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Methods and applications of molecular beacon imaging for infectious disease and cancer detection

A molecular beacon, disease technology, applied in the field of infectious and/or expressed or mutated molecular beacons, can solve the problems of lack, difficult to treat diseases, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2009-03-11
ALVITAE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Lack of effective early pharmacogenomic testing often makes it difficult to treat many life-threatening diseases

Method used

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  • Methods and applications of molecular beacon imaging for infectious disease and cancer detection
  • Methods and applications of molecular beacon imaging for infectious disease and cancer detection
  • Methods and applications of molecular beacon imaging for infectious disease and cancer detection

Examples

Experimental program
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Effect test

Embodiment 1

[0126] Molecular beacon-target DNA fluorescence detection: The method used to measure the binding of molecular beacons to DNA templates (to measure MB specificity) is described below:

[0127] Materials include: Opti-MEM transfection solution (Invitrogen), Costar 96-well black plates (eBioscience cat. no. 44-2504-21), 1.7 mL Eppendorf tubes (Denville cat. no. C-2170), standard PCR tubes, molecular beacons (MWG -Biotech AG) and target DNA (MWG-Biotech AG).

[0128] Methods as below:

[0129] (1) Dilution of Molecular Beacons and Target DNA: Based on the MWG oligomerization synthesis report, Molecular Beacons and target DNA were diluted according to the specified "volume to 100 pmol / μl" transfection solution. Vortex and spin. The same amount of oligomeric solution was separated and placed in a -20°C refrigerator in the dark.

[0130] (2) Preparation for fluorescence detection: Dilute each molecular beacon at 1:10 (1 μl molecular beacon solution plus 9 μl transfection solution...

Embodiment 2

[0136] As shown in Table 2, molecular beacons (MBs) for detecting FluA, FluB, FluAH5 and FluAN1 were designed based on specific DNA sequences identified by bioinformatics, respectively. The formation of the hairpin loop is designed to have 5 or 6 (mostly 5) base pairs. A common method for preparing MB is disclosed by Peng et al. (18). MBs were then synthesized by contractor MWG Biotech, Inc., located in North Carolina. The 5' end (or 3' end) fluorescent group can be any fluorescent protein, and the 3' end (or 5' end) quencher can be any quencher capable of quenching the corresponding fluorescent group. Figure 28 shows the conserved sequences identified by bioinformatics that are specific to influenza virus types FluA and FluB, and strains FluAH5 and FluAN1.

[0137] As shown in Table 3, the sequences identified by bioinformatics were specific to influenza virus types FluA and FluB as well as FluAH5 and FluAN1 strains.

[0138] Table 2: Molecular Beacons and SEQ ID NOs

[0...

Embodiment 3

[0144] Infectious Disease Detection: Influenza detection molecules of the invention show specific binding to the target. Molecules such as ALV-Flu A, ALV-Flu A H5, ALV-Flu AN1 and ALV-Flu B are designed to specifically detect Flu A, Flu A H5, Flu A N1 and Flu B, respectively. As shown in Figure 6, these molecules bound their respective targets with very low background specificity.

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Abstract

Molecular beacon for detecting an infection and / or expression or a mutation of a disease marker for diagnostics and pharmacogenomics. The molecular beacon is capable of hybridizing a disease-related RNA or DNA of a disease marker in a specimen obtained from a living subject, thereby emitting a signal detectable without a need for signal amplification. The disease marker includes a genetic sequence specific to a pathogen including a flu virus, a cancer cell marker, and a drug resistance-related genetic mutation marker for a drug resistant cancer and infectious pathogen. To detect a disease cell, a specimen containing one or more cells is obtained from a living subject, and fixed by an organic solvent. A molecular beacon is then added to the specimen, followed by staining nuclei of the cells in the specimen. The signal is detectable with a microscope, FACS scan, ELISA plate reader, Scanner, or any combinations thereof.

Description

[0001] This application is a PCT international patent application filed on December 22, 2006. For all countries except the United States, the applicant is the American national company name Alvitae Pharmaceuticals (ALVitae Pharmaceuticals), and only for the designated United States The applicants are Augustine Lin, a U.S. citizen, and Pan-Chyr Yang and Cheng-Chung Chou, R.O.C. Taiwanese citizens. [0002] Cross References to Related Patent Applications [0003] This application claims U.S. Provisional Patent Application Serial No. 60 / 753,651 filed December 23, 2005 by Augustine Line, Pan-Chyr Yang, and Cheng-Chung Chou under 35 U.S.C. § 119(e), entitled " Methods and Uses of Molecular Beacon Imaging for Infectious Disease and Cancer Detection," and U.S. Provisional Patent Application Serial No. 60 / 753,960, filed December 23, 2005, by Augustine Line, entitled "For the Identification and Methods and Applications of Molecular Beacon Imaging for Identification of Genomic Targets an...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/70
Inventor 奥古斯丁·林杨泮池周成春
Owner ALVITAE PHARMA
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