CN101845503B - Kit for detecting drug resistant mycobacterium tuberculosis (MDR-TB) - Google Patents
Kit for detecting drug resistant mycobacterium tuberculosis (MDR-TB) Download PDFInfo
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Abstract
The invention relates to a method adopting double-label probe detection and melting curve analysis for diagnosing the infection of multi-drug resistant mycobacterium tuberculosis and a kit which utilizes the method to detect multiple gene mutations related to drug resistant tuberculosis at the same time, and the invention belongs to the life science and biological technical field. The kit of the invention contains a primer designed for multiple gene mutations related to drug resistant, a double-label oligonucleotide probe capable of detecting multiple common gene mutation sites related to drug resistant tuberculosis and a DNA polymerases with heat stability and without 5' nuclease activity, and the kit can be used to detect at least 16 common gene mutation sites related to drug resistant tuberculosis under proper PCR reaction conditions. The detection method and kit of the invention can be used in the early diagnosis of multi-drug resistant tuberculosis and overcome the problems of the existing technology that the detection time is long, a great deal of manpower and large material resources are needed, the detection cost is high, etc.
Description
Technical field
The present invention relates to a kind of microorganism detection medical science vitro diagnostic techniques, particularly relate to the detection method that a kind of substance of medicines-resistant branched tubercle bacillus infects, and utilize described method to detect simultaneously multiple and the test kit Drug Resistance for Tuberculosis associated gene mutation.
Background technology
Tuberculosis remains serious public health problem, especially in developing country.Having Mycobacterium tuberculosis (mycobacterium tuberculosis, MTB) to infect according to 1/3 of WHO statistics world population, is the second largest killer in the whole world infectious diseases.And the resistance of Mycobacterium tuberculosis presents significant rising tendency in recent years.The whole world approximately has the patient after 4.3% new cases and the treatment that multidrug resistant occurs, and the multidrug resistant rate of part hotspot is up to more than 10%.China is one of heavier country of tuberculosis epidemic situation, occupies second in the high burden of 22 tuberculosis country of the whole world.The Drug Resistance for Tuberculosis patient sum of China, India and three countries of Russia accounts for 62% of the whole world.Recent Chinese monitoring of infectious disease result shows, the tuberculosis patient number occupies the first place of various transmissible diseases always, and the whole nation approximately has 5.5 hundred million people to infect the tuberculosis germ, and approximately 4,500,000 people suffer from tuberculosis, 2,000,000 people are that open tuberculosis is sick, and annual number because of tuberculosis death is up to 130,000.Retrospective review is the result show, total resistant rate of Chinese MTB is 27.8%, and anti-multiple medicines rate is 10.7%.This has become global common problem, and has caused the generally attention of society.
Yet the foundation of quick diagnosis lungy and optimized individual treatment plan remains one of significant challenge in the Control.Because the mycobacterium tuberculosis poor growth, the resistance measuring method of nowadays widespread use all is to be based upon on the basis of bacterial strain, although used new liquid nutrient medium, but still consuming time longer, postponed the time that obtains the bacterial strain responsive type.Therefore, develop a kind of anti-multiple medicines detection method lungy of quick and precisely diagnosing, for the propagation of early discovery, blocking-up resistant tuberculosis, the standard direction of medication usage, the curative ratio that improves resistant tuberculosis has very important significance.Reported in recent years the molecular biology method of many detection Drug Resistance for Tuberculosis sudden changes, yet these methods often need a large amount of manpowers and the substratum that grows fine.Recently, successfully set up the method for some real-time PCR Rapid detection of Mycobacterium tuberculosis clinical separation strain medicament-resistant mutations.But these methods often detect seldom several mutator gene types relevant with Drug Resistance for Tuberculosis only simultaneously, reach clinically may occur undetected.
Polymerase chain reaction (polymerase chain reaction, PCR) technological invention is nearly 20 years so far, technology has obtained continuous development in the meantime, real-time fluorescence quantitative PCR (the Real-time quantitative PCR) technology that occurs has in recent years realized the leap of PCR from qualitative to quantitative, it with its high specificity, highly sensitive, good reproducibility, quantitatively accurately, the advantages such as fast, the totally-enclosed reaction of speed become the important tool in the molecular biology research.The employed fluorescence chemical method of real-time PCR (also claiming Q-PCR) mainly contains five kinds at present, be respectively: DNA is in conjunction with dyeing, hydrolysis probes (TaqMan probe), molecular beacon (molecular beacon), fluorescent dye primer, hybridization probe.They can be divided into again the special and non-specific detection two large classes of extension increasing sequence.
The basis of the non-specific detection method of extension increasing sequence is the fluorescence molecule of DNA combination, such as fluorescence dyes such as SYBR GreenI.Real-time PCR development is exactly to use the simplest this method in early days, in the PCR reaction system, adds excessive SYBR Green I fluorescence dye, after SYBR Green I fluorescence dye mixes the dna double chain specifically, and the emitting fluorescence signal.The advantage of fluorescence dye is that it can monitor the amplification of any dsDNA sequence, does not need the design of probe, makes detection method become easy, has also reduced the cost that detects simultaneously.Yet just because of fluorescence dye can and any dsDNA combination, so it also can with non-specific dsDNA (such as primer dimer) combination, make easily generation false positive signal of experiment.
The extension increasing sequence method for detecting specificity is to utilize the gene specific oligonucleotide probe of mark fluorescent dyestuff to detect product in the PCR reaction, and it can be divided into direct method and indirect method again.Round-about way is exactly the strategy that utilizes hydrolysis probes.The most widely used TaqMan system has used this principle exactly in real-time PCR at present.When adding pair of primers, add a specific fluorescent probe during pcr amplification, this probe is an oligonucleotide, two ends are mark report fluorophor and a cancellation fluorophor respectively, so probe can't detect the fluorescence that this probe 5 ' end fluorophor sends when complete.But in pcr amplification, during low-temperature annealing, primer is combined with template with probe simultaneously after the template sex change in the liquation.Under the mediation of primer, extend to forward the probe junction along template, (this activity is double-stranded specific to 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme, free single-stranded probe is unaffected) hold the fluorophor that connects to cut down from probe probe 5 ', be free in the reaction system, thereby the shielding that breaks away from 3 ' end fluorescent quenching group, and send fluorescent signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form Complete Synchronization.
The probe that direct method refers to mark fluorescent is combined the rear fluorescence that namely directly produces with amplified production, molecular beacon (molecular beacon) just belongs to this class.It is a kind of neck ring double-tagging oligonucleotide probe that is hairpin structure in essence, when probe molecule is hairpin structure, is marked at upper the approaching of fluorophor distance at its two ends, so that the generate energy trans effect, and fluorescence does not occur.When complementary sequence occurs, probe and DNA hybridization, probe is transformed into the structure of an opening, be linear, the report fluorophor spatially produces enough separating each other with the cancellation fluorophor, and reporter group has broken away from the impact of quenching group, thereby produces the fluorescence that can be detected.Fluorescent dye primer is a kind of associating molecular probe system from the conception deriving of molecular beacon, and it directly combines the sequence of the hairpin structure of fluorophor mark with the PCR primer, thereby the fluorescent mark group is directly mixed in the pcr amplification product.Mainly contain at present two kinds: sunrise primer (sunrise primes) and scorpion primer (scorpion primes).Hybridization probe (hybridization probe) uses two special probes, and 3 ' end of the probe of its middle and upper reaches is marked with donor fluorescein (donor), and 5 ' end of the probe in downstream is marked with acceptor fluorescence element (acceptor).The template annealing stage in PCR, two probes are hybridized with amplified production simultaneously, and form the form of end to end combination, make donor and acceptor fluorescence element distance very approaching, both produce FRET (fluorescence resonance energy transfer) (FRET, this effect is opposite with the mode of said hydrolyzed probe) so that the acceptor fluorescence group sends fluorescence; When two probes are in unbound state, produce without fluorescence.Owing to having used two probes in the reaction, therefore increased the specificity of method, but also greatly improved testing cost simultaneously.Also can utilize in addition fluorescently-labeled oligonucleotide probe melting curve (melting curve) that the sequence of being combined with oligonucleotide probe is analyzed, therefrom obtain Useful Information.
Summary of the invention
The purpose of this invention is to provide the detection method that a kind of substance of medicines-resistant branched tubercle bacillus infects, and utilize described method to detect simultaneously multiple and the test kit Drug Resistance for Tuberculosis associated gene mutation.
For solving the problems of the technologies described above, the present invention takes following technical scheme:
At first, the present invention relates to a kind of method that detects Drug-Resistant Mycobacterium tuberculosis, comprising:
Amplification gene group zone in an amplified reaction, wherein, the sudden change in the described genome area has caused the resistance of mycobacterium tuberculosis.Amplification system in the amplified reaction comprises the oligonucleotide probe of at least one mark, at least one pair of amplimer (forward primer and reverse primer) and a heat-staple archaeal dna polymerase.
Produce one in the melting curve analysis that the oligonucleotide probe of mark and the hybridization of the genome area of amplification form and melt spectrum.
Described oligonucleotide probe is the double-tagging probe, and it comprises a report mark and a quencher mark.
When the double-tagging oligonucleotide probe is in not single stranded conformational with the genome area hybridization of amplification, the fluorescence that the quencher mark can quencher report mark sends; When the genome area of double-tagging oligonucleotide probe and amplification is hybridized, form duplex structure, the fluorescence of report mark can not be by quencher, fluorescence intensity when at this moment, the fluorescence intensity of report mark will not be in the strand state with the genome area hybridization of amplification far above this double-tagging oligonucleotide probe.
Described amplification is asymmetric amplification, and the concentration of primer is unbalanced in the amplification, and namely the concentration of a primer is higher than an other concentration of matching with it primer in the amplification.
Preferred described archaeal dna polymerase is the archaeal dna polymerase with 5 ' nuclease.
Described genome area is selected from one or more of following gene, comprising putting down relevant RpoB gene with Nai Lifu (RIF), the KatG gene relevant with anti-vazadrine (INH), mabA (fabG1)-inhA promotor and oxyR-ahpC intergenic region, the embB gene relevant with anti-Tibutol (EMB), the pncA gene relevant with anti-pyrazinoic acid amide (PZA), the rpsL relevant with anti-Streptomycin sulphate (STR) and rr s gene, the gyrA gene relevant with anti-levofloxacin; Sudden change in the described genome area comprises, 511,513,515,516,518,519,522,526,531 and 533 sites of rpoB gene, 306 sites of embB gene, 315 sites of katG gene,-15 and-8 sites of inhA gene, 18 in ahpC gene-6 ,-9 ,-10 and-12 sites etc. or multi-mutant site more.
The quencher mark can be non-quenching of fluorescence group, and it is marked at 3 ' end of oligonucleotide probe, and described report mark can be fluorochrome label, and it can be marked on the nucleotide residue of oligonucleotide probe inside.
Described report mark can also be marked at 5 ' end of oligonucleotide probe.
In the melting curve analysis of labeled oligonucleotide, produce the step that melts spectrum and also comprise when probe separates with target nucleic acid sequence, determine that the amplified production fluorescence intensity changes negative first order derivative (df/dT) peaked step.
Target nucleic acid sequence and the hybridization zone of probe will comprise a sudden change relevant with Drug Resistance for Tuberculosis at least.Wherein, probe and wild-type sequence coupling, perhaps probe and mutant sequences match.
Described amplification is Multiple detection reaction, has at least two probes with different genes group area hybridization in the reaction.
Every of described at least two probes can be all and the wild-type sequence coupling, also can every all with the mutant sequences match, can also be wherein one with the wild-type sequence coupling, other one with the mutant sequences match.
Described two probes can comprise the same or analogous dye marker that can detect in the single signal sense channel, perhaps also can be included in the different pigment marks that detect in the unlike signal sense channel.The present invention can also be designed to, the base mutation that the PCR reaction detection is all, and the probe with different dyes mark can be placed in the PCR reaction, adopts multiplex PCR, and the probe of different dyes mark can detect in different sense channels simultaneously.
Its middle probe can comprise not the Nucleotide with the extension increasing sequence complementation, this probe is hybridized the formation that has not stoped probe self hairpin structure with the Nucleotide of extension increasing sequence complementation that comprises in the above-mentioned probe with the 81bp nucleus of the RpoB gene that comprises 9bp palindrome sequence.
This method also comprises take the wild-type sample as contrast, clinical sample is produced the analysis that melts spectrum.If the change of at least 0.2 ℃ of appearance is compared in the position that melts peak in the spectrum with wild-type, this just indicates the existence that suddenlys change in the sample.
This method can in a reaction tubes, increase two or more genome area.
Described primer is selected from SEQ ID NO:1 in the sequence table of this specification sheets～SEQ ID NO:16.
Described probe is selected from SEQ ID NO:17 in the sequence table of this specification sheets～SEQ ID NO:36.
Secondly, the invention still further relates to a kind of probe that detects Drug-Resistant Mycobacterium tuberculosis:
Wherein, a probe is comprised of 25～50 Nucleotide, and under the condition of hybridization, it can be hybridized with the nucleus of mycobacterium tuberculosis RpoB gene 81bp.Above-mentioned probe comprises that one is marked at 3 ' terminal quencher mark and one and is marked on the inner nucleotide residue or 5 ' terminal report mark.Described probe hybridization is in the zone that comprises RpoB gene 526,531 and 533 codons, and probe contains the Nucleotide not complementary with extension increasing sequence; Described probe is hybridized the formation that has not stoped probe self hairpin structure with the Nucleotide of extension increasing sequence complementation that comprises in the above-mentioned probe with the 81bp nucleus of the RpoB gene that comprises 9bp palindrome sequence.Described probe is selected from the SEQ ID NO:17 in the sequence table, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:27.
Again, the present invention also provides a test kit that detects Drug-Resistant Mycobacterium tuberculosis from sample, comprising:
The oligonucleotide probe of at least one pair of amplimer and at least one mark.This oligonucleotide probe comprises a report mark and a quencher mark.When the oligonucleotide probe of mark is in not single stranded conformational with target sequence hybridization, the fluorescence that the quencher mark can quencher report mark sends;
When the hybridization of oligonucleotide probe and target sequence, form duplex structure, the fluorescence of report mark can not be by quencher, at this moment, the fluorescence intensity the when fluorescence intensity of report mark will not be in the strand state with target sequence hybridization far above this oligonucleotide probe;
Wherein, the quencher mark is non-quenching of fluorescence group, and it is marked at 3 ' end of probe.
Wherein, the report mark is fluorophor, and it is marked on the nucleotide residue of the inside of oligonucleotide probe or is marked at 5 ' end of probe.
Also comprise heat-staple and do not have the archaeal dna polymerase nucleic acid of 5 ' nuclease and required other each component commonly used of sequence amplification.
Wherein, described primer is selected from SEQ ID NO:1 in the sequence table～SEQ ID NO:16.
Wherein, described probe is selected from SEQ ID NO:17 in the sequence table～SEQ ID NO:36.
" oligonucleotide " refers to natural or through the monomer modified or the linear oligomer of polymkeric substance, and it comprises deoxynucleoside, ribonucleoside, protein nucleic acid nucleosides and by the interaction of base pairing and the ability of herbicide-tolerant polynucleotide specific combination.
" fluorophor " refers to absorb luminous energy under a definite excitation wavelength, the part of emission luminous energy under another different wave length of determining.For example fluorescent mark comprises, also be not limited only to this: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyestuff (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, and BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, and BODIPY 581/591, and BODIPY 630/650, BODIPY 650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, CascadeYellow, Cyanine dyestuff (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4 ', 5 '-Dichloro-2 ', 7 '-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green, 2 ', 4 ', 5 ', 7 '-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethyl rhodamine (TAMRA), Texas Red and Texas Red-X.
Distance is enough closely the time between described " quencher group " and the fluorescent mark, the fluorescently-labeled energy that can absorb or dissipate and excite.A quencher group can be the Quenching of fluorescence group, also can be non-Quenching of fluorescence group.If fluorophor listed above is contiguous with the another one fluorophor, also can play the part of the role of a quencher group, FRET quencher or contact quencher occur this moment.Preferably select not send the non-quenching of fluorescence group of any visible light.Non-quenching of fluorescence mark comprises as follows, also be not limited only to this: DABCYL (4-(4 '-dimethylaminophenylazo) benzoic acid) succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl ester (QSY-7) and 4 ', 5 '-dinitrofluorescein carboxylic acid, succinirnidyl ester (QSY-33), quencherl, or " Black hole quenchers " (BHQ-1, BHQ-2 and BHQ-3), Iowa BlackFQ, Deep Dark Quencher, Eclipse Dark Quencher nucleotide analog, Nucleotide G residue, nanoparticle and golden particulate.
For example, oligonucleotide probe a 5 ' end mark FAM, 3 ' end mark BHQ1, when probe and target nucleic acid sequence hybridization, the FAM fluorescence intensity was 1.5 times when the fluorescence intensity of FAM surpassed probe strand state at least.
For example, oligonucleotide probe a 5 ' end mark Texas Red, 3 ' end mark BHQ2, when probe and target nucleic acid sequence hybridization, the TexasRed fluorescence intensity was 3 times when the fluorescence intensity of Texas Red surpassed probe strand state at least.
For example, mark FAM on the oligonucleotide probe inner core thuja acid, 3 ' end mark BHQ1, when probe and target nucleic acid sequence hybridization, the FAM fluorescence intensity was 1 times when the fluorescence intensity of FAM surpassed probe strand state at least.
In the melting curve analysis of labeled oligonucleotide, produce the step that melts spectrum and also comprise, when probe separates with target nucleic acid sequence, determine that the amplified production fluorescence intensity changes negative first order derivative (df/dT) peaked step.
" melt and compose " collection that refers to widow's (or many) Nucleotide and its complementary sequence observed value, what it was represented is few (or many) Nucleotide is become strand by two strands molecular conversion (perhaps inverse process).The transformation that nucleic acid becomes strand by two strands is described as " melting " of nucleic acid molecule usually in academic term, this transformation also can be described as being " sex change " or " dissociating " of nucleic acid.Therefore, the spectrum of the melting among the present invention also can be called " solution goes against accepted conventions ", " sex change spectrum ", " melting curve ", " dissociation curve ", " hybridization/solution goes against accepted conventions " etc.
Target nucleic acid sequence and probe hybridization zone will comprise at least sudden change Nucleotide, single nucleotide polymorphism (SNP), a disappearance or insert, or comprise two even the Nucleotide that more suddenlys change, single nucleotide polymorphism (SNP), disappearance or insertion.Probe and target nucleic acid sequence hybridization zone can comprise two or more medicament-resistant mutation Nucleotide.
The oligonucleotide probe of described mark and heat-staple archaeal dna polymerase are used in conjunction with, and what preferably use is the archaeal dna polymerase with 5 ' nuclease.
After finishing the target nucleic acid sequence amplification, in the determination and analysis of nucleotide sequence, also can use the probe among the present invention; Perhaps also can be used in the independent end point determination test of target amplification.The oligonucleotide probe of double-tagging usually hybridization amplified target sequence the primer between the position.
Detect in real time in the experimental system of fluorescent signal in each circulation, can during increasing, analyze.Certainly, the melting curve analysis after also can finishing by amplification is studied target nucleic acid sequence.Probe can be fluorescently-labeled, and is also can right and wrong fluorescently-labeled, the biological example element.
Rifampin and vazadrine are first-line drugs crucial in the tuberculotherapy scheme, also are the antitubercular agents that resistance the most easily occurs.Studies show that, 97% rifampin-resistant is because due to the rpoB transgenation, sudden change mainly concentrates on the 81bp zone of one section high conservative.The Isoniazid-resistant mainly ahpC transgenation with the katG gene of coding Catalase-peroxidase, the inhA gene of coding nicotinamide adenine dinucleotide reduced dependency enoyl-carrier proteins reductase enzyme and the alkyl peroxide enzyme of encoding is relevant.The embB306 site mutation is the genetic marker relevant with the anti-multiple medicines of tuberculosis of a high specific.Also have in addition the sudden change of some other gene also relevant with Drug Resistance for Tuberculosis, to detect frequency lower but it is clinical.The clinical diagnosis needs are a kind of simply, quick, the method for good reproducibility, can detect a series of transgenation relevant with Drug Resistance for Tuberculosis simultaneously.The Real-time round pcr is in conjunction with the advantages such as specificity of the susceptibility of PCR and DNA hybridization, has simple to operate, result and judges the characteristics such as directly perceived and pollution-free, has been widely used in the detection of multiple-microorganism pathogenic agent DNA in clinical diagnosis.
The present invention be more particularly directed to a kind of substance or multiple asymmetric PCR and fluorescent hybridization probe technique utilized, in a PCR reaction tubes, detect simultaneously fast, abreast a plurality of and the diagnostic method Drug Resistance for Tuberculosis associated gene mutation, and utilize described method to detect simultaneously a plurality of and the test kit Drug Resistance for Tuberculosis associated gene mutation.
General PCR only adopts the primer of a pair of identical concentration, produces the nucleic acid fragment of a two strands by pcr amplification.The present invention then adopts substance or multiple asymmetric PCR method (Multiplex Asymmetric PCR), in same PCR reaction system, add a pair of or primer more than two pairs, the upstream primer of every pair of primer is different with downstream primer concentration, and each increases for special separately target sequence to primer.In the PCR reaction system, comprise also that the oligonucleotide probe of at least one double-tagging and one are heat-staple and do not have the archaeal dna polymerase of 5 ' nuclease.In initial 10～15 circulations of PCR reaction, its amplified production mainly is double-stranded DNA, but after restricted primer (lower concentration primer) ran out of, the PCR of non-limiting primer (high density primer) guiding will produce a large amount of single stranded DNAs.By analyzing more special fluorescent mark oligonucleotide probe with strand PCR product complementary with it (wild-type) and existing the strand PCR product (mutant) of sudden change in conjunction with the variation of the melting curve Tm value that produces, detect sample DNA and whether have the transgenation relevant with Drug Resistance for Tuberculosis.
Method steps of the present invention comprises:
Design Auele Specific Primer a pair of or Drug Resistance for Tuberculosis genes involved more than two pairs, and contain whole mutational sites to be detected in the target sequence amplified production of guaranteeing to produce.
The double-tagging oligonucleotide probe of design more than one or two.Target nucleic acid sequence and probe hybridization zone will comprise a sudden change Nucleotide at least, or comprise two even the Nucleotide that more suddenlys change.And the mutational site of guaranteeing probe and wild-type or mutagenicity target nucleic acid sequence is complementary, and the sequence beyond in the mutational site can be complete complementary, also can contain not complementary base, such as disappearance, inserts or unpaired.Usually the length of probe is no more than 35 Nucleotide.When the length of probe during less than 25 Nucleotide, 5 ' end mark fluorescence report group of probe, the non-quenching of fluorescence group of 3 ' end mark.When probe length during greater than 25 Nucleotide, preferably the fluorescence report group is marked on the nucleotide residue of probe interior, non-quenching of fluorescence group is marked on 3 ' end of probe.Can the interval between report mark and the quencher mark less than 20 Nucleotide, perhaps less than 18 Nucleotide, perhaps less than 15 Nucleotide, perhaps less than 14 Nucleotide, but preferably be greater than 8 Nucleotide.The fluorescence report group that is used for label probe can be one or more of FAM, TET, JOE, ViC, HEX, ROX, TAMPA, CY3, CY3.5, CY5, CY5.5, OregonGreenTM, CALRedTM, Red640, Texas Red, also is not limited only to this.The non-quenching of fluorescence group that is used for label probe can be DABCYL (4-(4 '-dimethylaminophenylazo) benzoic acid) succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl ester (QSY-7) and 4 ', 5 '-dinitrofluorescein carboxylic acid, succinirnidyl ester (QSY-33), quencherl, or " Black hole quenchers " (BHQ-1, BHQ-2 and BHQ-3), Iowa BlackFQ, Deep Dark Quencher, Eclipse Dark Quencher nucleotide analog, Nucleotide G residue, nanoparticle and golden particulate also are not limited only to this.
Probe in the PCR reaction system, needs to add a kind of heat-staple archaeal dna polymerase, preferably do not have the archaeal dna polymerase of 5 ' nuclease, so that can keep complete in the reaction whole process.Heat-staple and the archaeal dna polymerase that do not have a 5 ' nuclease is the Taq polysaccharase through modifying, do not have 5 ' nuclease preferably, and in reaction process, probe can be hydrolyzed through the Taq polysaccharase of modifying.The present invention finds, adopts the better result of Taq polysaccharase that can obtain having than employing 5 ' nuclease through the Taq polysaccharase of modifying, do not have 5 ' nuclease.In addition, add the upstream primer of each primer pair different with downstream primer concentration.Amplification will be higher than with it another primer concentration of pairing with the primer concentration of probe complementary strand, the ratio of one couple of PCR primers is for being no more than 1: 2, perhaps be no more than 1: 3, perhaps be no more than 1: 4, perhaps be no more than 1: 5, best PCR primer ratio depends on concrete experiment, and the concentration ratio of the two is generally 3～50: 1.
Because probe can not be hydrolyzed, just can not be limited, so can be designed to desirable length yet.In the present invention, can use a long probe with high Tm value.The Tm value of designing probe can be also higher than elongating temperature, for example 72 ℃ or higher.In reaction, adopt the polysaccharase effect meeting with strand displacement activity better.
After the PCR reaction beginning, in initial 10～15 circulations, its amplified production mainly is double-stranded DNA, but after restricted primer (lower concentration primer) ran out of, the PCR of non-limiting primer (high density primer) guiding will produce a large amount of single stranded DNAs.In solution, the double-tagging oligonucleotide probe is " ball of string " shape usually when being in not single stranded conformational with target sequence hybridization, and this moment is because reporter group and quencher group distance are very near, the fluorescence that the quencher group can the quencher reporter group sends.When the annealing of probe and strand PCR product forms when double-stranded, because the fluorescence of reporter group can not be by quencher, thereby produce the fluorescent signal that can detect, can Real-Time Monitoring PCR reaction.
After pcr amplification finishes, carry out melting curve (Melting curve) and detect.In melting curve analysis, when gene is undergone mutation, special fluorescent mark oligonucleotide probe and its can not complete complementaries, and probe will be lower than probe with the Tm value of complete complementary wild-type strand PCR melting curve that product produces with it with the Tm value of mutant strand PCR melting curve that product produces.Perhaps in melting curve analysis, when gene is undergone mutation, special fluorescent mark oligonucleotide probe and its complementation, probe will be higher than the same with it Tm value of incomplete complementary wild-type strand PCR melting curve that product produces of probe with the Tm value of mutant strand PCR melting curve that product produces.Different by wild-type and mutant PCR product melting curve Tm value namely can be detected sample DNA and whether have the transgenation relevant with Drug Resistance for Tuberculosis.
Before detecting medicament-resistant mutation, comprise also whether detection exists the step of mycobacterium tuberculosis.This detects the step that whether has mycobacterium tuberculosis and comprises amplification 16SRNA gene, and uses probe in detecting.
The preparation process of test kit is described in detail in following step among the present invention.The present invention has done to further specify in following examples, but scope of the present invention is not subjected to the restriction of embodiment.Although the test kit that particularly prepares among the embodiment only comprises 511,513,515,516,518,519,522 of rpoB gene with primer and the detection of 5 kinds of Drug Resistance for Tuberculosis genes involveds of amplification, 526,531 and 533 sites, 306 sites of embB gene, 315 sites of ka tG gene,-15 and-8 sites of inhA gene, the double-tagging oligonucleotide probe in 18 mutational sites such as ahpC gene-6 ,-9 ,-10 and-12 sites, but can not be interpreted as that the present invention just is confined to the detection in 18 mutational sites of these 5 kinds of Drug Resistance for Tuberculosis genes involveds.Everyly utilize method among the present invention to detection and the test kit of Drug Resistance for Tuberculosis associated gene mutation, all belong to protection domain of the present invention.
Useful technique effect of the present invention: the present invention can detect a plurality of gene mutation sites relevant with Drug Resistance for Tuberculosis simultaneously in same reaction tubes, can greatly save time, reduce cost, clinical more more accurately diagnostic messages, more the approach clinic check demand of providing is provided.
Description of drawings
Fig. 1 is substance mycobacterium tuberculosis RpoB gene wild-type and mutant melting curve analysis figure.
Fig. 2 is substance mycobacterium tuberculosis RpoB gene wild-type and the report of mutant melting curve analysis.
Fig. 3 is multiple mycobacterium tuberculosis ahpC﹠amp; EmbB gene wild-type and mutant melting curve analysis figure.
Fig. 4 is multiple mycobacterium tuberculosis ahpC﹠amp; EmbB gene wild-type and the report of mutant melting curve analysis.
Fig. 5 is multiple mycobacterium tuberculosis KatG﹠amp; InhA gene wild-type and mutant melting curve analysis figure.
Fig. 6 is multiple mycobacterium tuberculosis KatG﹠amp; InhA gene wild-type and the report of mutant melting curve analysis.
Embodiment
Below in conjunction with accompanying drawing technical scheme of the present invention is elaborated.
Embodiment 1: the design of primer and probe is with synthetic
Rifampin and vazadrine are first-line drugs crucial in the tuberculotherapy scheme, also are the antitubercular agents that resistance the most easily occurs.Studies show that, 97% rifampin-resistant is because due to rpoB gene (511,513,515,516,518,519,522,526, the 531 and 533 site) sudden change, sudden change mainly concentrates on the 81bp zone of one section high conservative.The inhA gene (8 ,-15 site) of main katG gene (315 site), the coding nicotinamide adenine dinucleotide reduced dependency enoyl-carrier proteins reductase enzyme with the coding Catalase-peroxidase of Isoniazid-resistant and the ahpC gene of coding alkyl peroxide enzyme (6 ,-9 ,-10 with-12 sites) suddenly change relevant.The embB306 site mutation is the genetic marker relevant with the anti-multiple medicines of tuberculosis of a high specific.According to above-mentioned 5 kinds and Drug Resistance for Tuberculosis genes involved primers and probe, and contain whole mutational sites to be detected in the target sequence amplified production of guaranteeing to produce; The hybridization zone of target nucleic acid sequence amplified production and probe will comprise a sudden change Nucleotide to be checked at least.Forward primer can be primer and the combination thereof of the SEQ ID NO.1～5 kind genes relevant with Drug Resistance for Tuberculosis that SEQ ID NO.9 is corresponding in the sequence table, reverse primer can be primer and the combination thereof of the 5 kind genes relevant with Drug Resistance for Tuberculosis that SEQ ID NO.10～SEQ ID NO.16 is corresponding in the sequence table, and probe can be corresponding sequence and the combination thereof of SEQ ID NO.17～SEQ ID NO.36 in the sequence table
Primer and probe all entrust specialized company to synthesize, and wherein primer is the PAGE purifying, and probe is the HPLC purifying.The FAM fluorescence report group that removes RpoB probe 1 is marked on the 16th bit base dT, the FAM fluorescence report group of RpoB probe 2 is marked on the 17th bit base dT, the FAM fluorescence report group of RpoB probe 3 is marked on the 14th bit base dT, it is upper outside that the FAM fluorescence report group that the FAM fluorescence report group of RpoB probe 4 is marked at the 24th bit base dT and ahpc3 probe 3 is marked at the 12nd bit base dT, and all the other each probes are 5 ' end mark FAM fluorescence report group.3 ' terminal all mark BHQ1 quencher groups of all probes.The calmodulin binding domain CaM of RpoB probe 2 and target sequence contains the reversing repetitive sequence of 9 base pairs, and in order to prevent the formation of palindrome, RpoB probe 2 has been introduced unpaired base, respectively 11 and 22 bit base positions.RpoB probe 3, RpoB probe 4, inhA probe 3 have also been introduced unpaired base to reduce the Tm value.RpoB probe 4, inhA probe 4, inhA probe 5, and Ahpc probe 4 also introduced unpaired base, or be equivalent to wild-type base deletion is sequentially arranged, introducing disappearance on the one hand can the increase coverage territory, shortens on the other hand probe length.
Above probe can be used the FAM mark, also can use HEX, or the TexasRed mark, perhaps one group of probe FAM mark, such as the RpoB probe, another group probe Hex mark, such as embB and ahpc probe, another group probe TexasRed mark is such as inhA and KatG probe.The probe of isolabeling can not be placed in the PCR reaction, thereby reaches the purpose of all transgenations of a PCR reaction detection.
Embodiment 2: the extraction of sample DNA
Learning from else's experience is accredited as the clinical sample strain isolated of the mycobacterium tuberculosis positive after cultivating, and high-temperature inactivation carries out the extraction of sample DNA again with commercial genome DNA extracting reagent kit.The genomic dna that obtains is directly used in Drug Resistance for Tuberculosis detects or-20 ℃ of storages, stand-by.
Embodiment 3: the extraction of sample DNA
Get sputum 2ml, add 2.5 times of volume 4%NaOH, 37 ℃ of incubation 30min.With the sputum after the liquefaction, centrifugal, remove supernatant, the gained precipitation is used for the extraction of sample DNA.Carry out the extraction of sample DNA with commercial genome DNA extracting reagent kit.The genomic dna that obtains is directly used in Drug Resistance for Tuberculosis detects or-20 ℃ of storages, stand-by.
Embodiment 4: the selection of positive reference substance
The mycobacterium tuberculosis type strain that will come from Nat'l Pharmaceutical ﹠ Biological Products Control Institute spends the night and shakes bacterium, high-temperature inactivation.Bacterium liquid after the deactivation carries out DNA extraction with commercial genome DNA extracting reagent kit.After the DNA that obtains is quantitative, be diluted to 10ng/ μ l with TE Buffer, as the positive reference substance of wild-type.
Embodiment 5:PCR reaction composition
According to the form below (table 1～table 3) preparation PCR reaction solution also adds TB dna profiling (each experiment must comprise negative control and wild-type positive control).Vortex vibration mixing after preparation is finished, centrifugal rear upper machine.
Each becomes to be grouped into (master MixI) table 1PCR system
| Each component title | Final |
| PCR Buffer | |
| 1× | |
| MgCl2 | 2mM |
| Betaine | 1M |
| dNTPs | 0.2mM |
| The RpoB forward primer | 0.4μM |
| The RpoB reverse primer | 0.1 |
| RpoB probe | |
| 1 | 0.2 |
| RpoB probe | |
| 2 | 0.2μM |
| Taq enzyme 5 ' exo- | 0.5U |
| Aseptic ultrapure water | In right amount |
| Dna profiling | 1μl |
| Cumulative volume | 20μl |
Each becomes to be grouped into (master MixII) table 2PCR system
| Each component title | Final |
| PCR Buffer | |
| 1× |
[0088]
| MgCl2 | 2mM |
| Betaine | 1M |
| dNTPs | 0.2mM |
| The embB forward primer | 0.4μM |
| The embB reverse primer | 0.1μM |
| The ahpC forward primer | 0.4μM |
| The ahpC reverse primer | 0.1μM |
| The embB probe | 0.2μM |
| The ahpC probe | 0.2μM |
| Taq enzyme 5 ' exo- | 0.5U |
| Aseptic ultrapure water | In right amount |
| Dna profiling | 1μl |
| Cumulative volume | 20μl |
Each becomes to be grouped into (master MixIII) table 3PCR system
| Each component title | Final |
| PCR Buffer | |
| 1× | |
| MgCl2 | 2mM |
| Betaine | 1M |
| dNTPs | 0.2mM |
| The KatG forward primer | 0.1μM |
| The KatG reverse primer | 0.4μM |
| The inhA forward primer | 0.1μM |
| The inhA reverse primer | 0.4μM |
| The KatG probe | 0.2μM |
| The inhA probe | 0.2μM |
| Taq enzyme 5 ' exo- | 0.5U |
| Aseptic ultrapure water | In right amount |
| Dna profiling | 1μl |
| Cumulative volume | 20μl |
Embodiment 6: response procedures is set
The fluorescence detection channel of collecting the FAM fluorescent signal is set, the PCR reaction tubes is put into quantitative real time PCR Instrument begin amplification, response procedures following (take Rotor Gene Q as example):
Table 4PCR program setting
Embodiment 7: the result judges
Behind the quantitative real time PCR Instrument end of run, with its software kit the result of this test is carried out melting curve analysis.Take positive reference substance reaction tubes (wild-type) as reference, its melting curve and the identical sample of positive control QC peak type are wild type strain; Its melting curve and positive control QC peak type are discrepant to be saltant.After bacterial strain was undergone mutation, the Tm value of its melting curve was compared with wild type strain and can be decreased or improve.
Embodiment 8: quality control standard (table 5)
Table 5 quality control product standard testing result (take Rotor Gene Q as example)
Claims (2)
1. probe combinations that detects Drug-Resistant Mycobacterium tuberculosis, it is characterized in that: each probe in this probe combinations is comprised of 25～50 Nucleotide, under the condition of hybridization, it can with the nucleus hybridization of mycobacterium tuberculosis RpoB gene 81bp; Above-mentioned probe comprises that one is marked at 3 ' terminal quencher mark and one and is marked on the inner nucleotide residue or 5 ' terminal report mark; Described probe hybridization is in the zone that comprises RpoB gene 526,531 and 533 codons, and probe contains the Nucleotide not complementary with extension increasing sequence; Described probe is hybridized with the 81bp nucleus of the RpoB gene that comprises 9bp palindrome sequence; The formation that has not stoped probe self hairpin structure with the Nucleotide of extension increasing sequence complementation that comprises in the above-mentioned probe; Described probe is SEQ ID NO:17, SEQ ID NO:18, SEQID NO:26, SEQ ID NO:27, the combination of SEQ ID NO:31.
2. test kit that from sample, detects Drug-Resistant Mycobacterium tuberculosis, described test kit comprises the oligonucleotide probe of amplimer and mark, this oligonucleotide probe comprises a report mark and a quencher mark; When the oligonucleotide probe of mark is in not single stranded conformational with target sequence hybridization, the fluorescence that the quencher mark can quencher report mark sends;
When the hybridization of oligonucleotide probe and target sequence, form duplex structure, the fluorescence of report mark can not be by quencher, at this moment, the fluorescence intensity the when fluorescence intensity of report mark will not be in the strand state with target sequence hybridization far above this oligonucleotide probe;
Wherein said quencher mark is non-quenching of fluorescence group, and is marked at 3 ' end of probe; Wherein said report mark is fluorophor, and is marked on the nucleotide residue of the inside of oligonucleotide probe or is marked at 5 ' end of probe;
Also comprise heat-staple and do not have the archaeal dna polymerase of 5 ' nuclease and required other each component commonly used of amplification of nucleic acid sequences;
Wherein said primer is the combination of the SEQ ID NO:1～SEQ ID NO:16 in the sequence table;
Wherein said probe is the combination of the SEQ ID NO:17～SEQ I D NO:36 in the sequence table.
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