Dataset Information

Evaluation of Intra- and Interlaboratory Variations in SARS-CoV-2 Real-Time RT-PCR Through Nationwide Proficiency Testing.

ABSTRACT:

Objective

This study aimed to examine the intra- and interlaboratory variations of cycle threshold (Ct) values using the nationwide proficiency testing for SARS-CoV-2.

Methods

Triplicated strong-positive contrived samples duplicated weak-positive contrived samples, and 2 negative samples were transported to participating laboratories in October 2021.

Results

A total of 232 laboratories responded. All except 4 laboratories correctly answered. Six false-negative results, including 2 false-negatives with Ct values beyond the threshold and 1 clerical error, were noted from weak-positive samples. Intralaboratory variations of Ct values of weak-positive and strong-positive samples were not acceptable (Ct > 1.66) in 17 and 7 laboratories, respectively. High interlaboratory variations of Ct values (up to 7 cycles) for the 2 commonly used polymerase chain reaction (PCR) reagents were observed.

Conclusion

The overall qualitative performance was acceptable; intralaboratory variation was acceptable. However, interlaboratory variations of Ct values were remarkable even when the same PCR reagents were used.

SUBMITTER: Park K

PROVIDER: S-EPMC9214160 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Publications

Evaluation of Intra- and Interlaboratory Variations in SARS-CoV-2 Real-Time RT-PCR Through Nationwide Proficiency Testing.

Park Kuenyoul K Sung Heungsup H Chun Sail S Min Won-Ki WK

Laboratory medicine 20221101 6

<h4>Objective</h4>This study aimed to examine the intra- and interlaboratory variations of cycle threshold (Ct) values using the nationwide proficiency testing for SARS-CoV-2.<h4>Methods</h4>Triplicated strong-positive contrived samples duplicated weak-positive contrived samples, and 2 negative samples were transported to participating laboratories in October 2021.<h4>Results</h4>A total of 232 laboratories responded. All except 4 laboratories correctly answered. Six false-negative results, incl ...[more]

PMID: 35699488

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Project description:BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan(R) PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 +/- 0.33 Ct values. The coefficients for inter- (1.89 +/- 0.48%) and intra-assay variation (0.85 +/- 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 +/- 0.33, without significant differences between self-designed and ABI inventoried Taqman(R) gene assays. Only two of the tested Taqman(R) ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 +/- 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 +/- 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 +/- 0.74), albeit higher inter-assay CVs (5.38 +/- 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Dataset Information

Evaluation of Intra- and Interlaboratory Variations in SARS-CoV-2 Real-Time RT-PCR Through Nationwide Proficiency Testing.

Objective

Methods

Results

Conclusion

Publications

Evaluation of Intra- and Interlaboratory Variations in SARS-CoV-2 Real-Time RT-PCR Through Nationwide Proficiency Testing.

Similar Datasets

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