Materials and Reagents

1. Respiratory specimens (throat-swabs, sputum and tracheal aspirate) from patients with influenza-like illnesses
   
2. Madin-Darby canine kidney (MDCK) cells (Shanghai Institutes for Biological Sciences)
   
3. Eagle's Minimum Essential Medium (EMEM) (Life Technologies, Gibco^®, catalog number: 11095-080 )
   
4. Fetal bovine serum (Life Technologies, Gibco^®, catalog number: 10099 )
   
5. Viral transportation medium (Yocon Bio-technology, catalog number: MT0301-1 )
   
6. QIAsymphony Virus/Bacteria Mini Kit (QIAGEN, catalog number: 931036 )
   
7. Nuclease-free water (Life Technologies, catalog number: 10977 )
   
8. SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies, catalog number: 11736 )
   
9. The primer sequences used for detection of influenza A H7N9 virus
   
   

   Primera	Sequence (5’-3’)b	PCR amplicon (bp)
   H7F H7R N9F N9R	TGAAAATGGVTGGGAAGGYY TGCCGATTGRGTGCTYTTRT ACAGTGTACAAYAGCARRGT GTTTCGRGCCCAYGTRTTAA	103 165

   ^a F: Forward; R: Reverse
   ^b V: A/C/G; Y: C/T; R: A/G
   

Equipment

1. T-25 cell culture flask (Corning, catalog number: 430168 )
   
2. A biosafety 3 laboratory (BSL-3) (required for virus isolation)
   
3. QIAsymphony SP instrument (QIAGEN, catalog number: 9001297 )
   
4. ABI Prism 7900HT Sequence Detection System (Life Technologies, Applied Biosystems^®, catalog number: 4329001 )
   

Software

1. Sequence Detection System Software (v2.3) (Life Technologies, Applied Biosystems^®)

Procedure