pyrophosphate and Influenza--Human

Pyrosequencing is a high-throughput non-gel-based DNA sequencing method that was introduced in the late 1990s. It employs a DNA sequencing-by-synthesis approach based on real-time measurement of pyrophosphate released from incorporation of dNTPs. A cascade of enzymatic reactions proportionally converts the pyrophosphate to a light signal recorded in a form of peaks, known as pyrograms. Routinely, a 45-60-nucleotide sequence is obtained per reaction. Recent improvements introduced in the assay chemistry have extended the read to approximately 100 nucleotides. Since its advent, pyrosequencing has been applied in the fields of microbiology, molecular biology and pharmacogenomics. The pyrosequencing approach was first applied to analysis of influenza genome in 2005, when it played a critical role in the timely detection of an unprecedented rise in resistance to the adamantane class of anti-influenza drugs. More recently, pyrosequencing was successfully applied for monitoring the emergence and spread of influenza A (H1N1) virus resistance to oseltamivir, a newer anti-influenza drug. The present report summarizes known applications of the pyrosequencing approach for influenza genome analysis with an emphasis on drug-resistance detection.

Topics: Antiviral Agents; Diphosphates; Drug Resistance, Viral; Expressed Sequence Tags; Genome, Viral; Genotype; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Influenza, Human; Molecular Diagnostic Techniques; Orthomyxoviridae; Oseltamivir; Sequence Analysis, DNA

A new colorimetric method for monitoring of rolling circle amplification was developed. At first H5N1 target hybrids with padlock probe (PLP) and then PLP is circularized upon the action of T4 ligase enzyme. Subsequently, the circular probe is served as a template for hyperbranched rolling circle amplification (HRCA) by utilizing Bst DNA polymerase enzyme. By improving the reaction, pyrophosphate is produced via DNA polymerization and chelates the Mg(2+) in the buffer solution. This causes change in solution color in the presence of hydroxy naphthol blue (HNB) as a metal indicator. By using pH shock instead of heat shock and isothermal RCA reaction not only the procedure becomes easier, but also application of HNB for colorimetric detection of RCA reaction further simplifies the assay. The responses of the biosensor toward H5N1 were linear in the concentration range from 0.16 to 1.20 pM with a detection limit of 28 fM.

Topics: Animals; Biosensing Techniques; Birds; Colorimetry; Diphosphates; DNA, Complementary; Humans; Influenza A Virus, H5N1 Subtype; Influenza in Birds; Influenza, Human; Limit of Detection; Magnesium; Naphthalenesulfonates; Nucleic Acid Amplification Techniques; RNA, Viral