Ann Lab Med.  2016 Sep;36(5):450-456. 10.3343/alm.2016.36.5.450.

Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR

• Kim MN
• Ko YJ
• Seong MW
• Kim JS
• Shin BM
• Sung H

• KMID: 2373575
• DOI: http://doi.org/10.3343/alm.2016.36.5.450

Abstract

BACKGROUND
During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits.
METHODS
PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively.
RESULTS
The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition.
CONCLUSIONS
The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.

Keyword

MERS coronavirus; Real-time reverse transcription polymerase chain reaction; Validation; Lower respiratory specimen; Commercial kits

MeSH Terms

Coronavirus Infections/diagnosis/virology
Humans
Middle East Respiratory Syndrome Coronavirus/*genetics/isolation & purification
Nasopharynx/virology
Open Reading Frames/genetics
RNA, Viral/*analysis/metabolism
Reagent Kits, Diagnostic
*Real-Time Polymerase Chain Reaction
Viral Envelope Proteins/genetics
RNA, Viral
Reagent Kits, Diagnostic
Viral Envelope Proteins

Figure

• 

  Fig. 1 LOD of the six commercial kits for MERS-CoV RNA detection with 95% confidence intervals (CI). LOD was determined by probit regression analysis for the 16 replicate assays of each of six concentrations of upE and ORF1a RNA transcripts from 0.3-100 copies/reaction with 0.5 log dilution. (A) upE with PowerChek single, (B) upE with PowerChek duplex, (C) ORF1a with PowerChek duplex, (D) upE with DiaPlexQ single, (E) upE with DiaPlexQ triplex, (F) ORF1a with DiaPlexQ triplex, (G) upE with AccuPower single, (H) ORF1a with AccuPower single, (I) upE with LightMix single, (J) ORF1a with LightMix single, (K) upE with UltraFast single, and (L) ORF1a with UltraFast single. LODs of each assay were denoted in copies per test (blue letters).Abbreviations: LOD, limit of detection; MERS-CoV, Middle East Respiratory Syndrome coronavirus.

• 

  Fig. 2 Comparison of LOD of the six commercial kits for MERS-CoV RNA detection. The log LOD values of upE (A) and ORF1a (B) with 95% confidence intervals (CI) are shown, but there were no CI values for Anyplex single upE. The 95% CI values overlapped among the kits, except for the LOD of ORF1a with PowerChek duplex.Abbreviations: See Fig. 1.

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