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Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans

A technology for human parainfluenza virus and influenza virus, which is used in the field of probes and detection of most nucleic acid sequences, and can solve the problems of false positives and difficulty in isolating viruses.

Inactive Publication Date: 2008-01-16
INST DE SALUD CARLOS III
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] - The sample is contaminated with bacteria from the normal flora in the sampling area, which makes it difficult to isolate viruses by cell culture, which may cause false positives; at the same time

Method used

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  • Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans
  • Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans
  • Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Detection of reference strains of viral pathogens causing respiratory infections from cell cultures

[0133] Detection of reference strains of viral pathogens causing respiratory infections from samples of cell cultures inoculated with the above strains is performed by an assay based on the interaction of amplified and labeled genetic material with probes immobilized on nylon membranes For hybridization, the method already reported by Kaufhold et al. (Kaufhold et al., 1994, FEMS Microbiology Letters "FEMS Microbiology Letters" 119: 19-25) was used.

[0134] 1.1 Materials

[0135] 1.1.1 sample

[0136] Samples for this test were collected from cell cultured reference strains of viruses that cause respiratory tract infections. The reference strains are provided by well-known laboratories, and these reference strains are virus reference strains widely accepted by the international scientific community. The list is as follows:

[0137] Virus virus strain

[0138]...

Embodiment 2

[0183] Detection of Respiratory Viruses in Clinical Samples Isolated from Respiratory Tract Infected Cell Cultures

[0184] Detection of different respiratory viruses causing respiratory infections from cell cultures was performed by an assay based on the hybridization of fragments of biotinylated genetic material to probes immobilized on nylon membranes.

[0185] 2.1 Materials

[0186] 2.1.1 sample

[0187] The samples used in this assay are those that cause respiratory infections, isolated from respiratory viruses inoculated in cell culture respiratory clinical samples. The viruses are as follows: influenza A virus, influenza B virus, human respiratory syncytial virus type A (RSVA), human respiratory syncytial virus type B (RSVB), human adenovirus (ADV) (several serotypes), Human parainfluenza virus type 3 (PIV3).

[0188] To examine the sensitivity of the technique, serial 10-fold dilutions of virus isolated from cell cultures of patient respiratory samples were used. ...

Embodiment 3

[0213] Detection of Influenza A Viruses Emerging in Clinical Samples

[0214] The method used for the direct detection of influenza A virus present in clinical test samples is an assay that hybridizes amplified and labeled genetic material to probes immobilized on nylon membranes.

[0215] 3.1 Materials

[0216] 3.1.1 sample

[0217] The samples used for this test are taken from throat smears of patients with flu symptoms in order to identify the virus causing the symptoms.

[0218] 3.1.2 probe

[0219] The probes used in this assay (SEQ ID NO: 1 and SEQ ID NO: 2) were designed with a hexylated amino acid group at the 5' end as reported by Kaufhold et al. (FEMS Microbiol Lett. "FEMS Microbiology Letters" 1994 Jun 1;119(1-2):19-25). The presence of this hexylated amino group facilitates probe immobilization and spatial ordering of the probes on the membrane. In order to immobilize the probe on the membrane, the probe should be washed with fresh pH 8.4, 500mM NaHCO 3 So...

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Abstract

The invention relates to probes and assays which are used for the simultaneous detection, in a single assay sample, of a plurality of nucleic acid sequences of viruses that cause respiratory infections in humans, selected from among influenza virus type A, influenza virus type B, influenza virus type C, human respiratory syncytial virus type A, human respiratory syncytial virus type B, human adenovirus, human parainfluenza virus type 1, human parainfluenza virus type 2, human parainfluenza virus type 3, human parainfluenza virus types 4A and 4B, enterovirus, rhinovirus, human coronavirus type 229E, human coronavirus type OC43, coronavirus that causes severe acute respiratory syndrome (SARS), human metapneumovirus and combinations thereof.

Description

technical field [0001] The present invention relates to the detection of nucleotide sequences, in particular probes and assays for the simultaneous detection of multiple nucleic acid sequences in a single test sample. More specifically, the present invention relates to probes and methods for detecting most of the nucleic acid sequences of viruses that cause respiratory infections in humans, through hybridization, and finally reflected in a single test sample, viruses include: type A influenza virus, type B influenza virus Influenza virus, Influenza virus type C, Human respiratory syncytial virus type A, Human respiratory syncytial virus type B, Human adenovirus, Human parainfluenza virus type 1, Human parainfluenza virus type 2, Human parainfluenza virus type 3, 4A Human parainfluenza viruses type and 4B, enteroviruses, rhinoviruses, human coronaviruses 229E, human coronaviruses OC43, coronaviruses that cause severe acute respiratory syndrome (SARS), human metapneumoviruses, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6837C12Q1/701Y02A50/30C12Q1/6888
Inventor 皮勒·佩雷兹·布兰那玛利亚·特雷萨·考拉斯·洛佩兹印玛克拉达·卡萨斯·弗莱彻
Owner INST DE SALUD CARLOS III
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