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Peptide nucleic acid probe, composition containing peptide nucleic acid probe, and application of peptide nucleic acid probe

A technology of peptide nucleic acid and probe, which is applied in the field of peptide nucleic acid probes, peptide nucleic acid probes for detecting mycobacteria, especially mycobacterium tuberculosis complex flora, and preparing reagents for detecting tuberculosis, which can solve the problem of weak signals and affecting hybridization Efficiency, steric hindrance effects and other problems, to achieve rapid detection, high signal strength, high specificity

Inactive Publication Date: 2013-09-25
BEIJING GP MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Two kinds of peptide nucleic acid probes for detecting Mycobacterium tuberculosis have been reported in the literature (Stender et al., Fluorescence In situ hybridization assay using peptidenucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures. J Clin Microbiol., 1999, 37(9): 2760-2765; Lefmann et al., Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections. J Clin Microbiol. 2006; 44(10): 3760-3767. , but the signals after hybridization of these two probes are weak
Part of the possible reason is that the specificity of the probe sequence is not very good, and no linker is used to separate the fluorescein group from the PNA chain, resulting in steric hindrance when the PNA chain binds to the target sequence, affecting hybridization efficiency

Method used

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  • Peptide nucleic acid probe, composition containing peptide nucleic acid probe, and application of peptide nucleic acid probe
  • Peptide nucleic acid probe, composition containing peptide nucleic acid probe, and application of peptide nucleic acid probe
  • Peptide nucleic acid probe, composition containing peptide nucleic acid probe, and application of peptide nucleic acid probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Preparation of peptide nucleic acid probe sample 1

[0050] According to the base sequence shown in SEQ ID NO: 3, Shanghai Keyeptide Company was commissioned to synthesize peptide nucleic acid probe sample 1. Wherein, the linker is an O-linker, which is connected to the 5' end of the base sequence, and a lysine (lys) is connected between the base sequence and the O-linker. The fluorescent label is 6-TAMRA, which is attached to lys. The structure of the prepared peptide nucleic acid probe sample 1 is as follows:

[0051] (6-TAMRA)-lys-(O-linker)-TCCTGGTGCCCTACG.

Embodiment 2

[0052] Embodiment 2: Preparation of control probe 1 and control probe 2

[0053] According to the technical protocol disclosed in Stender et al., 1999, control probe 1 was prepared.

[0054] According to the technical scheme disclosed in Lefmann et al., 2006, control probe 2 was prepared.

Embodiment 3

[0055] Embodiment 3: Three kinds of methods detect the test of sputum sample

[0056] Sputum samples from patients with tuberculosis often contain the Mycobacterium tuberculosis complex. The following is the experimental method and results of using the peptide nucleic acid probe sample 1 prepared in Example 1 to detect 111 sputum samples (obtained from different subjects, including tuberculosis patients and normal people) by PNA FISH method. At the same time, as a comparison, the conventional acid-fast staining method was used to detect the same 111 cases of sputum samples as the Roche culture method and the experimental method and results.

[0057] 1. PNA FISH detection

[0058] 1. Reagent Preparation

[0059] The formula of the hybridization solution is as follows:

[0060] 10% (w / v) dextran sulfate, 10mM NaCl, 20%-60% (v / v) formamide, 0.1% (w / v) sodium pyrophosphate, 0.2% (w / v) polyvinylpyrrolidone, 0.2% (w / v) Ficoll, 5mM Na 2 EDTA, 0.1% (v / v) Triton X-100, 50 mM Tri...

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Abstract

The invention belongs to the field of molecular biology, and relates to a peptide nucleic acid probe, a composition containing the peptide nucleic acid probe, and application of the peptide nucleic acid probe. The peptide nucleic acid probe is provided with a detectable marker, and the base sequence of the peptide nucleic acid probe is 10 to 20 continuous basic groups in SEQ ID NO:1 or SEQ ID NO:2. The invention also relates to the composition containing the peptide nucleic acid probe, a test agent for detecting mycobacteria, a kit for detecting the mycobacteria, an in-situ hybridization method, a method for detecting the mycobacteria, application of the peptide nucleic acid probe to preparation of a reagent for detecting the mycobacteria, and application of the peptide nucleic acid probe to preparation of a reagent for detecting tuberculosis. The peptide nucleic acid probe is high in signal intensity and specificity and can detect mycobacteria and mycobacterium tuberculosis complex effectively and quickly.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a peptide nucleic acid probe, in particular to a peptide nucleic acid probe for detecting mycobacteria, especially mycobacterium tuberculosis complex flora. The present invention also relates to a composition containing the peptide nucleic acid probe, a detection agent for detecting mycobacteria, a kit for detecting mycobacteria, an in situ hybridization method, a method for detecting mycobacteria, The use of the peptide nucleic acid probe in preparing a reagent for detecting mycobacteria, and the use of the peptide nucleic acid probe in preparing a reagent for detecting tuberculosis. Background technique [0002] Tuberculosis is a chronic infectious disease transmitted through the respiratory tract, mainly caused by Mycobacterium tuberculosis Complex (MTC), the most important of which is Mycobacterium tuberculosis. [0003] At present, the most commonly used method for clinical de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 胡军吕瑛陈忠吴晓东
Owner BEIJING GP MEDICAL TECH