CN105907878A - Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology - Google Patents
Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology Download PDFInfo
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Abstract
The invention relates to drug resistance gene mutation detection of tuberculous bacillus (TB) based on a high-resolution melting curve technology and particularly relates to a PCR (Polymerase Chain Reaction) primer set for detecting drug resistance gene mutation of the tuberculous bacillus (TB) based on the high-resolution melting curve technology. The invention further relates to a kit and a micro-array, which comprise the PCR set and are used for detecting the drug resistance gene mutation of the tuberculous bacillus (TB) based on the high-resolution melting curve technology. The invention further relates to application of the PCR set to preparation of the kit and the micro-array, which are used for detecting the drug resistance gene mutation of the tuberculous bacillus (TB) based on the high-resolution melting curve technology. The invention further relates to a method for detecting the drug resistance gene mutation of the tuberculous bacillus (TB) in a sample by utilizing the PCR primer set.
Description
Technical field
The present invention relates to mycobacterium tuberculosis based on high-resolution fusion curve technology (Mycobacterium tuberculosis, TB) and drug resistance gene abrupt climatic change.Specifically, the present invention relates to based on high-resolution fusion curve
The PCR primer group of technology for detection mycobacterium tuberculosis (TB) drug resistance gene sudden change.The invention still further relates to comprise PCR primer group
For the test kit suddenlyd change based on high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance gene and microarray.
The invention still further relates to PCR primer group in preparation for based on high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance
Purposes in the test kit of property gene mutation and microarray.The invention still further relates to use the tuberculosis in PCR primer group detection sample
The method of mycobacteria (TB) drug resistance gene sudden change.
Background technology
High-resolution fusion curve (high-resolution melt, HRM) analytical technology is the PCR skill of a kind of Efficient robust
Art, is not limited to type by mutating alkali yl site, it is not necessary to sequence-specific probes, directly runs high-resolution and melt after PCR terminates
Solve, got final product paired samples sudden change, single nucleotide polymorphism-snp analysis.Its cardinal principle is the length according to DNA sequence, GC
Content and base complementrity sex differernce, apply high-resolution melting curve to be analyzed sample, and its high temperature is homogeneous
Property and temperature resolution make resolving accuracy can reach the differentiation to single base difference.HRM is to make use of the specific dyestuff can
To insert the characteristic in DNA double chain, by the knot of monitoring temperature-rise period double center chain DNA fluorescent dye in real time with pcr amplification product
Conjunction situation record high-resolution melting curve, thus sample is detected.As in the detection of SNP, SNP site is not due to
Joining double-stranded DNA can first untie in temperature-rise period, fluorescent dye discharges from the DNA molecular that unwinds of local, from fluorescence intensity with
It may determine that whether there is SNP on time graph, and whether etc. different SNP site, heterozygote all can affect melting curve
Peak shape, therefore HRM analyzes and can effectively distinguish different SNP site and different genotype.
The first-line drug that the pulmonary tuberculosis caused mycobacterium tuberculosis clinically is commonly used includes isoniazid (INH), rifampicin
(RFP), streptomycin (SM) etc..Its mechanism of action of isoniazid is swashed by mycobacterium tuberculosis hydrogen peroxide enzyme-peroxidase
Live, act on enoyl-carrier protein reductase, suppress Mycobacterial cell wall biosynthesis to sterilize.KatG gene is coding
The encoding gene of catalase-peroxidase, its gene mutation may result in Catalase-peroxidase activity decrease and even loses
Lose.KatG gene mutation accounts for the 50 ~ 70% of Isoniazid-resistant.InhA gene code NADH (NADH)
The enoyl reductase relied on, its gene mutation accounts for Isoniazid-resistant 10 ~ 35%.
Rifampicin is a kind of broad spectrum antibiotic, can be combined with the β subunit of the RNA polymerase that DNA relies on, and suppression RNA gathers
Synthase activity, and then suppression mRNA transcribes and antibacterial action is played in albumen synthesis.The rifampin-resistance bacterium of about 95% is all owing to compiling
Code RNA polymerase β subunit (rpoB) gene is undergone mutation, thus is no longer combined with rifampicin and produces drug resistance.RpoB gene
In the region of the 81bp that sudden change occurs mainly in 507-533 position 27 amino acid codes of coding.
There is now various biological detection technique and be applied to drug resistance of Mycobacterium tuberculosis detection, as phage biocontrol expands
Method, conventional drug sensitive test, DNA sequencing, gene chip, the analysis of PCR-restriction fragment length polymorphism, the detection of fast culture instrument
System etc..
Phages Lysis test and conventional drug sensitive test complex operation time-consumingly length are difficult to standardization.Gene chip sample
Preparation and labelling are cumbersome, expensive equipment, and testing cost is high.PCR-restriction fragment length polymorphism analysis can only be analyzed known
The gene mutation in sequence specific site.Fast culture instrument detecting system instrument and reagent dependence on import are costly.More simultaneously
Non-order-checking mode needs to rely on the gene mutation situation of the mode interpretation patient of enzyme action or cluster, there is bigger typing mistake
Probability.Although direct Sequencing accuracy is high, but the DNA fragmentation that sequencing reaction produces needs through capillary electrophoresis separation, thereafter
Fluorescence signal is detected by detecting system, the longest and testing cost is higher.
HRM technical operation is easy quickly, and use cost is low, and result is accurate, it is achieved that real stopped pipe operation.Therefore, HRM
The application that technology detects at M. tuberculosis drug resistant gene (inhA, katG, rpoB) will have great value with latent with promoting
Power.
Summary of the invention
One aspect of the present invention relates to based on high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance
The PCR primer group of gene mutation, described PCR primer group includes that at least one is selected from following primer sets:
(1) primer sets 1, it comprises and has the primer of the nucleotide sequence shown in SEQ ID NO:1 and have a SEQ ID NO:
The primer of the nucleotide sequence shown in 2；
(2) primer sets 2, it comprises and has the primer of the nucleotide sequence shown in SEQ ID NO:3 and have a SEQ ID NO:
The primer of the nucleotide sequence shown in 4；With
(3) primer sets 3, it comprises and has the primer of the nucleotide sequence shown in SEQ ID NO:5 and have a SEQ ID NO:
The primer of the nucleotide sequence shown in 6.
In one embodiment, described PCR primer group includes primer sets 1, and optionally comprises at least one selected from primer
Group 2 and the primer sets of primer sets 3.In another embodiment, described PCR primer group includes primer sets 2, and optionally comprise to
Few a kind of selected from primer sets 1 with the primer sets of primer sets 3.In still another embodiment, described PCR primer group includes primer sets
3, and optionally comprise at least one selected from primer sets 1 and the primer sets of primer sets 2.In another embodiment again, described PCR draws
Thing group includes primer sets 1, primer sets 2 and primer sets 3.
Another aspect of the present invention relates to based on high-resolution fusion curve technology for detection Drug Resistance of Mycobacterium Tuberculosis base
Because of test kit or the microarray of sudden change, it comprises the PCR primer group of the present invention.Another aspect of the invention relates to the PCR of the present invention
Primer sets is used for based on the Drug Resistance of Mycobacterium Tuberculosis gene mutation in high-resolution fusion curve technology for detection sample in preparation
Test kit or microarray in purposes.
In one embodiment, described sample is biological sample, and the most described sample is humoral sample or tissue sample,
The most described sample selected from biopsy samples, cell culture, cured process sample (such as paraffin embedding sample
Product), whole blood, blood plasma, serum, saliva, brains liquid, perspiration, expectorant, bronchoalveolar lavage fluid, urine, feces, juice, milk and peritoneum
Liquid.
Another aspect of the invention relates to based on Drug Resistance of Mycobacterium Tuberculosis in high-resolution fusion curve technology for detection sample
The method of gene mutation, described method includes:
(1) extract and the nucleic acid in optional purification of samples,
(2) primer sets of the present invention is used to carry out PCR amplification, and
(3) PCR primer of step (2) is carried out high-resolution fusion curve analysis；
Wherein said method is not used in diagnostic purpose, and whether the mycobacterium tuberculosis that such as can be used for detecting in In vitro culture becomes
Different, whether the mycobacterium tuberculosis sample that can be used for detecting environmental sources has drug resistance..
In one embodiment, described sample is biological sample, and the most described sample is humoral sample or tissue sample,
The most described sample selected from biopsy samples, cell culture, cured process sample (such as paraffin embedding sample
Product), whole blood, blood plasma, serum, saliva, brains liquid, perspiration, expectorant, bronchoalveolar lavage fluid, urine, feces, juice, milk and peritoneum
Liquid.In another embodiment, the condition of high-resolution fusion curve analysis is: 95 DEG C 2 minutes, with 0.1 DEG C/step from 83 DEG C
Scan to 96 DEG C or to 93 DEG C or scan to 94 DEG C from 80 DEG C from 82 DEG C of scannings.In one embodiment, an embodiment party
In case, PCR amplification can be qualitative or quantitative PCR amplification.In another embodiment, PCR amplification is quantitative pcr amplification.?
In further embodiment, PCR amplification expands for real-time fluorescence quantitative PCR.
Accompanying drawing explanation
After Fig. 1 is for using PCR primer group of the present invention (primer sets 3, SEQ ID NO:5 and 6) to expand rpoB gene
The result sectional drawing that the sudden change of this gene is detected by application high-resolution fusion curve analysis.
After Fig. 2 is for using PCR primer group of the present invention (primer sets 2, SEQ ID NO:3 and 4) to expand katG gene
The result sectional drawing that the sudden change of this gene is detected by application high-resolution fusion curve analysis.
After Fig. 3 is for using PCR primer group of the present invention (primer sets 1, SEQ ID NO:1 and 2) to expand inhA gene
The result sectional drawing that the sudden change of this gene is detected by application high-resolution fusion curve analysis.
Fig. 4 is the sectional drawing using different PCR primer group to expand rpoB gene.A: have with PCR primer group 3 of the present invention
There is identical reverse primer but there is on forward primer the PCR primer group (SEQ ID NO:8 and 6) of fraction difference base,
Result display NTC has serious non-specific amplification (Fig. 4 A Grey curves)；B: PCR primer group of the present invention (primer sets 3, SEQ ID
NO:5 and 6), result shows and there is not non-specific amplification.
Detailed description of the invention
Several aspects of the present invention it are described below with reference to the example application for explanation.It should be appreciated that statement
Many details, relation and method provide and fully understand the present invention.But, those of ordinary skill in the related art
Will readily appreciate that, the present invention can be implemented in the case of without one or more details or can come with additive method
Implement the present invention.
Present invention aim at solving in prior art the detection that mycobacterium tuberculosis (TB) drug resistance gene suddenlys change
Shortcoming, the biggest typing mistake, the longest or testing cost is higher.By the present invention in that by the PCR primer of the present invention
Group high-resolution fusion curve technology and solve the problems referred to above.
The high-resolution fusion curve technology using the PCR primer group of the present invention has the advantage that
1) instrument and reagent are by system optimization, can reach the Detection results of optimum, and easily standardization；
2) testing result is given by software automatically, it is to avoid result judges subjectivity；
3) operating process is easy and saves time, omnidistance 120 minutes of detection, and its Instrumental runs the time 100 minutes automatically, manual operations
20 minutes time；With
4) there is not non-specific amplification and detection accuracy is high.
Unless otherwise stated, all scientific and technical terminologies used herein have one skilled in the art of the present invention
The implication being generally understood that.The definition of the general term in Cytobiology and molecular biology may refer to: the gene that Yu Long etc. translate
VIII, International Standard Book Number: ISBN:978-7-03-014597-0, Science Press publishes (2005)；The cell that Zhang Jinfeng etc. translate with
Molecular biology, Zhong Xin publishing house publishes (2004), ISBN:978-7-50-860075-8；The bioid compiled with Wang Jingyan etc.
Learning, Higher Education Publishing House publishes (2002), ISBN:978-7-04-011088-3；Kendrew, J. et al. (compile),
The Encyclopedia of Molecular Biology, Blackwell Science Ltd. publish (1994),
ISBN 0-632-02182-9;And Meyers, R.A. (volume), Molecular Biology and Biotechnology:
A Comprehensive Desk Reference, VCH Publishers, Inc. publish (1995), ISBN 1-
56081-5698.Although implement the present invention time can use any method similar or equivalent to methods described herein and material and
Material, but this document describes concrete material and method.
Sample
Terms used herein " sample " includes any sample containing nucleic acid molecules.Sample can derive from biogenetic derivation (" biological sample
Product "), such as organize (such as biopsy samples), extract or include the culture of mycobacterium tuberculosis and biological or life
Reason fluid, the sample (such as paraffin-embedded sample) of the most cured process, whole blood, blood plasma, serum, saliva, brains liquid, perspiration,
Expectorant, bronchoalveolar lavage fluid, urine, feces, juice, milk, peritoneal fluid etc..Available from source sample or in pretreatment to improve sample
Sample after product feature (such as preparing blood plasma, dilution sputum etc. from blood) can be used directly.At certain aspects of the invention, sample
Product are expectorant or bronchoalveolar lavage fluid.
The sample that can be analyzed according to the present invention and/or use includes the polynucleotide of clinical source, such as DNA or
RNA。
The method extracting nucleic acid from sample is well-known in the art, and available such as phenol and chloroform carry out DNA and carries
Take, or use commercially available DNA extraction reagent to extract.Such as, post test kit (such as GENERATION (registrar can be used
Mark) Capture Column Kit Gentra) extract.
It should be understood that nucleic acid can carry out purification by the purification process that this area crowd is conventional, such as, use PrepSEQ
Method in test kit (from Applied Biosystems) and U.S. Patent number 5,234,809 etc..
Drug resistance, also known as Drug resistance, means the tolerance for chemotherapeutics effect of microorganism, parasite and tumor cell
Property.In the present invention, drug resistance refers to the drug resistance of mycobacterium tuberculosis, particularly relates to mycobacterium tuberculosis to clinically to knot
The first-line drug that the pulmonary tuberculosis that core mycobacteria causes is commonly used (includes isoniazid (INH), rifampicin (RFP), streptomycin (SM)
Deng) drug resistance.Drug resistance gene sudden change refer in some embodiments of the present invention mycobacterium tuberculosis gene inhA,
The sudden change of katG and/or rpoB.
Primer
" primer " used herein is often referred to the linear oligonucleotide with target complement sequence and annealing.The lower limit of primer length is by miscellaneous
Depending on friendship ability, because the shortest primer (e.g., less than 5 nucleotide) is formed without thermodynamics under most of hybridization conditions
Stable duplex.Primer length generally changes in 8-50 nucleotide.In certain embodiments, primer is between about
Between 15-25 nucleotide.Terms used herein " forward primer " refers to the oligonucleoside of a specific chain annealing with target DNA
Acid.Terms used herein " reverse primer " refers to the oligonucleotide of the opposite strand annealing with target DNA.In a word, forward primer and
Reverse primer is generally oriented on target DNA sequence in the way of being similar to PCR primer so that its 3' end more connects than its 5' end
Near target sequence.Naturally occurring nucleotide (especially guanine, adenine, cytosine and thymus pyrimidine, hereinafter referred to as " G ",
" A ", " C " and " T ") and nucleotide analog, can be used in the primer of the present invention.Terms used herein " PCR primer " is
Refer to the oligonucleotide primers for the initial PCR that nucleic acid is carried out reaction.
" PCR primer " used herein refers to from nucleic acid-templated, is expanded by nucleic acid PCR and the nucleic acid of amplification that produces.
Terms used herein " nucleotide analog " refers to the compound being structurally similar with naturally occurring nucleotide.
Nucleotide analog can have the phosphate backbones of change, sugar moieties, core base or a combination thereof.It is generally of the core base of change
Nucleotide analog especially give different base pairings and base stacking characteristic.There is the core of the phosphoric acid-sugar skeleton of change
Thuja acid analog (such as peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA)) the most especially changes chain characteristic, such as secondary structure and is formed.
For the PCR primer suddenlyd change based on high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance gene
The example of group and target gene such as table 1.
Primer sets used by table 1 present invention and corresponding target gene
The nucleotide sequence of the PCR primer of the present invention also includes its modified forms, as long as the amplification of described primer or HRM analyze effect
Fruit is the most significantly affected.Described modification can be such as in nucleotide sequence or two ends add one or more cores
Thuja acid residue, in nucleotide sequence, lack one or more nucleotide residue or by the one or more nucleoside in sequence
Acid residue replaces to other nucleotide residue, such as, A is replaced to T, C is replaced to G etc..It will be apparent to those skilled in the art that
The primer of described modified forms is also covered by within the present invention, particularly within scope of the claims.An enforcement
In scheme, the modified forms of the nucleotide sequence of PCR primer is the chemical reinforcing type primer as disclosed in CN103270174A.
The most general DNA synthesizer (394 types such as manufactured) can be used by Applied Biosystems, through chemistry
Method synthesizes each nucleotide in primer of the present invention.Any other method well-known in the art also can be used to synthesize widow
Nucleotide, such as PCR primer.
The genomic DNA that use is extracted from sample is as template, and uses PCR primer resistance to mycobacterium tuberculosis (TB)
Property of medicine gene mutation carries out amplified reaction, to obtain amplified production.Amplified reaction includes but not limited to polymerase chain reaction
(PCR), ligase chain reaction (LCP), automatically maintain sequence replicating (3SR), amplification based on nucleotide sequence (NASBA), chain
Displacement amplification (SDA), multiple displacement amplification (MDA) and rolling circle amplification (RCA), it is disclosed in and (draws work at this below with reference to document
Reference) in: Mullis etc., U.S. Patent No. 4,683, No. 195；No. 4,965,188；No. 4,683,202；4,800th,
159 (PCR) number；Gelfand etc., U.S. Patent No. 5,210, No. 015 (with " Taqman " or " Taq " [registered trade mark] probe
The real-time PCR carried out)；Wittwer etc., U.S. Patent No. 6,174, No. 670；Kacian etc., U.S. Patent No. 5,399, No. 491
(“NASBA”)；Lizardi, U.S. Patent No. 5,854,033；Aono etc., Japanese Patent Publication JP 4-262799 (rolling
Circle amplification)；Etc..
PCR method is preferably used target nucleotide is expanded.PCR method itself is well-known in the art.Term " PCR "
Including the derivative form of this reaction, it includes but not limited to reverse transcriptional PCR, real-time PCR, nested PCR, multiplex PCR and fluorescence
Quantitative PCR etc..Fluorescence quantitative PCR method is preferably used target nucleotide is carried out quantitative amplification.In one embodiment, at one
In embodiment, PCR method can be qualitative or quantitative PCR method.In another embodiment, PCR method is quantitative PCR method.Again
In one embodiment, PCR method is quantitative real-time PCR.
In the presence of primer, template DNA and hot resistant DNA polymerase, use and the primer (reverse primer) of sense strand hybridization
And with the primer (forward primer) of antisense strand hybridization, by making degeneration, anneal and extend the circulation of step repeat about 30 times～
50 times (such as 45 times) carry out PCR.In one embodiment, PCR is real-time fluorescence quantitative PCR.An embodiment
In, use saturated fluorescence dyestuff (such as LC Green, LC Green Plus, Eva Green and SYTO-9) to carry out PCR.One
In individual embodiment, PCR employs primer sets 1,2 and/or 3 and (the such as combination of primer sets 1 and 2 of their combination in any
(when target gene is inhA and katG), the combination (when target gene is inhA, katG and rpoB) etc. of primer sets 1 and 2 and 3).
The PCR primer group of the present invention can in any combination, and to detect for the different target genes in sample, such as primer sets 1 can
Combining with primer sets 2 and/or 3, primer sets 2 can combine with primer sets 1 and/or 3, and primer sets 3 can be with primer sets 1 and/or 2 groups
Close.Therefore, in one embodiment, the PCR primer group of the present invention can comprise primer sets 1, and optionally comprises at least one choosing
From primer sets 2 and the primer sets of primer sets 3.In another embodiment, the PCR primer group of the present invention includes primer sets 2, and
Optionally comprise at least one selected from primer sets 1 and the primer sets of primer sets 3.In still another embodiment, the PCR of the present invention draws
Thing group includes primer sets 3, and optionally comprises at least one selected from primer sets 1 and the primer sets of primer sets 2.
In the PCR of the present invention, the hot resistant DNA polymerase of various routine can be used to expand, include but not limited to
FastStart Taq archaeal dna polymerase (Roche), Ex Taq (registered trade mark, Takara), Z-Taq, AccuPrime Taq
Archaeal dna polymerase and HS Taq archaeal dna polymerase.
The method selecting suitable PCR reaction condition based on primer Tm is well-known in the art, the common skill in this area
Art personnel can select according to primer length, G/C content, desired specificities and sensitivity, the polymerase character etc. used
Good condition.Such as, following condition can be used to carry out PCR reaction: 95 DEG C 5 minutes, 95 DEG C 15 seconds, 65 DEG C 10 seconds, 72 DEG C
10 seconds, circulate 45 times.
After obtaining PCR primer, according to high-resolution solubility curve method, the mutational site of the genes of interest after amplification is entered
Row detection.In one embodiment, the condition of high-resolution fusion curve analysis is: 95 DEG C 2 minutes, with 0.1 DEG C/step from 83
DEG C scanning to 96 DEG C (for rpoB genes) or from 82 DEG C scanning to 93 DEG C (for katG genes) or from 80 DEG C scanning to 94 DEG C
(for inhA gene).In one embodiment, Rotor-gene Q PCR instrument is used to carry out high-resolution fusion curve analysis.
Test kit
The invention still further relates to a kind of for based on high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance gene
The test kit of sudden change, it contains the PCR primer group of the present invention.The invention still further relates to PCR primer group of the present invention in preparation for base
Purposes in the test kit of high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance gene sudden change.At one
In embodiment, described PCR primer group be selected from primer sets 1, primer sets 2 and primer sets 3 at least one (for example, at least 2 or
Whole 3 kinds) primer sets.In one embodiment, the PCR primer group of the present invention can comprise primer sets 1, and optionally comprises at least
A kind of selected from primer sets 2 with the primer sets of primer sets 3.In another embodiment, the PCR primer group of the present invention includes primer
Group 2, and optionally comprise at least one selected from primer sets 1 and the primer sets of primer sets 3.In still another embodiment, the present invention
PCR primer group include primer sets 3, and optionally comprise at least one selected from primer sets 1 and the primer sets of primer sets 2.
Test kit can comprise implements the material used by the inventive method or reagent (including PCR primer group).Test kit is permissible
Including storing reaction reagent (the such as primer in suitable vessel, dNTP, enzyme etc.) and/or support material (such as buffer, reality
Execute the description etc. of detection).Such as, test kit can include one or more containing respective reaction reagent and/or support material
Container (such as box).Such content can be delivered to set receiver together or separately.Such as, first container can
Containing the enzyme for measuring, second container contains saturated fluorescence dyestuff (such as LC Green, LC Green Plus, Eva
Green and SYTO-9) and the 3rd container contains PCR primer group.Described test kit also can containing be suitable for accommodate described reagent or
The compartment of container.As an example, test kit can contain PCR primer group, saturated fluorescence dyestuff (such as LC Green, LC
Green Plus, Eva Green and SYTO-9), PCR reaction buffer, operation instructions.Test kit also can containing polymerase and
DTNP etc..Test kit also can contain UNG, internal standard, the positive and negative control etc. for Quality Control.Test kit also can comprise for from
The reagent of nucleic acid such as DNA prepared by sample.Test kit of the present invention also can comprise other in addition to the PCR primer group of the present invention
Any PCR primer group, such as, can effectively detect the PCR primer group of mycobacterium tuberculosis (TB) drug resistance gene sudden change.Above
Example is it is not intended that restricted application is in the test kit of the present invention and content thereof.
Microarray
The invention still further relates to a kind of for based on high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance gene
The microarray of sudden change, it contains the PCR primer group of the present invention.The invention still further relates to the PCR primer group of the present invention preparation for
Purposes in microarray based on the sudden change of high-resolution fusion curve technology for detection mycobacterium tuberculosis (TB) drug resistance gene.One
In individual embodiment, described PCR primer group be selected from primer sets 1, primer sets 2 and primer sets 3 at least one (for example, at least 2
Or whole 3 kinds) primer sets.In one embodiment, the PCR primer group of the present invention can comprise primer sets 1, and optionally comprise to
Few a kind of selected from primer sets 2 with the primer sets of primer sets 3.In another embodiment, the PCR primer group of the present invention includes drawing
Thing group 2, and optionally comprise at least one selected from primer sets 1 and the primer sets of primer sets 3.In still another embodiment, this
Bright PCR primer group includes primer sets 3, and optionally comprises at least one selected from primer sets 1 and the primer sets of primer sets 2.
Microarray refers to the solid support with flat surfaces, and it has nucleic acid array, each member bag in array
Contain the identical copy being fixed on the oligonucleotide on the region or site spatially determined or polynucleotide, described region or position
Point is not overlapping with the region of other member in array or site；It is to say, described region or site are spatially discrete
's.Additionally, the hybridization site spatially determined can be " addressable ", because its position and the body of immobilized oligonucleotide thereof
Part is known or predetermined (being known or predetermined the most before its use).Generally oligonucleotide or polynucleotide
For strand and generally covalently bound with solid support by 5'-end or 3'-end.Microarray contains the nucleic acid in non-overlapped district
Density is typically larger than 100/cm2, more preferably greater than 1000/cm2.Microarray technology is disclosed in such as below with reference in document:
The Microarrays:A Practical Approach (IRL Press, Oxford, 2000) that Schena edits；
Southern, Current Opin. Chem. Biol., 2:404-410,1998, entire contents is incorporated by reference into
Herein.
Although being described above various embodiments of the present invention, it should be understood that it is the most by way of example
There is provided, and and unrestricted.The many change of disclosed embodiment can be carried out according to this disclosure, without carrying on the back
From the spirit or scope of the present invention.Therefore, range and the scope of the present invention should not be restricted by any of above embodiment and limited.
All documents mentioned above are the most incorporated herein by reference.The all publications and patents that the application quotes
File is all incorporated by reference for all purposes, and degree of quoting is as pointed out each publication or patent document one individually
Sample.
Embodiment
Unless otherwise indicated, otherwise the equal city of material used by embodiment hereof available from, for carrying out the various tools tested
Body experimental technique is the conventional experimental technique in this area and (see for example " the fine works molecular biology reality of the chief editors such as F. Ao Sibai
Test guide " (1999), Science Press, " the molecular cloning reality of the chief editor such as ISBN 7-03-006408-9 and J. Pehanorm Brooker
Test guide (third edition) " (2002), Science Press, ISBN 7-03-010338-6) or according to the step proposed by manufacturer
Rapid and condition, and can be determined the most routinely by those skilled in the art.Hereinafter some material and method are carried out in detail
State.
Embodiment 1: use the high-resolution fusion curve analysis that PCR primer group of the present invention is carried out
1. material and method
1.1. material
PCR reactant liquor 1(includes PCR primer group 1), PCR reactant liquor 2(include PCR primer group 2), PCR reactant liquor 3(include PCR
Primer sets 3) and HS Taq enzyme.
The each concentration of component of PCR reactant liquor is shown in Table 2.
Table 2
| Title | Final concentration | Source |
| Forward primer F | 0.3uM | Invitrogen |
| Reverse primer R | 0.3uM | Invitrogen |
| dNTP | 0.2mM | Roche |
| Fluorescent dye Eva Green | 1x | Biotium |
| 20x buffer | 1x | QIAGEN |
| MgCl2 | 2mM | PG |
1.2. sample
Wild type control plasmid wt(inhA-katG-rpoB gene wild plasmid (SEQ ID NO:7), by Invitrogen
Company synthesizes) and hospital clinical sputum sample nucleic acid.
1.3. equipment
QIAGEN company Rotor-gene Q PCR instrument.
1.4. method
Initially with PCR reactant liquor and HS Taq to M. tuberculosis genes group DNA of purification at Rotor-Gene Q platform
On expand, PCR reactant liquor comprises fluorescent dye, amplification procedure can embed ever-increasing PCR double-stranded products
In.The process of the most whole product enrichment can be monitored in real time by the software of Rotor-Gene Q, so that it is guaranteed that quality
PCR primer is analyzed for follow-up HRM reliably.Postrun result is automatically analyzed by software, decreases manual analysis
Burden and error.
By required PCR reaction tube number n (n≤36), reaction system is formulated as follows table 3:
Table 3
| PCR reactant liquor | HS Taq enzyme | |
| Reactant liquor | n×27.6 µL | n×0.4µL |
RpoB detects program:
● 95 DEG C: 5 minutes；
● 95 DEG C: 15 seconds, 65 DEG C: 10 seconds, 72 DEG C: 10 seconds, fluorescence signal collection was located at 65 DEG C, and signal collection passage is
" Green ", 45 circulations；
● HRM:95 DEG C: 2 minutes
83 DEG C to 96 DEG C (0.1 DEG C/step)
KatG detects program:
● 95 DEG C: 5 minutes；
● 95 DEG C: 15 seconds, 65 DEG C: 10 seconds, 72 DEG C: 10 seconds, fluorescence signal collection was located at 65 DEG C, and signal collection passage is
" Green ", 45 circulations；
● HRM:95 DEG C: 2 minutes
82 DEG C to 93 DEG C (0.1 DEG C/step)
InhA detects program:
● 95 DEG C: 5 minutes；
● 95 DEG C: 15 seconds, 65 DEG C: 10 seconds, 72 DEG C: 10 seconds, fluorescence signal collection was located at 65 DEG C, and signal collection passage is
" Green ", 45 circulations；
● HRM:95 DEG C: 2 minutes
80 DEG C to 94 DEG C (0.1 DEG C/step).
2. result and analysis
Analysis by two kinds of analytical models, it is possible to the sample substantially made a distinction with wild type control then judges that this part of sample exists
There is sudden change in gene test region, is otherwise judged as wild type.The results detailed in Fig. 1-3.Fig. 1 gives use PCR of the present invention and draws
Thing group (SEQ ID NO:5 and 6) applies high-resolution fusion curve analysis to detect sudden change after expanding rpoB gene
A result sectional drawing.Fig. 2 gives use PCR primer group of the present invention (SEQ ID NO:3 and 4) and expands katG gene
The result sectional drawing that sudden change is detected by high-resolution fusion curve analysis is applied after increasing.Fig. 3 gives use PCR of the present invention
Primer sets (SEQ ID NO:1 and 2) applies high-resolution fusion curve analysis to examine sudden change after expanding inhA gene
The result sectional drawing surveyed.
Knowable to Fig. 1-3, apply PCR primer group of the present invention can detect Drug Resistance of Mycobacterium Tuberculosis rapidly and accurately
Sudden change in gene mutation (inhA, katG and rpoB), it is possible to fairway is divided wild type control, heterozygous mutant and isozygotys prominent
Become, and consistent to testing result or the DNA sequencing result (result does not shows) of sputum sample with conventional drug sensitive test clinically.
Embodiment 2: the comparison between different PCR primer groups
Material, instrument and method used in the present embodiment and condition are all with embodiment 1, but employ table 4 below during PCR
Wild plasmid rpoB gene is expanded by two kinds of shown PCR primer groups.
Table 4
PCR primer group 3(primer sets of the present invention) and PCR primer group 4 between identical on reverse primer, forward primer is the least
Number of base is different.Identical wild plasmid sample uses the testing result quality of different PCR primer group (3 and 4) the most not
With, see Fig. 4.PCR primer group 4 result display NTC has serious non-specific amplification (Fig. 4 A Grey curves), and PCR primer group 3 is not
There is non-specific amplification.
Claims (10)
1. for PCR primer group based on high-resolution fusion curve technology for detection Drug Resistance of Mycobacterium Tuberculosis gene mutation, institute
State PCR primer group and include that at least one is selected from following primer sets:
(1) primer sets 1, it comprises and has the primer of the nucleotide sequence shown in SEQ ID NO:1 and have a SEQ ID NO:
The primer of the nucleotide sequence shown in 2；
(2) primer sets 2, it comprises and has the primer of the nucleotide sequence shown in SEQ ID NO:3 and have a SEQ ID NO:
The primer of the nucleotide sequence shown in 4；With
(3) primer sets 3, it comprises and has the primer of the nucleotide sequence shown in SEQ ID NO:5 and have a SEQ ID NO:
The primer of the nucleotide sequence shown in 6.
2. the PCR primer group of claim 1, wherein said PCR primer group includes primer sets 1, and optionally comprises at least one choosing
From primer sets 2 and the primer sets of primer sets 3.
3. the PCR primer group of claim 1, wherein said PCR primer group includes primer sets 2, and optionally comprises at least one choosing
From primer sets 1 and the primer sets of primer sets 3.
4. the PCR primer group of claim 1, wherein said PCR primer group includes primer sets 3, and optionally comprises at least one choosing
From primer sets 1 and the primer sets of primer sets 2.
5. one kind for test kit based on high-resolution fusion curve technology for detection Drug Resistance of Mycobacterium Tuberculosis gene mutation or
Microarray, it comprises the PCR primer group any one of claim 1-4.
6. the PCR primer group any one of claim 1-4 is used for based on high-resolution fusion curve technology for detection sample in preparation
In the test kit of Drug Resistance of Mycobacterium Tuberculosis gene mutation or purposes in microarray.
7. the purposes of claim 6, wherein said sample is biological sample, and the most described sample is humoral sample or tissue sample
Product, and the most described sample selected from biopsy samples, cell culture, cured process sample (such as paraffin embedding
Sample), whole blood, blood plasma, serum, saliva, brains liquid, perspiration, expectorant, bronchoalveolar lavage fluid, urine, feces, juice, milk and abdomen
Film liquid.
8. based on a method for Drug Resistance of Mycobacterium Tuberculosis gene mutation, institute in high-resolution fusion curve technology for detection sample
The method of stating includes:
(1) extract and the nucleic acid in optional purification of samples,
(2) primer sets any one of claim 1-4 is used to carry out PCR amplification, and
(3) PCR primer of step (2) is carried out high-resolution fusion curve analysis；
Wherein said method is not used in diagnostic purpose.
9. the method for claim 8, wherein said sample is biological sample, and the most described sample is humoral sample or tissue sample
Product, and the most described sample selected from biopsy samples, cell culture, cured process sample (such as paraffin embedding
Sample), whole blood, blood plasma, serum, saliva, brains liquid, perspiration, expectorant, bronchoalveolar lavage fluid, urine, feces, juice, milk and abdomen
Film liquid.
10. the method for claim 8 or 9, wherein the condition of high-resolution fusion curve analysis is: 95 DEG C 2 minutes, with 0.1 DEG C/
Walk and to 96 DEG C or to 93 DEG C or scan to 94 DEG C from 80 DEG C from 82 DEG C of scannings from 83 DEG C of scannings.
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