Applicability of a Real-Time Quantitative PCR Assay for Diagnosis of Respiratory Syncytial Virus Infection in Immunocompromised Adults

Elden, Leontine J. R. van; Loon, Anton M. van; Beek, A. van der; Hendriksen, Karin A. W.; Hoepelman, A. I. M.; Kraaij, M.G.J. van; Schipper, P; Nijhuis, Manon M. Oude

doi:10.1128/jcm.43.8.4308.2005

Cited by 51 publications

(71 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Amplification and detection were performed with a 7500 Real Time PCR System (Applied Biosystems). The N gene of RSV A and of RSV B was targeted with primers and probes, according to van Elden et al ., 21 with minor modifications of the reverse primer for RSV A. Each tube contained a 25 µL reaction mix which included 2.5 µL of isolated RNA, 0.9 µmol/L forward primer, 0.9 µmol/L reverse primer and 0.25 µmol/L probe.…”

Section: Methodsmentioning

confidence: 99%

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region

Mlinarić‐Galinović¹,

Tabain²,

Kukovec³

et al. 2012

Pediatrics International

View full text Add to dashboard Cite

show abstract

Section: Methodsmentioning

confidence: 99%

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region

Mlinarić‐Galinović¹,

Tabain²,

Kukovec³

et al. 2012

Pediatrics International

View full text Add to dashboard Cite

show abstract

“…Analysis of hMPV, RSV, and HPIV RNA standards with dynamic ranges from 10 7 to 10 1 copies/µL showed no sensitivity reduction in our new trial. The standard curve showed that each virus performs well in the monoplex and triplex reactions, with the same detection limit from 300 to 425 copies virus gene per reaction, while the literature reported 5 to 500 copies of the virus gene (HPIV‐1, HPIV‐2, HPIV‐3, RSV‐A, and hMPV) or tissue‐culture infective dose of 0.34 (RSV‐B) . Other curve parameters such as the R 2 and slope indicated good linearization and efficiency in the process of amplification.…”

Section: Discussionmentioning

confidence: 87%

“…For monoplex real‐time RT‐PCR to detection individual human paramyxovirus as previous reports, the above sets of primers and probes changed from three to one, but the concentrations modified and reaction conditions did not change. A commercial kit of the routine multiplex‐ligation‐NAT‐based assay, RespiFinder‐22 (RF‐22, 2Smart; PathoFinder) was applied in this study to compare the performance of our real‐time RT‐PCR assay for detection of human paramyxoviruses in the clinical setting.…”

Section: Methodsmentioning

confidence: 86%

“…The sequence information of primers and probes for each triplex real‐time RT‐PCR is presented in Supporting Information Table S1, along with their target genes, reaction concentrations, and fluorescent‐dye labels. We have screened and optimized six common respiratory paramyxovirus primers and probes from previous reports . Primer Express software (version 3.0; Applied Biosystems, Foster City, CA) was used to modify sequences, and Oligo (version 7.57; Molecular Biology Insights, Colorado Springs, CO) was used to ensure that primer complements and primer dimers did not exist among different viruses in the same tube.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Detection of six common human paramyxoviruses in patients with acute febrile respiratory symptoms using a novel multiplex real‐time RT‐PCR assay

Liu

Niu

Zhao³

et al. 2018

Journal of Medical Virology

View full text Add to dashboard Cite

Human metapneumovirus (hMPV), respiratory syncytial virus type A (RSV‐A), RSV‐B, and human parainfluenza viruses 1, 2, and 3 (HPIV‐1, HPIV‐2, and HPIV‐3) are common respiratory paramyxoviruses. Here, we developed a two‐tube triplex one‐step real‐time reverse‐transcription polymerase chain reaction (real‐time RT‐PCR) and evaluated its performance using clinical samples. The data showed that this novel assay was 100% consistent with the monoplex real‐time RT‐PCR assay (in‐house), which was superior to the commercial routine multiplex‐ligation‐NAT‐based assay. Meanwhile, the clinical nasopharyngeal swabs of 471 patients with the acute febrile respiratory syndrome (AFRS) were analyzed using the established method. The results showed that 52 (11.7%) cases were positive for paramyxovirus. Among them, HPIVs and RSV‐A had the highest detection rate. The age and seasonal distribution of human paramyxovirus infection were analyzed. In conclusion, we developed a novel multiplex real‐time RT‐PCR assay for the rapid detection of six common human paramyxoviruses, which were dominant in patients with AFRS in Qinghai.

show abstract

“…As soon as the DNA is denatured again during PCR cycling, intercalated dye molecules are released into the solution resulting in a drop of fluorescence. The fluorescence is recorded after each cycle at the end of the elongation Hu et al (2003), Mentel et al (2003), van Elden et al (2003) and Perkins et al (2005)…”

Section: Detection Formats and Chemistriesmentioning

confidence: 99%

Detection and monitoring of virus infections by real-time PCR

Watzinger

Ebner

Lion

2006

Molecular Aspects of Medicine

196

124

View full text Add to dashboard Cite

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.

show abstract

Applicability of a Real-Time Quantitative PCR Assay for Diagnosis of Respiratory Syncytial Virus Infection in Immunocompromised Adults

Cited by 51 publications

References 0 publications

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region

Detection of six common human paramyxoviruses in patients with acute febrile respiratory symptoms using a novel multiplex real‐time RT‐PCR assay

Detection and monitoring of virus infections by real-time PCR

Contact Info

Product

Resources

About