Comparison of three commercial RT-PCR systems for the detection of respiratory viruses

Butt, Shafiq A.; Maceira, Vincente P.; McCallen, M.E.; Stellrecht, Kathleen A.

doi:10.1016/j.jcv.2014.08.010

Cited by 49 publications

(46 citation statements)

References 26 publications

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“…Enzyme-linked immunosorbent assays (ELISAs) are commercially available [6,7], but they usually require long assay times and a labor-intensive sampling process. Recently, testing of ribonucleic acid (RNA) viral cultures using reverse transcription-polymerase chain reactions (RT-PCR) has also become common, whereby viral nucleic acids extracted from samples are transcribed into complementary deoxyribonucleic acid (cDNA) which is then amplified and analyzed by fluorescence or luminescence [8,9]. Despite the simplicity of this method, it cannot provide specific sequence information and produces higher false positive results arising from the amplification of non-specific sequences.…”

Section: Introductionmentioning

confidence: 99%

Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses

Shi

Sun

et al. 2015

BME

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Abstract.A surface plasmon resonance (SPR)-based biosensor was developed for specific detection of nine common respiratory virus, including influenza A and influenza B, H1N1, respiratory syncytial virus (RSV), parainfluenza virus 1-3 (PIV1, 2, 3), adenovirus, and severe acute respiratory syndrome coronavirus (SARS). The SPR biosensor was developed by immobilizing nine respiratory virus-specific oligonucleotides in an SPR chip. To increase the biosensor sensitivity, biotin was used to label the PCR primer and further amplify the signal by introducing streptavidin after hybridization. Throat swab specimens representing nine common respiratory viruses were tested by the innovative SPR-based biosensor to evaluate the sensitivity, specificity and reproducibility of this method. Results suggest that this biosensor has the potential to simultaneously identify common respiratory viruses.

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Section: Introductionmentioning

confidence: 99%

Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses

Shi

Sun

et al. 2015

BME

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“…Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3)(4)(5)(6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays.…”

mentioning

confidence: 99%

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

et al. 2016

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Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20-to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

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“…Exact pipetting is not required, making peripheral use on an HSCT ward or an emergency department practical. Overall sensitivity is 97% for all tested pathogens with a low failure rate of 1% [16]. The literature gives a specificity of greater than 98% for all tested pathogens [17,18].…”

Section: Discussionmentioning

confidence: 87%

“…The method is expensive, however, and compared to conventional RT-PCR and other automated nested PCR-systems, Film Array has the highest costs per test [16]. Furthermore, simultaneous multiple sample testing with one instrument is not currently possible, requiring multiple instruments for a higher sample throughput.…”

Section: Discussionmentioning

confidence: 99%

Case Report on the Prevention of an Endemic Outbreak of Influenza B on an Allogeneic Transplant Ward

Heidrich

Boldt²,

Stölzel³

et al. 2016

J Leuk

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Respiratory tract infections pose a threat to hematopoietic stem cell recipients, increasing morbidity and mortality rates. To address this, preventive measures are of great importance. We describe the early detection of influenza B in a patient who received a recent hematopoietic transplantation, and elucidation of the transmission path using a point-of-care multiplex polymerase chain reaction panel, leading to prevention of viral spread. The use of risk-score systems and point-of-care testing should be further evaluated.

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Comparison of three commercial RT-PCR systems for the detection of respiratory viruses

Abstract: In summary, these systems demonstrated excellent performance. Furthermore, each system has benefits which will ensure they will all have a niche in a clinical laboratory.

Cited by 49 publications

References 26 publications

Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses

Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

Case Report on the Prevention of an Endemic Outbreak of Influenza B on an Allogeneic Transplant Ward

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