CN108866213A - Mycobacterium tuberculosis detection of rifampin resistant diagnosis marker and its application - Google Patents
Mycobacterium tuberculosis detection of rifampin resistant diagnosis marker and its application Download PDFInfo
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- CN108866213A CN108866213A CN201810697101.7A CN201810697101A CN108866213A CN 108866213 A CN108866213 A CN 108866213A CN 201810697101 A CN201810697101 A CN 201810697101A CN 108866213 A CN108866213 A CN 108866213A
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- rifampin
- mycobacterium tuberculosis
- resistance
- rv0644c
- detection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses a kind of mycobacterium tuberculosis detection of rifampin resistant diagnosis markers, are made of Rv2243, Rv0644c, when Rv2243, Rv0644c expression quantity are lower than normal expression amount, judge mycobacterium tuberculosis to rifampin-resistance.The invention discloses the rapid clinical detections that the detection of rifampin resistant diagnosis marker of mycobacterium tuberculosis can realize mycobacterium tuberculosis detection of rifampin resistant, and provide rifampin-resistance and treat potential target spot.
Description
Technical field
The present invention relates to a kind of mycobacterium tuberculosis detection of rifampin resistant diagnosis marker and its applications.
Background technique
Resistant tuberculosis is the great difficult problem faced in global tuberculosis prevention and control, especially multi-drug resistance tuberculosis and wide
General resistant tuberculosis seriously threatens human health, resistance to more although 2013 annual reports show that new hair tuberculosis patient is reduced
Medicine diagnosis and treatment lungy are much not up to target.The timely and effective monitoring of resistant tuberculosis is the pass of tuberculosis prevention and treatment work
Key, tuberculosis laboratory in China's includes conventional proportional method susceptibility, 960 system of BACTEC MGIT to the diagnosis of resistant tuberculosis at present
System and the fast diagnosis method based on molecular biology etc..
The technology of existing detection mycobacterium tuberculosis rifampin-resistance mainly has including two major classes：Susceptibility inspection based on culture
It surveys, the quick medicine-sensitive detection based on molecular biology mechanism.The shortcomings that prior art mainly includes：Time-consuming, needs to establish
Susceptibility detection is carried out on the basis of being separately cultured, is separately cultured and needs 4-8 week, and it is solid to carry out subsequent tradition on this basis
The time of body rule of three (4-8 weeks) or liquid susceptibility (1-2 weeks)；Place limitation, two kinds of cultural methods be required to volume compared with
Incubator, to guarantee that bio-safety needs fixed culture room；Drug resistance target spot limitation, it is emerging based on molecular biology
Quick diagnosis technology substantially reduce interval between diagnosis 1-2 hours within, but limited by current research level, it is optimal at present
Xpert MTB/RIF rifampin-resistance detection target spot be confined to the mutational site of ropB gene；And clinic has Xpert MTB/
RIF reports the situation of rifampin-resistance but clinical rifampin sensitivity, prompts rifampin-resistance that may have the prominent of other gene locis
Become.
Summary of the invention
The purpose of the present invention is to provide a kind of detection of rifampin resistant diagnosis marker of mycobacterium tuberculosis and its applications.
The technical solution adopted by the present invention is that：
A kind of mycobacterium tuberculosis detection of rifampin resistant diagnosis marker, is made of Rv2243, Rv0644c.
Further, when Rv2243, Rv0644c expression quantity are lower than normal expression amount, judge mycobacterium tuberculosis to benefit
The flat drug resistance of good fortune.
It is a kind of for detecting the kit of mycobacterium tuberculosis detection of rifampin resistant, containing quantitative in the kit
The reagent of Rv2243, Rv0644c expression quantity.
Further, when Rv2243, Rv0644c expression quantity are lower than normal expression amount, judge mycobacterium tuberculosis to benefit
The flat drug resistance of good fortune.
A method of detection mycobacterium tuberculosis detection of rifampin resistant includes the following steps：
1) Rv2243, Rv0644c expression quantity in quantitative mycobacterium tuberculosis to be measured；
2) according to Rv2243, Rv0644c expression quantity, judge mycobacterium tuberculosis detection of rifampin resistant.
Further, when Rv2243, Rv0644c expression quantity are lower than normal expression amount, judge mycobacterium tuberculosis to benefit
The flat drug resistance of good fortune.
The beneficial effects of the invention are as follows：The invention discloses the detection of rifampin resistant diagnosis markers of mycobacterium tuberculosis can
It realizes the rapid clinical detection of mycobacterium tuberculosis detection of rifampin resistant, and provides rifampin-resistance and treat potential target spot.
Detailed description of the invention
Fig. 1 is the thermal map of rifampin-resistance bacterial strain and control group genomic methylation level；
Fig. 2 is the KEGG analysis result of the changed gene warp of methylation level during rifampin-resistance；
Fig. 3 is the thermal map of rifampin-resistance bacterial strain and control group subgenomic transcription group level；
Fig. 4 is the KEGG analysis result of the changed gene of expression during rifampin-resistance.
Specific embodiment
Based on current technology level, the present invention is using the new molecular marker of rifampin-resistance as target, with external rifampin
Clearly rifampin-resistance family is research object for the control that gradient concentration drug sieve is selected, horizontal by genomic methylation,
The detection of two groups of express spectra level, filters out that methylation level compared with the control reduces most obvious while to express level bright
Show new diagnosis marker of raised three genes as rifampin-resistance.Molecular marker of the invention derives from two groups
Horizontal screening, significant molecular marker can comprehensively be found by learning screening by group, but will increase the chance falsely dropped,
To solve this problem, we select two biological processes (methylation, gene turn being closely connected during developed by molecule
Record) carry out data and be mutually authenticated, find out that new, difference is most obvious and the gene supported mutually on two groups of data theories as
The diagnosis marker of rifampin-resistance.
The experimental procedure of Screening Diagnosis marker is as follows in the present invention：
The building of drug resistance family
H37Rv monoclonal bacterial strain is made choice of first, G0 is saved as after amplification for bacterial strain, while getting out the bacterium of 1 Maxwell turbidity
Liquid is inoculated on the gradient media of rifampin that (concentration of rifampin is respectively 2 in culture medium0、2-1、2-2、2-3、2-4μ g/ μ L),
It is cultivated 4 weeks or so in 37 DEG C of incubators, takes bacterium colony growth and bacterium amount meets the subsequent highest drug concentration for freezing and passing on
The bacterial strain of culture medium is G1 generation, and so on, until there is enough bacteriums raw in highest concentration (the resistance to concentration of WHO standard) culture
It is long, it is defined as the success of rifampin-resistant model construction.
Group credit analysis
It expands rifampin-resistance bacterial strain and parallel control bacterium is analysed, extract genome respectively, RNA carries out subsequent methylation group
It learns, transcription group sequencing analysis, methylation level is filtered out after analysis of biological information and increases most significant and expression reduction
The new molecular marker that most significant three genes are diagnosed as rifampin-resistance.Combination various genetic tests mature at present
Method can develop new rifampin-resistance quick diagnosis reagent kit.
Rifampin-resistance bacterial strain compared with the control group genomic methylation level thermal map analysis：
As a result such as Fig. 1, each ledgement represent a gene, can be clearly indicated out in two groups of samples by thermal map
The difference of methylation.In rifampin-resistance generating process, some gene methylation levels are increased, and corresponding gene is in control group
Middle methylation level reduces.
The signal path of differential gene is enriched with analysis：
The changed gene of methylation level during rifampin-resistance is analyzed through KEGG, Fig. 2 is as a result seen, can obtain
The cell-signaling pathways being enriched with to these differential genes.The quantity of circle size expression enrichment gene in figure, and color
The corresponding enrichment degree of correlation of the depth.
Rifampin-resistance bacterial strain compared with the control group subgenomic transcription group level thermal map analysis：
As a result see Fig. 3, for each experimental group because transcriptome analysis requires to need to do three repetitions, each ledgement represents one
A gene can clearly indicate out the difference of same gene expression in two groups of samples by thermal map.In rifampin-resistance
In generating process, some gene expression doses are increased, and corresponding gene expression in control group reduces.
The signal path of transcriptional level differential gene is enriched with analysis：
The changed gene of expression during rifampin-resistance is analyzed through KEGG, Fig. 4 is as a result seen, can be obtained
The cell-signaling pathways that these differential genes are enriched with.The quantity of circle size expression enrichment gene in figure, and the depth of color
Shallow corresponding enrichment degree of correlation.
By the Conjoint Analysis of two groups, finally screens and increase while expressing in rifampin-resistance process methylation level
Most significant two genes Rv2243 and Rv0644c that level reduces are the new target for molecular diagnosis of rifampin-resistance, and
It may be potential therapy target.
Embodiment 1
A kind of mycobacterium tuberculosis detection of rifampin resistant diagnosis marker, is made of Rv2243, Rv0644c, when Rv2243,
When Rv0644c expression quantity is lower than normal expression amount, judge mycobacterium tuberculosis to rifampin-resistance.
Embodiment 2
It is a kind of for detecting the kit of mycobacterium tuberculosis detection of rifampin resistant, containing quantitative in the kit
The reagent of Rv2243, Rv0644c expression quantity judges tuberculosis point when Rv2243, Rv0644c expression quantity are lower than normal expression amount
Branch bacillus is to rifampin-resistance.
Embodiment 3
A method of detection mycobacterium tuberculosis detection of rifampin resistant includes the following steps：
1) Rv2243, Rv0644c expression quantity in quantitative mycobacterium tuberculosis to be measured；
2) according to Rv2243, Rv0644c expression quantity, judge mycobacterium tuberculosis detection of rifampin resistant, when Rv2243,
When Rv0644c expression quantity is lower than normal expression amount, judge mycobacterium tuberculosis to rifampin-resistance.
Claims (6)
1. a kind of mycobacterium tuberculosis detection of rifampin resistant diagnosis marker, is made of Rv2243, Rv0644c.
2. mycobacterium tuberculosis detection of rifampin resistant diagnosis marker according to claim 1, it is characterised in that：When
When Rv2243, Rv0644c expression quantity are lower than normal expression amount, judge mycobacterium tuberculosis to rifampin-resistance.
3. a kind of for detecting the kit of mycobacterium tuberculosis detection of rifampin resistant, it is characterised in that：Contain in the kit
The reagent of quantitative Rv2243, Rv0644c expression quantity.
4. kit according to claim 3, it is characterised in that：When Rv2243, Rv0644c expression quantity are lower than normal expression
When amount, judge mycobacterium tuberculosis to rifampin-resistance.
5. a kind of method for detecting mycobacterium tuberculosis detection of rifampin resistant, includes the following steps：
1) Rv2243, Rv0644c expression quantity in quantitative mycobacterium tuberculosis to be measured；
2) according to Rv2243, Rv0644c expression quantity, judge mycobacterium tuberculosis detection of rifampin resistant.
6. according to the method described in claim 5, it is characterized in that：When Rv2243, Rv0644c expression quantity are lower than normal expression amount
When, judge mycobacterium tuberculosis to rifampin-resistance.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725434A (en) * | 2021-01-20 | 2021-04-30 | 首都医科大学附属北京胸科医院 | Rifampicin-resistant tuberculosis molecular marker, detection reagent and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030236393A1 (en) * | 2002-03-22 | 2003-12-25 | United States Of America Dept Of Vetrans Affairs | Virulence genes of M. marinum and M. tuberculosis |
| CN102165064A (en) * | 2008-07-25 | 2011-08-24 | 葛兰素史密丝克莱恩生物有限公司 | The tuberculosis RV2386C protein, compositions and uses thereof |
-
2018
- 2018-06-29 CN CN201810697101.7A patent/CN108866213A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030236393A1 (en) * | 2002-03-22 | 2003-12-25 | United States Of America Dept Of Vetrans Affairs | Virulence genes of M. marinum and M. tuberculosis |
| CN102165064A (en) * | 2008-07-25 | 2011-08-24 | 葛兰素史密丝克莱恩生物有限公司 | The tuberculosis RV2386C protein, compositions and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| LIANG CHEN等: "Genome-wide DNA methylation and transcriptome changes in Mycobacterium tuberculosis with rifampicin and isoniazid resistance", 《INT J CLIN EXP PATHOL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725434A (en) * | 2021-01-20 | 2021-04-30 | 首都医科大学附属北京胸科医院 | Rifampicin-resistant tuberculosis molecular marker, detection reagent and application thereof |
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Application publication date: 20181123 |