Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

Phaneuf, Christopher R.; Oh, Kyudam; Pak, Nikita; Saunders, D. Curtis; Conrardy, Christina; Landers, James P.; Tong, Suxiang; Forest, Craig R.

doi:10.1007/s10544-012-9720-1

Cited by 14 publications

(14 citation statements)

References 58 publications

(85 reference statements)

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“…Samples that were negative for EV and PeV were tested for other viruses using pan-viral family and genus PCR assays for the following viruses: alphaviruses, astroviruses, bornaviruses, bunyaviruses, coronaviruses, flaviruses, influenza viruses, paramyxoviruses, rhabdoviruses, adenoviruses, anelloviruses, and herpesviruses [9][10][11][12]. Positive bands of the expected size were Sanger sequenced with the PCR primers to determine virus species.…”

Section: Specimens and Molecular-based Testingmentioning

confidence: 99%

Picornavirus etiology of acute infections among hospitalized infants

Abedi

Messacar

Luong³

et al. 2019

Journal of Clinical Virology

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Background: Enteroviruses (EV) and parechoviruses (PeV) are ubiquitous viruses that cause a range of illness, including acute illness in children aged < 1 year. Objectives: We describe EV and PeV infections among children from 2 US study sites aged < 1 year and hospitalized with acute infections. For EV-and PeV-negative case-patients, we explored other viral etiologies. Methods: Participants were aged < 1 year, hospitalized during 2016, and had cerebrospinal fluid (CSF) collected for routine diagnostic testing. Demographic and clinical data were abstracted from medical charts, and residual specimens were sent to CDC for confirmatory testing and typing. Results: Of 472 eligible case-patients, CSF specimen was available for 319 (67.6%). Among those, 13 (4.1%) were positive for EV and 11 (3.4%) for PeV. Most case-patients (86.8%, n = 277) were aged < 2 months, as were all EV-or PeV-positive case-patients. None of the positive case-patients had underlying conditions, and the chief complaint for 91.7% (n = 22) was fever. Twelve positive case-patients were admitted to intensive care (ICU) and had brief hospital stays (median 2 days). Sequencing revealed a variety of EV types and the predominance of PeV-A3 among the PeV-positive case-patients. Conclusions: A range of EV and PeV types were associated with acute febrile illnesses leading to hospitalization in children aged < 2 months. Approximately half of EV and PeV case-patients were admitted to ICU, but length of hospital stay was brief and illnesses were generally self-limiting. Clinicians should consider EV and PeV infections in infants presenting with febrile illness.

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Section: Specimens and Molecular-based Testingmentioning

confidence: 99%

Picornavirus etiology of acute infections among hospitalized infants

Abedi

Messacar

Luong³

et al. 2019

Journal of Clinical Virology

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“…As described in the previous work, 25,31,32 the microchips were fabricated from PMMA substrates using a 3-axis vertical milling center (Haas) at a rate of two chips per minute. The 60 mm Â 20 mm substrates were laser cut from sheets of 1.5 mm thick cast PMMA.…”

Section: Microfluidic Devicementioning

confidence: 99%

“…Once verified, these reactions were then tested at 1 ll in our microchips using the custom, automated water bath thermocycler described in previous work. 32 For this, hold times were modified to accommodate slower sample equilibration. Following the 1 h reverse transcription, samples were cycled with a 1 min denaturation, 3 min annealing, and 3 min extension.…”

Section: Pcr and Rt-pcr Protocolsmentioning

confidence: 99%

“…While not demonstrated in this work, microfluidic PCR has been shown to have sensitivity down to 140 starting copies. 32 When thermal multiplexing was utilized for the dual amplification at the ideal conditions for each reaction, electropherograms (Fig 6, right) indicate efficient amplifications. Average yields for the multiplexed annealing conditions were 8.05 and 10.54 ng/ll for k-phage and EBV, respectively.…”

Section: Figmentioning

confidence: 99%

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Thermally multiplexed polymerase chain reaction

et al. 2015

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Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 ll, oil-encapsulated reactions, and closed-loop pulsewidth modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 C and 68 C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. V C 2015 AIP Publishing LLC.

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“…It is relatively difficult to find a completely conserved genomic region shared by different genotypes and subtypes for PCR primer design. Therefore, degenerate primers were often used for pan-genotype/subtype detection of RNA viruses [9,10]. Despite this, the presence of various variants may cause mismatch between the primers and the targets.…”

Section: Introductionmentioning

confidence: 99%

A Mismatch-Tolerant RT-Quantitative PCR: Application to Broad-Spectrum Detection of Respiratory Syncytial Virus

Wan

et al. 2019

BioTechniques

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Quantitative PCR (qPCR) is widely used to detect viruses. However, mismatches occurring in the 3′-end of the primers reduce amplification efficiency of qPCR and limit its capacity in detection of highly variable viruses. Here, we reported a mismatch-tolerant RT-qPCR with a small amount of additional high-fidelity DNA polymerase for simultaneous detection of RSV-A and RSV-B. The novel assay had higher amplification efficiency for various variants forming mismatches with the primers than the conventional RT-qPCR, and showed good specificity and sensitivity. It demonstrated a good correlation coefficient with a commercial RSV detection kit and had relatively lower Ct values than the kit for 16 of 20 RSV-positive samples. The mismatch-tolerant qPCR technique is a promising approach for sensitive detection of highly variable viruses.

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Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

Cited by 14 publications

References 58 publications

Picornavirus etiology of acute infections among hospitalized infants

Picornavirus etiology of acute infections among hospitalized infants

Thermally multiplexed polymerase chain reaction

A Mismatch-Tolerant RT-Quantitative PCR: Application to Broad-Spectrum Detection of Respiratory Syncytial Virus

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