Humans ; Molecular Diagnostic Techniques ; standards ; Quality Control

BACKGROUND: The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used. METHODS: The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/microL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient. RESULTS: The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R2=0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples. CONCLUSIONS: This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity.
Coronavirus Infections/*diagnosis/epidemiology/virology ; Disease Outbreaks ; Humans ; Middle East Respiratory Syndrome Coronavirus/*genetics/isolation & purification ; Molecular Diagnostic Techniques/*standards ; Quality Assurance, Health Care ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction ; Republic of Korea/epidemiology ; Surveys and Questionnaires

OBJECTIVETo establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).

METHODSThe probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed.

RESULTSThe sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01).

CONCLUSIONSThe method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase ; chemistry ; metabolism ; Animals ; DNA Probes ; chemistry ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B virus ; genetics ; Humans ; Molecular Diagnostic Techniques ; methods ; standards ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity