Method for the specific identification of orthopoxvirus with the aid of a miniature biological chip

• US 20060147905A1
• Filed: 11/26/2001
• Published: 07/06/2006
• Est. Priority Date: 11/26/2001
• Status: Abandoned Application

• Current Assignee: Gosudarstvenny Nauchny Tsentr Virusologii I Biotekhnologii "Vektor", Institut Molekulyarnoi Biologii IMVA Engelgardta Rossiiskoi Akademii NAUK
• Original Assignee: Gosudarstvenny Nauchny Tsentr Virusologii I Biotekhnologii "Vektor", Institut Molekulyarnoi Biologii IMVA Engelgardta Rossiiskoi Akademii NAUK
• Inventors: Mikheev, Maxim Vyacheslavovich, Schelkunov, Sergei Nikolaevich, Sandakhchiev, Lev Stepanovich, Mikhailovich, Vladimir Mikhailovich, Lapa, Sergei Anatolievich, Mirzabekova, Natalia Vladimirovna, Mirzabekov, Andrei Darievich
• Application Number:
  US10/495,882
• Publication Number:
  US 20060147905A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

The invention relates to an alternative express-method for specific identification of orthopoxviruses with the aid of microchips by hybridising DNA-DNA. Said method involves a two-stage PCR producing a single stained fluorescently labelled DNA fragment, a hybridisation on a biochip containing an original set of typing oligonucleotides and original procedures for recording and interpreting results.

Claims

1. A method for the identification of virus species of the Orthopoxvirus genus by hybridizing an appropriately treated as described in step (b) orthopoxvirus DNA isolated from a test sample to a miniature biological chip, comprising the steps of

(a) providing a biochip for species diagnostics in the form of a microarray of gel spots on a glass support with subsequent immobilization of typing oligonucleotides,

(b) amplifying a fragment of the crmB gene by a two-step asymmetrical PCR using a fluorescently labeled primer to obtain single-stranded DNA for hybridization,

(c) performing DNA-DNA hybridization on said biochip, whereby a hybridization mix is incubated in a sealed reaction chamber over the microchip allowing the duplex formation between said fragment of viral DNA and the corresponding complementary oligonucleotides on the chip,

(d) performing detection and analysis of the hybridization results, whereby fluorescent signals are detected from the labeled sample using a recording device and the resulting hybridization pattern is subsequently compared to reference patterns that allow an adequate interpretation of the results.

2. The method according to claim 1, wherein said analysis of the resulting hybridization pattern is performed by comparing it to a set of standardized reference patterns specific for each species.

3. A test kit for implementing the method according to claim 1, comprising:

(a) a microchip that contains the typing oligonucleotides and is used for the diagnostics of orthopoxvirus species, and

(b) optionally, reagents for DNA isolation, primers for PCR reactions (to amplify a specific fragment of the viral crmB gene), reagents for hybridization, and a device for the detection of test results (such as a portable chip reader that employs an exciting laser beam and is equipped with a photographic camera or a CCD camera).

4. A biological microchip for implementing the method according claim 1.

5. Discriminating oligonucleotides to be immobilized on the biological microchip according to claim 4, characterized by species-specificity (complementarity) to different species of the Orthopoxvirus genus.

6. A test kit for implementing the method according to claim 2, comprising:

(a) a microchip that contains the typing oligonucleotides and is used for the diagnostics of orthopoxvirus species, and

(b) optionally, reagents for DNA isolation, primers for PCR reactions (to amplify a specific fragment of the viral crmB gene), reagents for hybridization, and a device for the detection of test results (such as a portable chip reader that employs an exciting laser beam and is equipped with a photographic camera or a CCD camera).

7. A biological microchip for implementing the method according to claim 2.