Figure 1.
Sequence analysis and primer design.
The regions targeted by primer pairs are nucleotide 1047 to 1149 for HA sequence (A) and nucleotide 490 to 654 for NA sequence (B) of H7N9 virus. The underlined sequences were used for the primer design. Sequence alignments were conducted between the primers and 135 H7 and 36 N9 subtype full-length viral gene sequences from NCBI’s influenza virus resource database, some representatives of which are shown here. Nucleotides identical to those of A/Shanghai/1/2013 (H7N9) strain are indicated as dots. A mixed base was used close to the 3′ end of each primer when a different nucleotide was encountered. The modified primer sequences were listed in Table 1.
Table 1.
The primer sequences used for detection of influenza A H7N9 virus.
Figure 2.
Melting curve analysis and confirmation of H7- and N9-specific amplicons from SYBR green-based real time RT-PCR assays.
The specific peaks of H7 (A) and N9 (B) amplicons as well as primer dimers are shown. Capillary electrophoresis analysis was then carried out to confirm the lengths of specific amplicons (C).
Figure 3.
Detection limit and standard curve.
The detection limit was determined based on serial ten-fold dilutions of in-vitro transcribed viral RNA ranging from 106 to 100 copies/µl. Melting curves and amplification plots are indicated for H7 (A, B) and N9 (D, E) assays. The standard curves of H7 (C) and N9 (F) assays were constructed upon the Ct values against the amount of RNA copy number.
Table 2.
Known human respiratory viruses used for testing the specificity of the SYBR green real time RT-PCR assay.
Figure 4.
Real time RT-PCR results using respiratory specimens from seven laboratory-confirmed patients with H7N9 virus infection.
The Ct values obtained from H7 and N9 assays for every clinical sample are presented.