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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: The LODs for upE varied from 21.88 to 263.03 copies/reaction, and those of ORF1a varied from 6.92 to 128.82 copies/reaction (Fig. 1). According to the probit regression analysis, the 95% confidence intervals (CI) for upE and ORF1a were found to overlap among the tested kits, with the exception of ORF1a by the PowerChek kit (Fig. 2). The LODs for upE using both the single and multiple gene-targeting formats of the 2-step kits were 1.64 and 1.45 log copies/reaction for the PowerChek kit and 1.76 and 1.61 log copies/reaction for the DiaPlexQ kit (Fig. 2). The LOD for upE was 1.34 log copies/reaction for the Anyplex kit, but no CI value could be calculated because there were no positive reactions at 1.0 log copy/reaction, while all 16 replicate specimens were positive at 1.5 log copies/reaction. In contrast, although the LODs for upE and ORF1a using the LightMix kit were 2.11 log copies/reaction and 1.78 log copies/reaction, respectively, trailing of positives was observed at much lower concentrations than these LODs (Fig. 1). The LODs for upE of three different 1-step kits, the AccuPower, Light Mix, and Ultrafast kits, were >2.0 log copies/reaction, which was less sensitive than those of three different 2-step kits; however, this difference was not significant (Fig. 2). The LODs for ORF1a varied less—i.e., from 1.78 to 2.11 log copies/reaction—with the exception of the PowerChek kit, which was considerably lower at 0.84 log copies/reaction (Fig. 2). None of the kits tested in this study showed cross-reactivity with other respiratory viruses.


Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR
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.
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Figure 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.
Mentions: The LODs for upE varied from 21.88 to 263.03 copies/reaction, and those of ORF1a varied from 6.92 to 128.82 copies/reaction (Fig. 1). According to the probit regression analysis, the 95% confidence intervals (CI) for upE and ORF1a were found to overlap among the tested kits, with the exception of ORF1a by the PowerChek kit (Fig. 2). The LODs for upE using both the single and multiple gene-targeting formats of the 2-step kits were 1.64 and 1.45 log copies/reaction for the PowerChek kit and 1.76 and 1.61 log copies/reaction for the DiaPlexQ kit (Fig. 2). The LOD for upE was 1.34 log copies/reaction for the Anyplex kit, but no CI value could be calculated because there were no positive reactions at 1.0 log copy/reaction, while all 16 replicate specimens were positive at 1.5 log copies/reaction. In contrast, although the LODs for upE and ORF1a using the LightMix kit were 2.11 log copies/reaction and 1.78 log copies/reaction, respectively, trailing of positives was observed at much lower concentrations than these LODs (Fig. 1). The LODs for upE of three different 1-step kits, the AccuPower, Light Mix, and Ultrafast kits, were >2.0 log copies/reaction, which was less sensitive than those of three different 2-step kits; however, this difference was not significant (Fig. 2). The LODs for ORF1a varied less—i.e., from 1.78 to 2.11 log copies/reaction—with the exception of the PowerChek kit, which was considerably lower at 0.84 log copies/reaction (Fig. 2). None of the kits tested in this study showed cross-reactivity with other respiratory viruses.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus