Performance and Cost Evaluation of One Commercial andSix In-House Conventional and Real-Time ReverseTranscription-PCR Assays for Detection of Severe AcuteRespiratory SyndromeCoronavirus
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abstract
ABSTRACT
We
evaluated seven reverse transcription-PCR (RT-PCR) assays, including
six in-house assays and one commercial assay for the detection of
severe acute respiratory syndrome coronavirus (SARS-CoV) RNA in
clinical specimens. RT-PCR assays targeted different genomic regions
and included three conventional assays (one nested and two non-nested)
run on a conventional heat block and four real-time assays performed in
a LightCycler (LC; Roche Diagnostics). All in-house assays were
optimized for assay parameters, including MgCl
2
, primer, and
probe concentrations. The commercial assay was the RealArt HPA CoV
RT-PCR assay (Artus), which was run in the LC. Testing serial dilutions
of cultured SARS-CoV showed that the analytical sensitivity of the
assays ranged from 10
−8
to 10
−6
,
corresponding to 1 and 100 copies of viral RNA, respectively.
Significant differences in analytical sensitivities were observed
between assays (
P
< 0.01, probit regression analysis
for 50% sensitivity levels for the top two assays versus the
others). Testing 68 clinical specimens (including 17 respiratory tract
specimens, 29 urine samples, and 22 stools or rectal swabs)
demonstrated that six of the seven assays detected at least 17 of 18
positives (defined as positive in at least two assays), and two of the
assays had a sensitivity of 100%. There were no significant
differences in sensitivity between the assays (
P
= 0.5
[Cochrance Q test, least sensitive 15 of 18 versus 18 of
18]). The specificities of the assays ranged from 94.0 to
100% without significant differences (
P
= 0.25
to 0.5 [McNemar test]). The reagent and technologist cost of
performing the in-house PCR assays ranged from $5.46 to $9.81 Canadian
dollars (CDN) per test. The commercial assay cost was
considerably higher at $40.37 per test. The results demonstrated good
performance for all assays, providing laboratories that need to do SARS
RNA testing with a choice of assay
formats.
