CN114410811A - Method for detecting or identifying helicobacter pylori drug-resistant gene mutation, primer probe composition, application, kit and using method - Google Patents

Method for detecting or identifying helicobacter pylori drug-resistant gene mutation, primer probe composition, application, kit and using method Download PDF

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CN114410811A
CN114410811A CN202210075286.4A CN202210075286A CN114410811A CN 114410811 A CN114410811 A CN 114410811A CN 202210075286 A CN202210075286 A CN 202210075286A CN 114410811 A CN114410811 A CN 114410811A
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韩敏
韩晶
任鹏飞
王俊峰
王玉朗
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Beijing Xinji Yongkang Biotechnology Co ltd
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Abstract

The application relates to the technical field of mutation detection, in particular to a method for detecting or identifying helicobacter pylori drug-resistant gene mutation, a primer probe composition, application, a kit and a using method. The primer probe composition comprises ARMS primer probe composition and other sequence specific primer probe composition; ARMS primer probe compositions are used for amplifying target mutant genes, and other sequence specific primer probe compositions are used for amplifying other sequences or genes; the kit contains the primer probe composition; the use method of the kit comprises the step of combining the primer probe composition with the template DNA. The primer probe composition or the kit can be used for detecting or identifying the drug-resistant gene mutation of helicobacter pylori gyrA, and has the advantages of high throughput, high efficiency and high sensitivity.

Description

Method for detecting or identifying helicobacter pylori drug-resistant gene mutation, primer probe composition, application, kit and using method
Technical Field
The application relates to the technical field of mutation detection, in particular to a method for detecting or identifying helicobacter pylori drug-resistant gene mutation, a primer probe composition, application, a kit and a using method.
Background
Helicobacter Pylori (HP) is a gram-negative bacterium that is distributed mainly in the tissues of the gastric mucosa and is currently the only microbial species that can survive in the human stomach. Helicobacter pylori is closely related to various diseases such as stomach and duodenum, and particularly, infection thereof is related to upper gastrointestinal diseases including gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. Eradication of HP promotes ulcer healing, reduces ulcer recurrence, reduces the probability of gastric cancer, and thus eradication of helicobacter pylori treatment is considered to be an effective method for the treatment of helicobacter pylori-associated diseases.
Currently, triple or quadruple therapy with antibiotics is mainly used for the treatment of helicobacter pylori, wherein commonly used antibiotic drugs include clarithromycin, metronidazole, amoxicillin and levofloxacin. Levofloxacin is one of the antibiotics commonly used for clinically eradicating helicobacter pylori, but because the drug is widely used, the drug resistance rate of the helicobacter pylori is increased year by year, and the eradication rate of the helicobacter pylori is reduced year by year. It has been clarified that the quinolone resistance of helicobacter pylori is caused by mutation of gyrA gene in the Quinolone Resistance Determining Region (QRDR), and the amino acid changes at 87 th and 91 th positions are most common. These point mutations result in the disruption of the conformation of bacterial DNA gyrase and topoisomerase and inhibit the binding of levofloxacin thereto, failing to prevent the replication of bacterial DNA, and ultimately leading to drug resistance.
Before the individual administration scheme of removing helicobacter pylori is established, it is necessary to know the drug resistance of helicobacter pylori in an individual, so that sensitive antibiotics are selected for treatment, and the eradication rate is improved. The kit can rapidly and sensitively analyze whether the nucleic acid sample has known point mutation or not based on a qPCR technology platform, and provides a basis for quinolone drug resistance.
The existing detection method for helicobacter pylori resistance mainly comprises a drug sensitivity detection method, a direct sequencing method, a fluorescence PCR method and the like. The drug sensitivity detection method is a gold standard for detecting drug resistance of helicobacter pylori, micro-aerobic conditions are required for culturing the helicobacter pylori, and different antibiotics are selected to perform sterilization experiments on the cultured bacteria, so that which drug has sensitivity to the helicobacter pylori is judged. But the HP has strict requirements on culture conditions, low detectable rate and long culture time, so that the method is not beneficial to high-sensitivity rapid diagnosis and limits clinical popularization and application. The sequencing method is the 'gold standard' for gene detection, but the detection period is long, about one week is needed, the process is complex, the operation is complicated, and the cost is high, so that the application of the sequencing method in clinic is limited. The fluorescence PCR dissolution curve method requires PCR amplification, and melting curve analysis is performed after the PCR amplification; however, the method is long in time consumption, the change of the Tm value of a single probe is small, a high-resolution instrument is required, and the requirement on the instrument is severe. The fluorescent Taqman probe method requires a large amount of probes and is expensive to detect. The common ARMS-PCR method needs multi-tube operation, has low flux, fussy operation and high detection cost, and is not suitable for clinical detection.
Disclosure of Invention
In order to realize the detection of multiple site mutations in one tube, the application provides a method for detecting or identifying the drug-resistant gene mutation of helicobacter pylori, a primer probe composition, application, a kit and a using method.
In a first aspect, the present application provides a method for detecting or identifying a mutation in a helicobacter pylori resistance gene, comprising the steps of:
designing specific primer probe compositions of other sequences and target primer probe compositions for amplifying and detecting a plurality of target mutation sites, wherein the other sequences are conserved region sequences of the target mutation sites or conserved region sequences of other genes different from the sequences of the target mutation sites; and mixing the target primer probe composition and the other sequence specific primer probe compositions with template DNA of a sample to be detected, carrying out ARMS-PCR, and judging the drug-resistant gene mutation result of the helicobacter pylori by calculating the difference value of the Ct values of the target mutant gene and other sequences.
By adopting the technical scheme, when the primer probe is designed, the target primer probe composition is designed, and at least one group of other sequence-specific primer probe compositions are also designed. The design aims at realizing the simultaneous amplification of sequences of various target mutation sites and other sequences when the primer probe composition is combined with an ARMS-PCR method for gene detection and gene mutation judgment; and during interpretation, the mutation results of various target mutation sites are obtained by interpreting the difference value of the Ct values of the target mutation genes marked by different fluorescein and other sequences. Therefore, when the primer probe composition is used for mutation detection of related target sites, the mutation detection of multiple sites can be completed by one tube, and the primer probe composition has the advantages of high efficiency and high flux.
Optionally, when the other sequence is a conserved region sequence of the target mutation site, the regions of the target mutation site and the other sequence are separated by more than 500 bp.
By adopting the technical scheme, when other sequences are the conserved region sequences of the target mutation sites, the 'regions where the target mutation sites and the other sequences are respectively located are separated by more than 500 bp', so that the amplification of the sequences of the target mutation sites and the other sequences cannot be influenced mutually when the sequences of the target mutation sites and the other sequences are simultaneously amplified. Here, the applicant can also appropriately adjust the distance between the "regions where the target mutation site and the other sequences are respectively located" according to the actual situation, as long as the amplification of the sequence where the target mutation site is located and the amplification of the other sequences are achieved so as not to affect each other. This distance limitation may be absent when the other sequence is a conserved region sequence of another gene different from the sequence of the target mutation site.
In a second aspect, the present application provides a primer probe composition for detecting or identifying helicobacter pylori drug-resistant gene mutation, which implements the above method, and adopts the following technical scheme:
a primer probe composition for detecting or identifying helicobacter pylori drug-resistant gene mutation by implementing the method, wherein the drug-resistant gene is helicobacter pylori gyrA drug-resistant gene, and the target primer probe composition is ARMS primer probe composition; the ARMS primer probe composition is used for binding to a target mutation site, and the other sequence specific primer probe composition is used for binding to other sequences.
The primer probe set for mutation detection designed in the related art is designed only for the mutation site to be detected, and related primer probes are designed for related mutation detection. When the primer probe set is combined with an ARMS-PCR method to carry out gene detection and judge gene mutation, the method carries out interpretation by detecting the difference value of the Ct values of a mutation tube and a wild tube, thereby bringing about the problems of small detection flux, high cost and the like.
By adopting the technical scheme, when the primer probe composition is combined with an ARMS-PCR method to carry out gene detection and judge gene mutation, the simultaneous amplification of a plurality of target mutation sites and other sequences can be realized. And during interpretation, the difference value of the Ct values of the target mutant genes marked by different fluorescein and other sequences is interpreted to obtain the mutation results of various target mutant sites. Therefore, the method can complete mutation detection of multiple sites through one tube, and has the advantages of high efficiency and high flux.
Optionally, the mutation sites of the helicobacter pylori gyrA drug-resistant gene are quinolone Asn87-Lys/Ile, Asp 91-Gly/Asn/Tyr.
Optionally, the additional sequence is a 16S rRNA gene sequence; the other sequence specific primer probe composition is a 16S rRNA gene primer probe composition.
In this scheme, the other sequence is selected to be the 16S rRNA gene sequence, which is a "conserved region sequence of other genes different from the sequence in which the target mutation site is located"; the applicants can also select "a conserved region sequence of the sequence in which the target mutation site is located" as the other sequence according to the actual situation.
Alternatively, the 3 'end of the ARMS primer is located at the mutation region and the 2 nd, 3 rd, 4 th or other positions of the 3' end of the ARMS primer may introduce at least one mismatched base.
By adopting the technical scheme, the specificity of the primer can be increased, the mismatch extension when the primer is mismatched with the target DNA is reduced, and the primer can form multiple mismatch with a template to prevent the mismatch extension by introducing another one or more mismatched bases at the 3' end of the primer.
Optionally, the mutation site of the gyrA drug resistance gene of helicobacter pylori includes at least one of A260T, T261G, T261A, G271A, G271T and A272G.
Optionally, the ARMS primer probe composition comprises a universal downstream primer gyrA-R, a probe gyrA-P and an upstream primer composition, wherein the upstream primer composition comprises at least two of an upstream primer F1 at A260T, an upstream primer F2 at T261G, an upstream primer F3 at T261A, an upstream primer F4 and an upstream primer F5 at G271A, an upstream primer F6 at G271T, an upstream primer F7 and an upstream primer F8 and an upstream primer F9 at A272G;
wherein, the gene sequence of the upstream primer F1 at the site A260T is shown as SEQ ID NO 1;
an upstream primer F2 at the site of T261G, wherein the gene sequence is shown as SEQ ID NO 2;
an upstream primer F3 at a T261A site, wherein the gene sequence is shown as SEQ ID NO 3;
an upstream primer F4 and an upstream primer F5 at the G271A site, wherein the gene sequence of the upstream primer F4 is shown as SEQ ID NO 4, and the gene sequence of the upstream primer F5 is shown as SEQ ID NO 5;
upstream primers F6 and F7 at the G271T site, wherein the gene sequence of the upstream primer F6 is shown as SEQ ID NO 6, and the gene sequence of the upstream primer F7 is shown as SEQ ID NO 7;
upstream primers F8 and F9 at A272G site, wherein the gene sequence of the upstream primer F8 is shown as SEQ ID NO 8, and the gene sequence of the upstream primer F9 is shown as SEQ ID NO 9;
the gene sequence of the general downstream primer gyrA-R is shown as SEQ ID NO 10;
the gene sequence of the probe gyrA-P is shown as SEQ ID NO 11.
Optionally, the 16S rRNA gene primer probe composition comprises an upstream primer 16S-F, the gene sequence of which is shown in SEQ ID NO 12; the gene sequence of the downstream primer 16S-R is shown as SEQ ID NO 13; and a probe 16S-P, the gene sequence of which is shown as SEQ ID NO 14.
Optionally, the primer probe composition further comprises an internal standard gene primer probe set, wherein the internal standard gene is a human housekeeping gene RNP gene.
Optionally, the internal standard gene primer probe group comprises an upstream primer RNP-F, and the gene sequence of the upstream primer RNP-F is shown as SEQ ID NO 15; the downstream primer RNP-R has a gene sequence shown as SEQ ID NO 16; the gene sequence of the probe RNP-P is shown as SEQ ID NO 17.
Optionally, the 5 'end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a fluorescence quenching group.
Optionally, the fluorescent group is selected from any one of FAM, TET, VIC, HEX, ROX, CY3, CY5 and CY 5.5;
the fluorescence quenching group is selected from any one of NFQ-BHQ, BHQ1, BHQ2, BHQ3 and TAMRA.
Optionally, the 5 'end of the probe gyrA-P is labeled with FAM group as a fluorescent group, and the 3' end uses BHQ1 as a quenching group;
the 5 'end of the probe 16S-P is marked with a VIC group as a fluorescent group, and the 3' end of the probe uses BHQ1 as a quenching group;
the 5 'end of the probe RNP-P is marked with a CY5 group as a fluorescent group, and the 3' end of the probe RNP-P uses BHQ3 as a quenching group.
In a third aspect, the present application provides an application of the above method and/or the above primer probe composition in the preparation of a product for detecting or identifying a helicobacter pylori drug-resistant gene mutation, which adopts the following technical scheme:
the application of the method and/or the primer probe composition in preparing a product for detecting or identifying helicobacter pylori drug-resistant gene mutation is disclosed, and the product is a detection reagent, a detection kit or a detection chip.
In a fourth aspect, the present application provides a kit for detecting helicobacter pylori gyrA drug-resistant gene mutation, which adopts the following technical scheme:
a kit for detecting the drug-resistant gene mutation of helicobacter pylori gyrA contains the primer probe composition.
Optionally, the kit further comprises a PCR reaction solution for fluorescent PCR reaction, an enzyme mixed solution, a negative reference substance and a positive reference substance, wherein the PCR reaction solution contains the primer probe composition and a PCR buffer solution as a solvent, and the enzyme mixed solution contains 1-5U/reaction Taq enzyme and 1-5U/reaction UNG enzyme.
Optionally, in the PCR reaction solution, the concentration of each primer is 100-800 nM; the concentration of each probe is 100-300 nM.
By adopting the technical scheme, the specificity of the reagent and the sensitivity of the detection method are effectively improved.
Optionally, the PCR reaction solution further comprises 2-8 mM dNTPs.
Optionally, the positive control contains mutant plasmids, internal standard gene plasmids and other gene plasmids of each site of the gyrA gene; a plurality of mutant plasmids of each site of the gyrA gene respectively contain a certain mutant site of the gyrA gene; the internal standard gene plasmid contains an internal standard gene and can be used as a template DNA to be combined with an internal standard gene primer probe set; the other gene plasmid contains other genes and can be used as a template DNA to be combined with the other sequence-specific primer probe composition.
In a fifth aspect, the application provides a use method of the kit, which adopts the following technical scheme:
according to the using method of the kit, the primer probe composition further comprises an internal standard gene primer probe group, wherein the internal standard gene is a housekeeping gene RNP gene;
mixing the primer probe composition with the template DNA of the sample to be detected, and performing ARMS-PCR (autoregressive moving System-polymerase chain reaction) to simultaneously perform fluorescent quantitative PCR reaction on the sequence of the target mutation site and other sequences;
the ARMS primer probe composition is combined with a region where a target mutation site on the template DNA is located, other sequence-specific primer probe compositions are combined with regions where other sequences on the template DNA are located, Ct values are respectively obtained during detection, and the gene mutation of the sample to be detected is judged through the Ct value difference.
Optionally, the determination method for determining the gene mutation of the sample to be detected through the Ct value difference comprises the following steps:
s1 interpretation of whether there is helicobacter pylori infection
Ct value of amplification of internal standard gene<At 38, a subsequent interpretation can be performed: if HEX (VIC) channel has no obvious amplification signal or CtHEX(VIC)>37, the sample is free from helicobacter pylori infection; if Ct isHEX(VIC)If the content is less than or equal to 37, the helicobacter pylori infection exists in the sample, and the subsequent mutation result is judged;
s2, interpretation of mutation results
The FAM channel of the sample to be detected has obvious amplification signal and CtFAMWhen the value is less than or equal to 37, calculating the Ct of the FAM channelFAMValue and Ct of HEX (VIC) channelHEX(VIC)Difference in value Δ Ct, where Δ Ct = CtFAMvalue-CtHEX(VIC)The value, determined by the Δ Ct value, is: if the delta Ct is less than 9, the detection site of the gyrA gene is judged to be mutant, and if the delta Ct is more than or equal to 9, the detection site of the gyrA gene is judged to be wild type;
FAM channel of sample to be detected has no obvious amplification signal or CtFAMValue of>37, judging that the detection site of the gyrA gene is a wild type or is lower than the detection limit.
Alternatively, the procedure for fluorescent quantitative PCR reaction includes:
1）37℃ 5min；
2）95℃ 5min；
3）95℃ 15s；
4）60℃ 1min；
wherein 3) and 4) are performed for a total of 45 cycles, and the fluorescent signal is collected at the annealing stage of each cycle.
In summary, the present application has the following beneficial effects:
1. the application provides a primer probe composition for detecting helicobacter pylori gyrA drug-resistant gene mutation, which designs at least one group of other sequence-specific primer probe compositions while designing ARMS primer probe compositions; therefore, when the primer probe composition is combined with an ARMS-PCR method for gene detection and gene mutation judgment, the sequences of a plurality of target mutation sites and other sequences can be simultaneously amplified; and when the discrimination is carried out, the discrimination of the difference value of the Ct value of the target mutation gene marked by different fluorescein and other sequences is carried out to obtain the mutation result of a plurality of target mutation sites, so that the detection of the mutation of the plurality of sites can be completed by one tube, and the advantages of high efficiency and high flux are achieved.
2. The utility model provides a helicobacter pylori gyrA drug-resistant gene mutation detect reagent box can realize the while detection of a plurality of position point mutation types, can accomplish the accurate detection to 6 mutation types of 4 positions simultaneously even, and the mode that accessible Ct value calculated is interpreted after the PCR reaction, detects with low costs, and the result is accurate reliable, directly perceived, has better practicality.
3. The method overcomes the problem of harsh requirements on instruments in certain technologies, such as a dissolution curve method, can realize detection only by using a general instrument, and is convenient and efficient.
4. The method adopts a closed tube operation mode, does not need special treatment after PCR, reduces the possibility of PCR product pollution, and further ensures the accuracy of the detection result.
5. The kits provided herein include UNG enzyme to reduce the likelihood of contamination of PCR products.
6. The probe in the kit can be only connected with three fluorescent groups, so that simultaneous detection of 6 target mutation sites and 1 internal control gene is realized in a single tube, the detection flux is increased, and the cost is reduced.
Drawings
FIG. 1 is a graph showing the positive fluorescence results of the 271G > T mutation of the gyrA gene in the paraffin sample of gastric mucosa in example 3;
FIG. 2 is a positive sequencing diagram showing the 271G > T mutation of the gyrA gene in the paraffin sample of gastric mucosa in example 3;
FIG. 3 is a positive sequencing diagram showing 261T > A mutation of the gyrA gene in example 3;
FIG. 4 is a positive sequencing diagram showing 271G > A mutation of the gyrA gene in example 3;
FIG. 5 is a positive sequencing diagram showing the gyrA gene 272A > G mutation in example 3;
FIG. 6 is a positive sequencing diagram showing the gyrA gene 260A > T mutation in example 3;
FIG. 7 is a graph showing fluorescence results positive for the gyrA gene 261T > G mutation in the stool sample of example 4;
FIG. 8 is a graph showing a positive sequencing of the gyrA gene 261T > G mutation in the stool sample of example 4.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
Examples
Example 1 primer Probe composition
Finding out the drug-resistant site to be detected of the gene according to the gyrA gene sequence of the helicobacter pylori downloaded from NCBI, and designing a primer probe. Wherein the 3 'end of the upstream ARMS primer at the detection site is positioned at the mutation site and is consistent with the mutant gene, and in order to improve the specificity of the primer, a mismatched base can be introduced into the 2 nd, 3 rd, 4 th or other positions at the 3' end. The Tm value of the primer is maintained at about 60 ℃ and the Tm value of the probe is maintained at about 70 ℃.
The ARMS primer probe composition is designed as follows:
A260T site upstream primer F1: 5'-ATACCACCCCCATGGCGcTAt-3' (SEQ ID NO 1), upstream primer F2 at position T261G: 5'-ACCACCCCCATGGCGAgAAg-3' (SEQ ID NO 2), T261A site upstream primer F3: 5'-CACCCCCATGGCGATAta-3' (SEQ ID NO 3), G271A site upstream primer F4: 5'-CATGGCGATAATGCGGTTTATa-3' (SEQ ID NO. 4) and F5: 5'-CATGGCGATAACGCGGTTTATa-3' (SEQ ID NO. 5), G271T site upstream primer F6: 5'-ATGGCGATAATGCGGTTTgTt-3' (SEQ ID NO. 6) and F7: 5'-ATGGCGATAACGCGGTTTATt-3' (SEQ ID NO. 7), A272G site upstream primer F8: 5'-TGGCGATAATGCGGTaTATGg-3' (SEQ ID NO 8) and F9: 5'-TGGCGATAACGCGGTTaATGg-3' (SEQ ID NO 9). Wherein, the bases indicated by lower case letters in each upstream primer are mismatched bases artificially introduced at the time of primer design, and the positions and the number of the mismatched bases are selected according to the actual situation.
General downstream primers gyrA-R: 5'-GCTGCAGCGTTATCGCCATCAAT-3' (SEQ ID NO 10). Probe gyrA-P: 5'-AGCCAAAGTTGCCTTGCCCATCCACTA-3' (SEQ ID NO 1); FAM groups are marked at the 5 'end of the probe gyrA-P to be used as fluorescent groups, and BHQ1 is used as a quenching group at the 3' end. Since the primers involved a synonymous mutation T or C at position 261 when the primers were upstream at positions 271 and 272, both primers were involved here.
The 16S rRNA gene primer probe composition has the sequence as follows:
the upstream primer 16S-F: 5'-CAGCCATGTTGCGGTGAA-3' (SEQ ID NO 12), downstream primer 16S-R: 5'-AAACACAACTCCCATGGTGTGA-3' (SEQ ID NO 13), probe 16S-P: 5'-ACGTTCCCGGGTCTTGTACTCACCG-3' (SEQ ID NO 14); the 5 'end of the probe 16S-P is labeled with a VIC group as a fluorescent group, and the 3' end uses BHQ1 as a quenching group.
The human housekeeping gene RNP primer probe composition (i.e. internal standard gene primer probe composition) has the sequence as follows:
an upstream primer RNP-F: 5'-GACTTCAGCATGGCGGTGTTT-3' (SEQ ID NO 15), downstream primer RNP-R: 5'-TGTCTCCACAAGTCCGCGC-3' (SEQ ID NO 16), probe RNP-P: 5'-ACCTGAAGGCTCTGCGCGGAC-3' (SEQ ID NO 17); the 5 'end of the probe RNP-P is marked with a CY5 group as a fluorescent group, and the 3' end of the probe RNP-P uses BHQ3 as a quenching group.
Example 2 kit for detecting mutation of drug-resistant gene of helicobacter pylori gyrA
The kit comprises PCR reaction liquid for fluorescent PCR reaction, enzyme mixed liquid, a negative reference substance and a positive reference substance. Wherein, the PCR reaction solution contains the primer probe composition and dNTPs in the embodiment 1, and the solvent is PCR buffer solution. Further, the PCR buffer solution is 2 XBuffer; the concentration of each primer in the PCR reaction solution is 400nM, and the concentration of each probe is 200 nM; the concentration of dNTPs was 2.5 mM.
The enzyme mixture contained 2U/reaction Taq enzyme and 1U/reaction UNG enzyme.
The positive control contains mutant plasmids, internal standard gene plasmids and other gene plasmids of each site of the gyrA gene, and the plasmids are synthesized by Jinwei Biotech company. The mutant plasmids of each site of the gyrA gene include a plasmid with a site mutation of A260T, a plasmid with a site mutation of T261G, a plasmid with a site mutation of T261A, a plasmid with a site mutation of G271A, a plasmid with a site mutation of G271T and a plasmid with a site mutation of A272G. The internal standard gene plasmid contains internal standard genes: the human housekeeping gene RNP gene can be used as a template DNA to be combined with the human housekeeping gene RNP primer probe composition; other genes plasmids contain other genes: 16S gene, capable of binding as template DNA to a 16S rRNA primer probe composition.
Example 3 method of Using kit for detecting mutation of drug-resistant Gene of helicobacter pylori gyrA
A use method of a kit for detecting helicobacter pylori gyrA drug-resistant gene mutation comprises the following steps: the kit of example 2 was used to mix the primer probe composition with the template DNA of the sample to be tested for ARMS-PCR, allowing the target mutant gene and other genes to undergo fluorescent quantitative PCR simultaneously.
The ARMS primer probe composition is combined with a region where a target mutation site on the template DNA is located, the 16S rRNA primer probe composition is combined with a region where a 16S gene on the template DNA is located, Ct values are respectively obtained during detection, and the a gene mutation of the sample to be detected is judged through the Ct value difference.
The use method of the kit specifically comprises the following steps:
I. nucleic acid extraction of a sample to be detected:
taking 3-8 paraffin-embedded gastric mucosa tissue slices as required, and extracting by using a commercial paraffin sample nucleic acid extraction kit to obtain nucleic acid, wherein the nucleic acid extraction reagent is a magnetic bead method paraffin-embedded tissue genome extraction kit (product number: NMG 1411). The genome is extracted as a PCR amplification template.
Preparing a reagent: the PCR amplification reaction solution was prepared in the amount of 17. mu.L of the PCR reaction solution and 3. mu.L of the enzyme mixture solution times the number of samples to be tested. The volume of 20. mu.L per tube was equally distributed to 8 rows of tubes.
II. Add 10. mu.L PCR amplification template obtained by nucleic acid extraction to the above reagent, and amplify the positive control and negative control simultaneously, after a short time of centrifugation, to the amplification area, the amplification procedure is shown in Table 1.
TABLE 1 PCR amplification procedure
Figure DEST_PATH_IMAGE001
III, interpretation of results
The judgment method for judging the gene mutation of the sample to be detected through the Ct value difference comprises the following steps:
s1 interpretation of whether there is helicobacter pylori infection
The internal control CY5 signal of the sample to be tested should have obvious amplification signal and CtCY5<38, a subsequent interpretation can be performed: if HEX (VIC) channel has no obvious amplification signal or CtHEX(VIC)>37, the sample is free from helicobacter pylori infection; if Ct isHEX(VIC)If the content is less than or equal to 37, the helicobacter pylori infection exists in the sample, and the subsequent mutation result is judged;
s2, interpretation of mutation results
The FAM channel of the sample to be detected has obvious amplification signal and CtFAMWhen the value is less than or equal to 37, calculating the Ct of the FAM channelFAMValue and Ct of HEX (VIC) channelHEX(VIC)Difference in value Δ Ct, where Δ Ct = CtFAMvalue-CtHEX(VIC)The value, determined by the Δ Ct value, is: if the delta Ct is less than 9, the detection site of the gyrA gene is judged to be mutant, and if the delta Ct is more than or equal to 9, the detection site of the gyrA gene is judged to be wild type;
FAM channel of sample to be detected has no obvious amplification signal or CtFAMValue of>37, judging that the detection site of the gyrA gene is a wild type or is lower than the detection limit. The interpretation of the mutation results is shown in Table 2.
TABLE 2 interpretation criteria for mutation results
Figure 214047DEST_PATH_IMAGE002
Confirming that the correction fluorescence is not selected, setting a baseline according to the specific amplification condition of each channel of the sample, and preferably considering the automatic selection of the instrument or manual selection; the threshold was replicated by using the inflection point of the rise of the CY5 channel amplification curve of the positive control as a threshold line, and the threshold of FAM and hex (vic) channels was set to coincide with that of CY5 channel.
When the gyrA gene 271 locus of the sample is G > T, FIG. 1 is a paraffin sample amplification curve, and FIG. 2 is a sample sequencing result graph; in many sample assays, the amplification profile is similar to that of FIG. 1, except that the 271G > T sites, and the profiles of the remaining samples are shown in FIGS. 3-6, where heterozygous mutations are also present in the clinical sample, since the reagents are not amenable to typing.
Comparative example 1 method of use of sequencing (gold standard) kit
The kits of the present application were compared for consistency with the sequencing (gold standard) results.
200 cases of helicobacter pylori infection and the need of adopting paraffin-embedded gastric mucosa tissues are collected, the extraction kit mentioned in the embodiment 3 is used for extracting nucleic acid of the paraffin tissues, the detection is respectively carried out by using the kit and a gene sequencing method (gold standard), and the coincidence rate of the two methods is calculated by comparing the detection result of gyrA gene Asn87-Lys/Ile and Asp91-Gly/Asn/Tyr mutation sites of a sample by using the kit and the detection result of the same mutation sites of the sample by using the gene sequencing method. Compared with the gold standard, the high coincidence rate represents that the two methods have high consistency, and the kit has high accuracy of detecting the site. The low coincidence rate indicates that the kit is low in accuracy and is not applicable.
According to the results, the coincidence rate of the detection result of the kit and the detection result of the gene sequencing method is 100%, and the statistical results of the samples of each mutation site type are shown in table 3.
TABLE 3 statistics of polymorphism type samples of each mutation site type
Figure DEST_PATH_IMAGE003
Clinical sensitivity is 81/(81 + 3) × 100%, 96.4%;
clinical specificity 115/(1 + 115) × 100% — 99.1%;
positive predictive value 81/(81 + 3) × 100% ═ 96.4%;
negative predictive value 115/(1 + 115) × 100% — 99.1%;
the total coincidence rate is (81 + 115)/200 × 100%: 98%.
Example 4 method of Using kit for detecting mutation of drug-resistant Gene of helicobacter pylori gyrA
This example differs from example 3 in that the target sample is selected differently, and the test sample in this application is feces.
The use method of the kit comprises the following steps:
I. stool sample: feces were frozen or stored in feces storage solution as required, and extracted using a commercial feces extraction kit (cat # NMG 1211). The genome is extracted as a PCR amplification template.
Preparing a reagent: the same as in example 3.
Steps II and III are the same as in example 3.
When the 261 th site of the gyrA gene of the sample is T > G, the amplification curve of the fecal sample is shown in FIG. 7, and the sequencing result of the sample is shown in FIG. 8.
Comparative example 2 method of use of sequencing (gold Standard) kit
The present comparative example differs from comparative example 1 in that the same stool sample as in example 3 was selected as the test sample.
Collecting 200 cases of excrement samples infected by helicobacter pylori and needing to detect the drug resistance of the helicobacter pylori, storing the excrement samples by adopting nano-magnetic excrement storage solution, extracting nucleic acid of the excrement samples by using the extraction kit mentioned in the embodiment 2, respectively detecting by using the kit and a gene sequencing method, and calculating the coincidence rate of the two methods by comparing the detection result of gyrA gene Asn87-Lys/Ile and Asp91-Gly/Asn/Tyr mutation sites of the samples by using the kit and the detection result of the same mutation sites of the samples by using the gene sequencing method. Compared with the gold standard, the high coincidence rate represents that the two methods have high consistency, and the kit has high accuracy of detecting the site. The low coincidence rate indicates that the kit is low in accuracy and is not applicable.
According to the results, the coincidence rate of the detection result of the kit and the detection result of the gene sequencing method is 95%, and the statistical results of the samples of various mutation sites are shown in table 4.
TABLE 4 statistics of polymorphism type samples of each mutation site type
Figure 927925DEST_PATH_IMAGE004
Clinical sensitivity of 72/(72 + 5) × 100% of 93.5%;
clinical specificity 118/(5 + 118) × 100% — 95.9%;
positive predictive value 72/(72 + 5) × 100% ═ 93.5%;
negative predictive value 118/(5 + 118) × 100% — 95.9%;
the total coincidence rate is (72 + 118)/200 × 100% ═ 95%.
The comparison of the detection results of the kit for detecting 6 polymorphic sites and the gene sequencing method shows that: the total coincidence rate is high, and the kit has strong specificity, high sensitivity and high detection rate. The method has the advantages of relatively high flux, low detection cost, accurate, reliable and visual result and good practicability.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Sequence listing
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Claims (10)

1. A method for detecting or identifying a mutation in a drug-resistant gene of helicobacter pylori, comprising the steps of:
designing specific primer probe compositions of other sequences and target primer probe compositions for amplifying and detecting a plurality of target mutation sites, wherein the other sequences are conserved region sequences of the target mutation sites or conserved region sequences of other genes different from the sequences of the target mutation sites; and mixing the target primer probe composition and the other sequence specific primer probe compositions with template DNA of a sample to be detected, carrying out ARMS-PCR, and judging the drug-resistant gene mutation result of the helicobacter pylori by calculating the difference value of the Ct values of the target mutant gene and other sequences.
2. The method according to claim 1, wherein when the other sequence is a conserved region sequence of the sequence in which the target mutation site is located, the regions in which the target mutation site and the other sequence are located are separated by 500bp or more.
3. A primer probe composition for detecting or identifying a mutation in a helicobacter pylori drug-resistant gene by carrying out the method according to any one of claims 1 to 2, wherein the drug-resistant gene is a helicobacter pylori gyrA drug-resistant gene, and the primer probe composition of interest is an ARMS primer probe composition; the ARMS primer probe composition is used for binding to a target mutation site, and the other sequence specific primer probe composition is used for binding to other sequences.
4. The primer probe composition of claim 3, wherein the additional sequence is a 16S rRNA gene sequence; the other sequence specific primer probe composition is a 16S rRNA gene primer probe composition.
5. The primer probe composition of claim 3, wherein the ARMS primer probe composition comprises at least two of the universal downstream primer gyrA-R, the probe gyrA-P and the upstream primer composition, wherein the upstream primer composition comprises at least two of the upstream primer F1 at the A260T site, the upstream primer F2 at the T261G site, the upstream primer F3 at the T261A site, the upstream primer F4 and the upstream primer F5 at the G271A site, the upstream primer F6 at the G271T site, the upstream primer F8 and the upstream primer F9 at the A7 and 272G sites;
wherein, the gene sequence of the upstream primer F1 at the site A260T is shown as SEQ ID NO 1;
an upstream primer F2 at the site of T261G, wherein the gene sequence is shown as SEQ ID NO 2;
an upstream primer F3 at a T261A site, wherein the gene sequence is shown as SEQ ID NO 3;
an upstream primer F4 and an upstream primer F5 at the G271A site, wherein the gene sequence of the upstream primer F4 is shown as SEQ ID NO 4, and the gene sequence of the upstream primer F5 is shown as SEQ ID NO 5;
upstream primers F6 and F7 at the G271T site, wherein the gene sequence of the upstream primer F6 is shown as SEQ ID NO 6, and the gene sequence of the upstream primer F7 is shown as SEQ ID NO 7;
upstream primers F8 and F9 at A272G site, wherein the gene sequence of the upstream primer F8 is shown as SEQ ID NO 8, and the gene sequence of the upstream primer F9 is shown as SEQ ID NO 9;
the gene sequence of the general downstream primer gyrA-R is shown as SEQ ID NO 10;
the gene sequence of the probe gyrA-P is shown as SEQ ID NO 11.
6. The primer probe composition of claim 3, wherein the 16S rRNA gene primer probe composition comprises an upstream primer 16S-F, the gene sequence of which is shown in SEQ ID NO 12; the gene sequence of the downstream primer 16S-R is shown as SEQ ID NO 13; and a probe 16S-P, the gene sequence of which is shown as SEQ ID NO 14.
7. The primer probe composition of claim 3, further comprising an internal standard gene primer probe composition, wherein the internal standard gene is a human housekeeping gene RNP gene.
8. Use of the method according to claims 1-2 and/or the primer probe composition according to any one of claims 3-7 in the preparation of a product for detecting or identifying a mutation in a drug-resistant gene of helicobacter pylori, wherein the product is a detection reagent, a detection kit or a detection chip.
9. A kit for detecting helicobacter pylori gyrA drug-resistant gene mutation detection, which is characterized by comprising the primer probe composition of any one of claims 3 to 6.
10. A method of using the kit of claim 9, comprising the steps of:
the primer probe composition also comprises an internal standard gene primer probe group, wherein the internal standard gene is a human housekeeping gene RNP gene;
mixing the primer probe composition with the template DNA of the sample to be detected, and performing ARMS-PCR (autoregressive moving System-polymerase chain reaction) to simultaneously perform fluorescent quantitative PCR reaction on the sequence of the target mutation site and other sequences;
the ARMS primer probe composition is combined with a region where a target mutation site on the template DNA is located, other sequence-specific primer probe compositions are combined with regions where other sequences on the template DNA are located, Ct values are respectively obtained during detection, and the gene mutation of the sample to be detected is judged through the Ct value difference.
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