Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus

Abstract

First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoVIn clinical samples the median concentrations of R-and N-gene RNA, respectively, were 1.2 ؋ 10 6 and 2.8 ؋ 10 6 copies/ml (sputum and endotracheal aspirates), 4.3 ؋ 10 4 and 5.5 ؋ 10 4 copies/ml (stool), and 5.5 ؋ 10 2 and 5.2 ؋ 10 2 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n ‫؍‬ 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.

High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation

“…All described procedures use pre-formulated components, which can be purchased ready for use, making implementation in other laboratories easy. The limit of detection is close to the mathematical limitation of PCR sensitivity according to the Probit model [22,28,29]. Technical properties of our assay are comparable with that of major commercial products, e.g.…”

Ultrasensitive Monitoring of HIV-1 Viral Load by a Low-Cost Real-Time Reverse Transcription-PCR Assay with Internal Control for the 5′ Long Terminal Repeat Domain

“…After extension-PCR with primers LTRS3/As4, products were TA-cloned, purified, sequenced, and transcribed as described previously (15,21,22 ). The RNA/DNA ratio, as determined by PCR and RT-PCR, was 10 6 .…”

“…Plasmids were purified, sequenced, and reamplified with plasmid-specific primers (M13f-20 and M13r, from the reagent set) to lower the plasmid background in subsequent in vitro transcription. Reamplification products were transcribed into RNA with the MegaScript T7 in vitro transcription reagent set as described (Ambion) (15,21,22 ). After DNase I digestion, RNA transcripts were purified with Qiagen RNeasy columns and quantified spectrophotometrically.…”

Evaluation of sensitivities and specificities of SARS-CoV detection by real-time quantitative reverse transcription-PCR assays

“…This caused us much concern in using the primers and probe based on the S gene despite having the best sensitivity for detection. Therefore, people opted to choose primers and probes based on conserved genes, such as N or Replicase 1b (1,3,4,6,11). From our result and five other independent laboratories (1,3,4,6,11), the N gene was more sensitive than the Replicase 1b both in cell culture and patient materials, which makes it suitable for molecular detection of SARS-CoV.…”

“…It has been reported that N-gene-specific PCR provides stronger fluorescence signals and lower C T values than Replicase 1b gene PCR (1,3,4,6). A Taqman amplicon targeting the N gene is 5 log10 times more sensitive for SARS-CoV target RNA extracted from infected cells and 2.79 log10 times more sensitive for RNA extracted from patient material than an amplicon targeting the polymerase gene (11).…”