CN102618625B - Method and kit for detecting drug resistance of mycobacterium tuberculosis - Google Patents
Method and kit for detecting drug resistance of mycobacterium tuberculosis Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting drug resistance of mycobacterium tuberculosis. The invention provides special-purpose primers and special-purpose probes for identification of drug resistance of mycobacterium tuberculosis in a sample needing to be detected, wherein the special-purpose primers comprise multiple primer groups; each one of the primer groups comprises a commonprimer and multiple specific primers for amplification of alleles corresponding to all mutational sites of same-purpose genes; the special-purpose probes comprise multiple types of probes; and each one of the probes specifically binds with target sequences corresponding to the common primer and the specific primers of the primer group. Experiments of the invention prove that the special-purpose primers and the special-purpose probes can be combined by an efficient and sensitive duplex Taqman real-time fluorescent polymerase chain reaction (PCR) technology and a sequence specific primer (SSP) amplification technology so that drug resistance of mycobacterium tuberculosis in a detected sample can be detected in a short time.
Description
Technical field
The present invention relates to the chemical sproof method of a kind of detection mycobacterium tuberculosis and test kit.
Background technology
Tuberculosis is by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) a kind of communicable disease due to.According to the data that The World Health Organization (WHO) provides, world's situation lungy was in 2002: number of the infected is about 3,000,000,000, New Development tuberculosis case 8,800,000.Wherein, newly be coated with yang constipation nuclear case 3,900,000, sickness rate increases by 1.1% than calendar year 2001, and anti-multiple medicines ratio is 3.2% in the new cases, and death toll lungy reaches 1,800,000.Retrospective review is the result show, total resistant rate of Chinese MTB is 27.8%, and initial drug resistant rates and acquisition resistant rate are respectively 18.6% and 46.5%, and anti-multiple medicines rate is 10.7%, and wherein initial and acquired anti-multiple medicines rate is respectively 7.6% and 17.1%.Because the resistance tubercule bacillus is popular, tuberculosis is become by single microorganism cause main causes of death, accounts for 7% of world population number, accounts for and can prevent 26% of death toll.
Along with the development of molecular biology theory and technology, the resistance mechanism of MTB and the molecular basis of resistance are progressively illustrated, and have set up the method for rapid detection MTB drug resistant gene, for the sensitivity test of MTB quick medicament opens up a new way.Studies show that MTB produces mechanism of drug resistance and comprises medicine fermentoid, cell wall permeability changes, target structure transgenation and pathways metabolism change etc., and due to the sudden change that main mechanism is drug effect target gene in the bacterium.Rifampin and vazadrine are the topmost line medicines for the treatment of tuberculosis, by various biology tool the chemical sproof hereditary basis of these two kinds of medicines is furtherd investigate, the result shows, under normal circumstances, Rifampin (RFP) is combined with RNA polymerase B subunit (rpoB) specificity, the synthetic effect of playing bacteria growing inhibiting by blocking-up RNA, the rpoB gene is undergone mutation under the medicament selection pressure of Rifampin and is changed coded rpoB subunit conformation, thus the avidity of reduction and Rifampin.The rpoB transgenation generally occurs in the 81bp zone of a high conservative in this gene (27 amino acid code in 507-533 position).Wherein modal codon mutation point is 531 Serines (Ser) → leucine (Leu) (531TCG → TTG), 526 hyte propylhomoserins (His) → tyrosine (Tyr) (526CAC → GAC), in addition, common point mutation also has 511CTG → CCG, 533CTG → CCG (Taniguchi H.Molecularmechanisms of multidrug resistance in Mycobacterium tuberculosis[J] .J Uoch, 2000,22 (3): 269-282).
The chemical sproof molecular basis more complicated in vazadrine; found severally to relate to that active intermediate forms relevant gene in the internal metabolism process of vazadrine, wherein relevant with transgenations such as catalase-superoxide enzyme coding gene katG and/or enoyl-reductase enzyme encoding gene inbA.These two kinds of enzymes are the formation of direct or indirect participation vazadrine intermediate all.
The common codon mutation point of katG gene is the 315th undergo mutation (315AGC → ACC, 315AGC → AAC, Herrara L, Valverde A, Saiz P, et al.Molecular characterization ofisoniazid-resistant mycobacterium tuberculosis clinical strains isolated inthe Philippines[J] .Int J Antimicrob Agents, 2004,23 (6) 572-576.) its occurrence frequency is about 67.2%.The anti-vazadrine sudden change of inhA gene mainly occur in its promoter region-15T position deoxyribonucleotide (the inhA gene its promoter region-15C sports T, Basso LA, Zhang R, Musser JM, et al.Mechanisms of isoniazid resistance in Mycobacterium tuberculosis:enzymatic characterization of enoyl reductase mutants identified inisoniazid-resistant clinical isolates[J] .J Infect Dis, 1998,178 (3): 769-775.).
The clinical detection of mycobacterium tuberculosis mainly relies on drug sensitivity test all the time, but traditional drug sensitivity test cycle in that solid medium carries out is long.Susceptibility is low, has had a strong impact on patient's early diagnosis and therapy.
Biological method is that the rapid detection of resistant tuberculosis has been opened wide prospect.In many nucleic acid amplification methods, PCR method is widely used in the detection of clinical tuberculosis most.
Tubercle bacillus genic mutation detection method commonly used at present is as follows:
(1) dna sequencing method (DNA sequencing) directly checks order to the PCR product of drug resistant gene fragment, same dna fragmentation with the standard sensitive strain compares then, analyze position and the distribution of base mutation, but this method is more loaded down with trivial details, and required equipment focuses mostly in some big order-checking companies, is difficult to popularize.
(2) PCR-SSCP method PCR-SSCP, be polymerase chain reaction one single strand conformation polymorphism (polymerasechain reaction-singlestranded conforma-tional polymorphism, PCR-SSCP), should be less than 200bp but this method detects the dna fragmentation length of the variation of single base, and from small segment, can not obtain more complete resistance information, and descend with the increase sensitivity of DNA length.
(3) (restriction fragmant length polymorphism, RFLP) rflp analysis is the integrated application of restriction enzyme, nucleic acid electrophoresis, engram technology, probe hybridization technology in restriction fragment length polymorphism analysis.This method specificity height still can only be analyzed the transgenation of known array specific site.
(4) multiple row probe in detecting method (line probe assay, LiPA) LiPA cost costliness, difficult popularizing.
(5) though biochip technology has obtained remarkable progress, but still there is defective, such as may sneaking into by vicious Nucleotide when the synthesising probing needle.In addition, its complicated operating process, the material requested costliness has all hindered the popularization of this technology.
Therefore, it is significant with early diagnosis tuberculosis and control propagation lungy to set up a kind of detection method pair of suddenling change simply fast.
Summary of the invention
An object of the present invention is to provide a kind of primer special of identifying Mycobacterium tuberculosis drug-resistant.
Primer special provided by the invention, it is made up of an above primer sets, and each described primer sets is at a kind of drug resistant gene, and the target sequence of described each primer sets amplification is all allelotrope fragments that comprise the mutational site;
Each described primer sets comprises Auele Specific Primer and the general primer for the described target sequence of amplification.
Described drug resistant gene is the gene of anti-Rifampin the and/or anti-oxyr gene;
3 ' terminal bases of described Auele Specific Primer is identical or complementary with the base in the mutational site of described allelotrope fragment.
Described primer special is made up of following 3 primer sets:
It is that Auele Specific Primer and 1 general primer 1 of corresponding allelotrope fragment are formed respectively for 511CTG → CCG, 526CAC → GAC, 531TCG → TTG, 533CTG → CCG mutational site of the rpoB gene of NC_000962 759807-763325 position Nucleotide that the primer sets that is used for the amplification gene of anti-the Rifampin is used for amplification GenBank Accession Number by 4;
Described 4 be used for 511CTG → CCG, 526CAC → GAC that amplification GenBank Accession Number is the rpoB gene of NC_000962 759807-763325 position Nucleotide, 531TCG → TTG, 533CTG → CCG mutational site respectively the nucleotide sequence of the Auele Specific Primer of corresponding allelotrope fragment be respectively sequence table sequence 5,4,3 and 2;
The nucleotides sequence of described general primer 1 is classified sequence 1 in the sequence table as;
It is that Auele Specific Primer and 1 general primer 2 of allelotrope fragment of inhA-15T mutational site correspondence of the inhA gene of NC_000962 1674202-1675011 position Nucleotide formed that the primer sets A that is used for the anti-oxyr gene of amplification is used for amplification GenBank Accession Number by 1;
Described is that the nucleotides sequence of Auele Specific Primer of allelotrope fragment of inhA-15T mutational site correspondence of the inhA gene of NC_000962 1674202-1675011 position Nucleotide is classified sequence table sequence 7 as for amplification GenBank Accession Number;
The nucleotides sequence of described general primer 2 is classified sequence 6 in the sequence table as;
Primer sets B for the anti-oxyr gene of amplification is made up of 2 Auele Specific Primer and general primers 3 that are used for the allelotrope fragment of 315AGC → AAC, the 315AGC that amplification GenBank Accession Number is the katG gene of NC_000962 2153889-2156111 position Nucleotide → ACC mutational site difference correspondence;
Described for amplification GenBank Accession Number be NC_000962 2153889-2156111 position Nucleotide the katG gene 315AGC → AAC, 315AGC → ACC mutational site respectively the nucleotide sequence of the Auele Specific Primer of corresponding allelotrope fragment be respectively sequence table sequence 9 and sequence 10;
The nucleotides sequence of described general primer 3 is classified sequence 8 in the sequence table as.
In " 511CTG → CCG, 526CAC → GAC, 531TCG → TTG, 533CTG → CCG " 511,526,531,533 is the amino acid whose position (position of codon) of sudden change, " → " expression sudden change, " base of → front " is the amino acid whose codon that does not suddenly change, and " base of → back " is amino acid whose codon of sudden change.
315 amino acid whose positions (position of codon) for sudden change in " 315AGC → ACC, 315AGC → AAC ", " → " expression sudden change, " base of → front " is the amino acid whose codon that does not suddenly change, and " base of → back " is amino acid whose codon of sudden change.
Among the inhA-15T-the 15T bit representation sports Nucleotide T with the inhA gene at-15 Nucleotide C of its promoter region.
Second purpose of the present invention provides a kind of chemical sproof reagent that detects tubercule bacillus.
Reagent provided by the invention is made up of described primer special and application specific probe;
The number of described application specific probe is identical with the number of described primer sets, the target sequence specific combination of a primer sets amplification in a kind of described application specific probe and the described primer special.
Described resistance is anti-Rifampin and/or anti-vazadrine;
Described application specific probe is fluorescence labeling probe, and the marker of described fluorescence labeling probe is specially fluorescein; The marker of described fluorescence labeling probe is specially fluorescein; Described fluorescein is specially 6-Fluoresceincarboxylic acid (FAM) or chlordene fluorescein (HEX);
Described application specific probe is formed by following 3 kinds:
Application specific probe 1 is the probe with the target sequence specific combination of described primer sets amplification for the amplification gene of anti-the Rifampin, 5 ' end mark FAM of described application specific probe 1, and the nucleotides sequence of described application specific probe 1 is classified the sequence 11 in the sequence table as;
Application specific probe 2 is the probe with the target sequence specific combination of described primer sets A amplification for the anti-oxyr gene of amplification, 5 ' end mark HEX of described application specific probe 2, and the nucleotides sequence of described application specific probe 2 is classified the sequence 12 in the sequence table as;
Application specific probe 3 is the probe with the target sequence specific combination of described primer sets B amplification for the anti-oxyr gene of amplification, 5 ' end mark HEX of described application specific probe 3, and the nucleotides sequence of described application specific probe 3 is classified the sequence 13 in the sequence table as.
The 3rd purpose of the present invention provides a kind of chemical sproof PCR reagent that detects tubercule bacillus.
PCR reagent provided by the invention is by described reagent, dNTP, archaeal dna polymerase, Mg
2+And the PCR damping fluid is formed;
The concentration of described each primer of primer centering in described PCR reagent is 0.2 μ M;
The concentration of described dNTP in described PCR reagent is 200 μ M;
The concentration of described archaeal dna polymerase in described PCR reagent is 0.075U/ μ L;
Described Mg
2+Concentration in described PCR reagent is 200 μ M.
The test kit that contains described reagent or described PCR reagent also is the scope of protection of the invention.
The application in the resistance that detects tubercule bacillus of described primer special or described PCR reagent or described test kit also is the scope of protection of the invention; Described resistance is anti-Rifampin and/or vazadrine.
The 3rd purpose of the present invention provides the chemical sproof method of tubercule bacillus in a kind of auxiliary detection testing sample.
Method provided by the invention comprises the steps: with described primer special or described reagent or described PCR reagent or described test kit testing sample to be carried out the dual TaqmanPCR amplification of real-time fluorescence, if fluorescent PCR instrument FAM passage has amplification fluorescent, the anti-Rifampin of described testing sample then, as if fluorescent PCR instrument HEX passage amplification fluorescent is arranged, then the anti-vazadrine of described testing sample.
The dual TaqmanPCR technology of real-time fluorescence is respectively at the specific sequence designing probe of different tuberculosis drug resistant genes, and press the different fluorescence of not isolabeling of gene resistance kind (Rifampin/vazadrine), by detecting the fluorescent signal of different passages, the differentiation of carrying out the tuberculosis drug resistant gene detects.This technology is different with traditional real-time fluorescence PCR technology, and normally used real-time fluorescence PCR technology is only to use a kind of fluorescently-labeled probe in the PCR reaction system, and the result can only judge the amplification of a certain PCR product by the variation of fluorescent signal.If some locus has a plurality of allelotrope (as HLA-DRB site or thalassemia mutator gene etc.), just very numerous and diverse with this fluorescent method detection, even be difficult to finish.Dual Taqman fluorescent PCR technology then is at each allelotrope implementation sequence Auele Specific Primer, and between target sequence upstream primer and downstream primer the implementation sequence specific probe,
In the described pcr amplification, be template with the genomic dna of testing sample;
The annealing temperature of described pcr amplification is 62 ℃;
Described testing sample is for containing the sputum of tubercule bacillus (Mycobacterium tuberculosis).
Of the present invention experimental results show that, the primer of special use provided by the invention and special-purpose probe, can be by dual Taqman real-time fluorescence PCR technology and sequence specific primers amplification technique (the Sequence specificprimer of efficient and sensible, SSP) combine, detect the resistance of tubercule bacillus in the testing sample within a short period of time.Also provide and detected the chemical sproof method of mycobacterium tuberculosis and test kit in the clinical biological sample, clinical biological sample can be clinical sputum, bronchial perfusate, blood, ascites, cerebrospinal fluid etc.Mycobacterium tuberculosis genomic dna in inspection person's sputum, blood, bronchial perfusate, ascites and the cerebrospinal fluid.The present invention has also utilized dual Taqman real-time fluorescence PCR technology, presses the different fluorescence of not isolabeling of gene resistance kind (Rifampin/vazadrine), and by detecting the fluorescent signal of different passages, the differentiation of carrying out the tuberculosis drug resistant gene detects.Improve the sensitivity and the amplification efficiency that detect so greatly, used method of the present invention and test kit, only needed about 3 hours time from the processing of sample to interpretation genotype detection result.Therefore, method of the present invention and test kit are specially adapted to the rapid detection of clinical samples and the generaI investigation of large sample.
Description of drawings
Fig. 1 is real-time fluorescence quantitative PCR reaction pattern synoptic diagram
Fig. 2 is each target DNA amplified fragments sensitivity experiment result
Fig. 3 is the pattern detection result
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.
Primer and the probe of embodiment 1, detection mycobacterium tuberculosis drug-tolerant gene mutation
1, primer design and synthetic
For special, goal gene in the nucleic acid samples to be checked efficiently increases, from GenBank, access mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) complete genome DNA (BX842578) sequence, at rpoB (NC_000962 759807-763325 position Nucleotide), katG (NC_000962 2153889-2156111 position Nucleotide), inhA (NC_000962 1674202-1675011 position Nucleotide) gene polymorphism sites, according to the general principle of design of primers, adopt Primer Premier5.0 and Oligo6.0 software to design and synthesize used forward and the reverse primer of real-time fluorescence PCR respectively.
Primer sets 1:
RpoB_comF2:5 ' AGCCGATGACCACCCAGGAC 3 ' (sequence 1)
533_R3:5 ' AGACCGCCGAGCCCCG 3 ' (sequence 2)
531_Ri2:5 ' CCGAGCCCCAGCGCCA 3 ' (sequence 3)
526_R3:5 ' CGGCAGTCGGCGCTTGTC 3 ' (sequence 4)
511_W7:5 ' TTCTGGTCCATGAATAGGCTCG 3 ' (sequence 5)
Primer sets 2:
InhA_comR2:5 ' GCGTAACCCCAGTGCGAAAGTTCC 3 ' (sequence 6)
Inh_F12:5 ' GTCACCCCGACAACCTAGCA 3 ' (sequence 7)
Primer sets 3:
KatG_comR1:5 ' GCTCCCACTCGTAGCCGTACAGGAT 3 ' (sequence 8)
KatG (315g-a) F7:5 ' GTAAGGACGCGATCACGAA 3 ' (sequence 9)
KatG (315g-c) F6_2:5 ' AAGGACGCGATCACGAC 3 ' (sequence 10)
2, the synthetic and mark of probe
At rpoB, inhA, the katG gene polymorphism sites has designed and synthesized 3 probes.In order to guarantee that each probe all can successfully match with specific target DNA, must be strict controlled in Tm value and the GC content of probe in the specified range under same temperature.5 ' end mark fluorescent group (FAM/HEX) of probe, 3 ' end mark quenching group (Eclipse).The oligonucleotide probe that uses in the PCR reaction is respectively:
Probe 1:5 ' AGGCGATCACACCGCAGACGTTG 3 ' (sequence 11, flag F AM is at the rpoB gene)
Probe 2:5 ' CCGGAAATCGCAGCCACGTT 3 ' (sequence 12, mark HEX is at the inhA gene)
Probe 3:5 ' CTGTTGTCCCATTTCGTCGGGGTGT 3 ' (sequence 13, mark HEX is at the katG gene)
The condition of embodiment 2, real-time fluorescence PCR is groped
1, the preparation of template
According to NC_000962 759807-763325 position Nucleotide, NC_000962 1674202-1675011 position Nucleotide, NC_000962 2153889-2156111 position Nucleotide, respectively synthetic rpoB gene, inhA gene, katG gene, consistent with NC_000962 759807-763325 position Nucleotide, NC_000962 1674202-1675011 position Nucleotide, NC_000962 2153889-2156111 position Nucleotide respectively through the nucleotide sequence of the rpoB gene of order-checking synthetic, inhA gene, katG gene.
With the rpoB gene of synthetic, inhA gene, katG gene respectively at pGEM-T Easy Vector (Promega company, catalog number (Cat.No.): A1380) connect, obtain wild plasmid rpoB, wild plasmid inhA, wild plasmid katG respectively.
Through checking order:
Wild plasmid rpoB is for to be connected the plasmid that obtains with rpoB (NC_000962 759807-763325 position Nucleotide) gene with pGEM-TEasy Vector.
Wild plasmid inhA is for to be connected the plasmid that obtains with inhA (NC_000962 1674202-1675011 position Nucleotide) gene with pGEM-T Easy Vector.
Wild plasmid katG is for to be connected the plasmid that obtains with katG (NC_000962 2153889-2156111 position Nucleotide) gene with pGEM-T Easy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains rpoB mutational site (533) is template, rpoB_comF2,533_R3 are primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with rpoB (NC_000962 759807-763325 position Nucleotide) for the Nucleotide of this PCR product, and the CTG of the 533rd amino acids (codon) that different is sports CCG, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change 533CTG → CCG site mutation plasmid with pGEM-T Easy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains rpoB mutational site (531), rpoB_comF2,531_R12 are primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with rpoB (NC_000962 759807-763325 position Nucleotide) for the Nucleotide of this PCR product, and the TCG of the 531st amino acids (codon) that different is sports TTG, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change 531TCG → TTG site mutation plasmid with pGEM-T Easy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains rpoB mutational site (526), rpoB_comF2,526_R3 are primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with rpoB (NC_000962 759807-763325 position Nucleotide) for the Nucleotide of this PCR product, and the CAC of the 526th amino acids (codon) that different is sports GAC, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change 526CAC → GAC site mutation plasmid with pGEM-T Easy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains rpoB mutational site (511), rpoB_comF2,511_W7 are primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with rpoB (NC_000962 759807-763325 position Nucleotide) for the Nucleotide of this PCR product, and the CTG of the 511st amino acids (codon) that different is sports CCG, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change 511CTG → CCG site mutation plasmid with pGEM-T Easy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains katG gene 315AGC → AAC sudden change, katG_comR1, katG (g-a) F7 is primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with katG gene (NC_000962 2153889-2156111) for the Nucleotide of this PCR product, and the AGC of the 315th amino acids (codon) that different is sports AAC, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change 315AGC → AAC site mutation plasmid with pGEM-TEasy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains katG gene 315AGC → ACC sudden change, katG_comR1, katG (g-c) F6 is primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with katG gene (NC_000962 2153889-2156111) for the Nucleotide of this PCR product, and the AGC of the 315th amino acids (codon) that different is sports ACC, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change 315AGC → ACC site mutation plasmid with pGEM-TEasy Vector.
The sputum (being provided by the 309 hospital of PLA) that contains the inhA catastrophe point, inhA_comR2, inh_F12 are primer, carry out pcr amplification, obtain the PCR product, send to order-checking, the result compares with inhA gene (NC_000962 1674202-1675011 position Nucleotide) for the Nucleotide of this PCR product, and different is that the-15 Nucleotide C are sported T, and all the other are all identical; This PCR product is connected the plasmid that obtains is sudden change inhA-15T site mutation plasmid with pGEM-T Easy Vector.
2, real-time fluorescence PCR detection sensitivity
The reaction pattern synoptic diagram of real-time fluorescence PCR as shown in Figure 1, wherein, upstream primer is respectively the sequence 1,6,8 in the sequence table, downstream primer is respectively the sequence 2,3,4,5,7,9,10 in the sequence table, the Taqman probe is the sequence 11,12,13 in the sequence table.
The optimum response system of PCR reaction solution is as follows:
In the 20ul reaction system:
0.4ul primer (final concentration of primer is 0.2uM);
0.4ul Mg
2+(Mg
2+Final concentration be 200uM);
(1.6uldNTP the final concentration of dNTP is 200uM);
2ul Buffer (available from Qiagen China, catalog number is 203203),
0.3ul archaeal dna polymerase (final concentration of archaeal dna polymerase is 0.075U/ul);
0.4ul the probe of mark (final concentration of probe is 0.2uM);
Water is supplied volume.
The optimum reaction condition of PCR reaction is as follows: 95 ℃ of 15min (pre-sex change), 95 ℃ of 15s, 62 ℃ of 40s (annealing and extension merge) (40 circulations).
Take out above-mentioned best amplification reaction solution, place on the ice chest, sample is got 2ul, adds in the nucleic acid reaction pipe.Each detection should be established feminine gender and positive control, and yin and yang attribute reference substance add-on is 2ul.Positive control is wild plasmid rpoB, wild plasmid inhA and wild plasmid katG, and negative control is not for adding template.
Adopt above-mentioned system and condition, the template of step 3 preparation is carried out in various degree dilution, carry out real-time fluorescence quantitative PCR, primer is primer sets 1, primer sets 2 and primer sets 3, and probe is probe 1-3.
The result as shown in Figure 2, wherein, A is rpoB gene 511T → C site amplification figure, template is 511CTG → CCG site mutation plasmid; B is rpoB gene 526CAC → GAC site amplification figure, and template is 526CAC → GAC site mutation plasmid; C is rpoB gene 531TCG → TTG site amplification figure, and template is 531TCG → TTG site mutation plasmid; D is rpoB gene 533CTG → CCG site amplification figure, and template is 533CTG → CCG site mutation plasmid; E is inhA gene mutation site 15C → T site amplification figure, and template is inhA 15C → T site mutation plasmid; F is katG gene 315G → C site amplification figure, and template is katG315G → C site mutation plasmid; G is katG gene 315G → A site amplification figure, and template is katG315G → A site mutation plasmid.2,3,4,5 represent that respectively sample content is 10
2, 10
3, 10
4, 10
5Copy/ml, W are wild plasmid rpoB, wild plasmid inhA or wild plasmid katG, the negative contrast of N, and the amplification size is all at desired extent.
In " 511CTG → CCG, 526CAC → GAC, 531TCG → TTG, 533CTG → CCG " 511,526,531,533 is the amino acid whose position (position of codon) of sudden change, " → " expression sudden change, " base of → front " is the amino acid whose codon that does not suddenly change, and " base of → back " is amino acid whose codon of sudden change.
315 amino acid whose positions (position of codon) for sudden change in " 315AGC → ACC, 315AGC → AAC ", " → " expression sudden change, " base of → front " is the amino acid whose codon that does not suddenly change, and " base of → back " is amino acid whose codon of sudden change.
Among the inhA-15T-the 15T bit representation with the inhA gene its promoter region-15C sports T.
As can be seen from the figure, this method can make the sample DNA of low copy number obtain good expanding effect, and it detects lower limit and reaches 10
2Copy/ml reaction system, the system of optimization and PCR condition can improve specificity and the high efficiency of nucleic acid amplification, thereby improve detection sensitivity greatly, are conducive to prevent and reduce pollution.
The resistance of mycobacterium tuberculosis in embodiment 3, the test sample
1, different biological sample real-time fluorescence quantitative PCRs detect
Adopt embodiment 1 to obtain 3 primer sets and 3 kinds of probes, with embodiment 2 optimized conditions and system, 155 parts of clinical positive samples (sputum, the known mycobacterium tuberculosis that contains is provided by the 309 hospital of PLA) that are coated with are carried out the real-time fluorescence quantitative PCR detection.
For real-time fluorescence PCR instrument FAM passage amplification fluorescent is arranged if detect judging criterion, then be judged as a certain site mutation of mycobacterium tuberculosis rpoB gene, there is anti-rifampicin resistance in this clinical sample; If real-time fluorescence PCR instrument HEX passage has amplification fluorescent, then be judged as mycobacterium tuberculosis inhA gene or there is sudden change in a certain site of katG gene, there is anti-vazadrine resistance in this clinical sample.
The result is anti-Rifampin strain 1 example, anti-vazadrine strain 13 examples, and anti-Rifampin and vazadrine strain 33 examples, (annotate: the 106 examples mutator gene that does not have anti-Rifampin and anti-vazadrine), 1 example does not detect in the wild-type strain.Above-mentioned detected result and clinical smear results coincidence rate reach 99%.
The result of part real-time fluorescence PCR as shown in Figure 3, as can be seen from the figure, the FAM passage has the amplified fluorescence curve, and the HEX passage does not have the amplified fluorescence curve, judges that this sample is the sample of anti-the Rifampin.
In order to prove the accuracy that obtains 3 primer sets and 3 kinds of probe in detecting with embodiment 1, the PCR product of 155 routine clinical samples is wherein carried out sequencing, and the result shows that the PCR product sequencing result that uses embodiment 1 to obtain 3 primer sets and 3 kinds of probe in detecting 1 routine anti-Rifampin strains is as follows: the PCR product that is numbered 1 anti-Rifampin strain has the Nucleotide that rpoB gene (NC_000962 759807-763325 position Nucleotide) 531TCG → TTG obtains;
The PCR product sequencing result of 13 routine anti-vazadrine strains is as follows: the PCR product that is numbered 2 anti-Rifampin strain has the inhA gene Nucleotide that (NC_000962 1674202-1675011 position Nucleotide)-15C → T obtains; The PCR product that is numbered the anti-Rifampin strain of 3-14 has the Nucleotide that katG gene (NC_000962 2153889-2156111 position Nucleotide) 315G → C obtains;
The PCR product sequencing result of 33 routine anti-Rifampins and vazadrine strain is as follows: the PCR product that is numbered the anti-Rifampin strain of 15-20 namely has the inhA gene Nucleotide that (NC_000962 1674202-1675011 position Nucleotide)-15C → T obtains, and has the Nucleotide that rpoB gene (NC_000962 759807-763325 position Nucleotide) 531TCG → TTG obtains again; The PCR product that is numbered 21 anti-Rifampin strain namely has the Nucleotide that katG gene (NC_000962 2153889-2156111 position Nucleotide) 315G → C obtains, and has the Nucleotide that rpoB gene (NC_000962 759807-763325 position Nucleotide) 511T → C obtains again; The PCR product that is numbered the anti-Rifampin strain of 22-23 namely has the Nucleotide that katG gene (NC_000962 2153889-2156111 position Nucleotide) 315G → A obtains, and has the Nucleotide that rpoB gene (NC_000962 759807-763325 position Nucleotide) 531TCG → TTG obtains again; The PCR product that is numbered the anti-Rifampin strain of 24-47 namely has the Nucleotide that katG gene (NC_000962 2153889-2156111 position Nucleotide) 315G → C obtains, and has the Nucleotide that rpoB gene (NC_000962 759807-763325 position Nucleotide) 531TCG → TTG obtains again.
From above-mentioned sequencing result as can be seen with use embodiment 1 to obtain 3 primer sets and 3 kinds of probe in detecting results are identical substantially, prove that method of the present invention has very high accuracy.
2,13 kinds of common non-tuberculous mycobacteria real-time fluorescence quantitative PCR detected results are judged
Adopt embodiment 1 to obtain 3 primer sets and 3 kinds of probes, with embodiment 2 optimized conditions and system, the 13 kinds of common non-tuberculous mycobacterias (identifying that by Chinese pharmaceutical biological product institute provides) that concentration are 1mg/ml carry out the specificity test,
13 kinds of common non-tuberculous mycobacterias are as follows:
Mycobacterium avium (Mycobacterium avium subsp.avium Chester) ATCC 25291; Mycobacterium intracellulare (Mycobacterium intracellulare (Cuttino and McCabe) Runyon) ATCC13950; Mycobacterium scrofulaceum (Mycobacterium scrofulaceum Prissick and Masson) ATCC 19981; Mycobacterium marinum (Mycobacterium marinum Aronson) ATCC 927; Ape and monkey mycobacterium (Mycobacterium simiae Karassova et al.) ATCC 25275; Survey Sa Si mycobacterium (Mycobacterium kansasii Hauduroy) ATCC 12478; Mycobacterium fortuitum (Mycobacteriumfortuitum subsp.fortuitum da Costa Cruz) ATCC 6841; M. smegmatics (Mycobacterium smegmatis (Trevisan) Lehmann and Neumann) ATCC 19420; Cow mycobacterium (Mycobacterium bovis Karlson and Lessel) ATCC 19210; Pale yellow mycobacterium (Mycobacterium gilvum Stanford and Gunthorpe) ATCC 43909; Mycobacterium phlei (Mycobacterium phlei Lehmann and Neumann) ATCC 11758; Mycobacterium chelonei abscess subspecies (Mycobacterium abscessus) ATCC 19977.
The result is not for false positive all occurring.
Claims (7)
1. primer special of identifying Mycobacterium tuberculosis drug-resistant, it is made up of an above primer sets, and each described primer sets is at a kind of drug resistant gene, and the target sequence of each described primer sets amplification is all allelotrope fragments that comprise the mutational site;
Each described primer sets comprises Auele Specific Primer and the general primer for the described target sequence of amplification;
Described drug resistant gene is the gene of anti-Rifampin the and anti-oxyr gene;
Described primer special is made up of following 3 primer sets:
It is that Auele Specific Primer and 1 general primer 1 of corresponding allelotrope fragment are formed respectively for 511CTG → CCG, 526CAC → GAC, 531TCG → TTG, 533CTG → CCG mutational site of the rpoB gene of NC_000962 759807-763325 position Nucleotide that the primer sets that is used for the amplification gene of anti-the Rifampin is used for amplification GenBank Accession Number by 4;
Described 4 be used for 511CTG → CCG, 526CAC → GAC that amplification GenBank Accession Number is the rpoB gene of NC_000962 759807-763325 position Nucleotide, 531TCG → TTG, 533CTG → CCG mutational site respectively the nucleotide sequence of the Auele Specific Primer of corresponding allelotrope fragment be respectively sequence table sequence 5,4,3 and 2;
The nucleotides sequence of described general primer 1 is classified sequence 1 in the sequence table as;
It is that Auele Specific Primer and 1 general primer 2 of allelotrope fragment of inhA-15T mutational site correspondence of the inhA gene of NC_000962 1674202-1675011 position Nucleotide formed that the primer sets A that is used for the anti-oxyr gene of amplification is used for amplification GenBank Accession Number by 1;
Described is that the nucleotides sequence of Auele Specific Primer of allelotrope fragment of inhA-15T mutational site correspondence of the inhA gene of NC_000962 1674202-1675011 position Nucleotide is classified sequence table sequence 7 as for amplification GenBank Accession Number;
The nucleotides sequence of described general primer 2 is classified sequence 6 in the sequence table as;
Primer sets B for the anti-oxyr gene of amplification is made up of 2 Auele Specific Primer and general primers 3 that are used for the allelotrope fragment of 315AGC → AAC, the 315AGC that amplification GenBank Accession Number is the katG gene of NC_000962 2153889-2156111 position Nucleotide → ACC mutational site difference correspondence;
Described for amplification GenBank Accession Number be NC_000962 2153889-2156111 position Nucleotide the katG gene 315AGC → AAC, 315AGC → ACC mutational site respectively the nucleotide sequence of the Auele Specific Primer of corresponding allelotrope fragment be respectively sequence table sequence 9 and sequence 10;
The nucleotides sequence of described general primer 3 is classified sequence 8 in the sequence table as.
2. a chemical sproof reagent that detects tubercule bacillus is made up of the described primer special of claim 1 and application specific probe;
The number of described application specific probe is identical with the number of described primer sets, the target sequence specific combination of a primer sets amplification in a kind of described application specific probe and the described primer special of claim 1;
Described resistance is anti-Rifampin and anti-vazadrine.
3. reagent according to claim 2 is characterized in that:
Described application specific probe is fluorescence labeling probe, and the marker of described fluorescence labeling probe is fluorescein.
4. according to claim 2 or 3 described reagent, it is characterized in that:
Described fluorescein is 6-Fluoresceincarboxylic acid or chlordene fluorescein.
5. reagent according to claim 2 is characterized in that:
Described application specific probe is formed by following 3 kinds:
Application specific probe 1 is the probe with the target sequence specific combination of described primer sets amplification for the amplification gene of anti-the Rifampin, 5 ' end mark 6-Fluoresceincarboxylic acid of described application specific probe 1, and the nucleotides sequence of described application specific probe 1 is classified the sequence 11 in the sequence table as;
Application specific probe 2 is the probe with the target sequence specific combination of described primer sets A amplification for the anti-oxyr gene of amplification, 5 ' end mark chlordene fluorescein of described application specific probe 2, and the nucleotides sequence of described application specific probe 2 is classified the sequence 12 in the sequence table as;
Application specific probe 3 is the probe with the target sequence specific combination of described primer sets B amplification for the anti-oxyr gene of amplification, 5 ' end mark chlordene fluorescein of described application specific probe 3, and the nucleotides sequence of described application specific probe 3 is classified the sequence 13 in the sequence table as.
6. chemical sproof PCR reagent that detects tubercule bacillus is by arbitrary described reagent, dNTP, archaeal dna polymerase, Mg among the claim 2-5
2+And the PCR damping fluid is formed;
The concentration of described each primer of primer centering in described PCR reagent is 0.2 μ M;
The concentration of described dNTP in described PCR reagent is 200 μ M;
The concentration of described archaeal dna polymerase in described PCR reagent is 0.075U/ μ L;
Described Mg
2+Concentration in described PCR reagent is 200 μ M.
7. the test kit that contains arbitrary described reagent among the claim 2-5 or the described PCR reagent of claim 6.
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| CN103555828B (en) * | 2013-10-10 | 2016-04-06 | 上海市肺科医院 | Mycobacterium quick medicine-sensitive detection method and detection target |
| CN104278082A (en) * | 2014-05-15 | 2015-01-14 | 中国疾病预防控制中心传染病预防控制所 | Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof |
| CA2955771C (en) | 2014-07-24 | 2022-08-30 | Abbott Molecular Inc. | Compositions and methods for the detection and analysis of mycobacterium tuberculosis |
| US10087490B2 (en) * | 2014-12-18 | 2018-10-02 | Roche Molecular Systems, Inc. | Compositions and methods for detection of drug resistant Mycobacterium tuberculosis |
| EP3322483A4 (en) | 2015-07-14 | 2019-01-02 | Abbott Molecular Inc. | Compositions and methods for identifying drug resistant tuberculosis |
| CN106916877A (en) * | 2015-12-24 | 2017-07-04 | 广州好芝生物科技有限公司 | A kind of mycobacterium tuberculosis rifampin-resistance mutation detection kit |
| CN109112226B (en) * | 2018-09-12 | 2022-03-15 | 江苏宏微特斯医药科技有限公司 | Method and kit for simultaneously detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation |
| CN111517947B (en) * | 2020-03-18 | 2022-05-24 | 西北师范大学 | Bilateral column [5] arene sensor molecule based on functionalization and synthesis and application thereof |
| CN113528541A (en) * | 2021-07-15 | 2021-10-22 | 清华大学深圳国际研究生院 | Novel rpoB resistance gene of rifampicin and application thereof |
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