CN104263834A - Nucleic acid membrane strip and kit for drug-resistant mutant gene detection of mycobacterium tuberculosis - Google Patents
Nucleic acid membrane strip and kit for drug-resistant mutant gene detection of mycobacterium tuberculosis Download PDFInfo
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Abstract
The invention relates to gene detection of disease pathogens and particularly relates to a nucleic acid membrane strip and a kit for drug-resistant mutant gene detection of mycobacterium tuberculosis. The kit comprises the nucleic acid membrane strip which comprises a substrate and a nucleic acid probe fixedly arranged on the substrate; the nucleic acid probe comprises probes with the base sequences as shown in SEQ ID NO: 1-26. The kit for drug-resistant mutant gene detection of mycobacterium tuberculosis can be used for comprehensively and quickly detecting the drug-resistant mutant gene of mycobacterium tuberculosis, can be used for detecting the drug resistance of five main antituberculosis drugs in one test and can be used for quickly and immediately determining drug-resistant information of mycobacterium tuberculosis on the main antituberculosis drugs, so that prophylactic treatment and diagnosis are favorably performed clinically in advance, thereby alleviating the burden of a patient.
Description
Technical field
The present invention relates to a kind of gene test of disease pathogen, particularly relate to a kind of nucleic acid film bar for drug resistant mutant genes of mycobacterium tuberculosis detection and test kit.
Background technology
Tuberculosis is that the one caused after invading human body by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) has strong communicable chronic wasting disease.Since the late nineteen eighties in last century, increased by floating population, the impact of the factor such as to increase of HIV/AIDS Epidemic and drug-resistant tuberculosis, tuberculosis spreads again in the whole world, has become global great public health problem and social concern.Claim according to the World Health Organization 2009 annual report: 2007, tuberculosis popular case in the whole world is about 1,370 ten thousand examples, wherein new cases have 9,270,000, higher than the level of 2006 (9,240,000 example), 2000 (8,300,000 examples) and nineteen ninety (6,600,000 is routine).The first five country of case load rank in 2007 is: India (2,000,000), China (1,300,000), Indonesia (530,000), Nigeria (460,000) and South Africa (460,000).
China is one of tuberculosis high burden country, and tuberculosis patient number occupies second place of the world, is only second to India.According to hygiene department of China statistics, total tuberculosis patient about 4,500,000 people in the whole nation, wherein newly-increased tuberculosis patient 1,300,000 at present, about 130,000 people die from tuberculosis every year.
Mycobacterium tuberculosis is mainly through the spittle through respiratory tract infection, and tuberculosis patient is when coughing, sneezing or talk loudly, and mycobacterium tuberculosis will gush out with phlegm foam, suspends in atmosphere, people can cause infection after sucking.The necessary long-term taking antitubercular agent for the treatment of lungy, at least 6 months.When antituberculosis therapy interruption or irregular treatment, tubercule bacillus can be survived down, constantly breeds, and even forms a kind of more dangerous drug resistant M bacterial strain, and treatment is got up more difficult.Therefore, early prevention, Timeliness coverage and Canonical management to be accomplished for control lungy.
Rimactazid, Tibutol, Streptomycin sulphate and fluoroquinolone are antitubercular agents the most frequently used clinically, but patient's long-term taking or treatment lack of standardization can cause mycobacterium tuberculosis producer to make a variation, reduce the susceptibility of medicine, clinical manifestation is Drug Resistance for Tuberculosis.WHO report of survey shows, and within 2009, there is tuberculosis 1,300,000 example in China, and wherein single resistant rate 21%, many resistances are 8.64%, and extensive resistant rate is 0.68%, total resistant rate 37.79%.The feature of China's resistant tuberculosis is that resistance number is many, distribution widely, the distribution of Drug-fast case tuberculosis based on rural area, the ratio that accounts for of person between twenty and fifty is higher, without obvious gender difference, and east, in, western different areas resistant rate there is no significant difference.
Research shows that Rifampin can not effectively be combined with RNA polymerase β subunit, makes bacterium produce tolerance to Rifampin when rpoB gene core region-rifampin-resistance determining area (RRDR) undergos mutation; And the generation of Isoniazid-resistant causes primarily of katG or inhA transgenation, Tibutol resistance causes primarily of the embB transgenation of encoding glycosyltransferases (catalysis pectinose is aggregated to Arabinogalactan), Streptomycin sulphate resistance causes primarily of encoding ribosomal proteins S12 (rpsL) transgenation, and fluoroquinolone resistance is mainly caused by gyrA transgenation.Therefore, the catastrophe detecting relevant drug resistant gene can obtain the tolerance of mycobacterium tuberculosis to relative medicine.
Resistance common detection methods at present for mycobacterium tuberculosis is susceptibility culture method, the method is cultivated in containing the substratum of antitubercular agent by M. tuberculosis strains, whether grows the resistance judging mycobacterium tuberculosis according to mycobacterium tuberculosis in pastille substratum.The main drawback of the method is that detection time is long, and common susceptibility culture experiment needs 6-8 consuming time week to report net result, even if adopt full-automatic susceptibility culture device also to need for 3 to 4 weeks could report result, very easily delays the golden hour of patient.Next is that susceptibility cultivates difficulty, and high to operational requirement, misoperation easily produces false negative after making inactivation of bacteria, causes erroneous judgement.
In the product of existing detection M. tuberculosis drug resistant gene related locus sudden change, just detect the resistance information of one or more of Rimactazid, Tibutol, Streptomycin sulphate and fluoroquinolone five kinds of antitubercular agents, detect not comprehensive.
Summary of the invention
Technical problem to be solved by this invention is: providing a kind of one-time detection can go out the tolerance of mycobacterium tuberculosis to species antitubercular agent, and detection time is short, the result accurately believable nucleic acid film bar for drug resistant mutant genes of mycobacterium tuberculosis detection and test kit.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: provide a kind of nucleic acid film bar detected for drug resistant mutant genes of mycobacterium tuberculosis, comprise substrate and be fixed on described suprabasil nucleic acid probe, described nucleic acid probe comprises the probe of following base sequence: SEQ ID NO:1-26.
Another technical scheme of the present invention is for providing a kind of test kit detected for drug resistant mutant genes of mycobacterium tuberculosis, described test kit comprises: the above-mentioned nucleic acid film bar for drug resistant mutant genes of mycobacterium tuberculosis detection and PCR reaction solution, and described PCR reaction solution comprises PCR reaction solution I and PCR reaction solution II;
Described PCR reaction solution I comprises the primer of following base sequence: SEQ ID NO:28-33;
Described PCR reaction solution II comprises the primer of following base sequence: SEQ ID NO:34-39.
Beneficial effect of the present invention: the present invention be used for drug resistant mutant genes of mycobacterium tuberculosis detect nucleic acid film bar and test kit can carry out drug resistant mutant genes of mycobacterium tuberculosis detection comprehensively, rapidly, can in single test, detect the tolerance of 5 kinds of Main Antituberculosis Drugs things, can fast timely clear and definite mycobacterium tuberculosis to the resistance information of Main Antituberculosis Drugs thing, be conducive to clinically making prophylactic treatment and diagnosis in advance, reduce the heavy burdens to patient.
Accompanying drawing explanation
Fig. 1 is the nucleic acid film bar detected result figure of test kit of the present invention;
Fig. 2 is the nucleic acid film bar detected result figure of test kit of the present invention;
Fig. 3 is the nucleic acid film bar detected result figure of test kit of the present invention.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, realized object and effect, accompanying drawing is coordinated to be explained in detail below in conjunction with embodiment.
The design of most critical of the present invention is: single test can fast timely clear and definite mycobacterium tuberculosis to the resistance information of 5 kinds of Main Antituberculosis Drugs things, be conducive to clinically making prophylactic treatment and diagnosis in advance, reduce the heavy burdens to patient.
Embodiment 1
The design and implementation of detection probes, primer and amplification reaction solution is carried out according to known M. tuberculosis genes group achievement in research.
Product of the present invention have employed the biochip technology that polymerase chain reaction (PCR) and reverse dot blot hybridization (RDB) combine.Gene chip is made up of nylon membrane and probe array fixed thereon, and the exploitation of membrane DNA chip then has the clear superiorities such as preparation is relatively simple, easy and simple to handle, with low cost.
The present invention is according to the known design carrying out detection probes, primer and amplification reaction solution in conjunction with Mycobacterium tuberculosis genes group achievement in research, special in conjunction with drug resistant mutant genes of mycobacterium sequence from choosing, Position Design gene order residing for drug resistant mutant genes site, as probe, adopts the mode of multiplex PCR to increase the DNA fragmentation of multiple drug resistant gene simultaneously.
The present invention detects 6 drug resistant genes corresponding to 5 kinds of antitubercular agents simultaneously, these 6 drug resistant gene distributions in M. tuberculosis genes group comparatively disperse, molecule is each other distant, also at a distance of more than 20,000 base between two nearest drug resistant gene rpoB and rpsL, adopt pair of primers increase simultaneously 6 goal gene be difficult to realize, therefore, the present invention is in order to evade above unfavorable factor, adopt the mode of separately design primer and probe, shorten the amplified fragments of goal gene, improve amplification and hybridization efficiency.Because M. tuberculosis genes group is larger, each drug resistant gene distribution compares dispersion again, when pcr amplification, by M. tuberculosis genes group considerable influence, it is less that goal gene is exposed to outer probability in genome, therefore, primer and goal gene sequence in conjunction with time be subject to space steric effect and affect comparatively large, in conjunction with more difficult.Therefore, in the present invention, design of primers difficulty is comparatively large, needs, through repeatedly groping and testing, could obtain the primer sequence that amplification efficiency is high.
In addition, what the present invention adopted is two pipe triple PCR amplification systems, namely the primer simultaneously containing three pairs of drug resistant genes in a PCR amplification system, the difficulty of triple PCR amplification system is larger, situation about suppressing mutually is easily there is between three pairs of primers, cause certain two pairs of primer can not coexist in a reaction system, need readjust and design primer pair; Three pairs of primers compete starting material mutually in amplification procedure, easily cause the amplification heterogeneity between primer; Multipair primer, in a reaction system, easily forms primer dimer thus the effective concentration of reduction primer, affects amplification efficiency.The present invention, through groping and comparing repeatedly, optimizes the best primer pair that can coexist, and form best primer pair proportioning by the consumption adjusting primer, achieving triple PCR amplification system also can homogeneous efficient amplification.
1, the amplimer that detects of drug resistance of Mycobacterium tuberculosis mutational site of the present invention and nucleic acid probe sequence as shown in table 1.
Table 1
2, the nucleic acid probe on nucleic acid film bar of the present invention puts in order as shown in table 2.
Table 2
| N1 | N2 | N3 | N4 | N5 | 315N | -15N | 90N | 94N |
| 511M | 516M | 522M | 526M | 531M | 315M | -15M | 90M | 94M |
| 43N | 88N | 306N | 43M | 88M | 306M1 | 306M2 | 306M3 | PC |
In above-mentioned table 2:
1) first row N represents this site is wild-type, and second row is mutational site.
Front 5 sites (D516V to S531L) of first row N1 to N5 and second row are rifampin-resistance genes involved rpoB detection probes; Other 315 and-15 sites are drug resistance related gene katG and the inhA detection probes of vazadrine respectively, and 90 and 94 sites are fluoroquinolone drug resistant gene gyrA detection probes.
2) 511M, 516M, 522M, 526M, 531M of second row represent amino acid mutation corresponding on rpoB gene respectively, 315M represents amino acid mutation corresponding on katG gene, 15M represents amino acid mutation corresponding on inhA gene, 90M and 94M represents amino acid mutation corresponding on gyrA gene.
3) the 3rd row 43N, 88N represents Streptomycin sulphate rpsL gene 43 and 88 codon site is wild-type, and 43M, 88M represent the corresponding site of Streptomycin sulphate rpsL gene and undergo mutation.
4) the 3rd row 306N represents Tibutol embB gene 306 site is wild-type, and 306M1,306M2,306M3 represent Tibutol embB gene locus and dissimilar sudden change occurs.
3, the confirmation of pcr amplification reaction system
Optimized by great many of experiments contrast, the PCR reaction solution formula that test kit of the present invention is finally determined is in table 3.
Table 3
Embodiment 2
For the test kit that drug resistant mutant genes of mycobacterium tuberculosis detects, described test kit comprises nucleic acid film bar and the pcr amplification reaction liquid of the drug resistant mutant genes detection in embodiment 1.Primer 5 ' in shown reaction solution holds mark vitamin H, and mark amino held by described probe 3 ', and described PC probe 5 ' end mark vitamin H 3 ' holds mark amino.
Test kit of the present invention have employed the biochip technology that polymerase chain reaction (PCR) and reverse dot blot hybridization (RDB) combine.Nucleic acid film bar is made up of nylon membrane and probe array fixed thereon, and the exploitation of membrane DNA chip has the clear superiorities such as preparation is relatively simple, easy and simple to handle, with low cost.
The use of test kit of the present invention:
Provide reagent for oneself:
20 × SSC, pH 7.0:NaCl 175.3g, Trisodium Citrate 88.2g, adds H
2o 750mL dissolves, and adjusts pH to 7.0, be finally settled to 1000mL with pH meter, and autoclaving is preserved.
10%SDS, pH 7.0:SDS 20g, adds H
2o 180mL dissolves, and adjusts pH to 7.0, be finally settled to 200mL with pH meter.
1M Trisodium Citrate, pH 5.0: Trisodium Citrate 294g, adds H
2o 700mL dissolves, and adjusts pH to 5.0, be finally settled to 1000mL with dense HCl.
A1 liquid (1 × SSC, 0.1%SDS, pH 7.4): 20 × SSC 50mL, 10%SDS 10mL, adds H
2o is settled to 1000mL.
B liquid (0.5 × SSC, 0.1%SDS, pH 7.4): 20 × SSC 25mL, 10%SDS 10mL, adds H
2o is settled to 1000mL.
C liquid (0.1M Trisodium Citrate, pH 5.4): 1M Trisodium Citrate 100mL, adds H
2o is settled to 1000mL.
Nitrite ion (Fresh uses, and is sequentially added into following solution): C liquid 19mL, TMB 1mL, 30%H
2o
2, 2 μ L.
1, clinical sample obtains: sputum sample
Leave and take dark expectoration sample 3-5ml in the Glass Containers of sterilization.
2, sample process
1) collect patient's sputum sample, add the reagent that reduces phlegm (NaOH of 4%) of equivalent, process 15 ~ 30 minutes of reducing phlegm, until sputum melts completely.
2) shift 1.2mL sputum sample Digestive system in 1.5mL centrifuge tube, centrifugal 5 minutes of 13000rpm, abandons supernatant.
3) the aseptic 1 × PBS of 1mL is added, mixing.Centrifugal 5 minutes of 13000rpm, abandons supernatant, repeats once.
4) add 1mL 10mM Tris solution, mix suspending sample, centrifugal 5 minutes of 13000rpm, abandons supernatant.
5) add 50 μ L lysates, break up precipitation block, boil in boiling water bath 10 minutes (notice that centrifuge tube lid may pop, carefully pollute), centrifugal 5 minutes of 13000rpm, namely supernatant liquor can be used as pcr template.
3, pcr amplification
Take out PCR reaction solution I and each pipe of PCR reaction solution II, tube wall carries out mark, after 5000rpm is centrifugal 2 seconds, adds 4 μ L sample supernatant as pcr amplification template.Each experiment, must arrange a positive control and a negative control, namely each with 4 μ L positive control dnas and lysate for template.
Increase by following condition:
4, hybridize
Get 15mL plastic centrifuge tube, put into the film bar (should of film bar jiao with Pencil marks) indicating patient code, add A1 liquid 5-6mL and all two pipe (each 25 μ L) PCR primer, lid is tightened, mixing PCR primer, slightly unscrewed by centrifuge tube lid, during to avoid heating, pipe lid pops again.Centrifuge tube is put into boiling water bath heating 10 minutes (guaranteeing that hybridization solution liquid level is positioned under boiling water bath liquid level completely), take out and tighten lid, put into hybridization 59 DEG C, case hybridization 1.5 hours, but be no more than 4 hours.
Get 50mL plastics tubing, add 40mL B liquid and carry out being preheated to 59 DEG C in hybridization case or water bath.
Contrast: separately get two film bars and hybridize with positive control and negative control PCR primer respectively, method is the same.
5, film is washed
Take out film bar, move in the 50mL pipe that preheating B liquid is housed, wash 15 minutes (often pipe 40mL solution can wash 4 films at most) in 59 DEG C of jogs simultaneously.
6, develop the color
With the POD solution (singly do two films and only need 4 μ LPOD, be mixed with 8mL and use liquid, doing four films with 6 μ LPOD, can be mixed with 12mL and use liquid) of A1 liquid preparation 1:2000, room temperature jog soaks 30 minutes, discards POD solution.Twice is washed, each 5 minutes with A1 liquid chamber temperature jog.Wash film 1-2 minute by C liquid chamber temperature, prepare nitrite ion (nitrite ion needs Fresh, joins method and sees page up) simultaneously.Film bar is soaked in lucifuge colour developing in nitrite ion and gets final product observations in 5 ~ 10 minutes.
7, nucleic acid film bar scanner uni of the present invention is preserved
Remove nitrite ion, rinse a film bar with clear water, film bar is placed on reading apparatus and scans, saving result.If film bar needs to preserve, wet film bar can be put into airtight sealed bag and be stored in 4 DEG C of refrigerators.
8, the nucleic acid film bar result figure obtained after using test kit of the present invention to detect, refers to Fig. 1, Fig. 2 and Fig. 3.
Fig. 1 shows sputum sample detected result: Rimactazid, fluoroquinolone, Streptomycin sulphate and Tibutol are all responsive;
Fig. 2 shows sputum sample detected result: rifampin-resistance (531M), vazadrine sensitivity, fluoroquinolone resistance (90M), Streptomycin sulphate and Tibutol sensitivity;
Fig. 3 shows sputum sample detected result: rifampin-resistance (531M), Isoniazid-resistant (315M), fluoroquinolone resistance (90M), Streptomycin sulphate resistance (43M) and Tibutol resistance (306M3).
9, product performance index
(1) accuracy: use test kit of the present invention to detect 100 routine clinical tuberculosis positive sample, result compares be shown as identical medicament-resistant mutation type with order-checking, and accuracy rate is 100%.
(2) specificity: it is all negative for using test kit of the present invention to detect 100 routine non-tuberculous mycobacteria sample results.
(3) sensitivity: the minimum detectability using test kit energy stable detection mycobacterium tuberculosis of the present invention is 1.0 × 10
4copies/mL.
(4) repeatability: use test kit of the present invention to detect 2.0 × 10
4the mycobacterium tuberculosis sample of copies/mL, parallel duplicate detection 10 times, colour developing result is homogeneous, and the resistance result of detection is consistent.
(5) stability: use reagent kit product validity period of the present invention to be 6 months, product performance are stablized before the deadline.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (5)
1., for the nucleic acid film bar that drug resistant mutant genes of mycobacterium tuberculosis detects, comprise substrate and be fixed on described suprabasil nucleic acid probe, it is characterized in that, described nucleic acid probe comprises the probe of following base sequence: SEQ ID NO:1-26.
2. the nucleic acid film bar detected for drug resistant mutant genes of mycobacterium tuberculosis according to claim 1, it is characterized in that, described substrate is nylon membrane.
3. the nucleic acid film bar detected for drug resistant mutant genes of mycobacterium tuberculosis according to claim 2, it is characterized in that, described nucleic acid probe also comprises internal reference nucleic acid probe, and its base sequence is as SEQ ID NO:27.
4. the test kit detected for drug resistant mutant genes of mycobacterium tuberculosis, it is characterized in that, described test kit comprises: the nucleic acid film bar for drug resistant mutant genes of mycobacterium tuberculosis detection as described in any one of claim 1-3 and PCR reaction solution, and described PCR reaction solution comprises PCR reaction solution I and PCR reaction solution II;
Described PCR reaction solution I comprises the primer of following base sequence: SEQ ID NO:28-33;
Described PCR reaction solution II comprises the primer of following base sequence: SEQ ID NO:34-39.
5. the test kit detected for drug resistant mutant genes of mycobacterium tuberculosis according to claim 4, is characterized in that, described primer 5 ' holds mark vitamin H, and described probe 3 ' holds mark amino.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109219667A (en) * | 2016-02-15 | 2019-01-15 | 安普诊断公司 | Diagnostic device and correlation technique |
| CN113025729A (en) * | 2020-12-24 | 2021-06-25 | 复旦大学 | Mycobacterium tuberculosis drug-resistance-related gene mutation site and application thereof |
| CN117187425A (en) * | 2023-11-03 | 2023-12-08 | 迪飞医学科技(南京)有限公司 | AS-RAPID tuberculosis drug-resistant duplex detection primer probe group, kit and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545011A (en) * | 2009-04-22 | 2009-09-30 | 亚能生物技术（深圳）有限公司 | Membrane strip and kit for detecting drug resistant mutant genes of mycobacterium tuberculosis |
| CN102229990A (en) * | 2011-05-25 | 2011-11-02 | 厦门大学 | Method and kit for detecting quinolone resistance mutation of Mycobacterium tuberculosis |
-
2014
- 2014-09-26 CN CN201410503311.XA patent/CN104263834B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545011A (en) * | 2009-04-22 | 2009-09-30 | 亚能生物技术（深圳）有限公司 | Membrane strip and kit for detecting drug resistant mutant genes of mycobacterium tuberculosis |
| CN102229990A (en) * | 2011-05-25 | 2011-11-02 | 厦门大学 | Method and kit for detecting quinolone resistance mutation of Mycobacterium tuberculosis |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109219667A (en) * | 2016-02-15 | 2019-01-15 | 安普诊断公司 | Diagnostic device and correlation technique |
| CN109219667B (en) * | 2016-02-15 | 2023-02-28 | 安普诊断公司 | Diagnostic device and related method |
| CN113025729A (en) * | 2020-12-24 | 2021-06-25 | 复旦大学 | Mycobacterium tuberculosis drug-resistance-related gene mutation site and application thereof |
| CN117187425A (en) * | 2023-11-03 | 2023-12-08 | 迪飞医学科技(南京)有限公司 | AS-RAPID tuberculosis drug-resistant duplex detection primer probe group, kit and application |
| CN117187425B (en) * | 2023-11-03 | 2024-02-20 | 迪飞医学科技(南京)有限公司 | AS-RAPID tuberculosis drug-resistant duplex detection primer probe group, kit and application |
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