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"The analytical sensitivity of the rRT-PCR assays contained in the CDC 2019 Novel Coronavirus (2019- nCoV) Real-Time RT-PCR Diagnostic Panel were determined in Limit of Detection studies.
Since no quantified virus isolates of the 2019-nCoV were available for CDC use at the time the test was developed and this study conducted, assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene; GenBank accession: MN908947.2) of known titer (RNA copies/μL) spiked into a diluent consisting of a suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen.
Samples were extracted using the QIAGEN EZ1 Advanced XL instrument and EZ1 DSP Virus Kit (Cat# 62724) and manually with the QIAGEN DSP Viral RNA Mini Kit (Cat# 61904). Real-Time RT-PCR assays were performed using the Thermo Fisher Scientific TaqPathTM 1-Step RT-qPCR Master Mix, CG (Cat# A15299) on the Applied BiosystemsTM 7500 Fast Dx Real- Time PCR Instrument according to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel instructions for use.( page 41)"
"Appendix B: Pooled Specimen Preparation and Processing( page 59)
Purpose:In response to strong demand for higher throughput testing approaches as well as a global shortage of nucleic acid extraction reagents causing significant delays in testing, the CDC has evaluated specimen pooling and determined that pooling of up to 4 specimens is suitable for use with the 2019-nCoV Real- Time RT-PCR Diagnostic Panel.Specimen pooling may cause a slight reduction in test sensitivity and therefore may be most appropriate for screening or diagnostic testing when laboratory staff, equipment or reagents are insufficient to accommodate testing demand. Specimen pooling only presents a throughput advantage when the disease prevalence is low. Therefore, laboratories should monitor specimen positivity rates over time to determine if pooling of specimens continues to provide a test throughput advantage over individual specimen testing."
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Precautions/Warnings/Limitations:• Pooling of specimens has the potential to decrease sensitivity. The specimens in a pooled procedure are diluted, which could result in a low concentration of viral genetic material below the limit of detection of a given test.• When pooling specimens, the laboratory cannot ensure the diagnostic integrity of an individual specimen because it is combined with other specimens before testing. Specimen integrity can be affected by the quality of swab specimen collection, which could result in some swabs having limited amounts of viral genetic material for detection. Inadequate individual specimens, including those with limited amounts of viral genetic material, might not be eliminated from the pooled specimen before testing and may be reported as negative through this process.• Performance of this specimen pooling process was evaluated with nasopharyngeal (NP) swabs. Pooling of other types of specimens for subsequent testing with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel has not been evaluated, however evidence suggests the Ct value distribution for other upper respiratory swab specimens is similar to that observed for NP swab specimens."
Source:https://www.fda.gov/media/134922/download