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Open Access The development of highly sensitive diagnostic methods of single nucleotide mutations by chemically-modified nucleic acid

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A research team at Osaka University in Japan has developed highly specific methods of detecting pathogenic virus sub-types and gene mutations, without the need for expensive laboratory facilities, that can be employed in the field by unskilled healthcare personnel.

The project is developing a diagnostic 'point-of-care' kit, which can combine the speed of immunochromatographic assays and the diagnostic specificity of amplified laboratory techniques. For such a kit to work at the bedside, or even in the field, it must be easy to use, require small volumes of reagent, work without the need for electricity, and be thermally stable. Kaihatsu is confident that his team is close to realising this goal: 'Our detection kit is currently tenfold-less sensitive than existing methods, but we have recently developed a means to improve our test's sequence specificity and have shown that we can discriminate single base polymorphism-induced drug resistant virus strains in 15 minutes.'

In this project, the team at Osaka University is using modified peptide nucleic acid (PNA) oligonucleotides. PNA is an artificially synthesised DNA mimic, in which the phosphate backbone of naturally occurring DNA has been replaced by one of a neutral amide backbone, such as a type of protein (peptide). Different forms of PNA can be used in a simple lateral flow assay and packaged in kit form with colour detection lines (such as in pregnancy kits for example) that clearly show when a target molecule is detected.

Neuraminidase inhibitors are drugs commonly used as antivirals, but certain virus strains such as the H1N1 strain of swine flu seen in 2008-09, have an SNP mutation, which renders them resistant. Kaihatsu says: 'We have created a PNA with a tolane intercalator which was able to detect this specific virus serotype.' Without an intercalator, which is an additional molecule included between the artificial DNA base pairs, the PNA interacted with non-drug resistant variants and thus produce some false-positives. The tolane mediated PNA improved sequence specificity and stability. The team has developed other PNAs, including azobenzene-tethered which also detects a sub-type of influenza A H1N1 and a double-stranded 'tail-clamped' variety which has demonstrated a high binding affinity and stability.
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Keywords: DIAGNOSTIC 'POINT-OF-CARE' KIT; DIAGNOSTIC SPECIFICITY; GENE MUTATIONS; H1N1; IMMUNOCHROMATOGRAPHIC ASSAYS; PATHOGENIC VIRUS SUB-TYPES; PEPTIDE NUCLEIC ACID (PNA) OLIGONUCLEOTIDES; SINGLE BASE POLYMORPHISM-INDUCED DRUG RESISTANT VIRUS STRAINS; TOLANE INTERCALATOR

Document Type: Research Article

Publication date: March 1, 2018

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