Application of a Fluorogenic PCR Assay for Typing and Subtyping of Influenza Viruses in Respiratory Samples

Abstract

A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5′–3′ nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997–1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998–1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997–1998 and 1998–1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.

A paucity of co-infecting respiratory viral pathogens in nasopharyngeal specimens from patients infected with H274Y-positive influenza A (H1N1) strains

“…Isolates of influenza A collected from Toronto, Ontario, Canada (estimated population 2.7 million) during the period November 14, 2007 to February 14, 2008 were screened by reverse transcriptase (RT)-PCR for the H1N1 subtype using primers described previously. 6 Strains of isolates were confirmed by Sanger sequencing and sequence alignment. Neuraminidase gene sequencing was undertaken and sequences were aligned using CLUSTALX.…”

Reverse transcription recombinase polymerase amplification with lateral flow dipsticks for detection of influenza A virus and subtyping of H1 and H3

“…Rapid influenza diagnostic tests (RIDTs) are widely used to identify IAVs in healthcare services because they are simple and swift [8][9][10] to 90%, the defect is an inconsistent sensitivity (10-80%) [8,9,11]. More recently, nucleic acid amplification tests (NAATs), such as reverse transcription polymerase chain reaction (RT-PCR) [12][13][14][15], real-time RT-PCR [16,17], and reverse transcription loop-mediated isothermal amplification (RT-LAMP) [18,19], have been used for rapid and sensitive diagnosis or subtyping of IAVs. Nevertheless, these methods require expensive equipment and/or skilled technicians, making them inappropriate for use in developing countries.…”

Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates

“…These were influenza A H1N1 positive isolates most similar genetically to the A/Brisbane/57/2007-like lineage collected during the 2007-2008 influenza season in Ontario, Canada. Upon arrival in the laboratory, neuraminidase (N1) was detected from specimens using the Quantitect Probe RT-PCR kit (Qiagen) and published primers and probes (Schweiger et al, 2000) (Table 1) using an MxPro3005 real-time PCR thermocycler (Stratagene). This N1 subtyping assay has been validated previously as a sensitive and specific method for the detection of N1 in clinical specimens (Schweiger et al, 2000).…”

“…Upon arrival in the laboratory, neuraminidase (N1) was detected from specimens using the Quantitect Probe RT-PCR kit (Qiagen) and published primers and probes (Schweiger et al, 2000) (Table 1) using an MxPro3005 real-time PCR thermocycler (Stratagene). This N1 subtyping assay has been validated previously as a sensitive and specific method for the detection of N1 in clinical specimens (Schweiger et al, 2000). It has also been used extensively in external quality assurance panels and classic phenotypic assays.…”

“…) were only obtained with oseltamivir-sensitive influenza A H1N1 nucleic acid, which was used as a positive control. Specificity of the N1 probe has been published previously (Schweiger et al, 2000) and was not repeated in this study.…”