Real-Time PCR for RSV Detection

The extracted nucleic acids (from a Roche MagNApure compact automated system) were used for 1-step real-time PCR for RSV detection, using a Qiagen 1-step RT-PCR kit. Real-time RT-PCR assays were performed with primers and probes for RSV, using the CDC protocol [11 ]. All assays were run with positive controls (RSV-RNA), negative controls (nuclease-free water), and internal positive controls (RNAseP) for quality-control purposes.

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Kazi A.M., Aguolu O.G., Mughis W., Ahsan N., Jamal S., Khan A., Qureshi H.M., Yildirim I., Malik F.A, & Omer S.B. (2021). Respiratory Syncytial Virus–Associated Mortality Among Young Infants in Karachi, Pakistan: A Prospective Postmortem Surveillance Study. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 73(Suppl 3), S203-S209.

Publication 2021

1-step RT-PCR kit

Assays Nucleic acids Primers Real time pcr Rt pcr

Corresponding Organization :

Other organizations : Aga Khan University, Yale University

Top 5 similar protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Detection of RSV

control variables

Positive controls (RSV-RNA)
Negative controls (nuclease-free water)
Internal positive controls (RNAseP)

Annotations

Based on most similar protocols

Use of positive controls (RSV-RNA) and negative controls (nuclease-free water) to ensure quality control of the real-time RT-PCR assays (Input protocol)

Use of internal positive controls (RNAseP) for normalization of sample concentrations (Input protocol)

Viral loads were normalized according to the formula: normalized sample Ct = (Ct sample) x (Ct RNase P of the sample)/ average Ct value of all RNase P samples) (Protocol 1)

Use of linear distribution (r = 0.99) between 10^1 and 10^8 copies of RSV-DNA for generating standard curve (Protocol 2)

Positive results defined as cycle threshold (Ct) value not higher than 35 for the TaqMan probe-based real-time PCR (qRT-PCR) assay (Protocol 3)

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