Diagnostic methods for influenza infection that are routinely performed such as virus isolation and antigen detection are both sensitive and specific. The presence of molecular techniques for detection of influenza virus provides advantages for the investigation of respiratory outbreaks and may be essential for further epidemiology purposes such as evolutionary studies. This is the first report describing the development of primer sets to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus that used Indonesian virus. As an archipelago country, the development of primer sets covering complete coding sequence of influenza A/H3N2 virus for further evolutionary studies has become a challenge in Indonesia. Maintaining the cold chain stability during specimen shipment become crucial for ILI surveillance in Indonesia.
The designed primer sets covering complete coding sequence of HA and NA gene of influenza A/H3N2 virus were developed based on the consideration that shorter PCR fragment gives effective PCR reaction compared with long fragment amplification. The most efficient amplification is in the 300–1000 base pair length. Given that not all of the samples of ILI surveillance were in good quality, efficient amplification of less than 1000 base pair fragment is become an important consideration in developing the primer sets to obtain complete coding sequence of HA and NA gene of H3N2 virus in Indonesia.
PCR has been recognized to have higher sensitivity and specificity compared to virus isolation and antigenic detection. Positive results of influenza virus in experimentally infected turkey was shown from day 3 to 10 post-challenge by virus isolation and from day 7 to 10 post-challenge by antigen detection using antigen capture enzyme immunoassay. In comparison, successful detection of influenza virus in the experimentally infected turkey using RT-PCR was made from day 3 to day 12 post-challenge, suggesting the higher sensitivity of RT-PCR compared to virus isolation and antigen detection .
Prior to sample examination, the RT-PCR primer sets used in this study were constructed based on the templates used for PCR amplification of the complete coding sequence of HA and NA genes . This primer sets were re-designed to generate two overlapping PCR products (Figs. 1 and 2). To prevent any primer mismatches, sequences located in conserved region within HA and NA genes were selected for primer binding locations as listed in Table 1. The design of these primer pairs for RT-PCR was based on simple rules for efficient primers that the primer pairs were only 18-20 nucleotide-long with 50–60 % G + C composition. Moreover, the primer pairs were also designed to prevent any complementary sequence at the 3′ ends between primer pairs that may promote the formation of primer-dimer artifacts and reduce the yields of the desired product [11, 12].
Possible reason of only 45.9 and 58.8 % successful sequenced samples in this study could be due to the low virus concentration in the samples. It is also possible that the virus titer had continued to decrease throughout the year during the samples storage as repeated freezing and thawing might reduce the possibility to recover the virus. The low yield of PCR product could also be due to the degradation of the viral RNA. The poor condition of cold-chain during specimen handling and shipment from local surveillance teams in many remote areas to laboratory in Jakarta was one of the possible reasons that might lower the samples quality.
To ensure the negative result in this study, we have performed additional RT-PCR using primer sets developed previously  to the 5 clinical samples that showed negative results using designed primers sets. The result including the real-time RT-PCR CT value (obtained from ILI surveillance result, not done in this study) was listed as Additional file 3. The conservation of the primer binding site is one of the issues that could cause primer mismatches and negative results. However, the designed primer sets aligned with HA and NA genes of H3N2 virus from 2005 to 2015 obtained from GISAID was conserved and illustrated in Additional files 4 and 5, respectively.
The annealing temperatures of 50 and 55 °C have been shown to generate optimum PCR products for HA and NA genes, respectively. Given that some samples were not amplified, annealing temperature of 50 °C was selected as the optimum annealing temperature. This was based on the consideration that lower temperature can increase primer binding to the template, although it may also increase the unintended extension of nucleotides at the 3′ end of the primers . Moreover, inner primers within PCR fragments were also employed to sequence the overlapping RT-PCR products in an attempt to obtain the intended length of sequences of each gene.
The limitation of this study is that the primer sets were validated using samples collected only in 2008–2009. However the alignment of the influenza A/H3N2 virus from 2010 until 2015 obtained from GISAID and the primer binding site both for HA and NA genes designed in this study showed that the primer binding site were still conserved (Additional files 4 and 5).