CN103060464A - Kit for detecting or assisting in detecting ethambutol resistance of multiple drugs resistant bacillus tuberculosis - Google Patents
Kit for detecting or assisting in detecting ethambutol resistance of multiple drugs resistant bacillus tuberculosis Download PDFInfo
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Abstract
The invention discloses a kit for detecting or assisting in detecting ethambutol resistance of multiple drugs resistant bacillus tuberculosis. The kit comprises a product for detecting an embB354 mutation site in an embB gene, wherein the nucleotide sequence of the embB gene is the sequence 1 shown in a sequence table; and the embB354 mutation site is that nucleotide GAC from the 1060th site to the 1062nd site at the 1-5' ends of the sequences in the sequence table is mutated into GCC. Tests show that the new mutation site embB354 of the embB gene can be used for detecting the ethambutol resistance of the multiple drugs resistant bacillus tuberculosis, and the mutation site embB354 is combined with another three mutation sites, namely, embB306, embB406 and embB497, to detect the ethambutol resistance of the multiple drugs resistant bacillus tuberculosis together so as to ensure higher diagnosis sensitivity.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the Tibutol resistance test kit of a kind of detection or auxiliary detection substance of medicines-resistant branched tubercle bacillus.
Background technology
Anti-multiple medicines (MDR) refers to that mycobacterium tuberculosis is at least to Rifampin (RIF) and vazadrine (INH) two kind of one line antitubercular agent generation resistance.Not only treatment cycle is long for anti-multiple medicines tuberculosis, drug side effect is large, and result for the treatment of is poor, curative ratio is low, mortality ratio is high, and medical expense is high, has brought very heavy economical load to patient.The resistance of Rapid﹠Early diagnosis mycobacterium tuberculosis is conducive to the treatment lungy of anti-multiple medicines and control.Compare based on the susceptibility detection method of solid, liquid culture with tradition, based on molecular biological susceptibility detection method (chemical sproof molecular diagnosis) have fast, sensitivity and the low advantage of cost.
Tibutol (EMB) is a line antitubercular agent of widespread use, and this medicine disturbs the synthetic of the Arabic semi-lactosi of mycobacterium cell wall constituent by the activity of the arabinofuranosyltransferase of inhibition embB genes encoding.The EmbB gene is considered to the Tibutol drug resistance related gene, and the sudden change of its 306 bit codon (embB306) is considered to the molecule marker of Tibutol resistance quick diagnosis.
More present commercial quick diagnosis products, GenoType MTBDRsl (Hain Lifescience for example, Germany) and QIAplex TB assay (Genaco), predict the resistance of tubercule bacillus by the sudden change that detects the embB306 Single locus, but because the drug resistance related gene mutation type of the popular bacterial strain in different areas is different with frequency, the sensitivity of these instruments is also different in different areas with specific degree, has approximately clinically in 30～70% the Tibutol Resistant strain to detect this site mutation.Therefore the sensitivity of Tibutol resistance molecular diagnosis needs further to improve, in the urgent need to a test kit that can be applicable to the more effective Tibutol resistance molecular diagnosis of different areas.
Summary of the invention
An object of the present invention is to provide the Tibutol resistance reagent of a kind of detection or auxiliary detection substance of medicines-resistant branched tubercle bacillus.
Reagent provided by the invention comprises the product for detection of embB354 mutational site in the embB gene; The wild-type nucleotides sequence of described embB gene is classified sequence 4 in the sequence table as;
Described embB354 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 1060-1062 position Nucleotide; The wild-type nucleotides sequence in described mutational site is classified GAC as, and the mutant nucleotides sequence is classified GCC as.
In the mentioned reagent, described product for detection of embB354 mutational site in the embB gene comprises comparison card and is used for the described embB gene fragment primer pair that amplification comprises described embB354 mutational site:
Record contains the nucleotide sequence of the fragment in described embB354 mutational site on the described comparison card.In an embodiment of the present invention, this comparison card is A) the comparison card, be described below nucleotide sequence (embB354 mutational site): in the sequence table sequence 1 from 5 ' terminal 851-1962 position Nucleotide, wherein, 1060-1062 position Nucleotide GAC or GCC; 1489-1491 position Nucleotide CAG; 1216-1218 position Nucleotide GGC; 916-918 position Nucleotide is ATG.
In the mentioned reagent, the described fragment that contains described embB354 mutational site also contains at least a in embB306 mutational site, embB406 mutational site and the embB497 mutational site; Described embB306 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 916-918 position Nucleotide; Described embB306 mutational site sequence is ATG, GTG, TTG, ATA, ATC or ATT; Described embB306 mutational site wild-type nucleotides sequence is classified ATG as, and the mutant nucleotides sequence is classified GTG, TTG, ATA, ATC or ATT as;
Described embB406 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 1216-1218 position Nucleotide; Described embB406 mutational site sequence is GGC, AGC, TGC, GAC or GCC; Described embB406 mutational site wild-type nucleotides sequence is classified GGC as, and the mutant nucleotides sequence is classified AGC, TGC, GAC or GCC as;
Described embB497 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 1489-1491 position Nucleotide; Described embB497 mutational site sequence is CAG, AAG, CCG or CGG; Described embB497 mutational site wild-type nucleotides sequence is classified CAG as, and the mutant nucleotides sequence is classified AAG, CCG or CGG as.
In an embodiment of the present invention, the comparison card is B) the comparison card, be described below four groups of nucleotide sequences:
1) in the sequence table sequence 1 from 5 ' terminal 851-1962 position Nucleotide, wherein, 1060-1062 position Nucleotide GAC or GCC, 1489-1491 position Nucleotide CAG, 1216-1218 position Nucleotide GGC, 916-918 position Nucleotide are the ATG(embB354 mutational site);
2) sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table, and wherein, 916-918 position Nucleotide is ATG or GTG or TTG or ATA or ATC or ATT; The 1060-1062 position is that GAC, 1216-1218 position are that GGC, 1489-1491 position are the CAG(embB306 mutational site);
3) sequence 1 is from 5 ' terminal 851-1962 position Nucleotide; Wherein, 1216-1218 position Nucleotide GGC, AGC, TGC, GAC or GCC; 916-918 position Nucleotide is ATG; The 1060-1062 position is that GAC, 1489-1491 position are the CAG(embB406 mutational site);
4) sequence 1 is from 5 ' terminal 851-1962 position Nucleotide; Wherein, 1489-1491 position Nucleotide CAG, AAG, CCG, CGG or; 1216-1218 position Nucleotide GGC; 916-918 position Nucleotide is ATG; The 1060-1062 position is the GAC(embB497 mutational site).
In the mentioned reagent, be used for amplification and comprise that the described embB gene fragment primer in described embB354 mutational site is to being comprised of the single strand dna shown in the sequence 3 in the single strand dna shown in the sequence table sequence 2 and the sequence table;
The described nucleotides sequence that contains the fragment in described embB354 mutational site is classified in the sequence table sequence 1 as from 5 ' terminal 851-1962 position nucleotide sequence.
The application of above-mentioned reagent in preparation detection or the chemical sproof product of auxiliary detection substance of medicines-resistant branched tubercle bacillus Tibutol.Described product is test kit.
The application of above-mentioned reagent in preparation detection or the chemical sproof product of auxiliary detection tuberculosis patient Tibutol; Described tuberculosis patient specifically infects substance of medicines-resistant branched tubercle bacillus.Described product is test kit.
The application of above-mentioned reagent in detection or auxiliary detection embB gene mutation site.
In the above-mentioned application, described embB gene mutation site is at least a among embB306, embB406, embB497 and the embB354.
The embB354 mutational site is as detecting or the chemical sproof target of auxiliary substance of medicines-resistant branched tubercle bacillus Tibutol or to detect mark also be the scope of protection of the invention.
The mutational site group that embB354 mutational site, embB306 mutational site, embB406 mutational site and embB497 mutational site form is as detecting or the chemical sproof target of auxiliary detection substance of medicines-resistant branched tubercle bacillus Tibutol or to detect mark also be the scope of protection of the invention.
Substance of medicines-resistant branched tubercle bacillus in the above-mentioned substance of medicines-resistant branched tubercle bacillus refers to from patient's sputum specimen success Isolation and culture, is defined as simultaneously mycobacterium tuberculosis clinical separation strain to anti-Rifampin (RIF) and vazadrine (INH) through acid-fast stain and identification of strains experiment and by drug sensitive experiment.
Of the present invention experimental results show that, reagent provided by the invention, can be used for detecting or the Tibutol resistance of auxiliary detection substance of medicines-resistant branched tubercle bacillus or tuberculosis patient, the present invention finds that the embB354 mutational site of embB gene can be used for detecting tubercule bacillus Tibutol resistance, and with itself and embB306, embB406 and embB497 detect tubercule bacillus Tibutol resistance together at four sites that combine, can obtain higher diagnostic sensitivity, can more effectively carry out the chemical sproof Rapid﹠Early diagnosis of substance of medicines-resistant branched tubercle bacillus Tibutol and formulate corresponding effective treatment plan.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The pathogenic bacteria of the tuberculosis patient among the following embodiment is the MDR mycobacterium tuberculosis, and is anti-Tibutol through medical verification, and the patient all knows the inside story.
The acquisition of the Tibutol resistance test kit of embodiment 1, detection or auxiliary detection substance of medicines-resistant branched tubercle bacillus
1, the acquisition in substance of medicines-resistant branched tubercle bacillus Tibutol drug resistance related gene mutational site
(1) extract genomic dna, specific as follows:
With 110 tuberculosis patient patient sputum specimens of 61 tuberculosis patient patients of Beijing tuberculosis control institute of anti-multiple medicines (being accredited as anti-Rifampin (RIF) and vazadrine (INH)), disease prevention and control center, Shanghai carry out smear, cultivation, separate, purifying etc., these tuberculosis infect substance of medicines-resistant branched tubercle bacillus (MDR mycobacterium tuberculosis) per capita, obtain respectively substance of medicines-resistant branched tubercle bacillus (elongated slightly crooked bacillus, size 1～4X0.4 μ m), save backup;
The MDR mycobacterium tuberculosis that the above-mentioned separation and purification of recovery obtains in 3 grades of laboratories of Biosafety, (1 week BA7005C-2), was cultivated for for 37 ℃ by Bei Suo company to be inoculated in the acid modified Russell medium of 2 5ml.Corresponding 2 5ml improvement Luo Shi culture tube is got 2 1.5ml centrifuge tubes, numbers, and adds respectively the TE of 1ml.With the bacterium colony on the inclined-plane in the transfering loop scraping Luo Shi culture tube (scraping complete) as far as possible, move into and be equipped with in the centrifuge tube of TE, fully stirring makes thalline suspend as far as possible, covers tightly the centrifuge tube lid, 85 ℃ of deactivations 1 hour.Centrifuge tube after the deactivation is moved to outside 3 grades of laboratories of Biosafety, and 100 ℃ were boiled 15 minutes, placed immediately afterwards 5 minutes on ice, and centrifugal 5 minutes of 13000rpm moves into new 1.5ml centrifuge tube with supernatant (containing genomic dna), obtains genomic dna.
(2) the embB gene order (sequence 4) of the tubercule bacillus laboratory standard strain H37Rv that provides according to Pasteur Institut tuberculist website (http://genolist.pasteur.fr/Tuberculist), use the PCR primer of primer-design software Primer Premier5.0 design drug resistant gene embB:
Be used for the primer of PCR reaction and sequencing primer and be embB-1 (5 '-TATTCGGCTTCCTGCTCTGG-3 ', sequence 2), embB-2 (5 '-CACACCGTAGCTGGAGACAT-3 ', sequence 3).Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(3) genomic dna that obtains with above-mentioned (1) is as template, take embB-1 and embB-2 as primer carries out pcr amplification, obtains the PCR product.
Wherein the PCR reaction system is:
Wherein, 5xHFbuffer, DMSO, dNTP and DNA Polymerase all adopt
Composition in Hot StartHigh-Fidelity DNA Polymerase (F549L, Finnzymes, the USA) test kit.
Wherein the PCR reaction conditions is:
Pcr amplification instrument: DNA engine cycler PTC-200 (BIORAD, USA) wherein
(4) get the PCR product that 5 μ l above-mentioned (3) obtain and carry out agarose gel electrophoresis, obtain the fragment of 1112bp size.If PCR product band is high-visible, and size meets, and then send company to check order.If PCR product band is weak or without band, then adjust PCR system and condition, again PCR.
(5) application software Seqman pro (version7.1, DNAstar Lasergene, Inc., USA) the embB gene order (sequence 4) of PCR product sequencing result and H37Rv reference culture is compared, obtains the mutational site mutant of following several embB genes: embB306 mutant, embB406 mutant, embB497 mutant and embB354 mutant:
The embB306 mutant be in the sequence table sequence 4 from mutational site that 5 ' terminal 916-918 position Nucleotide ATG carries out respectively following any sudden change: ATG → GTG, ATG → TTG; ATG → ATA, ATG → ATC, ATG → ATT;
The embB354 mutant is for to carry out following sudden change: GAC → GCC with sequence in the sequence table 4 from 5 ' terminal 1060-1062 position Nucleotide GAC;
The embB406 mutant for sequence in the sequence table 4 from mutational site that 5 ' terminal 1216-1218 position Nucleotide GGC carries out following any sudden change: GGC → AGC, GGC → TGC; GGC → GAC, GGC → GCC;
The embB497 mutant for sequence in the sequence table 4 from mutational site that 5 ' terminal 1489-1491 position Nucleotide CAG carries out following any sudden change: CAG → AAG; CAG → CCG, CAG → CGG.
2, the Tibutol resistance test kit of detection or auxiliary detection substance of medicines-resistant branched tubercle bacillus
The Tibutol resistance test kit of detection or auxiliary detection substance of medicines-resistant branched tubercle bacillus comprises that following primer is to blocking with comparison;
Above-mentioned primer is to as follows:
EmbB-1 (5 '-TATTCGGCTTCCTGCTCTGG-3 ', sequence 2)
EmbB-2 (5 '-CACACCGTAGCTGGAGACAT-3 ', sequence 3)
The comparison card is following A) or B):
A) the comparison card is described below nucleotide sequence (embB354 mutational site): sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table, and wherein, 1060-1062 position Nucleotide is GAC or GCC; 1489-1491 position Nucleotide is CAG; 1216-1218 position Nucleotide is GGC; 916-918 position Nucleotide is ATG.Wherein, 1060-1062 position Nucleotide is that GAC is the wild-type in embB354 mutational site; 1060-1062 position Nucleotide is that GCC is the mutant in embB354 mutational site.
B) the comparison card is described below four groups of nucleotide sequences:
1) nucleotide sequence in embB354 mutational site: sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table, wherein, 1060-1062 position Nucleotide GAC or GCC, 1489-1491 position Nucleotide CAG, 1216-1218 position Nucleotide GGC, 916-918 position Nucleotide are the ATG(embB354 mutational site).Wherein, 1060-1062 position Nucleotide GAC is the wild-type in embB354 mutational site, and 1060-1062 position Nucleotide GCC is the mutant in embB354 mutational site.
2) nucleotide sequence in embB306 mutational site: sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table, and wherein, 916-918 position Nucleotide is ATG or GTG or TTG or ATA or ATC or ATT; The 1060-1062 position is that GAC, 1216-1218 position are that GGC, 1489-1491 position are the CAG(embB306 mutational site).Wherein, 916-918 position Nucleotide is that ATG is the wild-type in embB306 mutational site, and 916-918 position Nucleotide is that GTG or TTG or ATA or ATC or ATT are the mutant in embB306 mutational site.
3) nucleotide sequence in embB406 mutational site: sequence 1 is from 5 ' terminal 851-1962 position Nucleotide; Wherein, 1216-1218 position Nucleotide is GGC, AGC, TGC, GAC or GCC; 916-918 position Nucleotide is ATG; The 1060-1062 position is that GAC, 1489-1491 position are the CAG(embB406 mutational site).Wherein, 1216-1218 position Nucleotide is that GGC is embB406 mutational site wild-type, and 1216-1218 position Nucleotide is that AGC, TGC, GAC or GCC are embB406 mutational site mutant.
4) nucleotide sequence in embB497 mutational site: sequence 1 is from 5 ' terminal 851-1962 position Nucleotide; Wherein, 1489-1491 position Nucleotide is CAG, AAG, CCG, CGG or CAC; 1216-1218 position Nucleotide is GGC; 916-918 position Nucleotide is ATG; The 1060-1062 position is the GAC(embB497 mutational site).Wherein, 1489-1491 position Nucleotide is that CAG is embB497 mutational site wild-type, and 1489-1491 position Nucleotide is that AAG, CCG or CGG are embB497 mutational site mutant.
The application of the Tibutol resistance test kit of embodiment 2, detection or auxiliary detection substance of medicines-resistant branched tubercle bacillus
Sample is: 61 of Beijing tuberculosis control institute have identified that the chemical sproof tuberculosis patient patient of Tibutol and 21 have identified that with the chemical sproof tuberculosis patient patient of Tibutol, disease prevention and control center, Shanghai 110 have identified that the chemical sproof tuberculosis patient patient's of Tibutol sputum specimen and 45 have identified not have the chemical sproof tuberculosis patient patient of Tibutol; These tuberculosis infect substance of medicines-resistant branched tubercle bacillus per capita, and namely after testing all anti-Rifampin (RIF), anti-vazadrine (INH) carry out following experiment patient and all know the inside story.
The Tibutol resistance is detected in one: 4 mutational site of method
1, respectively above-mentioned each patient's of each department isolated from sputum purifying is obtained substance of medicines-resistant branched tubercle bacillus, every patient's separation and purification goes out a strain substance of medicines-resistant branched tubercle bacillus, extract the genomic dna of substance of medicines-resistant branched tubercle bacillus, the separation and extraction method is seen 1 (1) of embodiment 1;
2, take above-mentioned 1 genomic dna that obtains as template, take embB-1 and embB-2 as primer carries out pcr amplification, all obtain the 1112bpPCR product;
3, above-mentioned each PCR product is checked order respectively, each PCR product sequence and the sequence of comparing the upper record of card is compared:
In actual applications, if the nucleotides sequence of PCR product is classified in the sequence table sequence 1 as from 5 ' terminal 851-1962 position Nucleotide, and embB354 mutational site, embB306 mutational site, embB406 mutational site and embB497 mutational site are wild-type, then the not anti-Tibutol of testing sample candidate;
If the nucleotides sequence of PCR product is classified in the sequence table sequence 1 as from 5 ' terminal 851-1962 position Nucleotide, and at least one is mutant for embB354 mutational site, embB306 mutational site, embB406 mutational site and embB497 mutational site, then the anti-Tibutol of testing sample candidate.
Method two: mutational site embB354 detects the Tibutol resistance
1,1 identical with method one;
2,2 identical with method one;
3, above-mentioned each PCR product is checked order respectively, with each PCR product sequence with compare card and go up sequence and compare:
In actual applications, sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table if the nucleotides sequence of PCR product is classified as, and wherein, the embB354 mutational site is wild-type, then the not anti-Tibutol of testing sample candidate;
Sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table if the nucleotides sequence of PCR product is classified as, and wherein, the embB354 mutational site is mutant, then the anti-Tibutol of testing sample candidate.
Method three: (contrast) detected in the embB306 mutational site
1,1 identical with method one;
2,2 identical with method one;
3, above-mentioned each PCR product is checked order respectively, each PCR product sequence and the sequence of comparing on the card is compared respectively:
In actual applications, sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table if the nucleotides sequence of PCR product is classified as, and wherein, the embB306 mutational site is wild-type, then the not anti-Tibutol of testing sample candidate.
Sequence 1 is from 5 ' terminal 851-1962 position Nucleotide in the sequence table if the nucleotides sequence of PCR product is classified as, and wherein, the embB306 mutational site is mutant, then the anti-Tibutol of testing sample candidate.
The result of above-mentioned three kinds of methods is as shown in table 1:
Table 1 is embB transgenation prediction patient or the corresponding chemical sproof sensitivity of substance of medicines-resistant branched tubercle bacillus Tibutol
The embB transgenation situation of the substance of medicines-resistant branched tubercle bacillus that Tibutol resistance patient (EMB resistance patient) separates is specific as follows:
One, Beijing
In the patient of 61 Tibutol resistances:
1, only detect the result in embB306 mutational site: the embB306 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of 32 patient's separation in the embB gene is mutant: wherein the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 12 patient's separation is GTG, the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 18 patient's separation is ATA (wherein the embB406 sudden change occurs 1 patient simultaneously), the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 1 patient's separation is ATC, the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 1 patient's separation is ATT; The embB306 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and the sequence in embB306 mutational site is ATG.
2, only detect the result in embB354 mutational site: the embB354 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of 2 patient's separation in the embB gene is mutant, and the mutational site sequence is GCC; The substance of medicines-resistant branched tubercle bacillus embB354 site that all the other patients separate is wild-type, and the sequence in embB354 mutational site is GAC;
3, detect the result of embB306, embB406, embB497 and embB354 mutational site group: it is mutant that embB306, embB406, embB497 and the embB354 mutational site in the substance of medicines-resistant branched tubercle bacillus genomic dna that 41 patients separate in the embB gene has a mutational site at least: the sudden change situation in (1) embB306 site and embB354 site as mentioned above.EmbB406 site in the substance of medicines-resistant branched tubercle bacillus genomic dna that (2) 6 patients separate in the embB gene is mutant: wherein 1 patient substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence of separating be the substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence that AGC, 3 patients separate be GAC(wherein 1 patient the embB306 sudden change occurs simultaneously), 2 patients substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence of separating is GCC; The embB406 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and embB406 mutational site sequence is GGC; EmbB497 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of (3) 2 patient's separation in the embB gene is mutant: wherein the substance of medicines-resistant branched tubercle bacillus embB497 mutational site sequence of 1 patient's separation is AAG, and 1 patient embB497 mutational site sequence is CGG; The substance of medicines-resistant branched tubercle bacillus embB497 mutational site that all the other patients separate is wild-type, and embB497 mutational site sequence is CAG.
Two, Shanghai
In the substance of medicines-resistant branched tubercle bacillus that the patient of 110 Tibutol resistances separates:
1, only detect the result in embB306 mutational site: the embB306 site in the substance of medicines-resistant branched tubercle bacillus genomic dna that the substance of medicines-resistant branched tubercle bacillus of 52 patient's separation separates in the embB gene is mutant: wherein the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 36 patient's separation is GTG, the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 1 patient's separation is TTG, the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 7 patient's separation is ATA (wherein the embB497 sudden change occurs the substance of medicines-resistant branched tubercle bacillus of 1 patient's separation simultaneously), the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 2 patient's separation is ATC, and the substance of medicines-resistant branched tubercle bacillus embB306 mutational site sequence of 6 patient's separation is ATT; The embB306 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and the sequence in embB306 mutational site is ATG.
2, only detect the result in embB354 mutational site: the embB354 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of 7 patient's separation in the embB gene is mutant, and the mutational site sequence is GCC; The embB354 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and the sequence in embB354 mutational site is GAC;
3, detect the result of embB306, embB406, embB497 and embB354 mutational site group: it is mutant that embB306, embB406, embB497 and the embB354 mutational site in the substance of medicines-resistant branched tubercle bacillus genomic dna that 80 patients separate in the embB gene has a mutational site at least.(1) the sudden change situation in embB306 site and embB354 site as mentioned above.EmbB406 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of (2) 12 patient's separation in the embB gene is mutant: wherein the substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence of 2 patient's separation is AGC, the substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence of 2 patient's separation is TGC, the substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence of 3 patient's separation is GAC, and the substance of medicines-resistant branched tubercle bacillus embB406 mutational site sequence of 5 patient's separation is GCC; The embB406 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, embB406 mutational site sequence is that the embB497 site in the embB gene is mutant in the substance of medicines-resistant branched tubercle bacillus genomic dna that separates of (3) 10 patients of GGC.: wherein 2 patients substance of medicines-resistant branched tubercle bacillus embB497 mutational site sequence of separating is AAG, the substance of medicines-resistant branched tubercle bacillus embB497 mutational site sequence of 2 patient's separation is CCG (wherein the embB306 sudden change occurs the substance of medicines-resistant branched tubercle bacillus of 1 patient's separation simultaneously), and the substance of medicines-resistant branched tubercle bacillus embB497 mutational site sequence of 6 patient's separation is CGG; The substance of medicines-resistant branched tubercle bacillus embB497 mutational site that all the other patients separate is wild-type, and embB497 mutational site sequence is that embB306, embB406, embB497 and the embB354 site in the embB gene of the substance of medicines-resistant branched tubercle bacillus that separates of all the other patients of CAG. is wild-type.
EmbB transgenation situation is specific as follows in the substance of medicines-resistant branched tubercle bacillus that responsive (not resistance) patient of Tibutol separates:
One, Beijing
In the substance of medicines-resistant branched tubercle bacillus that the patient of 21 Tibutol resistances separates:
1, only detect the result in embB306 mutational site: the embB306 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of 2 patient's separation in the embB gene is mutant: the mutational site sequence is ATA.The embB306 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and the sequence in embB306 mutational site is ATG.
2, only detect the result in embB354 mutational site: the embB354 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of 1 patient's separation in the embB gene is mutant: the mutational site sequence is GCC; The embB354 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and the sequence in embB354 mutational site is GAC;
3, detect the result of embB306, embB406, embB497 and embB354 mutational site group: it is mutant that embB306, embB406, embB497 and the embB354 mutational site in the substance of medicines-resistant branched tubercle bacillus genomic dna that 4 patients separate in the embB gene has a mutational site at least.(1) the sudden change situation in embB306 site and embB354 site as mentioned above.EmbB406 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of (2) 1 patient's separation in the embB gene is mutant, and the mutational site sequence is GCC; The embB406 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, embB406 mutational site sequence is that the embB497 mutational site of the substance of medicines-resistant branched tubercle bacillus of all patients' separation of GGC. (3) is wild-type, and the mutational site sequence is CAG.EmbB306, embB406, embB497 and embB354 site in the embB gene of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate are wild-type.
Two, Shanghai
In the substance of medicines-resistant branched tubercle bacillus that the patient of 45 Tibutol resistances separates:
1, only detect the result in embB306 mutational site: the embB306 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of 2 patient's separation in the embB gene is mutant: the embB306 site that the mutational site sequence is the substance of medicines-resistant branched tubercle bacillus of all the other patients' separation of GTG. is wild-type, and the sequence in embB306 mutational site is ATG.
2, only detect the result in embB354 mutational site: the embB354 site of the substance of medicines-resistant branched tubercle bacillus that all patients separate is wild-type, and the sequence in embB354 mutational site is GAC;
3, detect the result of embB306, embB406, embB497 and embB354 mutational site group: it is mutant that embB306, embB406, embB497 and the embB354 mutational site in the substance of medicines-resistant branched tubercle bacillus genomic dna that 4 patients separate in the embB gene has a mutational site at least.(1) the sudden change situation in embB306 site and embB354 site as mentioned above.EmbB406 site in the substance of medicines-resistant branched tubercle bacillus genomic dna of (2) 1 patient's separation in the embB gene is mutant, the mutational site sequence is GAC, the embB406 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, embB406 mutational site sequence is GGC.(3) embB497 site in the substance of medicines-resistant branched tubercle bacillus genomic dna that separates of 1 patient in the embB gene is for being mutant, the mutational site sequence is CGG, the embB497 site of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate is wild-type, and embB497 mutational site sequence is CAG.EmbB306, embB406, embB497 and embB354 site in the embB gene of the substance of medicines-resistant branched tubercle bacillus that all the other patients separate are wild-type.
The above results can be found out, detects separately the sudden change in embB354 site, can detect Tibutol Resistant strain and resistance patient; If detect separately the sudden change in embB306 site, 52.5% and 47.3% Tibutol Resistant strain can only be predicted in Beijing and Shanghai, and sensitivity is lower, and prediction effect is undesirable.By taking multidigit point joint-detection, be Tibutol resistance or the patient's resistance that embB306, embB406, embB497 and four mutational site groups of embB354 are predicted the MDR bacterial strain, the sensitivity in three areas all is significantly improved, the sensitivity of Beijing and District of Shanghai reaches 67.2% and 72.7%, is significantly improved.
Claims (10)
1. one kind is detected or the Tibutol resistance reagent of auxiliary detection substance of medicines-resistant branched tubercle bacillus, comprises the product for detection of embB354 mutational site in the embB gene; Described embB354 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 1060-1062 position Nucleotide; The nucleotides sequence in described mutational site is classified GAC or GCC as.
2. reagent according to claim 1 is characterized in that: described product for detection of embB354 mutational site in the embB gene comprises the comparison card and is used for the described embB gene fragment primer pair that amplification comprises described embB354 mutational site:
Record contains the nucleotide sequence of the fragment in described embB354 mutational site on the described comparison card.
3. reagent according to claim 2 is characterized in that:
The described fragment that contains described embB354 mutational site also contains at least a in embB306 mutational site, embB406 mutational site and the embB497 mutational site; Described embB306 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 916-918 position Nucleotide; Described embB306 mutational site sequence is ATG, GTG, TTG, ATA, ATC or ATT;
Described embB406 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 1216-1218 position Nucleotide;
Described embB406 mutational site sequence is GGC, AGC, TGC, GAC or GCC;
Described embB497 mutational site is that sequence 1 in the sequence table is from 5 ' terminal 1489-1491 position Nucleotide;
Described embB497 mutational site sequence is CAG, AAG, CCG or CGG.
4. it is characterized in that according to claim 2 or 3 described reagent:
Be used for amplification and comprise that the described embB gene fragment primer in described embB354 mutational site is to being comprised of the single strand dna shown in the sequence 3 in the single strand dna shown in the sequence table sequence 2 and the sequence table;
The described nucleotides sequence that contains the fragment in described embB354 mutational site is classified in the sequence table sequence 1 as from 5 ' terminal 851-1962 position nucleotide sequence.
5. the application of arbitrary described reagent in preparation detection or the chemical sproof product of auxiliary detection substance of medicines-resistant branched tubercle bacillus Tibutol among the claim 1-4.
6. the application of arbitrary described reagent in preparation detection or the chemical sproof product of auxiliary detection tuberculosis patient Tibutol among the claim 1-4; Described tuberculosis patient specifically infects substance of medicines-resistant branched tubercle bacillus.
7. the application of arbitrary described reagent in detection or auxiliary detection embB gene mutation site among the claim 1-4.
8. application according to claim 7 is characterized in that: described embB gene mutation site is at least a among embB306, embB406, embB497 and the embB354.
9.embB354 the mutational site is as detecting or the chemical sproof target of auxiliary substance of medicines-resistant branched tubercle bacillus Tibutol or detection mark.
10.embB354 the mutational site group that mutational site, embB306 mutational site, embB406 mutational site and embB497 mutational site form is as detecting or the chemical sproof target of auxiliary detection substance of medicines-resistant branched tubercle bacillus Tibutol or detection mark.
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| CN102010906A (en) * | 2010-11-09 | 2011-04-13 | 广州益善生物技术有限公司 | Emb B gene mutation detection specific primers and liquid phase chip |
| CN102229989A (en) * | 2011-05-25 | 2011-11-02 | 厦门大学 | Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis |
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| CN102010906A (en) * | 2010-11-09 | 2011-04-13 | 广州益善生物技术有限公司 | Emb B gene mutation detection specific primers and liquid phase chip |
| CN102229989A (en) * | 2011-05-25 | 2011-11-02 | 厦门大学 | Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis |
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| Title |
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| YOUNG KIL PARK, ET AL.: "Correlation of the phenotypic ethambutol susceptibility of Mycobacterium tuberculosis with embB gene mutations in Korea", 《JOURNAL OF MEDICAL MICROBIOLOGY》 * |
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