Cyril C Y Yip1, Siddharth Sridhar2, Andrew K W Cheng1, Ami M Y Fung1, Vincent C C Cheng1, Kwok-Hung Chan2, Kwok-Yung Yuen3. 1. Department of Microbiology, Queen Mary Hospital, Hong Kong Special Administrative Region. 2. Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region. 3. Department of Microbiology, Queen Mary Hospital, Hong Kong Special Administrative Region; Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region; State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong Special Administrative Region; Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region; Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region; The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: kyyuen@hku.hk.
Abstract
BACKGROUND: HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar® HHV-6 PCR Kit. METHOD: The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. RESULTS: Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 copies/ml and a coefficient of determination (R2) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar® HHV-6 PCR Kit (R2=0.926; P<0.0001), with an average bias of -0.24 log10 copies/ml. CONCLUSIONS: The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar® HHV-6 PCR Kit.
BACKGROUND:HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar® HHV-6 PCR Kit. METHOD: The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. RESULTS: Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 copies/ml and a coefficient of determination (R2) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar® HHV-6 PCR Kit (R2=0.926; P<0.0001), with an average bias of -0.24 log10 copies/ml. CONCLUSIONS: The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar® HHV-6 PCR Kit.
Authors: Samson S Y Wong; Cyril C Y Yip; Siddharth Sridhar; Kit-Hang Leung; Andrew K W Cheng; Ami M Y Fung; Ho-Yin Lam; Kwok-Hung Chan; Jasper F W Chan; Vincent C C Cheng; Bone S F Tang; Kwok-Yung Yuen Journal: Virol J Date: 2018-09-27 Impact factor: 4.099
Authors: Cyril C Y Yip; Siddharth Sridhar; Kit-Hang Leung; Andrew K W Cheng; Kwok-Hung Chan; Jasper F W Chan; Vincent C C Cheng; Kwok-Yung Yuen Journal: Biomed Res Int Date: 2019-10-09 Impact factor: 3.411