Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel

Abstract

The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories.

Comparison between Pernasal Flocked Swabs and Nasopharyngeal Aspirates for Detection of Common Respiratory Viruses in Samples from Children

“…We predict that the flocked swab failed to collect enough infected cells or cell-free virus because the health care provider did not follow our standard specimen collection protocols. The rapid and accurate detection of influenza viruses is of utmost importance so that appropriate antiviral therapy may be started and infected patients may be isolated to prevent nosocomial infections (8,12). All 48 samples found to be influenza A virus positive by the use of NPAs were also found to be positive by use of the flocked swabs.…”

Development of Multiplex Real-Time Reverse Transcriptase PCR Assays for Detecting Eight Medically Important Flaviviruses in Mosquitoes

“…Other published studies have used either four different reactions in four separate reaction tubes (2, 6, 11, 13) or a two-color reaction in one tube (10,21,24,25). The replacement of a fluorescent light quencher, such as 6-carboxy-N,N,NЈ,NЈ-tetramethylrhodamine, with a nonfluorescent thermoquencher (BHQ) allows the fluorescent reporters to use greater regions of the spectrum, thus allowing additional reporters to be incorporated into additional probes specific for additional viral RNA types.…”

Killing of Avian and Swine Influenza Virus by Natural Killer Cells

“…To determine the virus titer, the lungs were thawed and homogenized with an OMNI homogenizer, RNA was extracted from the lungs homogenate, and the virus titer was measured by a real-time reverse transcription-PCR (RT-PCR) assay. The virus titer was determined based on the viral genome copy numbers (15).…”