CN105331709A - Kit for detecting mycobacterium tuberculosis pncA gene mutation - Google Patents
Kit for detecting mycobacterium tuberculosis pncA gene mutation Download PDFInfo
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- CN105331709A CN105331709A CN201510802642.8A CN201510802642A CN105331709A CN 105331709 A CN105331709 A CN 105331709A CN 201510802642 A CN201510802642 A CN 201510802642A CN 105331709 A CN105331709 A CN 105331709A
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Abstract
The invention discloses a kit for detecting mycobacterium tuberculosis pncA gene mutation. The kit comprises reagents for detecting mutation sites, namely -12T>C, -11A>G, -7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and +1-+18del, of mycobacterium tuberculosis pncA genes. The kit can be used for detecting the mutation sites of the pncA genes of a patient simply, conveniently, quickly and accurately and can be accordingly used for diagnosis and treatment of the pyrazinamide-resistant tuberculous patient.
Description
Technical field
The present invention relates to a kind of for detecting the test kit causing 7, mycobacterium tuberculosis pyrazinoic acid amide drug resistant gene pncA transgenation hot zone to suddenly change, belonging to drug resistance of tuberculosis detection in Gene Mutation technical field.
Background technology
Tuberculosis is the chronic infectious disease that mycobacterium tuberculosis causes.Since Ke He (Koch) finds tubercule bacillus, tuberculosis threatens human health always, and treat vaccine lungy, medicine and diagnostic techniques be always the mankind constantly seek solve target.In recent years, in recent years, the reasons such as abuse of antibiotics, environmental pollution and acquired immune deficiency syndrome (AIDS) cause, and tuberculosis is staged a comeback, M & M rebound significantly trend, has become with malaria and AIDS and one of three large killers becoming transmissible disease.More severe, tubercule bacillus becomes more resistance to existing medicine.Remain one of comparatively serious transmissible disease of developing country.According to the World Health Organization (WorldHealthOrganization, WHO) statistics shows, in 2011, the whole world have 8,700,000 active tuberculosis new cases (wherein 13% relate to simultaneously infect human immunodeficiency virus) and 1,400,000 examples dead, 430,000 examples comprised in HIV patient are dead, there are 310,000 multi-drug resistance tuberculosis the first cases, by least to caused by the microorganism of vazadrine and rifampin-resistance.These patients more than 60% are in China, India, the Russian Federation, Pakistan and South Africa.Have the extensive resistant tuberculosis case of 84 national reports, there is the highest incidence of active tuberculosis per capita Sub-Saharan Africa, mainly due to HIV popular caused by.The maximum person of case absolute number is from Asia, and India and China have the heaviest Disease Spectrum in the whole world.At present, China is one of Present Global tuberculosis country occurred frequently, and drug resistance of tuberculosis situation is quite serious, and Nontuberculous mycobacterial infections also presents the trend increased year by year.The existing active tuberculosis patient 4,510,000 of China, bacterium inspection positive Tuberculosis patient patient 1,960,000, the total resistant rate of annual death toll about 130,000 tubercule bacillus is 27.8%, initial drug resistant rates is 18.6%, acquired resistance rate is 46.5%, Drug-fast case rate is 10.7%, and wherein vazadrine, Rifampin and Streptomycin sulphate resistance situation are comparatively serious.
Pyrazinoic acid amide usually can treat tuberculosis with the first-line drug conbined usage such as Rifampin and vazadrine; the treatment cycle 9-12 month of patient can be made to foreshorten to 6 months; efficiently can kill the thalline of (pH >=5.5) in acid-resistant environment in body, and other medicine is difficult to kill this kind of bacterium.Under the pyrazinamidase effect of pncA genes encoding, intracellular pyrazinoic acid amide changes pyrazine acid (pyrazinoicacid into, POA), arrival surface of cell membrane is acted on by Passive diffusion and outer row, protonated pyrazine acid impels tenuigenin acidifying to form potential difference, and the transdermal delivery pncA transgenation affecting cell causes pyrazinoic acid amide resistance, and this mrna length is 561bp, coding pyrazinamidase, the frequency that in the bacterial strain of resistance to pyrazinoic acid amide, this gene is undergone mutation is 68%-95%.
Mycobacterium tuberculosis resists the mechanism of pharmaceutical activity, approximately through low membrane passage and efflux pump mechanism, produces degraded or deactivation enzyme, the change of drug target.Or tubercule bacillus cannot obtain resistance by the mediation of plasmid from other bacteriums, therefore the resistance of Chromosome-encoded is the main foundation that MTB produces resistance.At present the research of MTB drug-resistance mechanism is mainly concentrated in the target site of medicine and the sudden change of genes involved thereof.Therefore, the discovery of new drug resistant gene locus provides effective means and strategy for quick, high-throughput, easy detection.
By literature search, have no and detect the identical open report in mutational site with the present invention.
Summary of the invention
The object of the present invention is to provide a kind of method detecting mycobacterium tuberculosis pncA transgenation.
The invention provides for detect the tuberculosis medicine of resistance to pyrazinoic acid amide pncA gene 7 mutational sites (-12T>C ,-11A>G ,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del) test kit.
The present invention is by detecting from 7 sudden changes that whether there is pncA gene in the sample of the mycobacterium tuberculosis strain of resistance to pyrazinoic acid amide, thus judge the drug-resistance mechanism of this patient's medicine of resistance to pyrazinoic acid amide, wherein-12T>C,-11A>G,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del the sudden change of pncA gene is sported with+1-+18del, these sudden changes cause the 7th, upstream, pncA gene coding region respectively, 11 and the 12 bit base (-12T>C,-11A>G,-7T>C), the 3rd, coding region and the 233rd bit base are (c.3G>A, c.233G>A) change, c.408InsA and c.538-561del phase shift mutation is caused with+1-+18del.These sudden changes have impact on pncA genetic expression, change the amino acid composition of this gene, or extend or shorten this gene coded protein, thus play the effect of its anti-pyrazinoic acid amide, and then make this bacterial strain produce resistance.
The method of contriver's candidate gene screening, detect in the Chinese 16 strain mycobacterium tuberculosis strains of resistance to pyrazinoic acid amide, the pncA gene new mutant 7 relevant to resistance to pyrazinoic acid amide is found in six strain Resistance Mycobacterium Tuberculosis kinds, wherein 5 is new mutant (-12T>C,-11A>G,-7T>C, c.408InsA, c.538-561del with+1-+18del), the frequency of this sudden change is 43.7%, this illustrates in the mycobacterium tuberculosis strain of resistance to pyrazinoic acid amide, pncA transgenation has higher occurrence frequency, therefore pncA transgenation can as the diagnosis basis of clinical pyrazinoic acid amide Resistant strain drug-resistance mechanism.And at present, at the international and domestic report being showed no wherein 5 sudden changes.
The present invention is for detecting pncA gene 7 sudden change (-12T>C,-11A>G,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del) test kit, comprise for the reagent needed for detect pncA gene 7 sudden changes and the reagent for the pncA gene that increases that can select and PCR primer.
For detecting pncA gene 7 sudden change (-12T>C,-11A>G,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del) test kit, comprise the combination of one or more reagent following:
(1) from measuring samples, extract the reagent of DNA;
(2) for the pncA gene 7 of the tubercule bacillus sample DNA that the increases PCR primer of suddenling change and the PCR reaction reagent of being correlated with;
(3) PCR primer purified reagent;
(4) PCR primer is carried out to the reagent of direct Sequencing.
For detecting pncA gene 7 sudden change (-12T>C,-11A>G,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del) test kit, PCR primer used is:
pncA-F：5’-TGTCGCTCACTACATCACC-3’
pncA-R：5’-TCGTAGGTCATAGCGTAGG-3’
For detecting pncA gene 7 sudden change (-12T>C,-11A>G,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del) test kit, the reagent of described detection pcr amplification product is selected from order-checking detection reagent, restriction enzyme length polymorphism detection reagent, sequence specific primers detection reagent, probe hybridization detection reagent and SNP typing detection reagent.
Adopt the method for pcr amplification-direct Sequencing to detect the catastrophe of sample, concrete operation step is as follows:
(1) gather the sample of test individual, be the tubercule bacillus sample cultivated, extract complete genome DNA;
(2) with the DNA extracted for template, carry out the amplification of DNA sequence dna with the primer for pncA gene coding region and upstream and downstream region of the present invention's design, obtain corresponding pcr amplification product;
(3) by after the PCR primer purifying that obtains, carry out direct Sequencing analysis, the normal sequence of measured sequence and pncA gene and upstream and downstream thereof is compared, determine whether 7 sudden changes exist;
(4) whether be the M. tuberculosis strains of resistance to pyrazinoic acid amide that pncA transgenation causes according to above interpretation patient;
(5) according to normal coding sequence reading frame, mutant nucleotide sequence is translated, determine the existence of missense mutation and phase shift mutation further.
Contriver carries out in the process of testing at collection Drug-Resistant Mycobacterium tuberculosis, collects the mycobacterium tuberculosis strain of the resistance to pyrazinoic acid amide of 16 strain, under the prerequisite obtaining patient's agreement, carries out gene test to the mycobacterium tuberculosis of patient infection.Meanwhile, we also have collected essential information and the clinical information of patient, have inquired that it infects and pathogenic process in detail, have established the Sample Storehouse of drug resistance of Mycobacterium tuberculosis strain.By frozen for the mycobacterium tuberculosis of deactivation in-80 DEG C of refrigerators.Extract the genomic dna of tubercule bacillus by the method for post absorption, be stored in-40 DEG C of refrigerators, every part of DNA sample has corresponding patient data and resistance information.Design pcr amplification primer with primer-design software Primer5 and Oligo6, include the DNA fragmentation of upstream 161, tubercule bacillus pncA gene coding region bit base to downstream, coding region 321 bit base, for pcr amplification.Pcr amplification product directly carries out forward and reverse order-checking (order-checking instrument is ABI company 3730 type DNA sequencer) by PCR primer.Sequence in the sequence obtained and GenBank is compared, determines the existence of pncA transgenation.
Nucleotide or the aminoacid sequence of pncA transgenation are as follows:
In the Nucleotide of pncA transgenation and aminoacid sequence, what mark with square frame is base or the amino acid of sudden change, and what go out by open collimation mark is amino acid after phase shift mutation, and what mark with horizontal line is initiator codon; Be positioned at the 7(T>C before pncA gene coded sequence), 11(A>G) and 12(T>C) sudden change of bit base, the regulating and controlling sequence of pncA upstream region of gene is changed, cause pncA albumen transcribe and translation changes, and then cause the resistance of pyrazinoic acid amide; Be positioned at the sudden change of pncA gene coding region (c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del) cause that the Argine Monohydrochloride of this genes encoding forms, structure or length there occurs change, thus changes the function of this albumen.The detection of pncA transgenation can be carried out, as PCR(polymerase chain reaction with any one point mutation detecting method in hereditary field)-RFLP method (restriction enzyme segment polymorphism), PCR-sequencing, DNA probe hybrid method, Allele-specific diagnostic PCR method, PCR-dHPLC(denaturing high-performance chromatography) and PCR-SSCP(single strand conformation polymorphism) method etc.
The PCR primer obtained in aforesaid method can also detect with additive method, as DNA hybridization probe method.Probe used can be hybridize with sudden change pncA gene nucleotide series, and two probes also can be had to hybridize with pncA gene nucleotide series that is normal or that suddenly change respectively.Probe can select isotopic labeling, coloring matter to mark or fluorescent substance mark.In addition, sudden change can also be determined by the method for site-specific PCR primer, restriction enzyme or single strand conformation polymorphism.
The PCR primer used in aforesaid method designs according to known nucleotide sequence, normal length is 18-25 base, GC content is at ± 45-55%, and design pcr amplification primer with primer-design software Primer5 and Oligo6, the pcr amplification primer sequence designed in the present invention is:
Upstream primer: 5 '-TGTCGCTCACTACATCACC-3 '
Downstream primer: 5 '-TCGTAGGTCATAGCGTAGG-3 ';
The test kit of inspection pncA gene provided by the invention 7 sudden changes, test kit planted agent is equipped with the reagent for detecting pncA transgenation, provides that audit through governmental drug administration, about medicine or biological products manufacture, use and marketing information simultaneously.As the test kits adopting PCR-direct sequencing to detect pncA gene 7 sudden change, containing amplimer, dNTPs, for one or more of the archaeal dna polymerase of PCR reaction and damping fluid and the required reagent of order-checking.It is known to those skilled in the art that above component is only schematic composition, if amplimer is pair of primers pncA-F and pncA-R described in the present invention, the described archaeal dna polymerase for PCR reaction is the enzyme that can carry out pcr amplification.
The invention has the advantages that:
1, test kit can easy, fast, measure the mutational site of patient pncA gene accurately, thus in Diagnosis and Treat for resistance to pyrazinoic acid amide tubercular;
2, can be used in extensive examination pyrazinoic acid amide resistant rate in tubercular, whether the infection strain for diagnosis of tuberculosis mycobacterium patient has detection of pyrazinamide resistance provides service and reference;
3, accurate and simple method is provided for the tubercular of resistance to pyrazinoic acid amide carries out gene screening; And establish solid basis for utilizing this sudden change to carry out treatment as detection target spot for resistant tuberculosis in the future.
accompanying drawing illustrates:
Fig. 1 is the sequence chart that c.3G>A, c.233G>A mycobacterium tuberculosis pncA gene suddenlys change;
Fig. 2 be mycobacterium tuberculosis pncA gene c.408InsA suddenly change sequence chart;
Fig. 3 be mycobacterium tuberculosis pncA gene-1 2T>C ,-11A>G ,-7T>C sudden change sequence chart;
Fig. 4 be mycobacterium tuberculosis pncA gene c.538-561del and+1-+18del sudden change sequence chart;
Fig. 5 is pncA gene PCR product electrophoresis detection schematic diagram.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail; but scope is not limited to described content; method employing ordinary method if no special instructions in embodiment, the reagent use conventional commercial reagent if no special instructions of use or the reagent prepared according to a conventional method.
embodiment 1: acquisition testing sample
Collect the 16 strain mycobacterium tuberculosis strains of resistance to pyrazinoic acid amide, clinical patients is lunger, known through susceptibility pyrazinoic acid amide (PZA measures 1 μ g/ml) testing inspection result, obtains the 16 strain mycobacterium tuberculosis strains of resistance to pyrazinoic acid amide altogether.The resistance to pyrazinoic acid amide patient mean age is 40 years old.Male patient accounting 68.75%%(11/16), the age, the mean age was 40.3 years old between 15 ~ 69 years old; Female patient accounting 31.25%(5/16), the age between 21 ~ 49 years old, 39.4 years old mean age.
embodiment 2: the extraction of genomic dna
1, adopt disposable transfering loop scraping tubercule bacillus bacterium to cultivate bacterium colony and be placed in 1.5mlEP pipe (scraping of trying not is to substratum);
2, in the centrifuge tube of bacterial sediment thing, add 500 μ l cell suspending liquids (first check whether and added Lysozyme), use pipettor or vortex oscillator thoroughly suspension tubercule bacillus cell precipitation, 37 DEG C of temperature bath 30min put upside down mixing for several times every 5-10min.12000rpm(~ 13400 × g) centrifugal 2min, exhaust supernatant as far as possible;
3, in bacterial sediment, add 225 μ l buffer A, vibrate to thalline and thoroughly suspend;
4, Xiang Guanzhong adds 10 μ l Proteinase K Solution, puts upside down mixing;
5, add 25 μ l lysis buffer S, put upside down mixing; 20min is placed in 57 DEG C of water-baths, puts upside down mixing therebetween for several times.
6, add 250 μ l buffer B, vibration 5s fully mixes;
7, add 250 μ l dehydrated alcohols, fully vibration mixing 15s, now may occur flocks, brief centrifugation is to remove the globule of inside pipe wall;
8, previous step gained solution and flocks are all added (adsorption column puts into collection tube) 12000rpm(~ 13400 × g in an adsorption column) centrifugal 30s, outwells waste liquid, adsorption column is put into collection tube;
9, in adsorption column, 500 μ l damping fluid C are added, 12000rpm(~ 13400 × g) centrifugal 30s, outwells waste liquid, adsorption column is put into collection tube;
10, please first check whether add 700 μ l rinsing liquid W2(uses in adsorption column before and added dehydrated alcohol), 12000rpm(~ 13400 × g) centrifugal 30s, outwell waste liquid, adsorption column puts into collection tube;
11, in adsorption column, add 500 μ l rinsing liquid W2,12000rpm(~ 13400 × g) centrifugal 30s, outwell waste liquid, adsorption column is put back in collection tube, then 12000rpm(~ 13400 × g) centrifugal 2min, adsorption column is placed in a new 1.5ml centrifuge tube, room temperature places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material;
12, proceeded to by adsorption column in a clean centrifuge tube, the unsettled dropping 135 in the middle part to adsorption film μ l elution buffer TE, room temperature places 2-5min, 12000rpm(~ 13400 × g) centrifugal 2min, by solution collection in centrifuge tube, freeze in-40 DEG C, treat that experimental study uses.
embodiment 3:PCR increases, electrophoresis result
With the mycobacterium tuberculosis complete genome DNA extracted for template, carry out pcr amplification, amplification gene is pncA gene, the primer 5 '-TGTCGCTCACTACATCACC-3 ' and 5 '-TCGTAGGTCATAGCGTAGG-3 '.
PCR amplification system: 2 × PCR premixed liquid 25 μ L(contains rTaq enzyme, TAKARA), each 1 μM of forward and reverse primer, template DNA 50ng, adds 21 μ L deionized waters.
PCR reaction conditions is: 94 degree of sex change 5 minutes, (94 degree sex change 30 seconds, 50 degree annealing 30 seconds, 72 degree extensions 1 point 30 seconds) of then 35 circulations, finally last 72 degree of extensions at end 5 minutes.Amplified production length is 867bp, then carries out agarose gel electrophoresis detection, selects DL2000 model DNAmarker to contrast as pcr amplification product, and preparation gum concentration is the sepharose of 1.5%, under 120 volts of constant-pressure conditions, and electrophoresis 20-30 minute.After electrophoresis terminates, sepharose is put into EB dye liquor to dye 5-10 minute, observe electrophoretic band under being placed in ultraviolet lamp and to go forward side by side line item.Electrophoresis result is shown in Fig. 5.
embodiment 4: sequencing result
PCR primer send order-checking company to carry out sequencing, sequencing primer is 5 '-TGTCGCTCACTACATCACC-3 ', through contrasting with mycobacterium tuberculosis type strain sequence after order-checking, find mutational site-12T>C ,-11A>G ,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del, mutation map is shown in Fig. 1,2,3,4.
Sequence table
<110> Kunming University of Science and Technology
<120> is for detecting the test kit of mycobacterium tuberculosis pncA transgenation
<130>1
<160>2
<170>PatentInversion3.3
<210>1
<211>19
<212>DNA
<213> synthetic
<400>1
tgtcgctcactacatcacc19
<210>2
<211>19
<212>DNA
<213> synthetic
<400>2
tcgtaggtcatagcgtagg19
Claims (2)
1. for detecting a test kit for mycobacterium tuberculosis pncA transgenation, described test kit comprise for detect mycobacterium tuberculosis pncA gene-1 2T>C ,-11A>G ,-7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and+1-+18del sudden change reagent.
2. test kit according to claim 1, is characterized in that: described test kit comprises the primer of nucleotide sequence as shown in SEQIDNO:1 and SEQIDNO:2.
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| CN201510802642.8A CN105331709B (en) | 2015-11-19 | Kit for detecting mycobacterium tuberculosis pncA gene mutations |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510802642.8A CN105331709B (en) | 2015-11-19 | Kit for detecting mycobacterium tuberculosis pncA gene mutations |
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| CN105331709A true CN105331709A (en) | 2016-02-17 |
| CN105331709B CN105331709B (en) | 2018-08-31 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628619A (en) * | 2019-01-03 | 2019-04-16 | 首都医科大学附属北京胸科医院 | For identifying the SNP marker and method, Primer composition, kit and application of mycobacteria |
| CN111286551A (en) * | 2020-01-04 | 2020-06-16 | 昆明理工大学 | Primer and kit for rapidly detecting mycobacterium tuberculosis and using method thereof |
| CN113913542A (en) * | 2021-11-30 | 2022-01-11 | 广州达安基因股份有限公司 | Mycobacterium tuberculosis pyrazinamide drug-resistant mutation detection method and detection kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580879A (en) * | 2009-04-30 | 2009-11-18 | 广西医科大学 | Drug-resistance gene film chip for detecting mycobacterium tuberculosis |
| CN102174652A (en) * | 2011-02-28 | 2011-09-07 | 中国科学院武汉病毒研究所 | Detection method of mycobacterium tuberculosis pyrazinamide drug resistance |
| CN102925554A (en) * | 2012-09-18 | 2013-02-13 | 中国科学院武汉病毒研究所 | Joint detection method for drug resistance of mycobacterium tuberculosis and pyrazinamide in clinical sample |
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| CN101580879A (en) * | 2009-04-30 | 2009-11-18 | 广西医科大学 | Drug-resistance gene film chip for detecting mycobacterium tuberculosis |
| CN102174652A (en) * | 2011-02-28 | 2011-09-07 | 中国科学院武汉病毒研究所 | Detection method of mycobacterium tuberculosis pyrazinamide drug resistance |
| CN102925554A (en) * | 2012-09-18 | 2013-02-13 | 中国科学院武汉病毒研究所 | Joint detection method for drug resistance of mycobacterium tuberculosis and pyrazinamide in clinical sample |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109628619A (en) * | 2019-01-03 | 2019-04-16 | 首都医科大学附属北京胸科医院 | For identifying the SNP marker and method, Primer composition, kit and application of mycobacteria |
| CN111286551A (en) * | 2020-01-04 | 2020-06-16 | 昆明理工大学 | Primer and kit for rapidly detecting mycobacterium tuberculosis and using method thereof |
| CN113913542A (en) * | 2021-11-30 | 2022-01-11 | 广州达安基因股份有限公司 | Mycobacterium tuberculosis pyrazinamide drug-resistant mutation detection method and detection kit |
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Effective date of registration: 20231120 Address after: Room 101-1, 9th Floor, Standard Building No. 3, Biotechnology Incubator, Block B-5-28-3, East District, High-tech Zone, Kunming City, Yunnan Province, 650000 Patentee after: Yunnan Kecan Biotechnology Co.,Ltd. Address before: 650093 No. 253, Xuefu Road, Wuhua District, Yunnan, Kunming Patentee before: Kunming University of Science and Technology |