In vitro diagnostic kit for identification of human papillomavirus in clinical samples

A technology of human papillomavirus and samples, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve the problems of expensive equipment and laborious operation

Active Publication Date: 2009-03-04
GENOMICA SAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, the use of microarray technology still has disadvantages, which require expensive equipment and laborious operations

Method used

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  • In vitro diagnostic kit for identification of human papillomavirus in clinical samples
  • In vitro diagnostic kit for identification of human papillomavirus in clinical samples
  • In vitro diagnostic kit for identification of human papillomavirus in clinical samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Preparation of 'lattice-tube'

[0063] The 'array tube' of the present invention was fabricated at CLONDIAG Chip Technology GmbH (Jena, Germany) as follows. Standard reaction test tubes from Eppendorf made of polypropylene and having a specified receiving volume of 1.5 ml were modified by remelting, so that in the tubes molded with adhesive edges were used for microarray supports. Open recess.

[0064] Microarrays to be inserted into these tubes were produced by using a MicroGrid II Arrayer (BioRobotics, Cambridge, UK). A probe consisting of a 5' terminal amino-modified oligonucleotide having a sequence from the Sequence Listing was arranged at a defined site on the epoxy glass surface of a glass slide (slide size: 75mm x 25mm), and covalently fixed. A single microarray contains 12 x 10 = 120, or 12 x 11 = 132 specific positions where oligonucleotides can be placed. The positions are spaced 0.2mm apart so that the DNA libraries contained in each microarra...

Embodiment 2

[0067] Example 2: Preparation of DNA samples

[0068] 2.1. HPV DNA standard

[0069] HPV DNAs used to assess the specificity and sensitivity of type-specific probes were those containing amplified L1 regions (HPV types 6, 11, 13, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89) recombinant plasmids, Or DNAs extracted from clinical samples whose amplified L1 region was further characterized by DNA sequencing. Recombinant plasmids were constructed by molecular cloning techniques. Briefly, the L1 region amplified from each HPV type was cloned into Easy carrier. Purified preparations obtained from each recombinant plasmid were further characterized by sequence analysis. 1-10 pg of plasmid DNA was used for the evaluation of specificity experiments.

[0070] DNA from the K562 cell line (Catalog No. DD2011, Promega Corporation, Madison, WI, USA) was used to evaluate the specificity and sensitivity of...

Embodiment 3

[0078] Embodiment 3: PCR amplification

[0079] PCR amplification using consensus primers MY11 and MY09 was performed (Manos et al., Molecular Diagnostics of Human Cancer; Furth M, Greaves MF, eds.; Cold Spring Harbor Press. 1989, Vol. 7:209-214). Also included in the PCR reaction is a third primer, HMB01, which is typically used in combination with MY09 and MY11 to amplify HPV type 51 that cannot be amplified efficiently using only MY09 and MY11 (Hildesheim et al., J Infect Dis. Journal) 1994, 169; 235-240). Briefly, PCR amplification was performed in 50 μl final volume reactions containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1 mM MgCl 2 , 0.3 μM each of primers MY09 and MY11 (SEQ ID NO142 and 143), 0.03 μM of primer HMB01 (SEQ ID NO 144), 200 μM of each dNTP, 4 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 5 μl of each HPV DNA standard from Example 2.1. or clinical sample DNA from Example 2.2. To test the suitability of sample DNA, 0...

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Abstract

A method and kit for detection and typing of HPV in a sample are described, as is a reaction vessel for use in the method. Universal HPV primers are used to amplify a sample by PCR; the amplified sample is then hybridised to an array of HPV type-specific probes to determine the HPV type.

Description

field of invention [0001] The present invention relates to in vitro diagnostic kits and methods for the identification of human papillomavirus (HPV) in clinical samples. The invention also relates to devices for use in said kits and methods. [0002] More specifically, in a preferred embodiment of the present invention, it relates to an in vitro diagnostic kit for specifically detecting the genotype of human papillomavirus in a clinical sample using a probe for genotyping HPV, a combination comprising said probe A nucleic acid dot array and a standard laboratory reaction vial platform, a device for automatically processing results and a method for diagnosing HPV infection using the in vitro diagnostic kit. Background of the invention [0003] To date, about 100 types of human papillomavirus (HPV) have been described. An HPV type is considered novel when at least 10% of the gene sequence in the HPV regions E6, E7 and L1 differs from any previously known type. A subtype or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70
CPCC12Q1/708C12Q1/6837C12Q1/6888
Inventor 伊雷妮·加斯孔·埃斯科瓦尔比利亚埃尔莫萨·贾因·玛丽亚·路易莎
Owner GENOMICA SAU
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