Please utilize the bioinformatics tools to design the PCR primer(s) and probe set that can specifically detect the avian influenza viruses which become an outbreak among poultry in Thailand since 2003.

Then, please answer these following questions;

1. What are avain influenza virus gene(s)/segment(s) that you will choose as for your PCR primer and probe target? And why?

I used Hemagglutinin and Neuraminidase genes as for PCR primer and probe target.

Because these 2 parts are variable among strains and are used for differentiating avian influenza virus as standard method.

2. Please show the size of PCR product and the position of your primers and probe which could specifically identify the avian influenza virus isolated in Thailand since 2003.

Firstly I chose Hemagglutinin and Neuraminidase gene from avian influenza viruses (A/chicken/Thailand/CH-2/2004(H5N1)) as targets in designed primers and probe by using "Primer3" program.

Steps in "Primer3"

Input : enter PCR target sequence (FASTA) in box > mark left, right primers and probe. > click "Pick primers"

Output of Hemagglutinin gene

Product size : 153 bp

Position of Primers : Left 140-159, Right 273-292, Probe 208-227

Output of Neuraminidase gene

Product size : 194 bp

Position of Primers : Left 218-237, Right 392-411, Probe 295-314

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After I got Primers and probe sequences, I've checked that they should specifically bind to Avian influenza viruses H5 and N1subtype, respectively by using BLAST "Search for short nearly exact matches" option. No cross binding to other subtypes occurred.

3. Please extract Thailand-infected avian influenza virus sequences from the public database and tell us the relationship between avian influenza virus from Thailand and our neighbor countries.

From NCBI, I chose 12 avian influenza virus (H5N1), Hemagglutinin gene sequences to perform phylogenetic construction.

List of sequences included in my study

Influenza A virus (A/chicken/Thailand/CH-2/2004(H5N1)) Influenza A virus (A/Thailand/2(SP-33)/2004(H5N1)) Influenza A virus (A/chicken/Nakorn-Patom/Thailand/CU-K2/2004(H5N1)) Influenza A virus (A/quail/Vietnam/36/04(H5N1)) Influenza A virus (A/chicken/Vietnam/C58/04(H5N1)) Influenza A virus (A/chicken/Yamaguchi/7/2004(H5N1)) Japan Influenza A virus (A/Ck/YN/115/2004(H5N1)) China Influenza A virus (A/Ck/ST/4231/2003(H5N1)) China Influenza A virus (A/Dk/HN/101/2004(H5N1)) China Influenza A virus (A/peregrine falcon/HK/D0028/2004(H5N1)) Hong Kong Influenza A virus (A/Ck/Indonesia/5/2004(H5N1)) Influenza A virus (A/goose/China/F3/2004(H5N1))

 

Result from Phylogenetic construction

As picture below, red boxes are Thailand strains

From phylogenetic tree, the most related strain to Thailand is Influenza A virus from Vietnam (A/chicken/Vietnam/C58/04(H5N1)).

Influenza A virus (A/Thailand/2(SP-33)/2004(H5N1)) and Influenza A virus (A/chicken/Nakorn-Patom/Thailand/CU-K2/2004(H5N1)) are most related among strains from Thailand.

4. Please specify type/name of the (bioinformatics) programs that you have used to get these all answers.

1.NCBI database for nucleotide sequences of hemagglutinin and neuraminidase gene

2.Primer 3 for primers and probe design

3.BLAST "Search for short nearly exact matches" in NCBI for check specific binding of primers and probe

4.ClustalX for sequence alignment

5.BioEdit for editing sequences

6.PHYLIP for phylogenetic construction

7.TreeView for opening tree

 

 

 

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