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Rifampicin drug-resistant tuberculosis molecular marker, detection reagent and application thereof

A technology of molecular markers and detection reagents, which can be used in biological testing, microbial measurement/testing, disease diagnosis, etc., and can solve the problems of time-consuming and high cost

Active Publication Date: 2021-04-30
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, phenotypic culture-based methods are still the mainstay of drug susceptibility testing, but are time-consuming and require strict microbiological safety precautions
Although molecular techniques such as Xpert MTB / RIF or linear probes are fast and efficient, there are still certain false positive and false negative rates, and the cost is high, which has not yet been popularized in grassroots units

Method used

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  • Rifampicin drug-resistant tuberculosis molecular marker, detection reagent and application thereof
  • Rifampicin drug-resistant tuberculosis molecular marker, detection reagent and application thereof
  • Rifampicin drug-resistant tuberculosis molecular marker, detection reagent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Preparation of peripheral blood nucleated cells

[0049] (1) Collect 2 mL of peripheral blood from each subject on an empty stomach in the morning, put it in a blood collection tube containing sodium heparin anticoagulant, and prepare nucleated cells within 6 hours.

[0050] (2) 1 times the volume of fresh whole blood, add 3 times the volume of red blood cell lysate. In this example, 2 ml of fresh whole blood was added to 6 ml of erythrocyte lysate (solarbio, ID: R1010), and gently vortexed or inverted to mix.

[0051] (3) Place on ice for 15 minutes, during which time gently vortex and mix twice. After red blood cells are lysed, the solution should be clear and transparent.

[0052] (4) Cell collection: centrifuge at 450×g for 10 minutes at 4°C to pellet white blood cells, and carefully aspirate and discard the supernatant.

[0053] (5) Add twice the volume of erythrocyte lysate (4 ml in this example) to the leukocyte pellet, and gently vortex to fully res...

Embodiment 2

[0056] Example 2. Application of RT-qPCR method to detect expression changes of TRIM gene

[0057] 1. Extraction of RNA

[0058] (1) Thaw the cell lysate obtained in Example 1 at room temperature, add 200 μL of chloroform to each tube, mix by inversion for 20 seconds, a pink turbid solution without stratification occurs, and stand at room temperature for 10 minutes.

[0059] (2) Centrifuge at 12000g / min at 4°C for 15min, take out the centrifuge tube, and the sample is divided into three layers: a colorless supernatant aqueous phase, a white middle layer and a pink lower organic phase.

[0060] (3) Carefully transfer the colorless supernatant aqueous phase to a new RNase-free centrifuge tube, add 1 / 2 TRIZOL volume (500mL in this implementation) of isopropanol to the obtained supernatant aqueous phase, mix gently, and place at -20°C for 20min .

[0061] (4) Centrifuge at 12000g / min at 4°C for 10min, discard the supernatant, add 1mL of 75% ethanol (prepared with RNase-free wate...

Embodiment 3

[0079] Embodiment 3.TRIM gene is used for the judgment method of distinguishing RR-TB and DS-TB

[0080] According to the above experimental results, RR-TB and DS-TB patients can be identified by RT-qPCR. In the present invention, the relative expression levels of TRIM9, TRIM21 and TRIM56 are used to analyze the diagnostic efficacy of each and their combination by ROC curve using SPSS 24.0 software. details as follows:

[0081] Calculate the ROC curve of the relative expression of a single TRIM gene to identify rifampicin-resistant tuberculosis, including the area under the curve (AUC), sensitivity and specificity. The results show that the AUC of a single TRIM gene is 0.808, 0.704 and 0.526; are 66.7%, 71.4% and 95.2%, and the specificities are 87.8%, 65.9% and 19.5%, respectively. The results showed that the relative expression of a single TRIM gene was not ideal for the differential diagnosis of rifampicin tuberculosis.

[0082] The Logistic stepwise regression model was...

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Abstract

The invention provides a rifampicin drug-resistant tuberculosis molecular marker, a rifampicin drug-resistant tuberculosis detection reagent and application thereof, and relates to the technical field of biomedicine. The molecular marker comprises a TRIM9 gene, a TRIM21 gene, a TRIM56 gene and expression products of the TRIM9 gene, the TRIM21 gene and the TRIM56 gene. The expression of the TRIM9, TRIM21 and TRIM56 genes in a rifampicin drug-resistant tuberculosis patient is reduced. The rifampicin drug-resistant tuberculosis molecular marker can be used for identifying rifampicin drug-resistant tuberculosis patients more quickly, efficiently and sensitively, and can be used for screening rifampicin drug-resistant tuberculosis.

Description

technical field [0001] The invention relates to the technical field, in particular to a rifampicin drug-resistant tuberculosis molecular marker, detection reagent and application thereof. Background technique [0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis infection, and it is one of the top ten causes of death in the world. According to estimates by the World Health Organization (WHO), there were about 9.96 million new tuberculosis patients worldwide in 2019, which has remained at the same level in recent years. In 2019, about 3.3% of new patients and 18% of retreated patients were resistant to rifampicin among new tuberculosis patients worldwide. It is estimated that the number of rifampicin-resistant tuberculosis (RR-TB) patients in the world is about 465,000, of which multi-drug-resistant tuberculosis (MDR-TB) accounts for about 78%. The latest treatment outcome data for MDR / RR-TB patients shows that the global MDR / RR-TB ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686G01N33/68
CPCC12Q1/6883C12Q1/686G01N33/6893G01N2800/26C12Q2600/158C12Q2600/106C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 李传友刘盛盛任卫聪唐神结
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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