CN113604589B - Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor - Google Patents
Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor Download PDFInfo
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Abstract
The invention provides a kit for simultaneously detecting drug-resistant loci, virulence genotyping and metabolic genotyping of proton pump inhibitors of helicobacter pylori, in particular to a kit for detecting 16 drug-resistant locus variations, 2 virulence loci and 3 loci of 2 proton pump drug metabolism genes of 6 common helicobacter pylori antibiotics based on multiplex PCR-time-of-flight mass spectrometry, and provides a matched primer, a probe combination and a kit.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for simultaneously detecting helicobacter pylori drug resistance sites, virulence genotyping and proton pump inhibitor drug metabolism genotyping.
Background
Helicobacter pylori (h.pyri) is a microaerophilic gram-negative bacterium colonizing the gastric epithelium, infecting about 50% to 70% of the world's population, and some developing countries have rates of infection up to 80% or more, one of the most common pathogens worldwide, which can cause gastrointestinal diseases including peptic ulcers, gastric marginal zone/mucosa-associated lymphoid tissue (MALT) lymphomas, and gastric cancer. Helicobacter pylori has been identified by the world health organization and the international agency for research on cancer (IARC) as a class I carcinogen. It is counted that more than 6% of cancers and about 90% of non-cardiac gastric cancer cases worldwide are due to helicobacter pylori infection. According to World Health Organization (WHO) data, cancer caused by infection in 2018 was 24.2%, 45% of which was due to HP infection. Eradication of H.pylori will significantly reduce the incidence of gastric and peptic ulcers and reduce the costs associated with controlling these incidences, especially in highly prevalent populations, it is significantly cost effective. At present, eradication schemes for helicobacter pylori mainly include Proton Pump Inhibitors (PPI), gastric mucosa protectants and one or two antibiotics, constituting a triple or quadruple therapy. Antibiotics used to eradicate helicobacter pylori include clarithromycin, metronidazole, quinolones (levofloxacin), amoxicillin, tetracycline, rifampin, and the like. At the beginning of the 1990 s, the rate of eradication of H.pylori exceeded 80%. However, in recent years, as bacterial resistance of the global H.pylori strain to the most commonly used antibiotics has increased, antibiotic resistance in many countries has exceeded a threshold of 15-20% over the last 20 years, with resistance levels of clarithromycin, amoxicillin and metronidazole up to 50%, 30% and 95%, respectively. The occurrence of multidrug resistance significantly affects the efficacy of standard therapies for eradication of helicobacter pylori, resulting in a continuous decline in eradication rate of helicobacter pylori worldwide. To best optimize the management of helicobacter pylori infection, helicobacter pylori treatment should be based on local and individual antibiotic resistance patterns, and tailoring effective antibiotic treatment strategies to specific patients may greatly reduce treatment failure and reduce antibiotic resistance.
On the other hand, not all helicobacter pylori positive patients are recommended to be subjected to eradication treatment, and excessive treatment causes waste of medical resources, increases of drug resistance rate of the population, and risks of intestinal flora disorder caused by antibiotic use. A large number of researches show that CagA and VacA are main virulence factors of helicobacter pylori, wherein the CagA is divided into positive and negative, the positive virulence is larger than the negative, the VacA gene can be divided into four types of S1/M1, S1/M2, S2/M1 and S2/M2 according to the difference of gene sequences of a signal region (S region) and an intermediate region (M region), and the virulence degree can be sequentially weakened according to the sequence of S1/M1> S1/M2> S2/M1> S2/M2. Epidemiological investigation showed that vacA s1/m1 type was significantly associated with cagA positives, and vacA s1/m1 and cagA positive strains were significantly associated with gastric ulcers, the occurrence of gastric cancer. Therefore, according to the toxicity gene detection result, the method can combine clinical symptoms and doctor suggestions to determine whether to carry out HP eradication treatment, and provide powerful judgment basis for doctors.
Proton pump inhibitors (proton pump inhibitors, PPIs) are used as the drugs with the strongest acid inhibiting effect at present, have high specificity and long duration, and are widely applied to the treatment of digestive system diseases. However, inadequate knowledge of the adverse effects of PPI has led to current global PPI abuse. The use of PPIs is constrained due to the large inter-individual variability of the drug administration. The bioavailability and metabolism of PPI is mainly affected by the drug metabolizing enzyme CYP2C 19. Mutation of CYP2C19 coding gene can cause change of CYP2C19 enzyme metabolic activity, and further, difference of blood concentration in vivo and even different clinical reactions occur after different patients take medicines taking CYP2C19 as key metabolic enzyme. Currently, more than 38 CYP2C19 alleles are found, but mainly CYP2C19 x 2 and CYP2C19 x 3, which are all loss-of-function alleles, play a role in drug metabolism. In addition, CYP2C19 x 17 also plays a minor role in PPI metabolism with minimal probability of mutation of other alleles. CYP2C19 isozymes are divided into four different metabolic classes: ultrafast metabolic forms (ultrarapid metabolizers, UM) (. 1/. 17,. 17/. 17) may have significantly increased enzymatic activity due to abnormal replication or abnormal amplification of the functional region gene; fast metabolism (extensive metabolizer, EM) (×1/×1), functional region genes are normally expressed, normal active enzymes are expressed, EM is also the metabolic phenotype of normal population; an Intermediary Metabolic (IM) (. 1/. Times.2,. Times.1/. Times.3,. Times.17/. Times.2,. Times.17/. Times.3) may carry a defective or nonfunctional allele and the activity of the expressed drug metabolizing enzyme is slightly reduced than normal; slow metabolome (PM) (. Times.2/. Times.2,. Times.2/. Times.3,. Times.3/. Times.3) may carry two null alleles, resulting in a significant decrease in the activity of the expressed drug metabolizing enzyme. The PM isozyme gene is mutated to generate a stop code, so that the protein synthesis is stopped prematurely, thereby generating the non-active CYP2C19 enzyme and losing the hydroxylation metabolic capacity of the substance. Therefore, the genotyping detection of CYP2C19 can guide doctors to accurately dose PPI medicines.
In the field of drug resistance detection, endoscopic helicobacter pylori culture and phenotypic Drug Susceptibility Testing (DST) are gold standard techniques for detecting drug resistance. However, since invasive endoscopy is required to obtain a gastric biopsy from a patient, and strict conditions are required for H.pylori transport and cultivation, it is difficult and time consuming to perform bacterial cultivation and antibiotic susceptibility testing for at least 10 days, which is time consuming and costly, it is not recommended to perform a complete phenotypic DST prior to first-line treatment. With the continuous development and perfection of molecular detection technology, the molecular detection has increasingly wide clinical application in diagnosis and treatment monitoring of pathogenic microorganism infection, and can be used as an effective alternative method for drug sensitivity test of a culture method. The detection method of pathogenic microorganism molecules commonly used at present mainly comprises the following steps: electrophoresis based on PCR, real-time fluorescent quantitative PCR (qPCR), gene chip technology, sequencing technology (Sanger sequencing technology, pyrosequencing technology, high throughput NGS sequencing technology), etc. PCR is a molecular biological technology for amplifying specific DNA fragments, has the characteristics of high sensitivity, strong specificity, simplicity, rapidness, low purity requirement on specimens and the like, and is the etiology detection technology most widely applied at present. The current molecular detection methods for determining helicobacter pylori resistance mainly comprise restriction fragment length polymorphism PCR (PCR-RFLP), fluorescence in situ hybridization (fluorescence in situ hybridization, FISH), real-time fluorescence quantitative PCR, allele-specific PCR and the like, detection samples comprise biopsy specimens, gastric juice, bacterial colonies and even feces, and the methods show good sensitivity and specificity in detecting 23S rRNA and gyrA gene mutation to predict the drug resistance of clarithromycin and levofloxacin. Chinese patent No. 109797203A "helicobacter pylori detection system and detection kit and application" and No. CN111334592A "a nucleic acid composition for detecting helicobacter pylori drug resistance gene and kit and application" respectively disclose fluorescent quantitative PCR amplification system for identification, typing and antibiotic resistance of helicobacter pylori, but at present, fluorescent quantitative PCR method is limited by the technical limitations of the method, and one detection reaction can only realize simultaneous detection of a plurality of sites at most, and the detection flux is limited, so that the detection of a plurality of gene mutation sites is limited. On the other hand, the need for rapid detection of large sample volumes may not be met using conventional fluorescent quantitative PCR methods. Chinese patent CN105368825A ' kit and method for analyzing helicobacter pylori antibiotic drug resistance ', CN105506160A ' system for quantitative and virulence multiplex gene detection of helicobacter pylori, kit and application thereof all disclose analysis of helicobacter pylori common antibiotic drug resistance in the same reaction system based on multiplex PCR-capillary electrophoresis, but the capillary electrophoresis can only be used for typing according to fragment size, and has high requirements on uniformity and specificity of multiplex PCR amplification. In recent years, the NGS high-throughput sequencing technology and the chip technology are used as a high-throughput high-precision nucleic acid detection technology, and the limitation of the traditional microbiological detection is broken through as pathogen is not required to be separated and cultured, so that the method has a wide application prospect in the field of clinical microbiology research. Chinese patent CN103060455B discloses a helicobacter pylori infection individuation treatment detection gene chip and application, which is a detection gene chip for helicobacter pylori infection individuation treatment, and can detect human genome CYP4502C19 x 2, CYP4502C19 x 3 polymorphism and helicobacter pylori clarithromycin and quinolone drug resistant sites at the same time. However, since the equipment and the process of chip detection and high-throughput sequencing (next generation and third generation sequencing) are complex and expensive, the detection cost is high, the time consumption is long, and the method is mainly used for researching the correlation between genotype and phenotype drug resistance at present, and is rarely used in helicobacter pylori clinical molecular detection. Therefore, how to effectively utilize the means of nucleic acid molecule detection, and to rapidly, efficiently, high-throughput and accurately judge the drug resistance of helicobacter pylori is an urgent problem to be solved.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting helicobacter pylori drug-resistant genes (clarithromycin drug-resistant related 23S gene, quinolone drug-resistant related gyrA gene, metronidazole drug-resistant RdxA gene, amoxicillin drug-resistant related PBP1A, furazolidone drug-resistant genes oorD and porD gene), virulence genes (CagA and VacA genes) and proton pump inhibitor drug metabolism gene CYP2C19 typing sites by utilizing a multiplex PCR-time-of-flight mass spectrum, a matched primer/probe combination and kit, and application of the detection method in clinical HP eradication therapy drug administration guidance. The detection result of the virulence gene can be used for assisting doctors in evaluating the meaning of HP eradication treatment, the detection of the drug resistance gene is used for determining the type of selected antibiotics, and the detection of the metabolic gene of the proton pump inhibitor can accurately guide the dosage of PPI drugs.
In a first aspect of the invention, there is provided a kit for diagnosing helicobacter pylori infection using multiplex PCR-time-of-flight mass spectrometry, the kit comprising a first primer pair set comprising primers having sequences as shown in SEQ ID NO.1 to SEQ ID NO. 14.
In another preferred embodiment, the kit further comprises a second primer pair group comprising primers having sequences shown in SEQ ID NO.15 to SEQ ID NO. 30.
In another preferred embodiment, the kit further comprises a third primer pair group comprising primers having sequences shown in SEQ ID NO.31 to SEQ ID NO. 48.
In another preferred embodiment, the kit further comprises a first set of probes comprising the probes of sequences SEQ ID NO.49 through SEQ ID NO. 55.
In another preferred embodiment, the kit further comprises a second set of probes comprising the probes of sequences SEQ ID NO.56 to SEQ ID NO. 63.
In another preferred embodiment, the kit further comprises a third set of probes comprising the probes of sequences SEQ ID NO.64 to SEQ ID NO. 72.
In another preferred embodiment, the kit includes a first container containing the first primer pair set therein.
In another preferred embodiment, the kit comprises a second container containing the second primer pair set.
In another preferred embodiment, the kit includes a third container containing the third primer pair set.
In another preferred embodiment, the kit includes a fourth container containing the first probe set therein.
In another preferred embodiment, the kit includes a fifth container containing the second probe set therein.
In another preferred embodiment, the kit includes a sixth container, wherein the sixth container contains the third probe set.
In another preferred embodiment, the kit comprises a seventh container, wherein the seventh container contains a PCR premix solution; preferably, the PCR premix mainly comprises hot start Taq enzyme, dNTPs and Mg 2+ 。
In another preferred embodiment, the kit comprises an eighth container comprising Shrimp Alkaline Phosphatase (SAP) therein.
In another preferred embodiment, the kit comprises a ninth container, the ninth container containing an elongase.
In another preferred embodiment, the kit includes a tenth container containing ddNTP.
In another preferred embodiment, the kit comprises an eleventh container containing an extension reaction buffer.
In another preferred embodiment, the kit includes a twelfth container containing pure water.
In a second aspect of the invention, there is provided a method of diagnosing helicobacter pylori infection using multiplex PCR-time-of-flight mass spectrometry, the method comprising the steps of:
(1) Providing a sample to be detected, and carrying out PCR amplification on nucleic acid in the sample to be detected to obtain a target sequence amplification product in the sample to be detected;
(2) Treating the amplification product obtained in step (1) with Shrimp Alkaline Phosphatase (SAP);
(3) Carrying out single base extension reaction on the amplified product treated in the step (2) by using an extension probe to obtain an extension product;
(4) Purifying the extension product;
(5) Detecting the molecular weight of the purified extension product by using a matrix-assisted laser desorption ionization time of flight MASS spectrometry (MALDI-TOF-MASS) system, and determining whether a sample to be detected has a certain site drug resistance mutation according to a molecular weight mark;
wherein in the step (1), PCR amplification is performed using the first primer pair group, the second primer pair group, and the third primer pair group, respectively;
preferably, the first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
In another preferred embodiment, in the step (3), single base extension reaction is performed using the first probe set, the second probe set, and the third probe set, respectively;
Preferably, the first probe set comprises probes having sequences shown in SEQ ID NO.49 through SEQ ID NO. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe set includes probes having sequences shown in SEQ ID NO.64 through SEQ ID NO. 72.
In another preferred embodiment, the method is for non-diagnostic purposes. For example, environmental samples may be tested to identify resistant helicobacter pylori in the environmental samples.
In a third aspect of the invention, there is provided the use of a primer set for the preparation of a detection kit for the diagnosis of helicobacter pylori infection;
the primer pair group is one or more of a first primer pair group, a second primer pair group and a third primer pair group; wherein,
the first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
In a fourth aspect of the invention, there is provided the use of a set of probes for the preparation of a detection kit for the diagnosis of helicobacter pylori infection;
The probe sets are one or more probe sets of the first probe set, the second probe set and the third probe set;
the first probe group comprises probes with sequences shown as SEQ ID NO.49 to SEQ ID NO. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe set includes probes having sequences shown in SEQ ID NO.64 through SEQ ID NO. 72.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a mass spectrum peak diagram of nucleic acid mutated in Clarithromycin resistance pharmacopoeia type 23S_A2143G-C;
FIG. 1-1 is a nucleic acid mass spectrum peak diagram of the lowest detection limit 1000 copies/mL of the clarithromycin resistance pharmacopoeia type 23S_A2143G-C mutation;
FIGS. 1-2 are nucleic acid mass spectra peak diagrams of 23S_A2143G-C detection negative control;
FIG. 2 is a nucleic acid mass spectrum peak diagram of quinolone resistance pharmacopoeia type gyrA_T-C261A-G mutation;
FIG. 2-1 is a nucleic acid mass spectrum peak diagram of the minimum detection limit 1000 copies/mL of quinolone resistance pharmacopoeia type GyrA_T-C261A-G mutation;
FIG. 2-2 is a nucleic acid mass spectrum peak diagram of a GyrA_T-C261A-G detection negative control;
FIG. 3 is a nucleic acid mass spectrum peak diagram of amoxicillin resistant pharmacopoeia type PBP1A_CT1242AG mutation;
FIG. 3-1 is a nucleic acid mass spectrum peak diagram of the minimum detection limit 1000 copies/mL of the amoxicillin resistant pharmacopoeia type PBP1A_CT1242AG mutation;
FIG. 3-2 is a nucleic acid mass spectrum peak diagram of a PBP1A_CT1242AG detection negative control;
FIG. 4 is a nucleic acid mass spectrum peak diagram of the Metronidazole resistance Pharmacopeia type Rdx _G616A mutation;
FIG. 4-1 is a nucleic acid mass spectrum peak diagram of the minimum detection limit 1000 copies/mL of the Metronidazole resistance Pharmacopeia type Rdx _G616A mutation;
FIG. 4-2 is a nucleic acid mass spectrum peak diagram of Rdx _G616A detection negative control;
FIG. 5 is a nucleic acid mass spectrum peak diagram of the tetracycline resistance pharmacopoeia type 16S_926-928TTC mutation;
FIG. 5-1 is a nucleic acid mass spectrum peak diagram of the minimum detection limit 1000 copies/mL of the tetracycline resistance pharmacopoeia type 16S_926-928TTC mutation;
FIG. 5-2 is a nucleic acid mass spectrum peak diagram of 16S_926-928TTC detection negative control.
FIG. 6 is a nucleic acid mass spectrum peak diagram of furazolidone resistance pharmacopoeia type garD_G353A mutation;
FIG. 6-1 is a nucleic acid mass spectrum peak diagram of the lowest detection limit 1000 copies/mL of furazolidone resistance pharmacopoeia type garD_G353A mutation;
FIG. 6-2 is a nucleic acid mass spectrum peak diagram of a garD_G353A detection negative control.
FIG. 7 is a mass spectrum generated by the VS-ZP-F0 extension primer for detecting the virulence gene VacA without distinguishing the S1/S2 typing-specific extension primer peaks.
FIG. 8 is a mass spectrum generated by the VS-ZP-F1 extension primer for detecting S1 typing of VacA virulence genes.
FIG. 9 is a CagA virulence gene-specific site CaA-1 nucleic acid mass spectrum peak diagram of CagA-specific site 1 (CagA 1) mass spectrum peak diagram
FIG. 10 is a mass spectrum of CagA-2 nucleic acid at the specific site of the CagA virulence gene.
FIG. 11 is a mass spectrum peak diagram of CagA-3 nucleic acid at the specific site of CagA virulence gene.
FIG. 12 is a mass spectrum peak diagram of the nucleic acid of the combination of the control primer pair 1 and the control extension probe 1.
Detailed Description
Based on multiple PCR-time-of-flight mass spectrometry, the invention provides that three noninvasive tests can be performed simultaneously with fecal samples: the method for detecting the variation of drug-resistant gene loci of 6 common helicobacter pylori antibiotics (including clarithromycin, metronidazole, quinolones, amoxicillin, tetracycline and furazolidone), detecting 5 loci of 2 virulence genes and 3 loci of 2 proton pump drug metabolism genes and typing, and providing a matched primer/probe combination and a kit. Based on a Massary matrix assisted laser Desorption ionization time of flight mass spectrometry (MALDI-TOF) system (Sequenom, inc., san Diego, calif., USA), simultaneous detection and analysis of 5 virulence genotyping sites (CagA gene, S region of VacA gene, M region of VacA gene), 3 SNP sites (rs 496893, rs4244285 and rs 12248360) of the typing of the drug metabolism gene CYP2C19, 16 common HP drug resistant sites, 5 virulence genotyping sites and 3 typing simultaneous detection and analysis of CYP2C19 can be realized in the same reaction system, and the five drug resistant species are included: tetracycline resistance (2 sites for 16S), clarithromycin resistance (2 sites for 23S rRNA), quinolone resistance (3 sites for GyrA), amoxicillin resistance (2 sites for PBP 1A), metronidazole resistance (2 sites for RdxA), furazolidone resistance (2 sites for ood and 3 sites for gard). The detection method provided by the invention has the advantages of high precision, high sensitivity, high flux, low cost and rapid detection.
Multiplex PCR (multiplex PCR), also called multiplex primer PCR or multiplex PCR, is a PCR reaction in which more than two pairs of primers are added in the same PCR reaction system and simultaneously a plurality of nucleic acid fragments are amplified, and the reaction principle, reaction reagents and operation process are the same as those of the general PCR.
There are many factors that affect multiplex PCR reactions, such as:
(1) The imbalance of the reaction system results in rapid amplification of certain advantageous primers and templates thereof in the previous rounds of reaction, resulting in large amounts of amplified products which are also good inhibitors of DNA polymerase. Therefore, with the large amount of amplified products, the polymerization ability of the polymerase is more and more strongly inhibited, and therefore, the primer and its template, which are at a disadvantage in the early stage, are more difficult to react, eventually resulting in an amount of amplified products that is too small to be detected.
(2) Primer specificity, if the primer binds more strongly to other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, resulting in a decrease in amplification efficiency.
(3) The optimal annealing temperatures are not uniform, and a plurality of pairs of primers are placed in a system for amplification, so that the optimal annealing temperature of each pair of primers is required to be close because the annealing temperatures for carrying out PCR reactions are the same.
(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are also third party DNA mediated polymers, which, like non-specific primers, interfere with the competition of primers with the target binding sites, affecting amplification efficiency.
Although several factors affecting amplification efficiency are mentioned above, more factors are not yet clear. To date, there is no effective method by which amplification efficiency can be predicted explicitly.
The determination method is based on a multiplex PCR technology and a time-of-flight mass spectrum, and is used for designing 24 specific site primers for 24 common helicobacter pylori drug resistance sites, virulence gene sites and drug metabolism gene sites, selecting specific conserved sequences, designing PCR primers, carrying 10 base (ACGTTGGATG) sequences at the 5' end of the primers by using a pair of PCR primers, so that the total length reaches 29 bases or more, and distinguishing the primers from probes in terms of molecular weight. A single base extension probe is designed in the conserved sequence region in the amplified region, the probe length is 15-21 bases, and a designed base is allowed to extend at the 3' -end of the probe to be used as the genotype specific sequence marker.
The multiplex PCR-time-of-flight mass spectrometry detection technology can detect with ultra-high throughput, but has high quality requirements on PCR amplification yield. The inventor finds that the existing primer and probe capable of detecting by the multiplex fluorescence PCR method are directly applied to the multiplex PCR-time-of-flight mass spectrometry detection, and have many defects, such as false negative of the mass spectrometry detection caused by incapability of single base extension reaction, low sensitivity and poor repeatability, and are difficult to meet clinical application. Therefore, the inventor redesigns a plurality of pairs of primers and extension probes for each detection site, performs multiple combination detection verification under the condition that single-site detection can meet the requirement, and finally obtains a multiple PCR detection system and extension probes which have high sensitivity, good specificity and stable detection results and are suitable for time-of-flight mass spectrometry detection through a large number of test screening.
Thus, in a preferred embodiment, the present invention provides a method, primer and probe combination for simultaneous detection of helicobacter pylori drug resistance gene mutation, virulence genotyping and proton pump inhibitor drug metabolism genotyping, further comprising detection reagents required for the detection.
In another preferred embodiment, the present invention provides a detection method for simultaneously detecting 3 items from one stool sample based on multiplex PCR time-of-flight mass spectrometry, comprising the steps of:
1. and (3) PCR reaction: and obtaining a target sequence amplification product in the sample to be detected through the first round of PCR amplification.
2. Shrimp Alkaline Phosphatase (SAP) treatment: the unbound remaining nucleic acids are dephosphorylated (dNTPs) are inactivated, preventing interference with the next base extension reaction.
3. Base extension reaction: and (3) extending the 3' -end of the single-base extension probe in the second round of amplification, and extending one sequence specific single nucleotide to make molecular weight mark, wherein the molecular weight difference between the obtained extension product and the extension probe and other extension products is not less than 16Da.
4. Resin desalination: purifying the extension reaction product, and adsorbing Na and K ions in the system.
5. Mass spectrometry detection: and (3) detecting the molecular weight of the purified product by adopting a matrix-assisted laser desorption ionization time of flight MASS spectrometry (MALDI-TOF-MASS) system, determining whether a sample to be detected has a certain site drug resistance mutation according to a molecular weight mark, judging the type, and automatically processing a report judging result by software.
Preferably, a negative control, which is normal human blood DNA, is added to each reaction.
Compared with other existing technology or similar technology for detecting helicobacter pylori drug resistance, the technical scheme of the invention fully plays the advantages of simultaneous detection of a plurality of loci by PCR-mass spectrometry, and simultaneously can detect virulence gene loci and drug metabolism gene loci. The kit has the advantages of no need of fluorescent marking, no need of washing, reaction in a trace system, small sample loading amount (the concentration is larger than 1 ng/. Mu.L and can be detected), and the like, so that the reagent consumable cost is inversely proportional to the detection PCR weight, and the purposes of simultaneously detecting at most 16 drug-resistant gene locus variations, 5 loci of 2 virulence genes and 3 loci of 2 proton pump drug metabolism genes in one batch of reactions by using a plurality of probes are realized. The first round of PCR amplification region is a genotype specific fragment of 50-130bp, the second round of PCR amplification is carried out by adopting a specific probe end single-point method, more cycles can be allowed to be used, the detection sensitivity and the specificity are improved to the maximum extent, the detection sensitivity reaches a single copy level of 1aM level, and the false negative problem caused by detection missing diagnosis is avoided. Meanwhile, the specific fragment amplification design technology can thoroughly eliminate the false positive problem caused by PCR product pollution and homologous sequence probe mismatch, and provides a reliable experimental method for multiple detection of respiratory pathogens.
Primer and probe combinations for helicobacter pylori nucleic acid detection based on multiplex PCR time-of-flight mass spectrometry, said combinations being used to detect 16 common HP drug-resistant loci, including six drug-resistant species: tetracycline resistance (2 sites for 16S rRNA), clarithromycin resistance (2 sites for 23S rRNA), quinolone resistance (3 sites for GyrA), amoxicillin resistance (2 sites for PBP 1A), metronidazole resistance (2 sites for RdxA), furazolidone resistance (2 sites for ood, 3 sites for gard); 2 virulence genotyping (including CagA gene, and VacA gene s-fragment and m-fragment typing sites); proton pump inhibitor class drug metabolism genotyping (including CYP2C19 x 1, x2, x3, x17).
The primer and probe combination comprises,
1) Primer sequence combinations as shown in table 1; the PCR primer Panel was divided into 3 combinations of:
w1 multiplex primer combination: SEQ ID NO.1 to SEQ ID NO.14;
w2 multiplex primer combination: SEQ ID NO.15 to SEQ ID NO.30;
w3 multiplex primer combination: SEQ ID NO.31 to SEQ ID NO.48.
Wherein F is a forward primer and R is a reverse primer. In the amplification reaction, each reaction tube contains a multiplex primer combination.
2) Probe sequence combinations as shown in table 2; the extension primer Panel correspondence is also divided into 3 combinations:
w1: SEQ ID NO.49 through SEQ ID NO.55;
w2: SEQ ID NO.56 to SEQ ID NO.63;
w3: SEQ ID NO.64 to SEQ ID NO.72.
TABLE 1 primer sequences
TABLE 2 probe sequences
The inventor designs 24 specific site primers aiming at 24 common helicobacter pylori drug resistance sites, virulence gene sites and drug metabolism gene sites, selects specific conserved sequences, designs PCR primers, uses a pair of PCR primers, has 10 base (ACGTTGGATG) sequences at the 5' end of the primers, enables the total length to reach 29 bases or more, and distinguishes the primers from probes in terms of molecular weight. A single base extension probe is designed in the conserved sequence region in the amplified region, the probe length is 15-21 bases, and a designed base is allowed to extend at the 3' -end of the probe to be used as the genotype specific sequence marker.
The synthetic polynucleotides listed in tables 1 and 2 were synthesized using conventional polynucleotide synthesis methods. The purification mode is ePAGE.
In addition to the primers and probes, the invention also provides a helicobacter pylori drug-resistant locus nucleic acid detection kit, and the specific contents of the components in the detection kit are as follows:
TABLE 3 Table 3
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The invention has the main advantages that:
At present, most detection means mainly detect through fluorescence quantitative PCR, but can not detect a plurality of drug resistance sites, virulence genes and proton pump drug metabolism genes simultaneously, and sequencing means are used, but the cost is higher, and the invention has the following advantages:
(1) Through a large number of screening and deep researches, the invention finally obtains the multiplex primer probe combination suitable for the detection of the flight time mass spectrum, so that the detection of helicobacter pylori drug-resistant sites by multiplex PCR-flight time mass spectrum becomes possible; meanwhile, by adopting one sample of excrement, three detection items can be detected in a non-invasive way. Thereby greatly improving the detection efficiency and remarkably reducing the detection cost.
(2) 16 common HP drug-resistant sites can be detected simultaneously, including six drug-resistant species: tetracycline resistance (2 sites for 16S rRNA), clarithromycin resistance (2 sites for 23S rRNA), quinolone resistance (3 sites for GyrA), amoxicillin resistance (2 sites for PBP 1A), metronidazole resistance (2 sites for RdxA), furazolidone resistance (2 sites for ood, 3 sites for gard); 2 virulence genotyping (including CagA gene, and VacA gene s-fragment and m-fragment typing sites); proton pump inhibitor class drug metabolism genotyping (including CYP2C19 x 1, x2, x3, x17).
(3) The method has higher sensitivity and can reduce the detection concentration to 1000 copies/mL.
(4) The method has extremely high detection efficiency, and can detect hundreds of fluxes in the same batch, thereby greatly improving the detection efficiency.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Example 1
The method for simultaneously detecting/identifying helicobacter pylori drug-resistant sites, virulence genes and proton pump inhibitor drug metabolism genotyping provided by the embodiment of the invention comprises the following steps: the fluorescent quantitative detection is known to be positive fecal samples and negative fecal samples. The method comprises the following steps:
(1) Aiming at 24 common helicobacter pylori drug-resistant loci, virulence loci and drug metabolism loci, 24 specific locus primers are designed, a specific conserved sequence is selected, PCR primers are designed, a pair of PCR primers is used, 10 base (ACGTTGGATG) sequences are carried at the 5' end of the primers, the total length reaches 29 bases or more, and the primers are distinguished from probes in terms of molecular weight. A single base extension probe is designed in the conserved sequence region in the amplified region, the probe length is 15-21 bases, and a designed base is allowed to extend at the 3' -end of the probe to be used as the genotype specific sequence marker. 24-site probes are shown in Table 2.
(2) And obtaining a target sequence amplification product in the sample to be detected through PCR amplification.
Sample selection: selecting positive samples of clarithromycin resistance, quinolone resistance, amoxicillin resistance, metronidazole resistance, tetracycline resistance and furazolidone resistance as fluorescent quantitative results; a positive plasmid with partial site mutation was synthesized, and the plasmid was diluted to 1000 copies/mL.
The PCR reaction system was prepared as shown in Table 4.
TABLE 4 Table 4
| Reagent(s) | Final concentration | Volume 1× (μl) |
| 1mM PCR Primer Mix | 0.2mM | 1.00 |
| 2.5×PCR Mix | 1Unit | 2.00 |
| DNA Template(0.5-2ng/μL) | 1.00 | |
| RNase-Free Water | N/A | 1.00 |
| Total | 5.00 |
The PCR enzymes in Mix were first hot-started at 95℃for 10 min, followed by PCR cycling amplification. The reaction conditions are denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds and extension at 72 ℃ for 1 minute, 45 cycles are all performed; finally, the temperature was 72℃for 5 minutes, and the temperature was kept at 4℃after completion.
(3) Dephosphorylation and inactivation of unbound remaining nucleic acids (dNTPs) by Shrimp Alkaline Phosphatase (SAP) treatment prevents interference with the next base extension reaction. The SAP digestion enzyme reaction system is shown in Table 5.
TABLE 5
| Reagent(s) | Volume 1× (μl) |
| ddH 2 O | 1.75 |
| SAP Enzyme | 0.25 |
| Total | 2.00 |
Incubating for 10 minutes at 37 ℃ under the reaction condition, and removing the residual dNTPs; the SAP enzyme was then inactivated at 85℃for 5 minutes, and after completion the temperature was kept at 4 ℃.
(4) And (3) extending a sequence specific mononucleotide at the 3' end of the single-base extension probe through base extension reaction, and marking the molecular weight, wherein the molecular weight difference between the obtained extension product and the extension probe and between the extension products of all types is not less than 16Da, and the extension reaction system is shown in Table 6.
TABLE 6
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* The concentration of the extension reaction probe Mix was adjusted in a linear relationship with the molecular weight of each type.
The cyclic reaction was a 200 short step procedure, comprising two cyclic chimerism, beginning with denaturation at 95 ℃ for 30 seconds, followed by denaturation at 95 ℃ for 5 seconds, annealing at 52 ℃ for 5 seconds, extension at 80 ℃ for 5 seconds, for a total of 40 cycles, each insert fire off and extension for 5 small cycles; finally, the temperature was 72℃for 3 minutes, and the temperature was kept at 4℃after completion.
(5) The extension reaction product was desalted and purified using a resin.
(6) And detecting the molecular weight of the purified product by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry system, and determining whether a sample to be detected has a mutation at a certain site according to the molecular weight mark.
(7) In fig. 1, the abscissa indicates molecular weight, and the ordinate indicates peak intensity. The dashed line in the figure is the molecular weight position of the genotype extension probe, if the type probe peak is not present, the peak is unchanged; if the copy number of the detected sample is more than 1000, the probe can be completely consumed, and the left peak disappears and turns to the right; the dotted lines on the right represent the extension product molecular weight positions. And (3) reading the spectrogram by using software, automatically analyzing and reporting the result, and deriving data. The data is interpreted that the extension probes of each site or reference gene have corresponding molecular weight peaks at different mass positions of a mass spectrogram, single base extension products appear when the probes find that the target genes work, the molecular weight peaks of the probes are transferred into product molecular peaks, and the analysis result is reported as positive. Positive results are divided into four interpretation cases: A. the result is reliable; B. the medium degree is reliable; C. is generally reliable; D. and the low reliability is realized. The first three are regarded as effective extension reactions, and can be diagnosed that mutation occurs at the site, corresponding to certain drug resistance, and the fourth one needs manual auxiliary judgment, and whether the probe is consumed is observed, so that suspicious infection or negative result is judged. For suspected infection samples, a repeatability verification test can be performed if necessary.
(8) The test shows that the detection results are consistent with the fluorescence quantitative results by carrying out mass spectrum detection on positive samples of clarithromycin resistance, quinolone resistance, amoxicillin resistance, metronidazole resistance, tetracycline resistance and furazolidone, and corresponding drug resistant sites can be detected (shown in figures 1, 2, 3, 4, 5 and 6); the positive plasmid detection results are shown in figures 1-1, 2-1, 3-1, 4-1, 5-1 and 6-1; and negative control results at the time of detection are shown in FIGS. 1-2, 2-2, 3-2, 4-2, 5-2, 6-2.
(9) The VS-ZP-F0 extension primer can recognize the VacA gene, but does not distinguish between the S1/S2 types, the VS-ZP-F1 extension primer is used for detecting the S1 type, the S1 type is provided with an extension peak if the VS-ZP-F1 has no extension peak, the S2 type is provided with an extension peak if the VS-ZP-F0 has an extension peak, and the VacA gene is not detected if the VS-ZP-F0 and the VS-ZP-F1 have no extension peak.
(10) CagA-1, cagA-2 and CagA-3 are specific sites at 3 different positions designed in a relatively conserved region of the CagA gene, and because the sequence variability of the CagA gene is large, 3 sites are arranged, and any specific site is detected and is positive to the CagA gene
(11) The simultaneous detection of the VacA gene s1 and CagA positive as HP being a virulent strain strongly suggests eradication therapy under the direction of doctors.
(12) The invention carries out mass spectrum detection of proton pump inhibitor drug metabolism genotyping on the stool sample with positive fluorescence quantification, and the proton pump metabolism type and frequency, genotyping and frequency are finished to be in accordance with the routine (see table 7); the present invention also found that the ratios of the individual genotypes were similar to the data of the known database pharmGKB (East Asian) or literature (Yin Shengju et al, frequency of distribution of drug metabolism CYP2C19 and CYP2D6 genotypes for the Chinese population) (Table 8 below).
TABLE 7 metabolism and genotyping and frequency of proton pumps
TABLE 8 comparison of the distribution frequencies of the individual alleles of CYP2C19 (%)
Conclusion: the invention can simultaneously detect six drug-resistant gene mutations, virulence genotyping and proton pump inhibitor drug metabolism genotyping of helicobacter pylori, and a total of 24 detection sites. And the detection sensitivity is as low as 1000 copies/mL.
Example 2 specificity assay
The detection results of the multiplex PCR-time-of-flight mass spectrometry detection kit using escherichia coli, staphylococcus aureus, campylobacter jejuni, candida albicans, enterococcus faecium, streptococcus pneumoniae, salmonella paratyphi a and human genome DNA nucleic acid as templates show that no false positive result appears, and the kit has good specificity.
Example 3 clinical sample testing
The method for detecting and/or identifying the helicobacter pylori drug-resistant locus with high sensitivity is used for detecting nucleic acid samples of 100 cases of helicobacter pylori positive patients, and a commercial single fluorescent quantitative PCR detection kit is used for verification.
The results show that:
of 100 positive samples, 100 positive samples are detected by the single fluorescent quantitative PCR and the multiplex PCR-time-of-flight mass spectrometry detection method, and the positive detection rate is 100%. Wherein 11 cases of tetracycline resistance, 35 cases of clarithromycin resistance, 32 cases of quinolone resistance, 3 cases of amoxicillin resistance, 56 cases of metronidazole resistance, 3 cases of furazolidone resistance and the rest of samples are sensitive bacterial infection are identified.
Comparative example 1 screening of primer set and extension Probe
Through a large number of experiments, the inventor designs a large number of primers and extension probes manually, performs optimization selection and verification on the primers and the extension probes, and finally determines multiple PCR amplification primer sequences and extension probe sequences and combinations thereof which can be used for time-of-flight mass spectrometry detection.
This comparative example shows a primer and extension probe with partial unsatisfactory effect, taking the 23S_A2143G-C site as an example.
23S_A2143G-C site primer and extended probe sequence:
Control primer pair 1:
F-1：ACGTTGGATGGTGAAATTGTAGTGGAGGTG(SEQ ID NO.:73)
R-1：ACGTTGGATGTTCCCATTAGCAGTGCTAAG(SEQ ID NO.:74)
control primer pair 2:
F-2：ACGTTGGATGGCTGTCTCAACCAGAGATTC(SEQ ID NO.:75)
R-2：ACGTTGGATGCGCATGATATTCCCATTAGC(SEQ ID NO.:76)
control extension probe 1:
P-1：TCATACCCGCGGCAAGACGGA(SEQ ID NO.:77)
the primer pair of the invention: SEQ ID NOS.9 and 10
The invention extends the probe: SEQ ID NO.53
The specific method is the same as in example 1, wherein single base extension is performed by using a single PCR amplification and then using different extension probes, and then mass spectrometry detection is performed on the extension products. The control primer pair 1 is respectively combined with the control extension probe 1 and the extension probe (SEQ ID NO. 53) of the invention, and the detection sensitivity can only reach 1 multiplied by 10 5 copy/mL. The control primer pair 2 and control extension probe 1 combination works well in a single detection system, whereas positive results cannot be obtained by mass spectrometry in a multiplex system (as shown in fig. 12).
The combination of the primer pair (SEQ ID NO.9 and 10) of the present invention and the extension probe (SEQ ID NO. 53) of the present invention enables detection sensitivity of 1X 10 to be achieved 3 copy/mL.
Comparative example 2 construction of multiplex detection System
Efficient multiplex PCR amplification primer and single base extension probe combinations are difficult to obtain due to competitive inhibition between the primers of the multiplex reaction system, primer specificity differences, inconsistent annealing temperatures, primer dimers, and the like.
Thus, there is a need to multiplex the candidate primer pairs screened for each mutation site with the extension probe to optimize the multiplex detection system. This comparative example illustrates a multiplex assay system with partially non-ideal results.
Control multiplex detection system 1:
TABLE 9
| Sequence number | Site(s) |
| 1 | GyrA_T-C261A-G |
| 2 | GyrA_G271A-T |
| 3 | GyrA_A272G-T |
| 4 | 16S_926-928TTC |
| 5 | 16S_926-928AGA |
| 6 | 23S_A2142G-C |
| 7 | 23S_A2143G-C |
| 8 | RdxA_G565T |
| 9 | RdxA_G616A |
Control multiplex detection system 2:
table 10
| Sequence number | Site(s) |
| 1 | PBP1A_CT1242AG |
| 2 | PBP1A_C1667G |
| 3 | 23S_A2142G-C |
| 4 | 23S_A2143G-C |
| 5 | oorD_A041G |
| 6 | oorD_A122G |
| 7 | porD_G353A |
| 8 | porD_A356G |
| 9 | porD_C357T |
The specific method is the same as in example 1, and the control multiplex detection system 1 is a system before optimization of the W1 multiplex primer combination, wherein two sites of 16S_926-928TTC and 16S_926-928AGA have competition inhibition phenomena, so that no detection can be caused.
The control multiplex detection system 2 is a W3 multiplex primer combination optimization pre-system, wherein after two sites of 23S_A2143G-C and garD_G353A are formed by new combination, the molecular weight of the extension product of the individual site or the molecular weight difference between the extension probe and each type of extension product is larger than 16Da, and the system cannot work as a multiplex detection system.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Jiangsu kang is century Biotech stock Co., ltd
<120> kit for simultaneously detecting drug-resistant site of helicobacter pylori, virulence genotyping and metabolic genotyping of proton pump inhibitor
<130> 035003
<160> 77
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
acgttggatg gattggtaaa taccaccccc 30
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
acgttggatg gcatggaaaa atcttgcgcc 30
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
acgttggatg gcatggaaaa atcttgcgcc 30
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
acgttggatg gattggtaaa taccaccccc 30
<210> 5
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
acgttggatg gcatggaaaa atcttgcgcc 30
<210> 6
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
acgttggatg gattggtaaa taccaccccc 30
<210> 7
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
acgttggatg tgaaaattcc tcctacccgc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
acgttggatg atcctgcgca tgatattccc 30
<210> 9
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
acgttggatg tgaaaattcc tcctacccgc 30
<210> 10
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
acgttggatg atcctgcgca tgatattccc 30
<210> 11
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
acgttggatg atcaataagc ctaaaatcgc atg 33
<210> 12
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
acgttggatg gaaaacaccc ctaaaagagc g 31
<210> 13
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
acgttggatg atcaataagc ctaaaatcgc atg 33
<210> 14
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
acgttggatg gaaaacaccc ctaaaagagc g 31
<210> 15
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
acgttggatg gactgtaagt ggtttctcag 30
<210> 16
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
acgttggatg aacatcagga ttgtaagcac 30
<210> 17
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
acgttggatg caaatttgtg tcttctgttc 30
<210> 18
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
acgttggatg ggatttgagc tgaggtcttc 30
<210> 19
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
acgttggatg tctttcttgc ctgggatctc 30
<210> 20
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
acgttggatg ctctttccat tgctgaaaac g 31
<210> 21
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
acgttggatg acaccgcaaa atcaatcgc 29
<210> 22
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
acgttggatg ccaacaatgg ctggaatgat cac 33
<210> 23
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
acgttggatg acaccgcaaa atcaatcgc 29
<210> 24
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
acgttggatg ccaacaatgg ctggaatgat cac 33
<210> 25
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
acgttggatg ggtatcaatc cagaatggat ttc 33
<210> 26
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
acgttggatg ttcaaggtcg ctttttgctt g 31
<210> 27
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
acgttggatg ggcctrctgg tggggattgg 30
<210> 28
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
acgttggatg tatgtcggtg gtagtagtgg c 31
<210> 29
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
acgttggatg gagtggctta agctcgtgaa 30
<210> 30
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
acgttggatg cttggtggaa aacttgaacg 30
<210> 31
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
acgttggatg aacagcgaac aaaaccacgc 30
<210> 32
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
acgttggatg tcttgcaagg ttacaagccc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
acgttggatg aaatggcaca gggagtttgg 30
<210> 34
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
acgttggatg taaagccaat gaaccaagcg 30
<210> 35
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
acgttggatg aactcaaagg aatagacggg 30
<210> 36
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
acgttggatg gtcaagccta ggtaaggttc 30
<210> 37
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
acgttggatg aactcaaagg aatagacggg 30
<210> 38
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
acgttggatg gtcaagccta ggtaaggttc 30
<210> 39
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
acgttggatg gcacaaagga gaatgaatgg 30
<210> 40
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
acgttggatg ccaagcaccc tttctttttc 30
<210> 41
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
acgttggatg gcacaaagga gaatgaatgg 30
<210> 42
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
acgttggatg ccaagcaccc tttctttttc 30
<210> 43
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
acgttggatg cgctatggat gtttgaagaa c 31
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
acgttggatg ccatcccaca cttctatctc 30
<210> 45
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
acgttggatg cgctatggat gtttgaagaa c 31
<210> 46
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
acgttggatg ccatcccaca cttctatctc 30
<210> 47
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
acgttggatg cgctatggat gtttgaagaa c 31
<210> 48
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
acgttggatg ccatcccaca cttctatctc 30
<210> 49
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
accaccccca tggcgataa 19
<210> 50
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
attctcacta gcgcat 16
<210> 51
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
gccattctca ctagcgca 18
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
tcctacccgc ggcaagacgg 20
<210> 53
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 53
aaggtccacg gggtct 16
<210> 54
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 54
ctgccaccct cttac 15
<210> 55
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 55
tgatcaagaa aatcaaaagt tgat 24
<210> 56
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 56
ttggccttac ctggat 16
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 57
tgtgtcttct gttctcaaag 20
<210> 58
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 58
tgggatctcc ctcct 15
<210> 59
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 59
caaagtcatg ccgcct 16
<210> 60
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 60
gcctttttca caaccgtga 19
<210> 61
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 61
tgatttcaaa aatggcaa 18
<210> 62
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 62
ttaacaaaaa acaatctt 18
<210> 63
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 63
agtatgataa aattg 15
<210> 64
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 64
aaacttgcga gaataatt 18
<210> 65
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 65
tattgttgtt agaagtcccg 20
<210> 66
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 66
taattcgaag ataca 15
<210> 67
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 67
taattcgatt ctaca 15
<210> 68
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 68
gagcgctcca gatggggtt 19
<210> 69
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 69
ggttcttggc atgggg 16
<210> 70
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 70
aagaacaaat tgagcct 17
<210> 71
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 71
gaagaacaaa ttgagcctgc t 21
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 72
gcggccattg agtgagagcg 20
<210> 73
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 73
acgttggatg gtgaaattgt agtggaggtg 30
<210> 74
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 74
acgttggatg ttcccattag cagtgctaag 30
<210> 75
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 75
acgttggatg gctgtctcaa ccagagattc 30
<210> 76
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 76
acgttggatg cgcatgatat tcccattagc 30
<210> 77
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 77
tcatacccgc ggcaagacgg a 21
Claims (8)
1. A kit for diagnosing helicobacter pylori infection by using multiplex PCR-time-of-flight mass spectrometry, which is characterized by comprising a first primer pair group, a second primer pair group and a third primer pair group, wherein the first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14; the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30; the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48;
the kit further comprises a first probe set, a second probe set and a third probe set, wherein the first probe set comprises probes with sequences shown as SEQ ID NO. 49-SEQ ID NO. 55; the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63; the third probe group comprises probes with sequences shown as SEQ ID NO.64 to SEQ ID NO.72
And, the kit includes a first container, a second container, a third container, a fourth container, a fifth container, and a sixth container, wherein the first container contains the first primer pair group therein; the second container contains the second primer pair group; the third container contains the third primer pair group; the fourth container contains the first probe set; the fifth container contains the second probe set; the sixth vessel contains the third probe set therein.
2. The kit of claim 1, wherein the kit comprises a seventh container comprising a PCR pre-mix solution; the PCR premix comprises hot start Taq enzyme, dNTPs and Mg 2+ 。
3. The kit of claim 2, comprising an eighth container comprising Shrimp Alkaline Phosphatase (SAP) therein.
4. A kit according to claim 3, comprising a ninth container containing an elongase.
5. A kit as claimed in claim 4, comprising a tenth container containing ddNTP.
6. The kit of claim 5, comprising an eleventh container comprising an extension reaction buffer.
7. The kit of claim 6, wherein the kit comprises a twelfth container, the twelfth container containing purified water.
8. The use of a primer set and a probe set for preparing a detection kit for diagnosing helicobacter pylori infection;
the primer pair group comprises a first primer pair group, a second primer pair group and a third primer pair group; wherein,
The first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48; the probe set includes a first probe set, a second probe set, and a third probe set;
the first probe group comprises probes with sequences shown as SEQ ID NO.49 to SEQ ID NO. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe group comprises probes with sequences shown as SEQ ID NO.64 to SEQ ID NO. 72;
and, the kit includes a first container, a second container, a third container, a fourth container, a fifth container, and a sixth container, wherein the first container contains the first primer pair group therein; the second container contains the second primer pair group; the third container contains the third primer pair group; the fourth container contains the first probe set; the fifth container contains the second probe set; the sixth vessel contains the third probe set therein.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110677868.5A CN113604589B (en) | 2021-06-18 | 2021-06-18 | Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor |
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| CN113604589A CN113604589A (en) | 2021-11-05 |
| CN113604589B true CN113604589B (en) | 2024-03-29 |
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