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. 2013 Nov 20;8(11):e80028.
doi: 10.1371/journal.pone.0080028. eCollection 2013.

Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9) virus

Zheng Zhu  1 , Huan Fan, Xian Qi, Yuhua Qi, Zhiyang Shi, Hua Wang, Lunbiao Cui, Minghao Zhou
Affiliations

Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9) virus

Zheng Zhu et al. PLoS One. .

Abstract

Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR) was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (10(6) to 10(1) copies/ µl) with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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
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.
Figure 4
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.
See this image and copyright information in PMC

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Grants and funding

This study was supported in part by the Jiangsu Province Health Development Project with Science and Education (ZX201109), the Jiangsu Province Key Medical Talent Foundation (JKRC2011002, RC2011191), and the Science and Technology Pillar program of Jiangsu Province (BE2011796). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.