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Active tuberculosis diagnostic marker, kit and application thereof

A technology for diagnosing markers and active pulmonary tuberculosis, applied in the field of biomedicine, can solve the problems of poor specificity and low sensitivity, achieve the effects of good specificity, overcome low detection rate, and facilitate clinical application

Pending Publication Date: 2020-12-18
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the problems of poor specificity, low sensitivity, and time-consuming detection of existing active tuberculosis diagnostic markers, and provide a technical concept of MCEMP1 mRNA as an active tuberculosis diagnostic marker, for The diagnosis of active pulmonary tuberculosis provides a new target and has good clinical application value for the diagnosis of active pulmonary tuberculosis

Method used

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  • Active tuberculosis diagnostic marker, kit and application thereof
  • Active tuberculosis diagnostic marker, kit and application thereof
  • Active tuberculosis diagnostic marker, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Blood samples from patients with active pulmonary tuberculosis and healthy volunteers were obtained from Shanghai Public Health Clinical Center. The collected EDTA anticoagulated whole blood was 2000g within 4 hours, centrifuged for 10min, and 500μL of plasma was taken and stored in a -80℃ refrigerator. The remaining blood samples were separated and extracted from whole blood with the QIAamp RNA Blood mini Kit kit from QIAGEN. Take 1 μL for Nanodrop 2000 detection to test the concentration and purity of the sample.

Embodiment 2

[0053] Using the whole blood total RNA obtained in Example 1 as a template, Takara's PrimeScriptRT reagentKit with gDNA Eraser (Perfect Real Time) reverse transcription kit was used to synthesize cDNA according to the operating instructions.

[0054] 1. Removal of Genomic DNA Reaction

[0055] Prepare the reaction mixture on ice according to the following components. In order to ensure the accuracy of the preparation of the reaction solution, prepare the Master Mix in the amount of +2 first, then divide it into each reaction tube, and finally add the RNA sample.

[0056]

[0057] 2min at 42°C (or 5min at room temperature)

[0058] Store at 4°C

[0059] 2. Reverse transcription

[0060] Please prepare the reaction solution on ice. In order to ensure the accuracy, the Master Mix was first prepared according to the amount of the number + 2, and then 10 μL was dispensed into each reaction tube. The reverse transcription reaction was performed immediately after gentle mixing...

Embodiment 3

[0066] Using cDNA as a template, Promega's qPCR Master Mix Kit, prepare the reaction mixture according to the table below. The specific upstream and downstream primers of the target gene MCEMP1 were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The primer sequence is: MCEMP-Forward Primer: 5'-CCATGCAAAGGGTGGTCATTC-3'; MCEMP-ReversePrimer: 5'-GCTTGTACGGAGTTTGAGACATT-3' (SEQ NO ID: 2-3).

[0067]

[0068]

[0069] Then PCR amplification was carried out in the reaction system shown in the table below.

[0070]

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Abstract

The invention provides an active tuberculosis diagnostic marker, a kit and application thereof. The diagnostic marker is characterized by comprising a blood biomarker MCEMP1 protein in a blood sample,the amino acid sequence of the MCEMP1 protein is as shown in the SEQ ID NO:1. The invention further provides the kit capable of rapidly detecting whether a subject suffers from active tuberculosis ornot. The kit comprises a total RNA extraction related reagent in a whole blood sample, a quantitative PCR related reagent and an ELISA related reagent. According to the active tuberculosis diagnosticmarker, the MCEMP1 is adopted as a biomarker for active tuberculosis detection for the first time, is used for clinical diagnosis and differential diagnosis of tuberculosis, and provides a new direction for clinical diagnosis of tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and more specifically relates to MCEMP1 (C19orf59) as a diagnostic marker for active pulmonary tuberculosis, a kit and application thereof. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection. Worldwide, tuberculosis is one of the top ten causes of death and the leading cause of death from a single infectious agent (higher than HIV / AIDS). In 2018, an estimated 10 million people worldwide became ill with tuberculosis and a total of 1.5 million people died of tuberculosis (including 251,000 people living with HIV). Two-thirds of new tuberculosis patients came from eight countries with a high burden of tuberculosis, among which India accounted for the highest proportion, followed by China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa. Multidrug-resistant tuberculosis (MDR-TB) remains a public he...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883G01N33/68
CPCC12Q1/6883G01N33/6893C12Q2600/158G01N2800/12G01N2800/26Y02A50/30
Inventor 宋言峥黄家颖温子禄牛良飞马辉张舒林王琳夏凡
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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