Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2011.07.006 ◽

2011 ◽

Vol 13 (6) ◽

pp. 695-700 ◽

Cited By ~ 10

Author(s):

Stella Laus ◽

Lawrence A. Kingsley ◽

Michael Green ◽

Robert M. Wadowsky

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Whole Blood ◽

Epstein Barr Virus ◽

Barr Virus ◽

Epstein Barr

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Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

10.26434/chemrxiv.14579127 ◽

2021 ◽

Author(s):

Sayantan Tripathy ◽

Arunansu Talukdar ◽

Goutam Pramanik ◽

P. V. Rajesh ◽

Souradyuti Ghosh

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Magnetic Particles ◽

Limited Resource ◽

Nucleic Acid Amplification ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Nucleic Acid Extraction ◽

Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

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Detection of human cytomegalovirus and Epstein-Barr Virus in symptomatic and asymptomatic apical periodontitis lesions by real-time PCR

Medicina Oral Patología Oral y Cirugia Bucal ◽

10.4317/medoral.18905 ◽

2013 ◽

pp. e811-e816 ◽

Cited By ~ 12

Author(s):

S. Ozbek ◽

A. Ozbek ◽

MS. Yavuz

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Apical Periodontitis ◽

Barr Virus ◽

Epstein Barr

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Comparison of Commercial Real-Time PCR Assays for Quantification of Epstein-Barr Virus DNA

Journal of Clinical Microbiology ◽

10.1128/jcm.43.5.2053-2057.2005 ◽

2005 ◽

Vol 43 (5) ◽

pp. 2053-2057 ◽

Cited By ~ 23

Author(s):

G. Ruiz ◽

P. Pena ◽

F. de Ory ◽

J. E. Echevarria

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Barr Virus ◽

Pcr Assays ◽

Epstein Barr

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Quantitation of Epstein-Barr virus mRNA using reverse transcription and real-time PCR

Journal of Medical Virology ◽

10.1002/jmv.20220 ◽

2004 ◽

Vol 74 (4) ◽

pp. 612-618 ◽

Cited By ~ 15

Author(s):

Birgit Weinberger ◽

Annelie Plentz ◽

Klaus M. Weinberger ◽

Joachim Hahn ◽

Ernst Holler ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Barr Virus ◽

Epstein Barr

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Heterogeneous, restricted patterns of Epstein–Barr virus (EBV) latent gene expression in patients with chronic active EBV infection

Journal of General Virology ◽

10.1099/0022-1317-82-10-2385 ◽

2001 ◽

Vol 82 (10) ◽

pp. 2385-2392 ◽

Cited By ~ 23

Author(s):

Mikio Yoshioka ◽

Nobuhisa Ishiguro ◽

Hiroaki Ishiko ◽

Xiaoming Ma ◽

Hideaki Kikuta ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Real Time ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Clinical Symptoms ◽

The Other ◽

Early Gene ◽

Barr Virus ◽

Epstein Barr

Epstein–Barr virus (EBV) has been shown to infect T lymphocytes and to be associated with a chronic active infection (CAEBV), which has been recognized as a mainly non-neoplastic T-cell lymphoproliferative disorder (T-cell LPD). The systemic distribution of EBV genomes was studied, by real-time PCR, in multiple tissues from six patients with CAEBV, including three patients with T-cell LPD, one patient with B-cell LPD and two patients with undetermined cell-type LPD. There were extremely high loads of EBV genomes in all tissues from the patients. This reflects an abundance of circulating and infiltrating EBV-infected cells and a wide variety of clinical symptoms in the affected tissues. We chose one sample from each patient that was shown by real-time PCR to contain a high load of EBV genomes and examined the expression of EBV latent genes by RT–PCR. EBER1 and EBNA1 transcripts were detected in all samples. Only one sample also expressed EBNA2, LMP1 and LMP2A transcripts in addition to EBER1 and EBNA1 transcripts. Two of the remaining five samples expressed LMP1 and LMP2A transcripts. One sample expressed LMP2A but not LMP1 and EBNA2 transcripts. Another sample expressed EBNA2 but not LMP1 and LMP2A transcripts. The other sample did not express transcripts of any of the other EBNAs or LMPs. None of the samples expressed the viral immediate-early gene BZLF1. These results showed that EBV latent gene expression in CAEBV is heterogeneous and that restricted forms of EBV latency might play a pathogenic role in the development of CAEBV.

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