Prospective application of reverse transcriptase polymerase chain reaction for diagnosing influenza infections in respiratory samples from a children's hospital

Abstract

A prospective clinical evaluation of the reverse transcriptase polymerase chain reaction (RNA PCR) for detection of influenza viruses was carried out with specimens from 342 patients of a children's hospital in The Netherlands. The RNA PCR, carried out directly on the specimens without an organic extraction, showed a sensitivity and specificity which are superior to those of direct immunofluorescence and comparable to those of cell culture combined with immunofluorescence (culture/IF). Negative results can be obtained within 2 days by the RNA PCR but may take up to 14 days by culture/IF. Because culturing is the standard technique for the detection of respiratory viruses, at this moment there are no strong arguments to replace culture/IF with RNA PCR for the detection of influenza A virus.

Rapid and Sensitive Method Using Multiplex Real-Time PCR for Diagnosis of Infections by Influenza A and Influenza B Viruses, Respiratory Syncytial Virus, and Parainfluenza Viruses 1, 2, 3, and 4

“…Multiplex real-time PCR was found to be more sensitive than cell culture on a range of different respiratory samples. These findings are consistent with those of other studies, which employed RT-PCR for the detection of viral infections (3,7,9,15,22,23,24). Conventional respiratory viral cell culture is limited by a lack of speed and therefore has little impact on patient care (1,27).…”

“…Realtime PCR was performed in 50 l of reaction mixture consisting of 10 l of 5ϫ one-step reverse transcription (RT)-PCR buffer (Qiagen one-step RT-PCR kit), All samples found to be positive for influenza A virus by the real-time PCR were tested by a second influenza A virus PCR to confirm the results. PCR amplification was performed with primers described by Claas et al (3), which target the same gene as the real-time PCR primers. Briefly, 5 l of isolated RNA was converted to cDNA and amplified for 40 cycles with influenza A virus primers, designed to the nonstructural gene segment sequence.…”

Simultaneous Detection of Influenza Viruses A and B Using Real-Time Quantitative PCR

“…To overcome this lack of sensitivity and to obtain rapid diagnostic results, PCR techniques have been developed for the specific detection and subtyping of influenza viruses. They have proven to be very sensitive and specific but, unfortunately, are often difficult to implement in a routine diagnostic setting and still require time-consuming sample handling and post-PCR analysis (1,3,5). Needless to say, better techniques are still needed.…”

Effectiveness of Reverse Transcription-PCR, Virus Isolation, and Enzyme-Linked Immunosorbent Assay for Diagnosis of Influenza A Virus Infection in Different Age Groups

“…For these reasons, rapid and highly sensitive influenza-specific reverse transcription (RT)-PCR assays have been developed (1,4) that may be used for typing and subtyping of influenza viruses (5,25) or for detection in a multiplex PCR format (12). Despite their higher in vitro sensitivity, RT-PCR assays exhibited no clear advantage over virus isolation in terms of sensitivity, specificity, and virus detection rates when applied to clinical samples from pediatric patients (6,20).…”