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SRNA marker for distinguishing mycobacterium tuberculosis and BCG vaccine and use thereof

A technology of mycobacterium tuberculosis and markers, applied in the field of medicine, can solve the problems of missed diagnosis of tuberculosis, misdiagnosis, acid-fast staining and low positive rate of tuberculosis culture, etc., and achieve the effect of easy acquisition and sequence optimization

Active Publication Date: 2019-08-06
HARBIN MEDICAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficult standardization of sample acquisition and the influence of the degree of lesion on the bacterial content in the lesion, the positive rate of acid-fast staining and tuberculosis culture is not high. The global average of tuberculosis detection and diagnosis rate is 50%, which is less than 30% in China. %, resulting in a lot of missed and misdiagnosed tuberculosis
Since BCG is generally inoculated in the population, BCG affects many laboratory test results of tuberculosis. Therefore, finding sRNA fragments that can effectively distinguish BCG from Mycobacterium tuberculosis is an important technical basis for establishing sRNA detection methods of Mycobacterium tuberculosis in peripheral blood and other body fluid samples in the future.

Method used

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  • SRNA marker for distinguishing mycobacterium tuberculosis and BCG vaccine and use thereof
  • SRNA marker for distinguishing mycobacterium tuberculosis and BCG vaccine and use thereof
  • SRNA marker for distinguishing mycobacterium tuberculosis and BCG vaccine and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Bacterial RNA Sequencing

[0035] 1. Extraction of total RNA from bacteria

[0036] (1) BCG culture: Dissolve one BCG powder (0.25 mg / branch) in 200 μL of normal saline, inoculate it in 5 mL of 7H9 medium containing 10% ADC, and culture at 37° C. with shaking at 210 r / min for 16-30 days.

[0037] (2) Extraction of total RNA: collect the cells by centrifugation, add lysozyme at a final concentration of 4 mg / ml, mix well and let stand for 5 min, then sonicate for 30 cycles at 5 s / 3 s. Add 500 μL Trizol, and extract bacterial total RNA routinely according to the manual.

[0038] 2. Sequencing

[0039] Whole-transcriptome sequencing was performed on the extracted total RNA to detect and screen sRNA species in bacteria.

[0040] RNA sequencing analysis revealed the type and abundance of sRNA in BCG cells. The detected sRNAs include MTS2823, Asdes, G2, AS1890, mcr7, Aspks, MTS1338, C8, MTS1082, mcr3, mcr15, etc. See Table 1.

[0041] Table 1 sRNA species and ab...

Embodiment 2

[0043] Embodiment 2 target sRNA primer design

[0044] Using bioinformatics analysis method, different oligonucleotide primer pairs were designed according to the sequence and structural characteristics of each sRNA in Mycobacterium tuberculosis and BCG. The primer sequences are shown in Table 2.

[0045] Table 2 detects the primer sequence of sRNA

[0046]

Embodiment 3

[0047] Embodiment 3 establishes real-time fluorescent quantitative PCR detection system

[0048] 1. Bacterial total RNA extraction: Use RNA extraction reagents to extract bacterial total RNA by conventional methods.

[0049] 2. The total RNA was reverse transcribed into cDNA. After exploration, the following reaction system and reaction conditions were determined.

[0050] (1) Reaction system:

[0051] Take 13 μL of total RNA, 4 μL of random primers, and make up the volume to 20 μL with deionized water.

[0052] (2) Reaction conditions:

[0053] 15min at 37°C, 5s at 85°C, and stored at 4°C, it is cDNA.

[0054] 3. Real-time fluorescent quantitative PCR system and determination of reaction conditions

[0055] (1) Real-time fluorescent quantitative PCR system: EvaGreen 2xqPCR MasterMix 10 μL, upstream and downstream primers in Table 2 0.6 μL each, cDNA template 2 μL, add deionized water to make up the volume to 20 μL.

[0056] (2) Real-time fluorescent quantitative PCR reac...

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Abstract

The invention discloses an sRNA marker for distinguishing mycobacterium tuberculosis and a BCG vaccine and a use thereof. For solving the problem that mycobacterium tuberculosis infection and BCG vaccination are difficult to distinguish, the type of sRNA in mycobacterium tuberculosis is detected and screened by bacterial RNA sequencing firstly, a specific primer and kit capable of detecting the sRNA of mycobacterium tuberculosis are designed according to the screened target sRNA sequence, and a corresponding real-time fluorescence quantitative PCR detection method is established. After free sRNA is extracted from bacterial bodies and cDNA is synthesized, the content of sRNA is detected by the real-time fluorescent quantitative PCR method. The experiment proves that the method can be used for distinguishing the mycobacterium tuberculosis and the BCG vaccine, has the advantages of high sensitivity, good specificity, low cost, convenience in operation and the like, and is hopeful to further meet the clinical and laboratory needs for rapid diagnosis of the mycobacterium tuberculosis and the BCG vaccine.

Description

technical field [0001] The present invention relates to an sRNA marker for distinguishing Mycobacterium tuberculosis and BCG vaccine and its application, and also relates to a primer and a kit for distinguishing Mycobacterium tuberculosis infection and BCG vaccination based on the sRNA marker. The invention belongs to the technical field of medicine. Background technique [0002] Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis, which can invade various organs of the body, and tuberculosis is the most common. Tuberculosis is a worldwide public health problem, ranking first in the mortality rate of infectious diseases. About 250,000 people die from tuberculosis in China every year, more than twice the total number of deaths from various infectious diseases. At present, the important challenge of TB prevention and control in China is the low rate of etiological diagnosis and low detection rate of MDR-TB. [0003] The gold standard for clinical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/32
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 付英梅张凤民宋武琦李爱梅刘鑫一房勇
Owner HARBIN MEDICAL UNIVERSITY
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