CN104164491A - ARMS-qPCR detection method and kit for helicobacter pylori 23S rDNA gene mutation subtype - Google Patents
ARMS-qPCR detection method and kit for helicobacter pylori 23S rDNA gene mutation subtype Download PDFInfo
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- CN104164491A CN104164491A CN201410347808.7A CN201410347808A CN104164491A CN 104164491 A CN104164491 A CN 104164491A CN 201410347808 A CN201410347808 A CN 201410347808A CN 104164491 A CN104164491 A CN 104164491A
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Abstract
The invention discloses an ARMS-qPCR detection method and kit for helicobacter pylori 23S rDNA gene mutation subtype. The invention provides a DNA molecular composition for identifying the helicobacter pylori 23S rDNA genotype, and the composition comprises a DNA molecular composition A and a DNA molecular composition B, which are individually used. The experiments have improved that the provided primer and kit comprising the primer can rapidly, accurately, and high-sensitively detect the mutations in specific sites of 23S rDNA gene of helicobacter pylori in gastric mucosa tissues. The kit has a high sensitivity, can be used to detect the biopsy tissues from gastric mucosa, and can also be used to detect the free DNA in wax-embedded tissue slices.
Description
Technical field
The invention belongs to biological technical field, relate in particular to detection method and test kit for the ARMS-qPCR of helicobacter pylori 23S rDNA gene mutation typing.
Background technology
Helicobacter pylori (Helicobaeter pylori, Hp) is a kind of common micro-aerobasilus of Grain-negative that can be colonizated in for a long time mankind's gastric mucosa, has closely and contacts with many disease of upper digestive tract.The whole world has the people who exceedes half to infect Hp, and is 55% at the infection rate of China.The infection of Hp causes great misery to patient, increases the weight of medical treatment burden.WHO Hp classify as with B-mode, hepatitis C is the same, is all biological carcinogens.Triple therapy with proton pump inhibitor (proton pump inhibitor, PPI) in conjunction with amoxycilline Trihydrate bp and clarithromycin (or metronidazole) is the anti-Hp treatment of infection combination of a line of generally acknowledging both at home and abroad.Clarithromycin belongs to macrolide antibiotics, has in vitro powerful germicidal action, and oral administration biaavailability is high, stable to acid.The antibacterial mechanisms of clarithromycin is that medicine penetrates in somatic cells, combines closely with rrna, acts on the polypeptide transfer loop of 23S rDNA V functional zone, suppress polypeptide transferring enzyme, affect ribosomal displacement process, stop peptide elongation, thus the protein synthesis of anti-bacteria.But, due to the continuous rising of the resistance of carat element, the decline gradually of the success ratio of a roentgenism x.
The resistance of bacterium can be divided into primary drug resistance and Secondary cases resistance.Primary drug resistance is because bacterium lacks the target position to medicaments insensitive, or bacterium has the natural cover for defense and make medicine cannot enter thalline, and Secondary cases resistance refers to due to antibiotic and be widely used, and bacterium obtains drug resistant gene, becomes Resistant strain from sensitive strain.Resistance that it is generally acknowledged helicobacter pylori mostly is Secondary cases resistance.Helicobacter pylori has following several to the resistance mechanism of clarithromycin: 1, rDNA methylase causes clarithromycin inactivation; 2, cell membrane permeability decline reduces the medicine that enters bacterium; 3, clarithromycin is discharged increases; 4, transgenation: be mainly the V district origination point sudden change of helicobacter pylori 23S rDNA, cause rrna allosteric, the binding site of clarithromycin changes, and the avidity of helicobacter pylori and clarithromycin is weakened, and can not stop the synthetic resistance that produces of albumen of bacterium.Comprehensive above some, Chinese scholars generally believes that helicobacter pylori is due to 23S rDNA transgenation to the main mechanism of clarithromycin resistance, remaining machine-processed relativity a little less than.The people such as Taylor in 1997 studies confirm that the 2142nd (wherein adenine nucleotide replaces by cytosine(Cyt) or by guanine) in the peptidyl transferase district of encoding in the structural domain V that is located at 23S rDNA gene, or 3 point mutation of the 2143rd (wherein VITAMIN B4 is replaced by guanine), these sudden changes are closely related with clarithromycin resistance mechanism in this microorganism.Various countries scholar also unanimously finds that the sudden change of the 23S rDNA gene of the Helicobacter pylori Strains of clarithromycin resistance the most often appears as the sudden change of A2143G, A2142G, A2142C, also has report to find G2115A, T2182C sudden change.Raymond etc. find French 138 strain clarithromycin persister researchs: the mutation rate of A2143G, A2142G and A2142C accounts for respectively 81.9%, 14.5% and 3.6%.In addition, Liu etc. studies show that Beijing 65 strain clarithromycin persisters: the mutation rate of A2143G, A2142G and A2142C accounts for respectively 84.6%, 6.2% and 1.5%.Therefore, detect A2143G, A2142G and A2142C sudden change in Hp23SrDNA, the more than 90% clarithromycin resistance Hp strain of China can be detected.So A2143G, the A2142G in Hp bacterial strain 23rDNA and the sudden change of A2142C type can be used as the target spot of domestic detection Hp clarithromycin resistance.
Method for clarithromycin Drug Resistance Detection is a lot, comprises as medicament sensitivity test, DNA direct Sequencing, restriction fragment length polymorphism analysis (RFLP-PCR), fluorescence quantitative PCR method etc.The gold standard that wherein drug sensitive experiment is Drug Resistance Detection, but these class methods are to be based upon on microbial culture basis, its shortcoming is that the microbial culture time is long, fostering requirement is higher, clinically more difficult accomplishing.In recent years, quantitative fluorescent PCR is fast and convenient with it, specificity good, highly sensitive, can quantitatively, effectively solve PCR pollution problem and level of automation advantages of higher, aspect medical field, microorganism, the quarantine of animals and plants disease, be widely used.
Amplification refractory mutation system (amplification refractory mutation system ARMS) was set up in 1989, for known mutations gene is detected.This method is by the design ARMS primer that suddenlys change, and its 3 ' terminal bases is mated with the mutating alkali yl of saltant type to be detected, for specific amplification sudden change template.The detection sensitivity of ARMS depends on the specificity of ARMS primer and reaction conditions as enzyme, the optimization of magnesium ion concentration etc.In order to increase the specificity of primer, reduce the mispairing of primer and the wrong timing of target DNA and extend, can, by introducing another one or two base mismatch in 2-3 base of primer 3 ' end, make it to form multiple mispairing to stop mistake to be extended between template.
ARMS-qPCR technology, based on Real-time PCR platform, detects two kinds of technology in conjunction with ARMS sudden change enrichment and Taqman specificity fluorescent.Utilize ARMS primer pair sudden change target sequence to carry out pcr amplification, Taqman probe carries out the detection of specificity site to amplified production, on Real-time PCR basis, identifies specific sudden change.
Lock nucleic acid (locked nucleic acid, LNA) be a kind of novel few nucleic acid derivative, the 2-0 of B-D-ribofuranose in structure, 4C position forms annular Oxymethylene bridge, sulphur methylene bridge or amine methylene bridge by shrink effect, the structure of furanose is locked in the N configuration of type in C3, has formed the condensation structure of rigidity.LNA, as a kind of new antisense nucleic acid, has the advantage such as nontoxicity in the hybridization avidity powerful with DNA/RNA, antisense activity, nuclease-resistant ability, good water solubility and body.Thereby it can be combined specifically and can prepare LNA probe with DNA and RNA, compared with DNA probe, the stability of its hybridization and specificity increase, and can greatly improve efficiency and the sensitivity of gene diagnosis.
Summary of the invention
An object of the present invention is to provide the primer sets that whether contains mutational site ARMS-qPCR for the identification of helicobacter pylori 23S rDNA.
Primer sets provided by the invention is following 1) or 2):
1) formed by ARMS primer, the ARMS primer of A2142G saltant type of 23S rDNA and the ARMS primer of the A2142C saltant type of 23S rDNA of the A2143G saltant type of the 23S rDNA using separately;
2) formed by ARMS primer, the ARMS primer of A2142G saltant type of 23S rDNA and the ARMS primer of the A2142C saltant type of 23S rDNA of the A2143G saltant type of 23S rDNA;
The nucleotides sequence of the ARMS primer of the A2143G saltant type of described 23S rDNA is classified sequence 3 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142G saltant type of described 23S rDNA is classified sequence 4 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142C saltant type of described 23S rDNA is classified sequence 5 in sequence table as.
An object of the present invention is to provide the DNA molecular composition that whether contains mutational site ARMS-qPCR for the identification of helicobacter pylori 23S rDNA.
DNA molecular composition provided by the invention is following 1) or 2):
1) the DNA molecular composition shown in comprises DNA molecular composition A, DNA molecular composition B and the DNA molecular composition C of independent use;
Described DNA molecular composition A is made up of the ARMS primer of the A2143G saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR and 23S rDNA;
Described DNA molecular composition B is made up of the ARMS primer of the A2142G saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR and 23S rDNA;
Described DNA molecular composition C is made up of the ARMS primer of the A2142C saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR and 23S rDNA;
2) the DNA molecular composition E shown in is made up of ARMS primer, the ARMS primer of A2142G saltant type of 23S rDNA and the ARMS primer of the A2142C saltant type of 23S rDNA of the A2143G saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR, 23S rDNA;
The nucleotides sequence of described lock nucleic acid retardance probe is classified sequence 1 in sequence table as;
The nucleotides sequence of described universal TaqMan probe is classified sequence 2 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2143G saltant type of described 23S rDNA is classified sequence 3 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142G saltant type of described 23S rDNA is classified sequence 4 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142C saltant type of described 23S rDNA is classified sequence 5 in sequence table as;
The nucleotides sequence of the general downstream primer of described PCR is classified sequence 7 in sequence table as.
Above-mentioned DNA molecular composition also comprises described DNA molecular composition D;
Described DNA molecular composition D is made up of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR and internal control primer;
The nucleotides sequence of described internal control primer is classified sequence 6 in sequence table as.
Described mutational site is A2143G, A2142G or A2142C.
The nucleotides sequence of helicobacter pylori 23S rDNA wild type gene is classified the sequence 8 in sequence table as;
The A2143G mutational site of helicobacter pylori 23S rDNA is that the sequence 8 in sequence table sports G from the A of the 97th of 5 ' end;
The A2142G mutational site of helicobacter pylori 23S rDNA is that the sequence 8 in sequence table sports G from the A of the 96th of 5 ' end;
The A2142C mutational site of helicobacter pylori 23S rDNA is that the sequence 8 in sequence table sports C from the A of the 96th of 5 ' end;
In above-mentioned DNA molecular composition, in described DNA molecular composition A, the mol ratio of the ARMS primer of the A2143G saltant type of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described 23S rDNA is 4:2:5:5;
In described DNA molecular composition B, the mol ratio of the ARMS primer of the A2142G saltant type of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described 23S rDNA is 4:2:5:5;
In described DNA molecular composition C, the mol ratio of the ARMS primer of the A2142C saltant type of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described 23S rDNA is 4:2:5:5;
In described DNA molecular composition D, the mol ratio of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described internal control primer is 4:2:5:5.
Second object of the present invention is to provide the PCR reagent for the identification of the genotypic ARMS-qPCR of helicobacter pylori 23S rDNA.
PCR reagent provided by the invention is 1) or 2):
1) the PCR reagent shown in comprises PCR reagent A, PCR reagent B and PCR reagent C composition;
Described PCR reagent A is made up of pcr amplification reaction liquid, described DNA molecular composition A and water;
Described PCR reagent B is made up of pcr amplification reaction liquid, described DNA molecular composition B and water;
Described PCR reagent C is made up of pcr amplification reaction liquid, described DNA molecular composition C and water;
The final concentration of the ARMS primer of the A2143G saltant type of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described 23SrDNA in described PCR reagent A is followed successively by respectively: 0.4 μ M, 0.2 μ M, 0.5 μ M, 0.5 μ M;
The final concentration of the ARMS primer of the A2142G saltant type of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described 23SrDNA in described PCR reagent B is followed successively by respectively: 0.4 μ M, 0.2 μ M, 0.5 μ M, 0.5 μ M;
The final concentration of the ARMS primer of the A2142C saltant type of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, described 23SrDNA in described PCR reagent C is followed successively by respectively: 0.4 μ M, 0.2 μ M, 0.5 μ M, 0.5 μ M.
2) the PCR reagent shown in is PCR reagent E, and it is by pcr amplification damping fluid, 2) shown in DNA molecular composition and water form.
Described above 1) the PCR reagent shown in or described 2) shown in PCR reagent all also comprise PCR reagent D;
Described PCR reagent D is made up of pcr amplification damping fluid, described DNA molecular composition D and water;
Described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR, the final concentration of described internal control primer in described PCR reagent D are followed successively by respectively: 0.4 μ M, 0.2 μ M, 0.5 μ M, 0.5 μ M.
Described mutational site is A2143G, A2142G or A2142C.
In reaction system, the ARMS primer of the A2142C saltant type of the ARMS primer 2 3S rDNA of the ARMS primer of the A2143G saltant type of above-mentioned 23S rDNA, the A2142G saltant type of 23S rDNA is equimolar ratio; Its final concentration is 0.25 μ M in an embodiment of the present invention.
The 3rd object of the present invention is to provide the PCR test kit for the identification of the genotypic ARMS-qPCR of helicobacter pylori 23S rDNA.
PCR test kit provided by the invention, comprises it being above-mentioned DNA molecular composition or above-mentioned PCR reagent.
In above-mentioned DNA molecular composition or above-mentioned PCR reagent or above-mentioned PCR test kit, described mutational site is A2143G, A2142G or A2142C.
Whether above-mentioned DNA molecular composition or above-mentioned PCR reagent or above-mentioned PCR test kit contain the application that sports in site or are also the scope of protection of the invention in the application whether characterization or assistant identification helicobacter pylori 23S rDNA contain in the product of mutational site at qualification or assistant identification helicobacter pylori 23S rDNA;
Or whether the helicobacter pylori 23S rDNA that contains in qualification or assistant identification sample to be tested of above-mentioned DNA molecular composition or above-mentioned PCR reagent or above-mentioned PCR test kit contains the application in mutational site or whether the helicobacter pylori 23S rDNA that contains in characterization or assistant identification sample to be tested contains the application in the product in mutational site;
Or application in above-mentioned DNA molecular composition or above-mentioned PCR reagent or the above-mentioned PCR test kit product in the whether resistance to clarithromycin of sample to be tested that characterization or assistant identification contain helicobacter pylori;
Or application in above-mentioned DNA molecular composition or above-mentioned PCR reagent or the above-mentioned PCR test kit product in characterization or the whether resistance to clarithromycin of assistant identification helicobacter pylori to be measured.
In above-mentioned application, described mutational site is A2143G, A2142G or A2142C.
The 4th object of the present invention is to provide the method whether helicobacter pylori 23SrDNA that a kind of qualification or assistant identification sample to be tested contain contains mutational site ARMS-qPCR.
Method provided by the invention, comprises the steps:
1) method shown in comprises the steps:
Respectively sample to be tested is carried out to ARMS-qPCR by described PCR reagent A, described PCR reagent B, described PCR reagent C and described PCR reagent D, detect amplified production;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition A amplification S type curve and itself and DNA molecular composition D amplification curve Δ Ct≤7, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained A2143G mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition B amplification S type curve and itself and DNA molecular composition D amplification curve Δ Ct≤7, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained A2142G mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and amplification curve Δ Ct≤7 of DNA molecular composition C amplification S type curve and itself and DNA molecular composition D, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained A2142C mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition A, B and C are all without amplification curve; Or DNA molecular composition D amplification S type curve and Ct≤32, and the amplification curve of DNA molecular composition A, B and C and DNA molecular composition D amplification curve Δ Ct > 7; In sample to be tested, helicobacter pylori 23S rDNA does not contain or candidate is not contained mutational site;
2) method shown in comprises the steps:
Respectively sample to be tested is carried out to ARMS-qPCR by described PCR reagent E and described PCR reagent D, detect amplified production;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition E has amplification curve Δ Ct≤7 of S type amplification curve and itself and DNA molecular composition D, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained 23S rDNA mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition E is without the amplification curve Δ Ct > 7 of S type amplification curve and DNA molecular composition D, in sample to be tested, helicobacter pylori 23S rDNA does not contain or candidate is not contained 23S rDNA mutational site;
Described mutational site is A2143G, A2142G or A2142C.The oligonucleotide of probe used in the present invention two ends difference mark fluorescent reporter groups (R) and fluorescent quenching group (Q) is the TaqMan probe of 5 ' end FAM mark.In the time that probe is complete, i.e. random state and during without PCR product hybridization state, the fluorescence that reporter group sends is quenched group and absorbs.In ARMS-qPCR amplification procedure, in the time of special PCR product and TaqMan probe generation hybridization, 5 ' of Taq enzyme end 5 prime excision enzyme activity is simultaneously also probe cracking, and the photofluorometer that the fluorescence that reporter group discharges just can be built in instrument detects.PCR is every through a circulation, and fluorescent signal is also the same with object fragment, the process that has sync index to increase, the power of fluorescent signal just represented template DNA copy number number.Therefore the present invention not only can be used for simple qualitative detection, also can be used as the detection by quantitative of the concrete content of sample.
3 ' section of P04 of lock nucleic acid retardance probe mark.
Described sample to be tested is in vitro helicobacter pylori Stomach in Patients mucosal tissue sample.
Of the present invention experimental results show that, helicobacter pylori 23S rDNA gene specific locus mutation in quick, accurate, the high sensitive detection mucosa tissue of test kit energy of the primer of the present invention's design and formation thereof, highly sensitive, can further predict that it is to clarithromycin resistance.
The lock nucleic acid retardance probe that the present invention adopts, its sequence is mated completely with the wild template of helicobacter pylori 23S rDNA gene, part base lock nucleination, for the Hp23S rDNA wild-type DNA of specific binding template DNA, because of its terminal phosphate, thereby cannot extend the amplification of mutation inhibiting district wild-type DNA, greatly increase sensitivity and the specificity that sudden change detects.
Therefore, the present invention has following advantage for detection of 23S rDNA transgenation:
1, high specificity: the ARMS primer of design is respectively for the three kinds of mutational site sequences of A2143G, A2142G and A2142C that comprise 23S rDNA gene, can the corresponding sudden change template DNA of specific amplification; 3 ' end 2-3 reciprocal position at ARMS primer increases one or two base mispairings, can increase the specificity of ARMS primer; The technology of the present invention adds the lock nucleic acid retardance probe of wild-type 23S rDNA gene specific in PCR reaction system, thereby by suppressing the amplification of wild type gene group DNA cloning enrichment mutant DNA, has further improved the specificity detecting.
2, susceptibility is high: the technology of the present invention adds wild-type 23S rDNA gene LNA retardance probe in PCR reaction solution, by suppressing specifically the amplification of wild-type template, thereby reaches the inrichment to trace sudden change template, improves detection sensitivity.This technology for detection 23S rDNA transgenation susceptibility reaches 0.1-1% (being that the 0.1-1% that mutator gene group DNA reaches the total DNA of gene can detect); And direct sequencing detects the susceptibility of 23S rDNA sudden change and is about 10% (be mutator gene group DNA reach 10% of the total DNA of gene just can detect).
3, testing process is stopped pipe reaction, greatly reduces the possibility of pollution and result error.
4, simple to operate quick, can in 3 hours, complete to obtaining a result from specimen transfer.And direct sequencing detecting step is loaded down with trivial details: censorship sample → extraction DNA → pcr amplification → checking PCR product (electrophoresis) → purified pcr product → direct Sequencing, and the electrophoresis process of two PCR products of its experience, opportunities for contamination is large, is not suitable for carrying out on a large scale in hospital.
5, result interpretation is clear and definite, objective; Can carry out accurate somatotype to different mutation types fast, if desired also can carry out quantitative analysis to result.
6, high-throughput, once can detect at most 48 examples.
7, safety: in whole system, do not comprise hazardous and noxious substances, without the aftertreatment of PCR product, to operator and environment all without harm.
Brief description of the drawings
Fig. 1 is the amplification curve diagram that detects the 23S rDNAA2143G genic mutation type of Helicobacter pylori sample 1 with ARMS-qPCR detection kit somatotype
Fig. 2 is the amplification curve diagram that detects the 23S rDNAA2142G genic mutation type of Helicobacter pylori sample 2 with ARMS-qPCR detection kit somatotype
Fig. 3 is the amplification curve diagram that detects the 23S rDNAA2142C genic mutation type of Helicobacter pylori sample 3 with ARMS-qPCR detection kit somatotype
Fig. 4 is the amplification curve diagram that detects the 23S rDNA gene wild-type of Helicobacter pylori sample 4 with ARMS-qPCR detection kit somatotype
Fig. 5 is the amplification curve diagram that mixes the 23S rDNA genic mutation type that detects Helicobacter pylori sample 1-3 by ARMS-qPCR detection kit
Fig. 6 is the amplification curve diagram that mixes the 23S rDNA gene wild-type that detects Helicobacter pylori sample 4 by ARMS-qPCR detection kit
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The design of embodiment 1, ARMS-qPCR detection kit and primer special and probe
The nucleotides sequence of helicobacter pylori 23S rDNA wild type gene is classified the sequence 8 in sequence table as;
The A2143G mutational site of helicobacter pylori 23S rDNA is that the sequence 8 in sequence table sports G from the A of the 97th of 5 ' end;
The A2142G mutational site of helicobacter pylori 23S rDNA is that the sequence 8 in sequence table sports G from the A of the 96th of 5 ' end;
The A2142C mutational site of helicobacter pylori 23S rDNA is that the sequence 8 in sequence table sports C from the A of the 96th of 5 ' end;
One, primer and probe
Use sequence area designing probe and the primer of Primer Express Version3 software two sudden changes.Designing and screen can specific detection the 2143rd and one article of the ARMS primer of three kinds of (A2143G, A2142G, A2142C) base substitution mutations in the 2,142 two site each one article and internal control primer, design one of general downstream primer, design and screen one of universal TaqMan probe, design and screen one of lock nucleic acid retardance probe, each probe, primer sequence are distinguished specific as follows:
Lock the nucleotide sequence of nucleic acid retardance probe as shown in sequence in sequence table 1, its 3 ' end mark PO4;
The nucleotide sequence of universal TaqMan probe as shown in sequence in sequence table 2, its 5 ' end flag F AM, 3 ' end mark BHQ1;
For detection of the saltant type ARMS primer of 23S rDNA by the ARMS primer of the A2143G saltant type for detection of 23S rDNA, form for detection of the ARMS primer of the A2142G saltant type of 23S rDNA with for detection of the ARMS primer of the A2142C saltant type of 23S rDNA, wherein
For detection of the Nucleotide of the ARMS primer of the A2143G saltant type of 23S rDNA as shown in sequence 3;
For detection of the Nucleotide of the ARMS primer of the A2142G saltant type of 23S rDNA as shown in sequence 4;
For detection of the Nucleotide of the ARMS primer of the A2142C saltant type of 23S rDNA as shown in sequence 5;
Internal control primer is the upstream primer of can simultaneously increase 23S rDNA wild-type and a mutated genes group DNA, and its Nucleotide is as shown in sequence 6;
The Nucleotide of the general downstream primer of PCR is as shown in sequence 7.
Transfer to Synesis Company synthetic the primer designing and probe sequence, generally adopt automation equipment to learn synthetic, synthetic qualification test report need be provided.
Two, the reagent of helicobacter pylori quantitative fluorescent PCR or test kit preparation
1, the optimization of reaction system
(1) optimization of primer concentration: in the situation that other condition of reaction system is identical, primer concentration is done to multiple proportions serial dilution from 0.1 μ M to 1.0 μ M respectively, by the analysis comparison to test-results, determine that best primer concentration is 0.5 μ M.
(2) optimization of concentration and probe concentration: in the situation that other conditions of reaction system are identical, concentration and probe concentration is done to multiple proportions serial dilution from 0.05 μ M to 0.5 μ M respectively, by the analysis comparison to test-results, determine that best concentration and probe concentration is 0.2 μ M.
(3) optimization of enzyme: add various enzymes in reaction system, by the analysis comparison to test-results, determine that best enzyme is EXTaq enzyme.
(4) optimization of magnesium ion concentration: in the situation that other condition of reaction system is identical, qPCR reaction solution component magnesium ion concentration is done to multiple proportions serial dilution from 0.5mM to 4.0mM respectively, by the analysis comparison to test-results, determine that best magnesium ion concentration is 2mM.
(5) optimization of annealing temperature: in the situation that other condition of reaction system is identical, carry out grads PCR (56-62 DEG C of annealing temperature), by the analysis comparison to test-results, determine that optimum annealing temperature is 58 DEG C.
Through optimizing, the PCR reagent whether suddenling change for detection of the 23S rDNA in sample to be tested is 1) or 2):
1) formed by PCR reagent A-D:
PCR reagent A is A2143G detector tube reaction system: 20 μ L, are made up of ARMS primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2143G.
PCR reagent B is A2142G detector tube reaction system: 20 μ L, are made up of ARMS primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2142G.
PCR reagent C is A2142C detector tube reaction system: 20 μ L, are made up of ARMS primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2142C.
PCR reagent D is the reaction system of interior keyholed back plate, identical with detector tube, and different is that ARMS primer is replaced with to internal control primer.
2) formed by PCR reagent E and D:
The PCR reagent whether suddenling change for detection of sample to be tested 23S rDNA comprises PCR reagent E and PCR reagent D;
PCR reagent E is that the reaction system of detector tube is 20 μ L, is made up of the each 0.5 μ L (final concentration is) of ARMS primer, the ARMS primer of A2142G and the ARMS primer of A2142C, universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2143G.
Above-mentioned qPCR hybrid reaction fluid component and final concentration thereof are in table 1.
Table 1 is qPCR reaction solution component
| Component | Final concentration |
| MgCl 2 | 2mM |
| Taq archaeal dna polymerase | 5U/μL |
| dNTPs | 2.5mM |
| Reaction buffer | 10× |
2, test kit composition
Can using above-mentioned lock nucleic acid retardance probe, universal TaqMan probe, for the ARMS primer of the A2143G of 23S rDNA or for the ARMS primer of the A2142G of 23S rDNA or for ARMS primer, internal control primer, the general downstream primer of PCR and the water of the A2142C of 23S rDNA the chief component as test kit, build and obtain test kit;
In test kit, specifically contain following reagent and material:
(1) primer solution: a, lock nucleic acid retardance probe (sequence 1); B, universal TaqMan probe (sequence 2); C, ARMS primer (sequence 3 or sequence 4 or sequence 5 or sequence 3+ sequence 4+ sequence 5); D, internal control primer (sequence 6); The general downstream primer of e, PCR (sequence 7).
(2) qPCR reaction solution: comprise 10 × PCR damping fluid; DNTPs; MgCl
2solution; EXTaq enzyme.
(3) positive quality control: be 23S rDNA Gene A 2143G, the A2142G of synthetic and the mixing plasmon DNA of tri-kinds of saltant types of A2142C.
(4) negative Quality Control: aseptic double-distilled water
(5) aseptic double-distilled water
The genotypic ARMS-qPCR sudden change of embodiment 2, helicobacter pylori 23S rDNA detection method
One, detect
1, sample preparation
From in vitro people's mucosa tissue sample, obtain DNA, the business-like test kit of recommendation extracts genomic dna as sample 1-4;
Measure DNA concentration and purity, require concentration to be greater than 10ng/ μ L; OD260nm/OD280nm=1.8～2.0.
2, fluorescent PCR detects
1) ARMS primer somatotype detects
React respectively four pipes corresponding A 2143G detector tube (ep manages A), A2142G detector tube (ep manages B), A2142C detector tube (ep manages C), interior keyholed back plate (ep manages D) respectively at four pipe A-D for each sample standard deviation in sample 1-4.In detector tube (A-C), reaction adds qPCR reaction solution, LNA retardance probe, universal TaqMan probe, general downstream primer, template DNA and corresponding ARMS primer; (D) is basic identical for interior keyholed back plate, and difference is to add internal control primer to substitute ARMS primer, specific as follows:
Ep pipe A (PCR reagent A): 20 μ L reaction systems, are made up of ARMS primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2143G;
Ep pipe B (PCR reagent B): 20 μ L reaction systems, are made up of ARMS primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2142G;
Ep pipe C (PCR reagent C): 20 μ L reaction systems, are made up of ARMS primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2142C;
Ep pipe D (PCR reagent D): 20 μ L reaction systems, are made up of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), internal control primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and water.
2) ARMS primer mixes detection
Each sample standard deviation of sample 1-4 reacts at two pipes E, D respectively, and every pipe is corresponding detector tube (ep manages E), interior keyholed back plate (ep manages D) respectively.Detect inner reaction tube and add qPCR reaction solution, LNA retardance probe, universal TaqMan probe, general downstream primer, template DNA and three kinds of ARMS primers; (D) is basic identical for interior keyholed back plate, and difference is to add internal control primer to substitute ARMS primer, specific as follows:
Ep pipe E (PCR reagent E): 20 μ L reaction systems, are made up of ARMS primer 0.5 μ L (final concentration 0.25 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and the water of ARMS primer 0.5 μ L (final concentration 0.25 μ M), the A2142C of ARMS primer 0.5 μ L (final concentration 0.25 μ M), the A2142G of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), A2143G;
Ep pipe D (PCR reagent D): 20 μ L reaction systems, are made up of qPCR mixed reaction solution 10 μ L, general downstream primer 1 μ L (final concentration 0.5 μ M), internal control primer 1 μ L (final concentration 0.5 μ M), universal TaqMan probe 0.4 μ L (final concentration 0.2 μ M), lock nucleic acid retardance probe 0.8 μ L (final concentration 0.4 μ M), template DNA 5 μ L (final concentration 1-300ng/ μ L) and water.
PCR reaction conditions is: 95 DEG C of denaturations 10 minutes, 95 DEG C 15 seconds, 58 DEG C 40 seconds, amplified reaction 40 circulates, and collects fluorescence 58 DEG C of 40 second stages.
ARMS-qPCR carries out on ABI7500 detector.
3, result interpretation:
If internal control primer pipe amplification curve feminine gender or Ct > 32, point out this sample extraction failure, need to again extract.
ARMS primer somatotype detected result as Figure 1-4, can find out,
For sample 1, internal control primer pipe D amplification S type curve and Ct≤32, ARMS primer pipe A has amplification curve Δ Ct≤7 of amplification S type curve and ARMS primer pipe A and internal control primer pipe D, and sample to be tested contains or candidate is contained A2143G mutational site (Fig. 1 is 3 ARMS primer detector tubes of sample 1 and the amplification figure of interior keyholed back plate);
For sample 2, internal control primer pipe D amplification S type curve and Ct≤32, ARMS primer pipe B has amplification curve Δ Ct≤7 of amplification S type curve and ARMS primer pipe B and internal control primer pipe D, and sample to be tested result contains or candidate is contained A2142G mutational site (Fig. 2 is 3 ARMS primer detector tubes of sample 2 and the amplification figure of interior keyholed back plate);
For sample 3, internal control primer pipe D amplification S type curve and Ct≤32, ARMS primer pipe C has amplification curve Δ Ct≤7 of amplification S type curve and ARMS primer pipe C and internal control primer pipe D, and sample to be tested contains or candidate is contained A2142C mutational site (Fig. 3 is 3 ARMS primer detector tubes of sample 3 and the amplification figure of interior keyholed back plate);
For sample 4, internal control primer pipe D amplification S type curve and Ct≤32, three kinds of ARMS primer pipe A, B, C are all without amplification curve, sample to be tested does not contain or candidate is not contained an arbitrary mutational site in A2143G, A2142G, A2142C, and it is 23S rDNA wild type gene (Fig. 4 is 3 ARMS primer detector tubes of sample 4 and the amplification figure of interior keyholed back plate);
Respectively taking the genomic dna of sample 1-4 as template, with 5 '---the 23S rDNA gene object fragment of ATGAATGGCGTAACGA---3 ' primer forward order-checking sample 1-4, result is consistent with method of the present invention, proves that method of the present invention is correct.
ARMS primer mixes detected result as shown in Fig. 5-6, can find out,
For sample 1-3, internal control primer pipe D amplification S type curve and Ct≤32, in ARMS primer mixing tube E, have amplification S type curve and amplification curve Δ Ct≤7 with internal control primer pipe D, sample to be tested result is or candidate is 23S rDNA mutated genes (Fig. 5 is the ARMS detector tube of sample 1-3 and the amplification figure of interior keyholed back plate);
For sample 4, internal control primer amplification S type curve and Ct≤32, ARMS primer mixing tube E without amplification curve or with the amplification curve Δ Ct > 7 of internal control primer, sample to be tested result for or candidate be 23S rDNA wild type gene (Fig. 6 is the ARMS detector tube of sample 4 and the amplification figure of interior keyholed back plate).
Two, specific detection
38 strains of preserving with embodiment 1ARMS primer genotyping detection method testing laboratory are increased through the 23S rDNA sequence of the helicobacter pylori clinical separation strain of order-checking known mutations type, result is except negative control, all the other are to corresponding DNA mutation or do not suddenly change and form the amplification that is positive, and reach 100% with sequencing result coincidence rate.
Three, sensitivity detects
Be connected respectively to pMD18-T carrier as template taking the corresponding sequence helicobacter pylori 23S rDNA wild type gene of synthetic, A2143G type mutated genes, the A2142G type mutated genes of helicobacter pylori 23S rDNA and the A2142C type mutated genes of helicobacter pylori 23S rDNA of helicobacter pylori 23S rDNA, it is 10～10 that concentration gradient is all diluted
6cFU is template.
ARMS primer genotyping detection method according to above-mentioned one carries out real-time PCR, and each template concentrations gradient is done 3 Duplicate Samples, and each template detects with detector tube corresponding to its mutation type, when system standard template amount is 10～10
6when CFU, 3 Duplicate Samples of each concentration gradient positive that all increases, the detection lower limit of three kinds of ARMS primer pipe A, B, C is 10CFU, and its sensitivity is 10CFU.
Whether therefore, can detect helicobacter pylori 23S rDNA gene in sample to be tested with the PCR reagent A-D for detection of whether 23S rDNA suddenlys change in sample to be tested in embodiment 1 suddenlys change or detects the whether resistance to clarithromycin of sample to be tested; And can carry out specificity somatotype to sudden change positive sample.
If internal control primer amplification S type curve and Ct≤32, and for detection of amplification curve Δ Ct≤7 of the A2143G saltant type ARMS primer amplification S type curve of 23S rDNA and itself and internal control primer, in sample to be tested, helicobacter pylori 23SrDNA contains or candidate is contained A2143G mutational site, sample to be tested be or candidate for to clarithromycin resistance;
If internal control primer amplification S type curve and Ct≤32, and for detection of the A2142G saltant type ARMS primer amplification S type curve of 23S rDNA and itself and internal control primer amplification curve Δ Ct≤7, in sample to be tested, helicobacter pylori 23SrDNA contains or candidate is contained A2142G mutational site, sample to be tested be or candidate for to clarithromycin resistance;
If internal control primer amplification S type curve and Ct≤32, and for detection of amplification curve Δ Ct≤7 of the A2142C saltant type ARMS primer amplification S type curve of 23S rDNA and itself and internal control primer, in sample to be tested, helicobacter pylori 23SrDNA contains or candidate is contained A2142C mutational site, sample to be tested be or candidate for to clarithromycin resistance;
If internal control primer amplification S type curve and Ct≤32, and for detection of A2143G saltant type ARMS primer, A2142G saltant type ARMS primer and the A2142C saltant type ARMS primer of 23S rDNA all without amplification curve or with the equal Δ Ct of the amplification curve > 7 of internal control primer, sample to be tested 23S rDNA does not contain or candidate is not contained an arbitrary mutational site in A2143G, A2142G, A2142C, shows as clarithromycin sensitivity.
Whether also can mix helicobacter pylori 23S rDNA gene in detection sample to be tested by the PCR reagent E of whether suddenling change for detection of 23S rDNA in sample to be tested in embodiment 1 and PCR reagent D suddenlys change or detects the whether resistance to clarithromycin of sample to be tested.
If internal control primer amplification S type curve and Ct≤32, and there are S type amplification curve and amplification curve Δ Ct≤7 with internal control primer for detection of the ARMS mix primer of 23S rDNA, sample to be tested 23S rDNA contains or candidate is contained mutational site, described mutational site is A2143G, A2142G or A2142C, and sample to be tested shows as clarithromycin resistance;
If internal control primer amplification S type curve and Ct≤32, and for detection of the ARMS mix primer of 23S rDNA without amplification curve or with the amplification curve Δ Ct > 7 of internal control primer, sample to be tested 23S rDNA does not contain or candidate is not contained mutational site, and sample to be tested shows as clarithromycin sensitivity.
The clinical application of embodiment 3, ARMS-qPCR test kit
1, sample preparation
The 50 routine patients' (patient's feelings, are numbered 1-50) that collection Clinicopathologic Diagnosis is Helicobacter pylori the section of paraffin embedding mucosa tissue or freezing mucosa tissue piece extract genomic dna from tissue.
Measure DNA concentration and purity, require concentration to be greater than 10ng/ μ L; OD260nm/OD280nm=1.8～2.0.
2, fluorescent PCR detects
Detect and carry out fluorescent PCR detection according to the ARMS primer somatotype in one 2 the method for embodiment 2.
Result is as follows:
In the 50 routine patients that are Helicobacter pylori at Clinicopathologic Diagnosis, detect altogether the A2143G saltant type positive sample (being numbered 1-13) of 13 routine 23S rDNA, the A2142G saltant type sudden change positive sample (being numbered 14) of 1 routine 23S rDNA, all the other are wild-type sample (being numbered 15-50), because the mutation rate of the A2142C of 23S rDNA is lower, therefore, in this experiment, do not detect the A2142C saltant type positive sample of 23S rDNA, other each sudden changes or wild-type are through sequence verification, and result is consistent.
Above-mentioned wild-type sample, A2143G sudden change positive sample, A2142G sudden change positive sample are carried out to clarithromycin culture method Drug Resistance Detection, and result A2143G sudden change is positive, all resistance to clarithromycins of the A2142G sudden change positive, wild-type sample clarithromycin sensitivity.
The above results shows to apply primer of the present invention, probe in detecting mucosa tissue 23S rDNA transgenation is highly sensitive, can detect the trace sudden change template (0.1-1%) in clinical sample; Reliable results, with the result coincidence rate > 95% (being 100%) of sudden change detection " gold standard " direct sequencing.In addition the method detection speed is fast, simple to operate, and result interpretation is objective, and stopped pipe reaction is polluted few, is very suitable for carrying out on a large scale in clinical.
Claims (10)
1. whether containing the primer sets of mutational site ARMS-qPCR for the identification of helicobacter pylori 23S rDNA, is following 1) or 2):
1) formed by ARMS primer, the ARMS primer of A2142G saltant type of 23S rDNA and the ARMS primer of the A2142C saltant type of 23S rDNA of the A2143G saltant type of the 23S rDNA using separately;
2) formed by ARMS primer, the ARMS primer of A2142G saltant type of 23S rDNA and the ARMS primer of the A2142C saltant type of 23S rDNA of the A2143G saltant type of 23S rDNA;
The nucleotides sequence of the ARMS primer of the A2143G saltant type of described 23S rDNA is classified sequence 3 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142G saltant type of described 23S rDNA is classified sequence 4 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142C saltant type of described 23S rDNA is classified sequence 5 in sequence table as.
2. whether containing the DNA molecular composition of mutational site ARMS-qPCR for the identification of helicobacter pylori 23S rDNA, is following 1) or 2):
1) the DNA molecular composition shown in comprises DNA molecular composition A, DNA molecular composition B and the DNA molecular composition C of independent use;
Described DNA molecular composition A is made up of the ARMS primer of the A2143G saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR and 23S rDNA;
Described DNA molecular composition B is made up of the ARMS primer of the A2142G saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR and 23S rDNA;
Described DNA molecular composition C is made up of the ARMS primer of the A2142C saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR and 23S rDNA;
2) the DNA molecular composition shown in is made up of ARMS primer, the ARMS primer of A2142G saltant type of 23S rDNA and the ARMS primer of the A2142C saltant type of 23S rDNA of the A2143G saltant type of locking nucleic acid retardance probe, universal TaqMan probe, the general downstream primer of PCR, 23S rDNA;
The nucleotides sequence of described lock nucleic acid retardance probe is classified sequence 1 in sequence table as;
The nucleotides sequence of described universal TaqMan probe is classified sequence 2 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2143G saltant type of described 23S rDNA is classified sequence 3 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142G saltant type of described 23S rDNA is classified sequence 4 in sequence table as;
The nucleotides sequence of the ARMS primer of the A2142C saltant type of described 23S rDNA is classified sequence 5 in sequence table as;
The nucleotides sequence of the general downstream primer of described PCR is classified sequence 7 in sequence table as.
3. DNA molecular composition according to claim 2, is characterized in that: described DNA molecular composition all also comprises described DNA molecular composition D;
Described DNA molecular composition D is made up of described lock nucleic acid retardance probe, described universal TaqMan probe, the general downstream primer of described PCR and internal control primer;
The nucleotides sequence of described internal control primer is classified sequence 6 in sequence table as.
4. according to the DNA molecular composition described in claim 2 or 3, it is characterized in that:
Described mutational site is A2143G, A2142G or A2142C.
5. the PCR reagent that whether contains mutational site ARMS-qPCR for the identification of helicobacter pylori 23S rDNA is 1) or 2):
1) the PCR reagent shown in comprises PCR reagent A, PCR reagent B and PCR reagent C composition;
Described PCR reagent A is made up of pcr amplification damping fluid, described DNA molecular composition A and water;
Described PCR reagent B is made up of pcr amplification damping fluid, described DNA molecular composition B and water;
Described PCR reagent C is made up of pcr amplification damping fluid, described DNA molecular composition C and water;
2) the PCR reagent shown in is PCR reagent E, and it is by pcr amplification damping fluid, 2) shown in DNA molecular composition and water form.
6. PCR reagent according to claim 5, is characterized in that: the PCR reagent described 1) or described 2) shown in PCR reagent all also comprise PCR reagent D;
Described PCR reagent D is made up of pcr amplification damping fluid, described DNA molecular composition D and water;
Described mutational site is A2143G, A2142G or A2142C.
7. whether contain the PCR test kit of mutational site ARMS-qPCR for the identification of helicobacter pylori 23S rDNA, comprise the PCR reagent described in arbitrary described DNA molecular composition or claim 5 or 6 in primer sets claimed in claim 1, claim 2-4.
8. according to PCR reagent or PCR test kit claimed in claim 7 described in arbitrary described DNA molecular composition or claim 5 or 6 in claim 2-4, it is characterized in that: described mutational site is A2143G, A2142G or A2142C.
9. in primer sets claimed in claim 1, claim 2-4, whether the PCR reagent described in arbitrary described DNA molecular composition or claim 5 or 6 or PCR test kit claimed in claim 7 contain the application sporting in site or whether contain the application in the product of mutational site at characterization or assistant identification helicobacter pylori 23S rDNA in qualification or assistant identification helicobacter pylori 23S rDNA;
Or whether the helicobacter pylori 23S rDNA that in primer sets claimed in claim 1, claim 2-4, the PCR reagent described in arbitrary described DNA molecular composition or claim 5 or 6 or PCR test kit claimed in claim 7 contain in qualification or assistant identification sample to be tested contains the application in mutational site or whether the helicobacter pylori 23S rDNA that contains in characterization or assistant identification sample to be tested contains the application in the product in mutational site;
Or the application in the PCR reagent described in arbitrary described DNA molecular composition or claim 5 or 6 or PCR test kit claimed in claim 7 product in the whether resistance to clarithromycin of sample to be tested that characterization or assistant identification contain helicobacter pylori in primer sets claimed in claim 1, claim 2-4;
Or the PCR reagent described in arbitrary described DNA molecular composition or claim 5 or 6 or the application in the product of PCR test kit claimed in claim 7 in characterization or the whether resistance to clarithromycin of assistant identification helicobacter pylori to be measured in primer sets claimed in claim 1, claim 2-4;
Described mutational site is specially A2143G, A2142G or A2142C.
10. whether the helicobacter pylori 23S rDNA that qualification or assistant identification sample to be tested contain contains a method of mutational site ARMS-qPCR, is following 1) or 2):
1) method shown in comprises the steps:
Respectively sample to be tested is carried out to ARMS-qPCR by described PCR reagent A, described PCR reagent B, described PCR reagent C and described PCR reagent D, detect amplified production;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition A amplification S type curve and itself and DNA molecular composition D amplification curve Δ Ct≤7, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained A2143G mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition B amplification S type curve and itself and DNA molecular composition D amplification curve Δ Ct≤7, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained A2142G mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and amplification curve Δ Ct≤7 of DNA molecular composition C amplification S type curve and itself and DNA molecular composition D, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained A2142C mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition A, B and C are all without amplification curve; Or DNA molecular composition D amplification S type curve and Ct≤32, and the amplification curve of DNA molecular composition A, B and C and DNA molecular composition D amplification curve Δ Ct > 7; In sample to be tested, helicobacter pylori 23S rDNA does not contain or candidate is not contained mutational site;
2) method shown in comprises the steps:
Respectively sample to be tested is carried out to ARMS-qPCR by described PCR reagent E and described PCR reagent D, detect amplified production;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition E has amplification curve Δ Ct≤7 of S type amplification curve and itself and DNA molecular composition D, in sample to be tested, helicobacter pylori 23S rDNA contains or candidate is contained 23S rDNA mutational site;
If DNA molecular composition D amplification S type curve and Ct≤32, and DNA molecular composition E is without the amplification curve Δ Ct > 7 of S type amplification curve and DNA molecular composition D, in sample to be tested, helicobacter pylori 23S rDNA does not contain or candidate is not contained 23S rDNA mutational site;
Described mutational site is A2143G, A2142G or A2142C.
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