, and 7.6% for nonrespiratory specimens. Conclusion. CA-PCR is a reliable assay that enables specialists to start treatment promptly on a positive test result. Lower value for specificity in a group of nonrespiratory specimens is a consequence of an extremely small number of mycobacteria in some of them.

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Identification of Salmonella spp and serovars Typhimurium, Enteritidis by qPCR

Naukovij vìsnik veterinarnoï medicini ◽

10.33245/2310-4902-2020-154-1-21-31 ◽

2020 ◽

pp. 21-31

Author(s):

N. Rublenko

Keyword(s):

Real Time ◽

High Sensitivity ◽

High Specificity ◽

Analytical Sensitivity ◽

Reaction Products ◽

Salmonella Spp ◽

Bacterial Dna ◽

E Coli ◽

Polymerase Chain ◽

Primer Sets

This article presents the results of the identification of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fluorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specificity of the primers was evaluated on Salmonella strains of different serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientific Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specificity of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confirmed the specificity of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecific reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specificity. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and differentiate Enteritidis and Typhimurium serovars. Keywords: Salmonella, bacteria, polymerasechainreaction, DNA, qPCR.

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A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

The Scientific World JOURNAL ◽

10.1100/2012/989514 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 20

Author(s):

Andrea Balboni ◽

Laura Gallina ◽

Alessandra Palladini ◽

Santino Prosperi ◽

Mara Battilani

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Screening Tool ◽

High Sensitivity ◽

Pcr Assay ◽

Viral Ecology ◽

Epidemiological Surveys ◽

Epidemiological Evaluation ◽

Polymerase Chain ◽

Horseshoe Bat

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.

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