CN107201410A - ARMS qPCR methods and kit for helicobacter pylori individuation genetic test - Google Patents
ARMS qPCR methods and kit for helicobacter pylori individuation genetic test Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The present invention discloses a kind of ARMS qPCR Primer compositions for helicobacter pylori individuation genetic test, including primer combination A, primer combination B, primer combination C, primer combination D, primer combination E and primer combination F.The invention also discloses the reagent comprising above-mentioned Primer composition or kit.The ARMS qPCR methods for helicobacter pylori individuation genetic test are further disclosed again.The present invention is based on ARMS qPCR platforms, designs primer for the mutational site of the 23S rRNA genes and gyrA genes of people CYP2C19 genes and helicobacter pylori, improves the specificity of amplification；The present invention, so as to be enriched with to micro template, improves the sensitivity of detection by specific amplification of nucleic acid DNA, can detect the sample DNA of the mutation in Hp gene-correlations site 0.1 1% and as little as 1ng people's CYP2C19 genes.
Description
Technical Field
The invention relates to the field of biotechnology. More particularly, relates to an ARMS-qPCR method and a kit for detecting helicobacter pylori individuation genes.
Background
Helicobacter pylori (Hp) is a microaerophilic Helicobacter, and Hp infection is closely associated with diseases such as chronic active gastritis, peptic ulcer and gastric cancer. Nearly half of the world population is reported to be infected by Hp, China also belongs to a high Hp incidence area, and the average infection rate is up to 40-80%. At present, factors influencing the curative effect of individualized eradication therapy of Hp infection mainly comprise two aspects: host and antibiotic resistance. The host mainly comprises the genetic polymorphism of CYP2C19, which can obviously influence the metabolism level and the acid inhibition effect of Proton Pump Inhibitors (PPI) and has influence on the curative effect of eliminating Hp; the Hp antibiotic resistance mainly takes the resistance of clarithromycin and levofloxacin in triple therapy as main drugs, wherein point mutations related to the resistance of clarithromycin mainly comprise A2142G and A2143G sites of 23S rRNA gene; amino acid substitutions at two sites of N87K and D91N/G/Y of the gyrA gene are main sites for causing Hp to resist levofloxacin.
Amplification hindered mutation system (ARMS) is the first established method by Newton et al for detecting known mutations, in which the designed mutation primer is matched with the 3' end mutation base of the site to be detected for specific detection of mutation. To improve primer specificity, one or two mismatched bases can be introduced at the 2 nd and 3 rd bases of the 3' end of the primer to prevent extension of the wild type.
The ARMS-qPCR technology is based on a Real-time PCR platform and combines two technologies of ARMS mutation enrichment and Taqman specific fluorescence detection. The ARMS primer is used for carrying out PCR amplification on the mutation target sequence, the Taqman probe carries out specific site detection on the amplification product, and the specific mutation is identified on the basis of Real-time PCR.
Therefore, it is required to provide an ARMS-qPCR method for efficiently detecting helicobacter pylori, which realizes high specificity and high sensitivity detection.
Disclosure of Invention
The first purpose of the invention is to provide an ARMS-qPCR primer combination for detecting the individual gene of the helicobacter pylori.
The second purpose of the invention is to provide an ARMS-qPCR reagent or a kit for detecting the helicobacter pylori individuation gene.
The third purpose of the invention is to provide an ARMS-qPCR method for detecting the helicobacter pylori individualized gene.
In order to achieve the purpose, the invention adopts the following technical scheme:
an ARMS-qPCR primer composition for detecting individual genes of helicobacter pylori, which comprises a primer combination A, a primer combination B, a primer combination C, a primer combination D, a primer combination E and a primer combination F, and is shown in the following 1), 2), 3), 4), 5) and 6):
1) the primer combination A consists of an upstream primer x 2, an upstream primer x 3, an upstream primer x 17, a CYP2C19 x 2G primer, a CYP2C19 x 3G primer, a CYP2C19 x 17C primer, a Taqman probe x 2, a Taqman probe x 3 and a Taqman probe x 17;
2) the primer combination B consists of an upstream primer x 2, an upstream primer x 3, an upstream primer x 17, a CYP2C19 x 2A primer, a CYP2C19 x 3A primer, a CYP2C19 x 17T primer, a Taqman probe x 2, a Taqman probe x 3 and a Taqman probe x 17;
3) the primer combination C consists of an upstream primer-1, an A2142G mutant primer, an A2143G mutant primer and a Taqman probe-1;
4) the primer combination D consists of a 87K-1 primer, a 87K-2 primer, a downstream primer-1 and a Taqman probe-2;
5) the primer combination E consists of a 91N primer, a 91G primer, a 91Y primer, a downstream primer-1 and a Taqman probe-2;
6) the primer combination F consists of an upstream primer-2, a downstream primer-2 and an external control probe;
wherein,
the nucleotide sequence of the upstream primer is shown in a sequence table SEQ ID NO. 1;
the nucleotide sequence of the CYP2C19 x 2G primer is shown in a sequence table SEQ ID NO. 2;
the nucleotide sequence of the CYP2C19 x 2A primer is shown in a sequence table SEQ ID NO. 3;
the nucleotide sequence of the Taqman probe x 2 is shown in a sequence table SEQ ID NO. 4;
the nucleotide sequence of the upstream primer is shown in a sequence table SEQ ID NO. 5;
the nucleotide sequence of the CYP2C19 × 3G primer is shown in a sequence table SEQ ID NO. 6;
the nucleotide sequence of the CYP2C 19X 3A primer is shown in a sequence table SEQ ID NO. 7;
the nucleotide sequence of the Taqman probe x 3 is shown in a sequence table SEQ ID NO. 8;
the nucleotide sequence of the upstream primer is shown in a sequence table SEQ ID NO. 9;
the nucleotide sequence of the CYP2C19 × 17C primer is shown in a sequence table SEQ ID NO. 10;
the nucleotide sequence of the CYP2C19 × 17T primer is shown in a sequence table SEQ ID NO. 11;
the nucleotide sequence of the Taqman probe 17 is shown in a sequence table SEQ ID NO. 12;
the nucleotide sequence of the upstream primer-1 is shown as a sequence table SEQ ID NO. 13;
the nucleotide sequence of the Taqman probe-1 is shown in a sequence table SEQ ID NO. 14;
the nucleotide sequence of the A2142G mutant primer is shown in a sequence table SEQ ID NO. 15;
the nucleotide sequence of the A2143G mutant primer is shown in a sequence table SEQ ID NO. 16;
the nucleotide sequence of the downstream primer-1 is shown as a sequence table SEQ ID NO. 17;
the nucleotide sequence of the Taqman probe-2 is shown in a sequence table SEQ ID NO. 18;
the nucleotide sequence of the 87K-1 primer is shown in a sequence table SEQ ID NO. 19;
the nucleotide sequence of the 87K-2 primer is shown in a sequence table SEQ ID NO. 20;
the nucleotide sequence of the 91N primer is shown as a sequence table SEQ ID NO. 21;
the nucleotide sequence of the 91G primer is shown in a sequence table SEQ ID NO. 22;
the nucleotide sequence of the 91Y primer is shown as a sequence table SEQ ID NO. 23;
the nucleotide sequence of the upstream primer-2 is shown as a sequence table SEQ ID NO. 24;
the nucleotide sequence of the downstream primer-2 is shown as a sequence table SEQ ID NO. 25;
and the nucleotide sequence of the external control probe is shown as a sequence table SEQ ID NO. 26.
Preferably, the molar ratio of the upstream primer 2, the upstream primer 3, the upstream primer 17, the CYP2C19 2G primer, the CYP2C19 3G primer, the CYP2C19 17C primer, the Taqman probe 2, the Taqman probe 3 and the Taqman probe 17 in the primer combination A is 3: 2: 1;
in the primer combination B, the molar ratio of an upstream primer 2 to an upstream primer 3 to an upstream primer 17 to a CYP2C19 to CYP 2A primer to CYP2C19 to CYP 3A primer to CYP2C19 to Taqman 17T primer to Taqman probe 2 to Taqman probe 3 to Taqman probe 17 is 3: 2: 1;
the molar ratio of the upstream primer-1, the A2142G mutant primer, the A2143G mutant primer and the Taqman probe-1 in the primer combination C is 6: 2: 3;
the molar ratio of the 87K-1 primer, the 87K-2 primer, the downstream primer-1 and the Taqman probe-2 in the primer combination D is 3: 1;
the molar ratio of the 91N primer, the 91G primer, the 91Y primer, the downstream primer-1 and the Taqman probe-2 in the primer combination E is 3: 1;
in the primer combination F, the molar ratio of the upstream primer-2 to the downstream primer-2 to the external control probe is 3: 2.
The primer combination A and the primer combination B are used for detecting the gene polymorphism of CYP2C19 in helicobacter pylori, the primer combination C and the primer combination F are used for detecting whether the 23S rRNA gene contains 23S rRNA mutation sites, and the primer combination D, the primer combination E and the primer combination F are used for detecting whether the gyrA gene contains N87K or D91G/Y/N mutations, and the primer combinations jointly complete the detection of the individual gene of the helicobacter pylori.
The invention further provides an ARMS-qPCR reagent for detecting the helicobacter pylori individualization gene, which comprises a PCR reagent A, PCR, a B, PCR reagent, a C, PCR reagent, a D, PCR reagent, and a PCR reagent, F, and is shown in the following 1), 2), 3), 4), 5) and 6):
1) the PCR reagent A consists of PCR amplification reaction liquid, the primer combination A, a freeze-drying protective agent and water;
2) the PCR reagent B consists of PCR amplification reaction liquid, the primer combination B, a freeze-drying protective agent and water;
3) the PCR reagent C consists of PCR amplification reaction liquid, the primer combination C, a freeze-drying protective agent and water;
4) the PCR reagent D consists of PCR amplification reaction liquid, the primer combination D, a freeze-drying protective agent and water;
5) the PCR reagent E consists of PCR amplification reaction liquid, the primer combination E, a freeze-drying protective agent and water;
6) the PCR reagent F consists of PCR amplification reaction liquid, the primer combination F, a freeze-drying protective agent and water.
The freeze-drying protective agent is prepared from the following raw materials: trehalose, mannitol, bovine serum albumin and water.
The preparation method of the freeze-drying protective agent comprises the following steps: weighing trehalose, mannitol and bovine serum albumin, adding into sterile water, mixing, and dissolving.
Wherein the mass concentration of trehalose in the freeze-drying protective agent is 2-10%;
the mass concentration of mannitol in the freeze-drying protective agent is 1% -8%;
the mass concentration of bovine serum albumin in the freeze-drying protective agent is 1% -8%.
The invention provides a preparation method of an ARMS-qPCR reagent, which comprises the following steps:
1) preparing a PCR reagent A, PCR reagent B, PCR reagent C, PCR reagent D, PCR reagent E and a PCR reagent F in the ARMS-qPCR reagent according to the proportion respectively and uniformly mixing;
2) and (5) freeze-drying to obtain the ARMS-qPCR reagent.
Wherein the freeze drying adopts freeze vacuum drying, and comprises the following steps:
1) a pre-freezing stage: the temperature of the plate layer is kept between minus 35 ℃ and minus 50 ℃ for 2 to 4 hours;
2) a sublimation stage: the vacuum degree reaches below 60Pa, and the temperature is kept at-35-10 ℃ for 3-8 h;
3) and (3) analysis and drying stage: keeping the temperature of 10-30 ℃ for 1-6 h.
The invention further provides an ARMS-qPCR kit for detecting the helicobacter pylori individualized gene, and the kit comprises the ARMS-qPCR reagent.
The ARMS-qPCR primer composition, the ARMS-qPCR reagent or the ARMS-qPCR kit are applied to the preparation of products for detecting the gene polymorphism of CYP2C19 in helicobacter pylori, detecting whether 23S rRNA genes contain 23S rRNA mutation sites or not and detecting whether gyrA genes contain N87K or D91G/Y/N mutations or not.
The invention also provides a method for detecting the helicobacter pylori individuation gene, which comprises the following steps:
respectively carrying out ARMS-qPCR detection on a sample to be detected by using the ARMS-qPCR primer composition, the ARMS-qPCR reagent or the ARMS-qPCR kit to obtain an amplification product for result interpretation:
if the FAM/ROX/CY5 three fluorescence channels in the tube A and the tube B have no amplification curve and no Ct value, or the Ct is more than 37, the reaction is invalid, and the sample to be tested needs to be extracted again for testing;
if Ct of at least one of two amplification tubes of the FAM/ROX/CY5 three fluorescence channels in the tube A and the tube B is less than or equal to 37, interpreting the results according to the following table:
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the C tube has an S-type amplification curve and delta Ct between the C tube and the F tube is less than or equal to 7, the helicobacter pylori 23S rRNA gene in the sample to be detected contains 23S rRNA mutation sites;
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the C tube has an S-type amplification curve and the amplification curve delta Ct between the C tube and the F tube is more than 7, the helicobacter pylori 23S rRNA gene in the sample to be detected does not contain 23S rRNA mutation sites;
if the F tube amplification curve is negative or Ct is more than 32, which indicates that the DNA extraction concentration of the sample to be detected is too low or exceeds the detection range of the method, the sample to be detected needs to be extracted again;
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the D tube has an S-type amplification curve and delta Ct between the S-type amplification curve and the amplification curve of the F tube is less than or equal to 7, the gyrA gene of the helicobacter pylori in the sample to be detected contains N87K mutation;
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the E tube has an S-type amplification curve and delta Ct between the E tube and the F tube is less than or equal to 7, the gyrA gene of the helicobacter pylori in the sample to be detected contains D91G/Y/N mutation;
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, the D tube and the E tube do not have S-type amplification curves, or the D tube and the E tube have S-type amplification curves and the amplification curve delta Ct between the D tube and the E tube and the F tube is more than 7, the gyrA gene of the helicobacter pylori in the sample to be detected does not contain N87K or D91G/Y/N mutation;
if the F tube amplification curve is negative or Ct is more than 32, which indicates that the DNA extraction concentration of the sample to be detected is too low or exceeds the detection range of the method, the sample to be detected needs to be extracted again.
The invention has the following beneficial effects:
the invention is based on ARMS-qPCR platform, aiming at the mutation sites of the 23S rRNA gene and the gyrA gene of the human CYP2C19 gene and the helicobacter pylori, the ARMS-qPCR primers are designed, thus improving the amplification specificity; the invention can enrich trace template through specific amplification of nucleic acid DNA, improve detection sensitivity, and detect 0.1-1% mutation of Hp gene related site and 1ng of human CYP2C19 gene sample DNA.
The reagent for detection is freeze-dried powder, can realize freeze drying, is simple to operate during detection, can realize the detection purpose only by one-step hydration, improves the stability of the reagent, and can be transported and/or stored at normal temperature.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the results of detection of the CYP2C19 x 2 polymorphic site in helicobacter pylori positive sample 2.
FIG. 2 shows the results of detection of the CYP2C19 x 3 polymorphic site in H.pylori positive sample 2.
FIG. 3 shows the results of detection of the CYP2C19 x 17 polymorphic site in H.pylori positive sample 2.
FIG. 4 is a graph showing the results of the kit for detecting the A2142G mutant form of the 23S rRNA gene in helicobacter pylori positive sample 1.
FIG. 5 is a graph showing the results of the kit for detecting the A2143G mutant form of the 23S rRNA gene of helicobacter pylori positive sample 2.
FIG. 6 is a diagram showing the results of the kit for detecting the wild type of the 23S rRNA gene of helicobacter pylori positive sample 3.
FIG. 7 shows the sequencing results of the mutant form of the 23S rRNA gene A2142G in the helicobacter pylori positive sample.
FIG. 8 shows the sequencing results of the mutant form of the 23S rRNA gene A2143G in the helicobacter pylori positive sample.
FIG. 9 shows the sequencing results of the wild-type 23S rRNA gene of a helicobacter pylori-positive sample.
FIG. 10 is a graph showing the results of the kit for detecting the N87K mutant form of the gyrA gene in H.pylori positive sample 1.
FIG. 11 is a graph showing the results of the detection of the D91G/Y/N mutant type of gyrA gene in H.pylori positive sample 2 by the kit.
FIG. 12 is a graph showing the results of the kit for detecting the N87K wild type gyrA gene of helicobacter pylori positive sample 3.
FIG. 13 is a graph showing the results of the kit for detecting the D91G/Y/N wild type of the gyrA gene of helicobacter pylori positive sample 3.
FIG. 14 is a graph showing the results of detecting the 23S rRNA gene of helicobacter pylori positive sample 2 after reconstitution of the liquid reagent with the lyophilized reagent.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1 primers and probes for detection of the individualized Gene of helicobacter pylori
1. Specific primers and probes are designed and screened according to sequences of three sites of the human CYP2C19 gene, and are used for detecting the polymorphism of the CYP2C19 gene, wherein the sequences are specifically as follows:
upstream primer 2: 5'-GATCAGGAAGCAATCAATAAAGTCC-3' (SEQ ID NO.1)
CYP2C19 × 2G primer: 5'-TCCCACTATCATTGATTATTTCCCG-3' (SEQ ID NO.2)
CYP2C19 × 2A primer: 5'-TCCCACTATCATTGATTATTTCGTA-3' (SEQ ID NO.3)
Taqman probe 2: 5 '-FAM-AATATCACTTTCCATAAAAGCAAGGT-BHQ 1-3' (SEQ ID NO.4)
Upstream primer 3: 5'-TCAAAAATGTACTTCAGGGCT-3' (SEQ ID NO.5)
CYP2C19 × 3G primer: 5'-CAGGATTGTAAGCACCCCCTAG-3' (SEQ ID NO.6)
CYP2C19 × 3A primer: 5'-GATTGTAAGCACCCCCTGA-3' (SEQ ID NO.7)
Taqman probe 3: 5 '-ROX-TGAAAACATCAGGATTGTAAGCAC-BHQ 2-3' (SEQ ID NO.8)
Upstream primer 17: 5'-CCTGTTTTATGAACAGGATGAA-3' (SEQ ID NO.9)
CYP2C19 × 17C primer: 5'-ATTATCTCTTACATCAGAGATG-3' (SEQ ID NO.10)
CYP2C19 × 17T primer: 5'-ATTATCTCTTACATCAGAGATA-3' (SEQ ID NO.11)
Taqman probe 17: 5 '-CY 5-TAACTAATGTTTGGAAGTTGTTTTGTT-BHQ 2-3' (SEQ ID NO.12)
Designing and screening specific mutation primers and probes according to the gene sequence of the mutation site of the 23S rRNA gene of the helicobacter pylori, and detecting whether the 23S rRNA gene of the helicobacter pylori contains the mutation site of the 23S rRNA gene, wherein the sequence is specifically as follows:
upstream primer-1: 5'-CTATAACGGTCCTAAGGTAGCGA-3' (SEQ ID NO.13)
Taqman probe-1: 5 '-FAM-AATTCCTCCTACCCGCGGCAAGAC-BHQ 1-3' (SEQ ID NO.14)
A2142G mutant primer: 5'-TAAAGGTCCACGGGGTCACC-3' (SEQ ID NO.15)
A2143G mutant primer: 5'-TAGTAAAGGTCCACGGGGTCGC-3' (SEQ ID NO.16)
Designing and screening specific mutation primers and probes according to the gene sequence of the mutation site of the gyrA gene of the helicobacter pylori, and detecting whether the gyrA gene of the helicobacter pylori contains N87K or D91G/Y/N mutation;
the sequence is specifically as follows:
downstream primer-1: 5'-ATTTCTTCACTCGCCTTAGTCATTC-3' (SEQ ID NO.17)
Taqman probe-2: 5 '-FAM-TGAGAATGGCGCAAGATTTTTCCAT-BHQ 13' (SEQ ID NO.18)
87K-1 primer: 5'-CACCCCCATGGCGATAAA-3' (SEQ ID NO.19)
87K-2 primer: 5'-CACCCCCATGGCGATATG-3' (SEQ ID NO.20)
A 91N primer: 5'-ATGGCGATAATGCGGTTTCTA-3' (SEQ ID NO.21)
91G primer: 5'-ATGGCGATAATGCGGTTTATAG-3' (SEQ ID NO.22)
91Y primer: 5'-ATGGCGATAATGCGGTTTACT-3' (SEQ ID NO.23)
Upstream primer-2: 5'-ATTTAGCTTATTCCATGAGCGTGAT-3' (SEQ ID NO.24)
Downstream primer-2: 5'-GCGACTTTTGAAGTAAGGCCTAATT-3' (SEQ ID NO.25)
An external control probe: 5 '-FAM-GCTTTACCGGACGCTAGAGATGGCT-BHQ 13' (SEQ ID NO.26)
Example 2 primer composition for detection of helicobacter pylori individualization gene
Primer compositions for detecting the helicobacter pylori individualized gene are 1), 2), 3), 4), 5) and 6) as follows:
1) the primer combination A consists of an upstream primer x 2, an upstream primer x 3, an upstream primer x 17, a CYP2C19 x 2G primer, a CYP2C19 x 3G primer, a CYP2C19 x 17C primer, a Taqman probe x 2, a Taqman probe x 3 and a Taqman probe x 17;
2) the primer combination B consists of an upstream primer x 2, an upstream primer x 3, an upstream primer x 17, a CYP2C19 x 2A primer, a CYP2C19 x 3A primer, a CYP2C19 x 17T primer, a Taqman probe x 2, a Taqman probe x 3 and a Taqman probe x 17;
3) the primer combination C consists of an upstream primer-1, an A2142G mutant primer, an A2143G mutant primer and a Taqman probe-1;
4) the primer combination D consists of a 87K-1 primer, a 87K-2 primer, a downstream primer-1 and a Taqman probe-2;
5) the primer combination E consists of a 91N primer, a 91G primer, a 91Y primer, a downstream primer-1 and a Taqman probe-2;
6) the primer combination F consists of an upstream primer-2, a downstream primer-2 and an external control probe.
EXAMPLE 3 preparation of PCR reagent and detection kit for detection of helicobacter pylori individualized Gene
1. PCR reagent for detecting helicobacter pylori individuation gene
PCR reagents for the detection of the individual gene of helicobacter pylori were 1), 2), 3), 4), 5) and 6) as follows:
1) the PCR reagent A consists of PCR amplification reaction liquid, the primer combination A, a freeze-drying protective agent and water;
2) the PCR reagent B consists of PCR amplification reaction liquid, the primer combination B, a freeze-drying protective agent and water;
3) the PCR reagent C consists of PCR amplification reaction liquid, the primer combination C, a freeze-drying protective agent and water;
4) the PCR reagent D consists of PCR amplification reaction liquid, the primer combination D, a freeze-drying protective agent and water;
5) the PCR reagent E consists of PCR amplification reaction liquid, the primer combination E, a freeze-drying protective agent and water;
6) the PCR reagent F consists of PCR amplification reaction liquid, the primer combination F, a freeze-drying protective agent and water.
2. Preparation of detection kit
2.1 preparation of Freeze-drying protective agent
5g of trehalose, 2.5g of mannitol and 1.2g of bovine serum albumin were weighed and added to 100mL of sterile water, mixed well and dissolved.
2.2 preparation of PCR reagents
PCR reagent A reaction system consisted of PCR amplification reaction solution 12.5. mu.l, forward primer 20.75. mu.l (final concentration 0.3. mu.M), forward primer 30.75. mu.l (final concentration 0.3. mu.M), forward primer 170.5. mu.l (final concentration 0.2. mu.M), CYP2C 19. mu.l 2G primer 0.75. mu.l (final concentration 0.3. mu.M), CYP2C 19. mu.3G primer 0.75. mu.l (final concentration 0.3. mu.M), CYP2C 19. mu.l 17C primer 0.5. mu.l (final concentration 0.2. mu.M), Taqman probe 20.5. mu.l (final concentration 0.2. mu.M), Taqman probe 30.5. mu.l (final concentration 0.2. mu.M), Taqman probe 170.25. mu.l (final concentration 0.1. mu.M), and freeze-dried protective solution 2. mu.l to 23. mu.l.
PCR reagent B reaction system, PCR amplification reaction solution 12.5 u l, upstream primer 20.75 u l (final concentration 0.3 u M), upstream primer 30.75 u l (final concentration 0.3 u M), upstream primer 170.5 u l (final concentration 0.2 u M), CYP2C19 x 2A primer 0.75 u l (final concentration 0.3 u M), CYP2C19 x 3A primer 0.75 u l (final concentration 0.3 u M), CYP2C19 x 17T primer 0.5 u l (final concentration 0.2 u M), Taqman probe 20.5 u l (final concentration 0.2 u M), Taqman probe 30.5 u l (final concentration 0.2 u M), Taqman probe 170.25 u l (final concentration 0.1 u M), the freeze-drying agent 2 u l, the protection system will be supplemented to 23 u l.
PCR reagent C reaction system consisted of PCR amplification reaction solution 12.5. mu.l, forward primer-11.5. mu.l (final concentration 0.6. mu.M), mutant primer A2142G 0.75. mu.l (final concentration 0.3. mu.M), mutant primer A2143G 0.75. mu.l (final concentration 0.3. mu.M), Taqman probe-10.5. mu.l (final concentration 0.2. mu.M), lyoprotectant 2. mu.l, and water was added to the reaction system to make up 23. mu.l.
The PCR reagent D reaction system consisted of 12.5. mu.l of PCR amplification reaction solution, 11.5. mu.l of downstream primer (final concentration 0.6. mu.M), 0.5. mu.l of 87K-1 primer (final concentration 0.2. mu.M), 0.5. mu.l of 87K-2 primer (final concentration 0.2. mu.M), and 20.5. mu.l of Taqman probe (final concentration 0.2. mu.M), and 2. mu.l of the above lyoprotectant was added to 23. mu.l with water.
PCR reagent E reaction system consisted of PCR amplification reaction solution 12.5. mu.l, downstream primer-11.5. mu.l (final concentration 0.6. mu.M), 91N primer 0.5. mu.l (final concentration 0.2. mu.M), 91G primer 0.5. mu.l (final concentration 0.2. mu.M), 91Y primer 0.5. mu.l (final concentration 0.2. mu.M), Taqman probe-20.5. mu.l (final concentration 0.2. mu.M), and the above mentioned cryoprotectant 2. mu.l, which was made up to 23. mu.l with water.
The PCR reagent F reaction system consisted of 12.5. mu.l of PCR amplification reaction solution, 21. mu.l of forward primer (final concentration 0.4. mu.M), 21. mu.l of reverse primer (final concentration 0.4. mu.M), 0.5. mu.l of external control probe (final concentration 0.2. mu.M), and 2. mu.l of the above-mentioned cryoprotectant, and the system was made up to 23. mu.l with water.
The total volume of the PCR reaction was 25. mu.l, containing 23. mu.l of the reaction system and 2. mu.l of the detection template.
2.3 lyophilization of PCR reagents
And (3) putting the prepared PCR reagent A-PCR reagent F into a freeze dryer, keeping the temperature of a plate layer at minus 35 ℃ to minus 50 ℃ for 2-4h, keeping the vacuum degree below 60Pa at minus 35 ℃ to 10 ℃ for 3-8h, and keeping the temperature at 10 ℃ to 30 ℃ for 1-6h to finally obtain the freeze-dried reagent.
2.4 kit Components
The kit adopts 8-linked PCR tube design, each 8-linked tube detects a sample, and the kit comprises the following components:
1) eight-linked PCR reaction tube: the tubes A to F are PCR freeze-drying reagents A to F, and the tubes G and H are empty tubes;
2) positive quality control product: artificially synthesized plasmid mixed freeze-dried powder.
Example 4 kit detection method
1. Sample genomic DNA extraction
Extracting DNA from human gastric mucosa tissue samples, and recommending the extraction of samples 1-3 by using a commercial extraction kit;
the concentration of the extracted DNA is measured by an ultraviolet spectrophotometer, the OD260/OD280 of the DNA is 1.8-2.0, the extracted DNA is recommended to be immediately detected, and otherwise, the extracted DNA is required to be stored below-20 ℃.
PCR detection
The PCR lyophilized reagents A-F were redissolved with 23. mu.l of purified water, then 2. mu.l of the extracted DNA was added, and samples 1-3 were reacted in tubes A-F, respectively.
The PCR amplification procedure was: reacting with UDG enzyme at 50 deg.C for 2min, pre-denaturing at 95 deg.C for 5min, denaturing at 95 deg.C for 15s, annealing and extending at 60 deg.C for 45s, and collecting fluorescence at 60 deg.C for 40 cycles.
3. Interpretation of results
1) Interpretation of human CYP2C19 Gene polymorphism
With reference to the accompanying drawings: fig. 1, 2, 3.
If none of the FAM/ROX/CY5 fluorescence channels in the tube A and the tube B has an amplification curve and no Ct value, or the Ct is larger than 37, the reaction is invalid, and a sample needs to be extracted again for testing.
If Ct of at least one of two amplification tubes of the FAM/ROX/CY5 fluorescence channels in the tube A and the tube B is less than or equal to 37, (the target gene channel has no amplification curve, and the Ct value is calculated according to 40), judging the result according to the following table:
2) interpretation of Hp 23S rRNA Gene mutation
If the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the C tube has an S-type amplification curve and delta Ct between the C tube and the F tube is less than or equal to 7, the helicobacter pylori 23S rRNA gene in the sample to be detected contains 23S rRNA mutation sites; referring to fig. 4, Δ Ct is 3, and fig. 5 Δ Ct is 0.5.
If the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the C tube has an S-type amplification curve and the amplification curve delta Ct between the C tube and the F tube is more than 7, the helicobacter pylori 23S rRNA gene in the sample to be detected does not contain 23S rRNA mutation sites; referring to fig. 6, Δ Ct is 12.
If the F tube amplification curve is negative or Ct is more than 32, the extraction concentration of the sample DNA is too low or exceeds the detection range of the method, and the sample is recommended to be extracted again.
The detection result of the kit is consistent with the sequencing result of the corresponding sample, and the sequencing result is shown in figure 7, figure 8 and figure 9.
3) Interpretation of Hp gyrA Gene mutation
With reference to the accompanying drawings: fig. 10, 11, 12, and 13.
If the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the D tube has an S-type amplification curve and delta Ct between the S-type amplification curve and the amplification curve of the F tube is less than or equal to 7, the gyrA gene of the helicobacter pylori in the sample to be detected contains N87K mutation;
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, and the E tube has an S-type amplification curve and delta Ct between the E tube and the F tube is less than or equal to 7, the gyrA gene of the helicobacter pylori in the sample to be detected contains D91G/Y/N mutation;
if the F tube has an amplification S-type curve and Ct is less than or equal to 32, the D tube and the E tube do not have S-type amplification curves, or the D tube and the E tube have S-type amplification curves and the amplification curve delta Ct between the D tube and the E tube and the F tube is more than 7, the gyrA gene of the helicobacter pylori in the sample to be detected does not contain N87K or D91G/Y/N mutation;
if the F tube amplification curve is negative or Ct is more than 32, the extraction concentration of the sample DNA is too low or exceeds the detection range of the method, and the sample is recommended to be extracted again.
EXAMPLE 5 clinical application of the kit
In this example, pieces of gastric mucosal tissue of 50 patients who were clinically and pathologically diagnosed as positive for helicobacter pylori were collected, and genomic DNA was extracted therefrom. The method for detecting the sample by using the individualized gene detection kit comprises the following specific steps:
1. sample genomic DNA extraction
Extracting the gastric mucosa tissue sample by using a commercial extraction kit according to instructions, extracting the genomic DNA of the sample to be detected, and measuring the concentration and the DNA quality of the extracted DNA sample by using an ultraviolet spectrophotometer, wherein the concentration requirement of the DNA is more than 10 ng/mu l, and the quality OD260/OD280 of the DNA is between 1.8 and 2.0.
2. Fluorescent PCR detection
The assay was performed as in example 4, method 2 and method 3 in interpretation.
3. Statistics of results
In the gastric mucosa sample, the polymorphism sites corresponding to the human CYP2C19 gene can be accurately typed, and the coincidence rate of the result and the sequencing result is up to more than 99%.
In the positive helicobacter pylori sample, 6 cases detected contained the 23S rRNA gene A2142G or A2143G mutation, and the mutant or wild type was consistent with the sequencing results. The wild type sample and the mutant type sample are compared with the result of the drug sensitivity experiment, and the result shows that the mutant type sample clarithromycin is resistant to the drug and the wild type sample clarithromycin is sensitive to the drug.
In 5 cases of helicobacter pylori positive samples, amino acid mutations at position 87 or 91 of the gyrA gene were detected, wherein the mutant or wild type was consistent with the sequencing results. The wild type sample and the mutant type sample are compared with the drug sensitivity experiment result, and the result shows that the mutant type sample levofloxacin is resistant to drugs and the wild type sample levofloxacin is sensitive.
In addition, the detection effect of the freeze-dried reagent and the liquid reagent on the helicobacter pylori positive sample is compared, and the result proves that the detection effect of the freeze-dried reagent on the helicobacter pylori positive sample after being redissolved is equivalent to the detection effect of the liquid reagent, and the result is shown in the attached figure 14.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
SEQUENCE LISTING
<110> hand-cranking cell
<120> ARMS-qPCR method and kit for detecting helicobacter pylori individualization gene
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<213> Artificial Synthesis of upstream primer 2
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gatcaggaag caatcaataa agtcc 25
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<213> Artificial Synthesis of CYP2C19 x 2G primer
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tcccactatc attgattatt tcccg 25
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<213> Artificial Synthesis of CYP2C19 x 2A primer
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tcccactatc attgattatt tcgta 25
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<213> Artificial Synthesis of Taqman Probe x 2
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aatatcactt tccataaaag caaggt 26
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<213> Artificial Synthesis of upstream primer x 3
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tgaaaacatc aggattgtaa gcac 24
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<213> Artificial Synthesis of upstream primer 17
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cctgttttat gaacaggatg aa 22
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<213> Artificial Synthesis of CYP2C19 x 17C primer
<400>10
attatctctt acatcagaga tg 22
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<213> Artificial Synthesis of CYP2C19 x 17T primer
<400>11
attatctctt acatcagaga ta 22
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taactaatgt ttggaagttg ttttgtt 27
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ctataacggt cctaaggtag cga 23
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<213> Artificial Synthesis of Taqman Probe-1
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<213> artificially synthesized A2142G mutant primer
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<213> artificially synthesized A2143G mutant primer
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tagtaaaggt ccacggggtc gc 22
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<213> Artificial Synthesis of downstream primer-1
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atttcttcac tcgccttagt cattc 25
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cacccccatg gcgataaa 18
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cacccccatg gcgatatg 18
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Claims (9)
1. An ARMS-qPCR primer composition for detecting individual genes of helicobacter pylori, which is characterized by comprising a primer combination A, a primer combination B, a primer combination C, a primer combination D, a primer combination E and a primer combination F as shown in the following 1), 2), 3), 4), 5) and 6):
1) the primer combination A consists of an upstream primer x 2, an upstream primer x 3, an upstream primer x 17, a CYP2C19 x 2G primer, a CYP2C19 x 3G primer, a CYP2C19 x 17C primer, a Taqman probe x 2, a Taqman probe x 3 and a Taqman probe x 17;
2) the primer combination B consists of an upstream primer x 2, an upstream primer x 3, an upstream primer x 17, a CYP2C19 x 2A primer, a CYP2C19 x 3A primer, a CYP2C19 x 17T primer, a Taqman probe x 2, a Taqman probe x 3 and a Taqman probe x 17;
3) the primer combination C consists of an upstream primer-1, an A2142G mutant primer, an A2143G mutant primer and a Taqman probe-1;
4) the primer combination D consists of a 87K-1 primer, a 87K-2 primer, a downstream primer-1 and a Taqman probe-2;
5) the primer combination E consists of a 91N primer, a 91G primer, a 91Y primer, a downstream primer-1 and a Taqman probe-2;
6) the primer combination F consists of an upstream primer-2, a downstream primer-2 and an external control probe;
wherein,
the nucleotide sequence of the upstream primer is shown in a sequence table SEQ ID NO. 1;
the nucleotide sequence of the CYP2C19 x 2G primer is shown in a sequence table SEQ ID NO. 2;
the nucleotide sequence of the CYP2C19 x 2A primer is shown in a sequence table SEQ ID NO. 3;
the nucleotide sequence of the Taqman probe x 2 is shown in a sequence table SEQ ID NO. 4;
the nucleotide sequence of the upstream primer is shown in a sequence table SEQ ID NO. 5;
the nucleotide sequence of the CYP2C19 × 3G primer is shown in a sequence table SEQ ID NO. 6;
the nucleotide sequence of the CYP2C 19X 3A primer is shown in a sequence table SEQ ID NO. 7;
the nucleotide sequence of the Taqman probe x 3 is shown in a sequence table SEQ ID NO. 8;
the nucleotide sequence of the upstream primer is shown in a sequence table SEQ ID NO. 9;
the nucleotide sequence of the CYP2C19 × 17C primer is shown in a sequence table SEQ ID NO. 10;
the nucleotide sequence of the CYP2C19 × 17T primer is shown in a sequence table SEQ ID NO. 11;
the nucleotide sequence of the Taqman probe 17 is shown in a sequence table SEQ ID NO. 12;
the nucleotide sequence of the upstream primer-1 is shown as a sequence table SEQ ID NO. 13;
the nucleotide sequence of the Taqman probe-1 is shown in a sequence table SEQ ID NO. 14;
the nucleotide sequence of the A2142G mutant primer is shown in a sequence table SEQ ID NO. 15;
the nucleotide sequence of the A2143G mutant primer is shown in a sequence table SEQ ID NO. 16;
the nucleotide sequence of the downstream primer-1 is shown as a sequence table SEQ ID NO. 17;
the nucleotide sequence of the Taqman probe-2 is shown in a sequence table SEQ ID NO. 18;
the nucleotide sequence of the 87K-1 primer is shown in a sequence table SEQ ID NO. 19;
the nucleotide sequence of the 87K-2 primer is shown in a sequence table SEQ ID NO. 20;
the nucleotide sequence of the 91N primer is shown as a sequence table SEQ ID NO. 21;
the nucleotide sequence of the 91G primer is shown in a sequence table SEQ ID NO. 22;
the nucleotide sequence of the 91Y primer is shown in a sequence table SEQID NO. 23;
the nucleotide sequence of the upstream primer-2 is shown as a sequence table SEQ ID NO. 24;
the nucleotide sequence of the downstream primer-2 is shown as a sequence table SEQ ID NO. 25;
and the nucleotide sequence of the external control probe is shown as a sequence table SEQ ID NO. 26.
2. An ARMS-qPCR reagent for detecting the individuation gene of helicobacter pylori, which is characterized by comprising a PCR reagent A, PCR, B, PCR, C, PCR, D, PCR, E and F as shown in the following 1), 2), 3), 4), 5) and 6):
1) the PCR reagent A consists of PCR amplification reaction liquid, a primer combination A as claimed in claim 1, a freeze-drying protective agent and water;
2) the PCR reagent B consists of PCR amplification reaction liquid, a primer combination B as claimed in claim 1, a freeze-drying protective agent and water;
3) the PCR reagent C consists of PCR amplification reaction liquid, a primer combination C as claimed in claim 1, a freeze-drying protective agent and water;
4) the PCR reagent D consists of PCR amplification reaction liquid, a primer combination D as claimed in claim 1, a freeze-drying protective agent and water;
5) the PCR reagent E consists of PCR amplification reaction liquid, a primer combination E as claimed in claim 1, a freeze-drying protective agent and water;
6) the PCR reagent F consists of a PCR amplification reaction solution, a primer combination F as claimed in claim 1, a freeze-drying protective agent and water.
3. The ARMS-qPCR reagent of claim 2, wherein the lyoprotectant is made from the following raw materials: trehalose, mannitol, bovine serum albumin and water.
4. The ARMS-qPCR reagent of claim 3, wherein the lyoprotectant is prepared by: weighing trehalose, mannitol and bovine serum albumin, adding into sterile water, mixing, and dissolving;
wherein the mass concentration of the trehalose is 2-10%;
the mass concentration of the mannitol is 1-8%;
the mass concentration of the bovine serum albumin is 1-8%.
5. A method of preparing the ARMS-qPCR reagent of claim 2, comprising the steps of:
1) respectively and uniformly mixing a PCR reagent A, PCR reagent B, PCR reagent C, PCR reagent D, PCR reagent E and a PCR reagent F in the ARMS-qPCR reagent;
2) and (5) freeze-drying to obtain the ARMS-qPCR reagent.
6. The method according to claim 5, wherein the freeze-drying is freeze-vacuum drying, comprising the steps of:
1) a pre-freezing stage: the temperature of the plate layer is kept between minus 35 ℃ and minus 50 ℃ for 2 to 4 hours;
2) a sublimation stage: the vacuum degree reaches below 60Pa, and the temperature is kept at-35-10 ℃ for 3-8 h;
3) and (3) analysis and drying stage: keeping the temperature of 10-30 ℃ for 1-6 h.
7. An ARMS-qPCR kit for detecting a helicobacter pylori individualized gene, which is characterized by comprising the ARMS-qPCR primer composition of claim 1 or the ARMS-qPCR reagent of claim 2.
8. The ARMS-qPCR primer composition of claim 1, the ARMS-qPCR reagent of claim 2 or the ARMS-qPCR kit of claim 7, for use in the detection of the gene polymorphism of CYP2C19 in H.pylori, the detection of whether the 23S SrRNA gene contains 23S rRNA mutation sites, and the detection of whether the gyrA gene contains N87K or D91G/Y/N mutations.
9. An ARMS-qPCR method for detecting a helicobacter pylori individualized gene, which is characterized by comprising the following steps:
performing ARMS-qPCR detection on a sample to be detected by using the ARMS-qPCR primer composition of claim 1, the ARMS-qPCR reagent of claim 2 or the ARMS-qPCR kit of claim 7 to obtain an amplification product, and judging the result.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055553A (en) * | 2018-08-23 | 2018-12-21 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs4244285 |
| CN110241231A (en) * | 2019-06-26 | 2019-09-17 | 湖南健基生物技术有限公司 | Detect composition, kit, method and the application of CYP2C19 gene pleiomorphism |
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| CN109055553A (en) * | 2018-08-23 | 2018-12-21 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs4244285 |
| CN111910011A (en) * | 2019-05-10 | 2020-11-10 | 江苏康为世纪生物科技有限公司 | Kit for detecting drug-resistant mutation sites of helicobacter pylori |
| CN110241231A (en) * | 2019-06-26 | 2019-09-17 | 湖南健基生物技术有限公司 | Detect composition, kit, method and the application of CYP2C19 gene pleiomorphism |
| CN111363837A (en) * | 2020-03-10 | 2020-07-03 | 中南大学 | Method for improving accuracy and efficiency of detection of drug-resistant mutation and drug-resistant gene of acne bacillus and matched kit thereof |
| CN114410811A (en) * | 2022-01-22 | 2022-04-29 | 北京新基永康生物科技有限公司 | Method for detecting or identifying helicobacter pylori drug-resistant gene mutation, primer probe composition, application, kit and using method |
| CN114592081A (en) * | 2022-04-26 | 2022-06-07 | 江苏康为世纪生物科技股份有限公司 | Kit for detecting helicobacter pylori drug-resistant gene mutation in fecal sample |
| CN116694786A (en) * | 2023-02-21 | 2023-09-05 | 广州凯普医药科技有限公司 | Primer probe combination for detecting helicobacter pylori drug-resistant mutant gene |
| CN116694786B (en) * | 2023-02-21 | 2024-01-05 | 广州凯普医药科技有限公司 | Primer probe combination for detecting helicobacter pylori drug-resistant mutant gene |
| CN117683919A (en) * | 2023-12-19 | 2024-03-12 | 深圳市海微生物科技有限公司 | Primer for detecting single base substitution of mutation site and application of primer in preparation of related products for detecting helicobacter pylori drug resistance genes |
| CN117683919B (en) * | 2023-12-19 | 2024-09-06 | 深圳市海微生物科技有限公司 | Primer for detecting single base substitution of mutation site and application of primer in preparation of related products for detecting helicobacter pylori drug resistance genes |
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