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Peripheral blood circular RNA markers and applications for non-invasive diagnosis of active pulmonary tuberculosis

A technology of active pulmonary tuberculosis and markers, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems that affect the sensitivity and clinical applicability of detection methods, difficult detection, and limit clinical transformation process, etc. problem, to achieve the effect of being suitable for popularization, high accuracy and good stability

Active Publication Date: 2020-09-11
顾万君 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some recent studies have shown that transcriptome markers in blood can be used to distinguish active tuberculosis from healthy individuals, and to predict the risk of tuberculosis; this method has the advantages of easy operation, low detection cost, and low background noise of the detected signal, but The expression of mRNA transcripts in peripheral blood may be affected by the blood collection process, making the stability and reproducibility of this method need to be further verified, thus limiting its clinical translation process
In addition, there are studies on the diagnosis and treatment monitoring of active pulmonary tuberculosis by quantitatively detecting Mycobacterium tuberculosis antigen polypeptides in the blood. Sensitivity and clinical applicability of the assay

Method used

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  • Peripheral blood circular RNA markers and applications for non-invasive diagnosis of active pulmonary tuberculosis
  • Peripheral blood circular RNA markers and applications for non-invasive diagnosis of active pulmonary tuberculosis
  • Peripheral blood circular RNA markers and applications for non-invasive diagnosis of active pulmonary tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Screening of active pulmonary tuberculosis diagnostic markers

[0034] In order to screen the diagnostic markers for active pulmonary tuberculosis, the present invention first analyzes the expression data of circRNA in PBMCs of a population used to discover the diagnostic markers for active pulmonary tuberculosis. As shown in Table 1, the population used to discover diagnostic markers included two active tuberculosis patients and two healthy controls matched in age and sex with active tuberculosis patients, including a group of young male paired case-controls and a group of A paired case-control case in older women.

[0035] Table 1

[0036]

[0037] The differences in the expression levels of PBMC circRNA in the paired case-control groups above were analyzed. Such as figure 1 As shown, the analysis results showed that the proportion of circRNA transcripts in the PBMC transcriptome of the two tuberculosis patients was increased.

[0038] Such as figu...

Embodiment 2

[0047] Example 2: Verification and application of "7-circRNA markers" in independent sample sets

[0048] In order to verify the effectiveness of "7-circRNA markers" in the diagnosis of active pulmonary tuberculosis, the expression data of the above 7 circRNAs in PBMCs of another verification population were obtained. This group was completely independent from the group used for marker discovery, including 11 healthy controls and 10 active tuberculosis patients, and there was no significant difference in age and gender between cases and controls (Table 4).

[0049] Table 4

[0050]

[0051] The sequence information of seven circRNAs was obtained from the circBase database, and specific back-to-back primers for amplifying each circRNA were designed according to the specific sequences on the circRNAs to distinguish linear RNAs. Expression levels of seven circRNAs in individual PBMCs. Among them, the forward primer and reverse primer of hsa_circ_0000681 are respectively show...

Embodiment 3

[0078] Example 3: Active tuberculosis diagnostic kit based on "7-circRNA marker"

[0079] Based on the "7-circRNA marker" screened and validated for the diagnosis of active pulmonary tuberculosis by the present invention, a diagnostic kit containing all the specific back-to-back primers for 7 circRNAs was further developed to obtain the expression levels of 7 circRNAs in human PBMC Determination and diagnosis of tuberculosis.

[0080] The production and operation process of the non-invasive diagnostic kit for active pulmonary tuberculosis is based on cell separation, RNA extraction, qRT-PCR and other technologies. The specific methods include: PBMC isolation; PBMC RNA extraction; "7-circRNA marker" detection and risk value calculation Wait.

[0081] The kit includes 7 circRNA-specific back-to-back primers;

[0082] Commonly used reagents for separating human PBMCs include: incomplete RPMI-1640 medium, human lymphocyte separation medium, etc.;

[0083] Common reagents for ex...

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Abstract

The invention discloses a peripheral blood circular RNA marker for noninvasive diagnosis of active pulmonary tuberculosis. A primer is designed according to the peripheral blood circular RNA marker; the designed primer can be used for preparing a noninvasive diagnosis kit used for the active pulmonary tuberculosis. The peripheral blood circular RNA marker can be used for effectivelydistinguishing patients with pulmonary tuberculosis from healthy people, and has the advantages of convenient and noninvasive detection, high accuracy and good stability, thus being suitable for being popularized and used.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to peripheral blood circular RNA markers and applications for non-invasive diagnosis of active pulmonary tuberculosis. Background technique [0002] Tuberculosis is one of the diseases that seriously endanger human health, and Mycobacterium tuberculosis is the pathogen of tuberculosis. Although the incidence, prevalence and mortality of tuberculosis have declined since 2000, the World Health Organization (WHO) estimated that there were still about 1.5 million deaths and 10.5 million new cases in the global population in 2015. TB cases, of which less than 63% are accurately screened and reported. China is one of the countries with a high burden of tuberculosis in the world, and the number of tuberculosis patients ranks third in the world, accounting for about 10% of the total number of tuberculosis patients in the world. [0003] At present, sputum smear examination is mostly used in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/113
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 顾万君钱中清
Owner 顾万君
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