Comparative Reproducibility of SYBR Green I and TaqMan Real-time PCR Chemistries for the Analysis of Matrix and Hemagglutinin Genes of Influenza A Viruses
Madhu Khanna,
Associate Professor,
Delhi University
Background: Influenza virus is epidemiological significant among all the respiratory viruses because of high mortality and morbidity rate. The outbreak of novel influenza A H1N1-2009 virus (pH1N1) and its rapid spread worldwide raised serious concern about pandemic preparedness. Real-time quantitative PCR (qPCR) is a leading edge technology for evaluate the expression of nucleic acids and has become a foremost need in diagnosis and research works. The high precision, sensitivity and specificity of qPCR assays have led to development of 20 different chemistries, of which TaqMan and/or SYBR Green chemistries are the most widely used. The TaqMan PCR exhibits the 5'–3' nuclease activity to cleave a sequence specific fluorescent labelled probe8 while SYBR Green assay is targeting intercalating fluorescent dyes between the minor grooves of the amplified DNA during the primer annealing and extension steps of each PCR cycle. TaqMan chemistry9 is recommended by World Health Organization (WHO) for detection of the pandemic H1N1 virus in human respiratory samples during the pandemic period10.
Objectives: The present study was designed to evaluate the sensitivity and specificity of two chemistries of real-time RT-PCR (with the use of fluorescent SYBR Green I dye and specific TaqMan probe) for detection of the matrix and hemagglutinin genes of human influenza A viruses.
Methods: Influenza A virus reference strains were used to perform the calibration curve analysis and the diagnostic accuracy of both the real-time RT-PCR formats were assessed on 110 clinical specimens from patients presenting with influenza-like-illness (ILI). Two different real-time RT-PCR assays, one with the fluorescent SYBR Green I dye and second with the specific TaqMan probe were used in the study. All the clinical samples were also screened for the detection of matrix gene for all type A and HA gene for pandemic H1N1-2009 infections by conventional RT-PCR. Statistical analysis was performed using Pri
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