CN105441582B - Helicobacter pylori is qualitative and quantitative multiplex genetic test system and its kit and application - Google Patents

Helicobacter pylori is qualitative and quantitative multiplex genetic test system and its kit and application Download PDF

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CN105441582B
CN105441582B CN201610070235.7A CN201610070235A CN105441582B CN 105441582 B CN105441582 B CN 105441582B CN 201610070235 A CN201610070235 A CN 201610070235A CN 105441582 B CN105441582 B CN 105441582B
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张艳梅
赵虎
项平
胡彬婕
赵付菊
周丽芳
吴勇
王诗雯
缪应新
方毅
季大年
黄任翔
陈洁
徐玲丽
孔咪咪
张景皓
姜文荣
陈飞
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Ningbo Haier Shi Gene Technology Co ltd
Huadong Hospital
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Huadong Hospital
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Abstract

The present invention relates to a kind of helicobacter pylori is qualitative and quantitative multiplex genetic test system and its kit and application.Helicobacter pylori is qualitative and quantitative multiplex genetic test system includes multipair primer, respectively for the strain idenfication gene of helicobacter pylori (16S rRNA) and quantitative analysis geneureCWithß‑globin.Helicobacter pylori of the invention is qualitative and quantitative detection system and its kit do not need to be separately cultured using Conventional bacteria, strain idenfication and quantitative analysis directly can be carried out to tissue samples in same reaction system, the accuracy of testing result significantly improves, accurate, inexpensive aetology foundation is provided for clinic at the first time, provides important references for the accurate Diagnosis and differential diaggnosis and observation of curative effect of helicobacter pylori infections.

Description

Qualitative and quantitative multiple gene detection system for helicobacter pylori, kit and application thereof
Technical Field
The invention relates to a multiple gene detection product and a detection system used by the product, belonging to the technical field of biology.
Background
Helicobacter pylori (H.pylori) Is a gram-negative, microaerophilic, campylobacter bacterium, which is mainly colonized in the human stomach. Helicobacter pylori infection is closely related to the occurrence and development of chronic atrophic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, gastric cancer and the like, thereby causing wide clinical applicationAttention is paid to the method. In 1994, the international agency for research on cancer IARC has listed it as a human class I carcinogen, being the only bacterial pathogenic microorganism listed as clearly carcinogenic to humans so far. Helicobacter pylori infection has been reported to be associated with coronary heart disease, rheumatoid diseases, liver and gallbladder diseases, pulmonary tuberculosis, vomiting of pregnancy, colorectal cancer, various skin diseases and the like. Epidemiology has shown that almost half of the world population is infected with this bacterium, even up to 60-70% in developing countries. Thus, H.pylori infection is a public health problem that needs to be faced in countries around the world.
The pathogenicity of H.pylori is closely related to the amount of infection. The existing helicobacter pylori detection and identification methods have limitations. For example: 1) and (3) isolation culture identification: after the helicobacter pylori is cultured into bacteria, the bacteria are identified by biochemical reaction. Because the culture of the helicobacter pylori needs microaerophilic conditions and has strict requirements on nutritional conditions, the detection rate is extremely low, the method is not easy to popularize as a conventional diagnosis means, and the culture of the helicobacter pylori needs a certain time, so that the rapid diagnosis is not facilitated. 2) Histopathological section staining method: the method comprises the steps of examining a biopsy tissue section of a patient by a gastroscope, and observing helicobacter pylori in the tissue after staining. The method is obviously influenced by the loading capacity of helicobacter pylori, is complicated to operate, is time-consuming, and is not suitable for detecting large-flux samples. 3) Urease-dependent test (urea breath test): according to the difference of markers13C breath test and14c breath test, clinical application is extensive. The defects are high cost, easy influence of bacteriostatic drugs and acid-inhibiting drugs and low sensitivity. 4) And (3) immunological examination: detecting antibodies (IgG) in serum or saliva and urine or directly detecting antigen components of helicobacter pylori in feces. The disadvantage is that symptomatic infection cannot be reflected. 5) Nucleic acid analysis method: including sequencing, PCR, oligonucleotide probe hybridization, etc., but these detection methods have few detection sites, low specificity, low flux, high cost and no quantitative analysis. 6) Quantitative analysis: the commonly used quantitative analysis method is real-time PCR, but the method has the advantages of single detection, small flux and high cost for analyzing a plurality of genes.
In conclusion, how to rapidly, accurately and comprehensively identify and quantitatively analyze helicobacter pylori is an urgent problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to provide a helicobacter pylori qualitative and quantitative multiple gene detection system and a kit thereof, which can be used for quickly, accurately and at low cost, and application of the detection system in the aspect of preparing diagnostic products.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a qualitative and quantitative multiple gene detection system for helicobacter pylori can be used for strain identification, quantification, virulence and drug resistance analysis in the same reaction system. Comprises identifying the gene for the strain16S rRNAPrimers for detection, respectively, of virulence genescagA、vacA-s1、vacA-s2、vacA-m1、vacA-m2、iceA1、iceA2、dupA、oipAAndluxSprimers for detection, respectively against drug-resistant genes23S rRNA2143 of (a),rdxAPosition 148 of,pbp1A1777 andgyrA261 locus, and genes for quantitative analysis of the copy number of helicobacter pylori, respectivelyureCAndß-globina primer for detection; the 5 'end of the forward primer of each primer is provided with a forward universal primer sequence, and the 5' end of the reverse primer of each primer is provided with a reverse universal primer sequence. And carrying out capillary electrophoresis analysis after PCR reaction of the detection system.
To is directed at16S rRNAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.1 and aims at16S rRNAThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 2;
to is directed atcagAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.3 and aims atcagAThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 4;
to is directed atvacA-s1OrvacA-s2The nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.5, aiming atvacA-s1OrvacA-s2The nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 6;
to is directed atvacA-m1The nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.7 and aims atvacA-m1The nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 8;
to is directed atvacA-m2The nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.9 and aims atvacA-m2The nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 10;
to is directed aticeA1The nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.11 and aims aticeA1The nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 12;
to is directed aticeA2The nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.13 and aims aticeA2The nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 14;
to is directed atdupAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.15 and aims atdupAThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 16;
to is directed atoipAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.17 and aims atoipAThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 18;
to is directed atluxSThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.19 and aims atluxSThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 20;
to is directed at23S rRNAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.21 and corresponds to23S rRNAThe nucleotide sequence of the reverse primer with the base A at the 2143 site of the gene is shown as SEQ ID No.22 and corresponds to23S rRNA2143 site of geneThe nucleotide sequence of the reverse primer of the base G is shown as SEQ ID No. 23;
to is directed atrdxAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.24 and corresponds tordxAThe nucleotide sequence of the reverse primer with the gene 148 site as the base C is shown as SEQ ID No.25 and corresponds tordxAThe nucleotide sequence of the reverse primer with the gene 148 site as the base T is shown as SEQ ID No. 26;
to is directed atpbp1AThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.27 and corresponds topbp1AThe nucleotide sequence of the reverse primer with 1777 locus as base A is shown in SEQ ID No.28, corresponding topbp1AThe nucleotide sequence of a reverse primer with the site of gene 1777 as base G is shown in SEQ ID No. 29;
to is directed atgyrAThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.30 and corresponds togyrAThe nucleotide sequence of the reverse primer of which the 261 site of the gene is base C or T is shown as SEQ ID No.31 and corresponds togyrAThe nucleotide sequence of a reverse primer of which the 261 site of the gene is a base G or A is shown as SEQ ID No. 32;
to is directed atureCThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.33 and aims atureCThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 34;
to is directed atß-globinThe nucleotide sequence of the forward primer of the gene is shown as SEQ ID No.35, and the primer aims atß- globinThe nucleotide sequence of the reverse primer of the gene is shown as SEQ ID No. 36;
the nucleotide sequence of the forward universal primer is shown as SEQ ID No. 37;
the nucleotide sequence of the reverse universal primer is shown as SEQ ID No. 38.
To is directed at16S rRNA、ureC、cagA、vacA-s1、vacA-m1、vacA-m2、iceA1、iceA2、dupA、oipAAndluxSgene forward primer on specimenThe final concentrations in the lines were all 200 nM; to is directed at16S rRNA、ureC、cagA、vacA-s1、vacA-m1、vacA-m2、iceA1、iceA2、oipA、luxSAnd, andß-globinthe final concentration of the reverse primers of the genes in the detection system is 100 nM; to is directed atdupAAndß-globinthe final concentration of the forward primers of the genes in the detection system is 100 nM;
to is directed at23S rRNA2143 sites of the gene,rdxA148 sites of the gene,pbp1A1777 site of Gene andgyrAthe final concentration of the forward primer at the 261 th site of the gene in a detection system is 100 nM; correspond to23S rRNAThe final concentration of the reverse primer with the base A at the 2143 locus of the gene in a detection system is 300 nM; correspond to23S rRNAThe final concentration of the reverse primer with the base G at the 2143 locus of the gene in a detection system is 350 nM; correspond tordxABase C at position 148 of gene, correspondingpbp1AAt 1777 position of gene, base A, correspondinggyrAReverse primer with 261 site of gene as base C or T and its corresponding applicationrdxABase T at position 148 of gene, correspondingpbp1AThe final concentration of the reverse primer with the gene 1777 site as the basic group G in the detection system is 400 nM; correspond togyrAThe final concentration of the reverse primer with the base G or A at the 261 site of the gene in the detection system is 450 nM.
The helicobacter pylori detection system also comprises PCR buffer solution and MgCl2Solution, dNTPs, hot start DNA polymerase, a fluorescent universal label mixture and a DNA template; the fluorescent universal label mixture comprises a reverse universal primer and a forward universal primer with a fluorescent label.
The above fluorescent label is CY5, CY3 or FAM, etc.
The helicobacter pylori detection system also comprises a positive control solution and a negative control solution; the positive control is a plasmid mixture comprising all target gene targets; the negative control solution was ultrapure water without nuclease.
In the reaction system of the helicobacter pylori detection systemThe components of (1) are 10 XPCR buffer, 1 volume of fluorescent universal label mixture and 2mmol/L dNTPs, and 25mmol/L MgCl2Volume 2 of solution, volume 1 of primer mixture, volume 1 of 5U/. mu.L hot start DNA polymerase, volume 2 of DNA template, and volume 2 of pure water.
The amount of the DNA template used is 5 to 50 ng/system.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: an application of the detection system in preparing helicobacter pylori detection and diagnosis products.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: a helicobacter pylori qualitative and quantitative multiple gene detection kit adopting the detection system.
The invention has the positive effects that:
(1) the qualitative and quantitative multiple gene detection system and kit for helicobacter pylori provided by the invention adopt multiple pairs of specific primers and universal primers to convert multiple PCR amplification reactions into single reaction, thereby effectively avoiding mutual interference among the primers and equivalently amplifying all target genes. The reaction system is optimized, capillary electrophoresis and fluorescence detection technologies are combined, the traditional gel electrophoresis analysis mode is different, non-specific amplification products, primer dimers and specific amplification products can be separated, and false positive is reduced to the maximum extent. The detection result of the kit has no miscellaneous peak and high specificity. Can detect pathogen with 10 copies and high sensitivity.
(2) The method has the advantages that the steps of conventional bacterial culture and the like of helicobacter pylori are not needed, strain identification and quantitative synchronous detection and analysis are directly carried out on the tissue sample in the same reaction system, all results are obtained by one-time detection, the defects of low flux, long consumed time, low detection rate and the like of a conventional detection method are overcome, the cost is low, the convenience is good, comprehensive, accurate and low-cost etiology basis is provided for clinic at the first time, and accurate diagnosis, differential diagnosis and curative effect observation of clinical helicobacter pylori infection are guided.
(3) Of helicobacter pylori16S rRNAThe gene has high conservation, and is used for strain identification.
(4) The pathogenic diversity of H.pylori is closely related to the amount of infection and the various virulence factors produced.
ureCThe gene is a single copy gene, combined with a human geneß-globinAs an internal reference, the method can realize quantitative analysis, obviously improve the accuracy of detection results, and monitor the infection amount and the treatment effect.
cagA、vacA-s1、vacA-s2、vacA-m1、vacA-m2、iceA1、iceA2、dupA、oipAAndluxSthe gene is used for judging the virulence of the bacteria. Wherein the CagA protein is on the pathogenic island cagPAI of helicobacter pyloricagAThe encoded product of the gene is an important effector protein for the inflammatory response of the host caused by helicobacter pylori infection.vacAThe gene is present in all H.pylori strains, but only about 50% of them have VacA peptone expression. VacA has a direct toxic effect on gastric epithelial cells, which can cause damage to the gastric mucosa and delay repair of gastric epithelium.vacAThe signal sequence region (s-region) and the middle region (m-region) are present in the gene structure. The s-block and the m-block are composed in different formsvacA5 chimeras of the genes, namely s1a/m1, s1a/m2, s1b/m1, s1b/m2 and s2/m 2. Detection ofvacA-s1、vacA-s2、vacA-m1、vacA-m2The expression quantity of the gene and the research on the classification of the gene are of great significance for judging the pathogenic intensity of the helicobacter pylori strains.iceAThere are two major allelic variations of a gene:iceA1andiceA2，iceA1gene expression means that the contact of H.pylori with epithelial cells is up-regulated and is closely associated with the development of ulcers.dupAThe gene is associated with the occurrence of duodenal bulbar ulcer.oipAEncoding pro-inflammatory outer membrane protein A, clinical symptoms following H.pylori infection, bacterial colonization density, severe neutrophil infiltration and higher end-of-year IL-8 levelsAre closely related andoipAopen state of gene signaling region andcagA、vacA、iceAthe existence of the genotype of the compound (A) is related to the existence of the genotype of the compound (B), and the compound (B) has certain directivity on duodenal ulcer and gastritis.luxSThe gene coding LuxS protease is the basis for synthesizing a signal molecule AI-2 which is highly conserved and can be recognized by different bacterial genera, and constitutes a LuxS/AI-2 QS system which is common to gram-positive bacteria and gram-negative bacteria in helicobacter pylori. AI-2 increased to a certain extent while specifically binding to luxS protein and activating transcription, regulating downstream gene expression, was highly correlated with the formation of specific biofilm of helicobacter pylori, which increased its pathogenicity and resistance to a certain extent. The detection of the helicobacter pylori infection amount and virulence related genes provides important reference for clinical diagnosis, treatment, prevention and the like.
(5) Conventional drugs for the clinical treatment of helicobacter pylori include clarithromycin, metronidazole, amoxicillin and levofloxacin. But the drug resistance phenomenon is increasingly serious and global, and the conventional drug resistance detection methods have the defects of long time consumption, low sensitivity and the like at present, thereby greatly influencing the clinical curative effect. Of helicobacter pylori23S rRNA、rdxA、pbp1AAndgyrAthe polymorphism of the gene is closely related to clinical drug resistance. Wherein,23S rRNAthe gene was used to determine resistance to Clarithromycin (Clarithromycin).rdxAThe gene was used to judge the resistance to Metronidazole (Metronidazole).pbp1AThe gene is used for judging the drug resistance of Amoxicillin (Amoxicillin).gyrAThe gene was used to judge resistance to Levofloxacin (Levofloxacin). The detection of the helicobacter pylori drug resistance related gene provides important reference for clinical individualized treatment.
Drawings
FIG. 1 is a diagram of the kit of example 1 of the present invention after performing a PCR reaction on a positive control and performing capillary electrophoresis analysis;
FIG. 2 is a diagram of the kit of example 1 of the present invention after performing a PCR reaction on a negative control and performing capillary electrophoresis analysis;
FIG. 3 is a diagram of a sample 1 subjected to a PCR reaction and then to capillary electrophoresis analysis using the kit of example 1 of the present invention;
FIG. 4 is a diagram of a sample 2 after a PCR reaction and a capillary electrophoresis analysis by the kit of example 1 of the present invention;
FIG. 5 is a diagram of a sample 3 after a PCR reaction and a capillary electrophoresis analysis by the kit of example 1 of the present invention;
FIG. 6 is a diagram showing a sample 4 subjected to a PCR reaction and then to capillary electrophoresis analysis by the kit of example 1 of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines, published by scientific Press 2002, edited by J. SammBruk et al, or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
Example 1
Composition of kit
The helicobacter pylori detection kit of the present embodiment includes: primer mixture, PCR buffer (10 XPCR buffer), MgCl2Solution, dNTPs, a universal label mixture with fluorescence, hot start DNA polymerase (Taq DNA polymerase), a positive control and a negative control. 2mmol/L dNTPs and the fluorescent universal label mixture are mixed together to form a one-tube reagent.
The PCR buffer, dNTPs and hot-start DNA polymerase were all from Takara (cat # R007A).
The positive control solution is a plasmid mixture including all target gene targets.
The negative control solution was ultrapure water without nuclease.
Inclusion of a pair in a primer mixture16S rRNAForward primer and target of gene16S rRNAReverse primer of gene, targetcagAForward primer and target of genecagAReverse primer of gene, targetvacA-s1 OrvacA-s2Forward primer and target of genevacA-s1 OrvacA-s2Reverse primer of gene, targetvacA-m1Forward primer and target of genevacA-m1Reverse primer of gene, targetvacA-m2Forward primer and target of genevacA-m2Reverse primer of gene, targeticeA1Forward primer and target of geneiceA1Reverse primer of gene, targeticeA2Forward primer and target of geneiceA2Reverse primer of gene, targetdupAForward primer and target of genedupAReverse primer of gene, targetoipAForward primer and target of geneoipAReverse primer of gene, targetluxSForward primer and target of geneluxSReverse primer of gene, target23S rRNAForward primer for gene, and correspondence23S rRNAReverse primer with base A at 2143 locus of gene and corresponding primer23S rRNAReverse primer and reverse primer aiming at base G at 2143 locus of generdxAForward primer for gene, and correspondencerdxAReverse primer with gene 148 site as base C and corresponding primerrdxAInversion of base T at site 148 of genePrimer and targetpbp1AForward primer for gene, and correspondencepbp1AReverse primer with gene 1777 site as base A and its corresponding processpbp1AReverse primer and reverse primer for gene 1777 site with base GgyrAForward primer for gene, and correspondencegyrAReverse primer with 261 site of gene as base C or T and its corresponding applicationgyrAReverse primer and reverse primer for gene 261 site with base G or AureCForward primer and target of geneureCReverse primer of gene, targetß-globinForward primer and target of geneß-globinReverse primer of gene. The characteristics of each primer are shown in Table 1.
TABLE 1 primer sequence characterization Table
The primers were synthesized by Shanghai Sangni Biotech Co., Ltd. Wherein, the 5 'end of each forward primer is provided with a forward universal primer sequence, and the 5' end of each reverse primer is provided with a reverse universal primer sequence.
The fluorescent universal label mixture comprises a proper amount of reverse universal primers and forward universal primers with fluorescent labels, wherein the fluorescent labels are CY5 (CY 3 or FAM and the like can also be used).
The nucleotide sequence of the forward universal primer is as follows: TGCATGACACTCGAGACTAG, as shown in SEQ ID No. 37.
The nucleotide sequence of the reverse universal primer is as follows: CATGACGACTCACTATACTA, as shown in SEQ ID No. 38.
Second, using method of kit
The specific detection steps of the helicobacter pylori detection kit of the embodiment are as follows:
1. sample collection
A gastric mucosa biopsy specimen of a helicobacter pylori patient is immersed in physiological saline, and a bacterial DNA extraction kit of Tiangen company is adopted to extract sample DNA, and the specific operation refers to the specification of the extraction kit product.
After sample DNA is obtained, the quality of the sample DNA is controlled by measuring the concentration and the ratio of OD260/OD280 through an ultraviolet spectrophotometer. The preferred concentration of sample DNA is 10 ng/. mu.L to 100 ng/. mu.L. The preferable range of the ratio of OD260/OD280 is 1.7-1.9.
PCR reaction
The reagents in the kit of this example were used to prepare PCR reaction systems with DNA templates (sample DNA, positive control solution, or negative control solution), respectively, the specific components of which are shown in table 2.
TABLE 2 PCR reaction System Components Table
The PCR reaction program was then performed on a PCR instrument (ABI Veriti 96 well), with the optimal reaction program as shown in Table 3.
TABLE 3 PCR reaction schedule
3. Capillary electrophoresis fragment analysis.
To each well of the 96-well Sample plate, 38.5. mu.L of formamide (Sample Loading Solution, ABsciex, cat # 608082) and 0.5. mu.L of an internal Standard (DNA Size Standard Kit-400 Base Pairs, ABsciex, cat # 608098) were added, and 1. mu.L of PCR product was added thereto, and one drop of mineral oil was added to prevent volatilization of the PCR product due to high temperature during electrophoresis.
About 250. mu.L of GenomeLab separation buffer was added to each well of another 96-well buffer plate.
The sample plate and buffer plate were placed into the machine according to the operating manual for capillary electrophoresis analyzer and the Frag-3 separation procedure was run. And executing a default analysis method, and finally saving the data.
And (3) obtaining capillary electrophoresis peak images aiming at different sizes of PCR product fragments of all genes, wherein the abscissa represents the fragment length, and the ordinate represents the fluorescence intensity.
4. Analysis of results
The capillary electrophoresis analyzer automatically performs data analysis.
Thirdly, judging the detection result of the kit
1. Kit validity determination
The result judgment can be carried out when the following conditions are met:
1) negative control: no fluorescence signal was detected between 138nt and 327 nt.
2) Positive control: one fluorescence signal was detected at each amplified fragment length and the fluorescence signal value was above 2000.
2. And (3) judging the validity of the sample:
between 138nt and 327 nt:
1) if the fluorescence signal value of the detected sample is at least higher than 200000, the sample is added in an excessive amount, and capillary electrophoresis detection is recommended after appropriate dilution of the PCR product.
2) If the fluorescence signal values of the detected samples are all lower than 2000, the sample addition amount is lower, and the PCR product addition amount or the PCR reaction cycle number can be properly increased; if the requirements are still not met, the sample is prepared again.
3. Criteria for determination of results
1) Identification of strains
16S rRNAThe target region of the gene shows corresponding peaks and the fluorescence signal values are all higher than 2000, and16S rRNAandureCthe corresponding specific peak area of the gene-ß-globinThe ratio of the corresponding specific peak areas is more than 0.1, and the helicobacter pylori infection can be judged.
2) Identification of virulence
cagA、vacA-s1、vacA-s2、vacA-m1、vacA-m2、iceA1、iceA2、dupA、oipA、luxSCorresponding peaks appear in the target fragment region of the gene, the fluorescence signal values are all higher than 2000, and the specific peak area corresponding to each virulence gene is-ß-globinThe ratio of the corresponding specific peak areas is greater than 0.1, indicating that the patient infected helicobacter pylori contains the virulence gene expression.
3) Drug resistance identification
(1) Clarithromycin resistance
When the ratio of the peak area appearing at 180 nt/the peak area appearing at 175nt was less than 0.25, it was demonstrated that23S rRNAThe 2143 locus of the gene is homozygous wild type when23S rRNAThe 2143 locus of the gene is homozygous wild type and has no drug resistance to clarithromycin. When the ratio of the peak area appearing at 180nt to the peak area appearing at 175nt is 0.25-0.7, the positive is determined to be weak positive, and when the ratio is less than the threshold value, the positive is determined to be weak positive23S rRNAWhen the 2143 locus is weakly positive, the gene has a small drug resistance to clarithromycin. When the ratio of the peak area appearing at 180nt to the peak area appearing at 175nt is 0.7-4, it indicates that23S rRNAThe 2143 locus of the gene is a heterozygous mutant, and when the ratio of the peak area appearing at 180nt to the peak area appearing at 175nt is more than 4, the result shows that23S rRNAThe 2143 site of the gene is homozygous mutant when23S rRNAThe 2143 locus of the gene is a homozygous mutant or a heterozygous mutant and has resistance to clarithromycin.
(2) Metronidazole resistance
When the ratio of the peak area appearing at 257.6 nt/the peak area appearing at 252.6nt was less than 0.25, it was demonstrated thatrdxAThe 148 locus of the gene is homozygous wild type whenrdxAThe 148 locus of the gene is homozygous wild type and has no drug resistance to metronidazole. When the ratio of the peak area appearing at 257.6nt to the peak area appearing at 252.6nt is 0.25-0.7, the positive is determined to be weak positive, and when the ratio is less than or equal to 0.25rdxAWhen the gene 148 locus is weak positive, the gene has a small amount of drug resistance to metronidazole. When the ratio of the peak area appearing at 257.6nt to the peak area appearing at 252.6nt is 0.7-4, the description shows thatrdxAThe 148 locus of the gene is a heterozygous mutant, and when the ratio of the peak area appearing at 257.6nt to the peak area appearing at 252.6nt is more than 4, the fact indicates thatrdxAThe 148 locus of the gene is a homozygous mutant whenrdxAThe 148 site of the gene is a homozygous mutant or a heterozygous mutant and has resistance to clarithromycin.
(3) Amoxicillin drug resistance
When the ratio of the peak area appearing at 158.9 nt/the peak area appearing at 153.8nt was less than 0.25, it was demonstrated thatpbp1AThe 1777 locus of the gene is homozygous wild typepbp1AThe 1777 locus of the gene is a homozygous wild type and has no drug resistance to amoxicillin. And when the ratio of the peak area appearing at 158.9nt to the peak area appearing at 153.8nt is 0.25-0.7, determining the test result as weak positive. When in usepbp1AWhen the 1777 site of the gene is weakly positive, the amoxicillin-resistant gene has little drug resistance. When the ratio of the peak area appearing at 158.9nt to the peak area appearing at 153.8nt is 0.7-4, it indicates thatpbp1AThe 1777 locus of the gene is a heterozygous mutant, and when the ratio of the peak area appearing at 158.9nt to the peak area appearing at 153.8nt is more than 4, the result shows thatpbp1AThe 1777 locus of the gene is homozygous mutant whenpbp1AThe 1777 locus of the gene is a homozygous mutant type or a heterozygous mutant type and has drug resistance to amoxicillin.
(4) Levofloxacin resistance
When the ratio of the peak area appearing at 311 nt/the peak area appearing at 306nt was less than 0.25, it was demonstrated thatgyrAThe 261 th site of the gene is homozygous wild type whengyrAThe 261 locus of the gene is homozygousWild type, no drug resistance to levofloxacin. And when the ratio of the peak area appearing at 311nt to the peak area appearing at 306nt is 0.25-0.7, determining the test piece to be weakly positive. When in usegyrAWhen the 261 locus of the gene is weak positive, the gene has little drug resistance to levofloxacin. When the ratio of the peak area appearing at 311nt to the peak area appearing at 306nt is 0.7-4, it indicates thatgyrAThe 261 locus of the gene is a heterozygous mutant type, and when the ratio of the peak area appearing at 311nt to the peak area appearing at 306nt is more than 4, the result shows thatgyrAThe 261 th site of the gene is homozygous mutant whengyrAThe 261 locus of the gene is homozygous mutant type or heterozygous mutant type and has drug resistance to levofloxacin.
4) Quantitative analysis
When in useureCThe peak area which appears at the corresponding specific fragment (138 nt) (-)ß-globinThe ratio of the peak areas appearing at the corresponding specific fragment (199 nt) is greater than 0.1, indicating that the patient is infected with H.pylori, the ratio is proportional to the number of copies of H.pylori, and a larger ratio indicates a more severe H.pylori infection. When in useureCThe peak area which appears at the corresponding specific fragment (138 nt) (-)ß-globinThe ratio of the peak area appearing at the corresponding specific fragment (199 nt) was less than 0.02, and the infection was judged to be negative. When in useureCThe peak area which appears at the corresponding specific fragment (138 nt) (-)ß-globinAnd when the ratio of the peak areas appearing at the corresponding specific fragment (199 nt) is 0.02-0.1, re-experiment is required.
4. Example of result judgment
FIG. 1 shows a chromatogram obtained by analyzing a positive control by capillary electrophoresis after PCR reaction using the kit of this example.16S rRNA、ureC、ß-globin、cagA、vacA-s1、vacA-m1、vacA-m2、iceA1、iceA2、dupA、oipA、luxS、vacA-s2、23S rRNA、rdxA、pbp1AAndgyrAcorresponding peaks appear in the target fragment region of the gene. The result is very visual, 1 strain identification gene, 2 quantitative genes, 10 virulence related genes and 4 drug resistance genesAll amplified well. Thus, the primers are not interfered with each other, and all target genes can be effectively amplified at the same time.
The spectrum of the negative control after PCR reaction and capillary electrophoresis analysis using the kit of this example is shown in FIG. 2, and no target gene peak appears, and only non-specific background fluorescence signal exists at less than 100 nt.
FIG. 3 shows a chromatogram obtained by analyzing a sample 1 by capillary electrophoresis after PCR reaction using the kit of this example.16S rRNA、ureC、cagA、vacA-s1、vacA-m1、iceA1、oipA、luxS、23S rRNA、rdxA、pbp1AAndgyrAcorresponding peaks appear in the target fragment region of the gene. According to the result judgment standard, the patient is infected with helicobacter pylori, expresses corresponding virulence factors, has drug resistance to clarithromycin and levofloxacin, and has no drug resistance to metronidazole and amoxicillin. The detection result is very intuitive.
FIG. 4 shows a chromatogram obtained by analyzing a sample 2 by capillary electrophoresis after PCR reaction using the kit of this example.16S rRNA、ureC、cagA、vacA-s1、vacA-m2、iceA2、oipA、luxS、23S rRNA、rdxA、pbp1AAndgyrAcorresponding peaks appear in the target fragment region of the gene. According to the result judgment standard, the patient is infected with helicobacter pylori, expresses corresponding virulence factors, has drug resistance to clarithromycin and metronidazole and has no drug resistance to amoxicillin and levofloxacin. The detection result is very intuitive.
FIG. 5 shows a chromatogram obtained by analyzing a sample 3 by capillary electrophoresis after PCR reaction using the kit of this example.16S rRNA、ureC、cagA、vacA-s1、vacA-m2、oipA、luxS、23S rRNA、rdxA、pbp1AAndgyrAcorresponding peaks appear in the target fragment region of the gene. Based on the result criteria, the patient is indicatedThe vaccine is infected with helicobacter pylori, expresses corresponding virulence factors, has drug resistance to amoxicillin and has no drug resistance to clarithromycin, metronidazole and levofloxacin. The detection result is very intuitive.
FIG. 6 shows a chromatogram obtained by analyzing a sample 4 by capillary electrophoresis after PCR reaction using the kit of this example.16S rRNA、ureC、cagA、vacA-s1、vacA-m1、iceA2、oipA、luxS、23S rRNA、rdxA、pbp1AAndgyrAno corresponding peak appears in the target fragment region of the gene, butß-globinThe corresponding peaks appear in the target fragment region of the gene. Based on the criteria for determination of the results, the patient is not infected with H.pylori. The detection result is very intuitive.
Example 2
The helicobacter pylori detection kit of this example is the same as the rest of example 1, except that: the primer mixture includes only the pair16S rRNAForward primer and target of gene16S rRNAReverse primer of gene, targetureCForward primer and target of geneureCReverse primer of gene, targetß-globinForward primer and target of geneß-globinReverse primer of gene.
It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be enumerated here, nor should they be enumerated. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.
SEQUENCE LISTING
<110> China east Hospital
<120> helicobacter pylori qualitative and quantitative multiple gene detection system, kit and application thereof
<130> do not
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<170> PatentIn version 3.3
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Claims (8)

1. A qualitative and quantitative multiple gene detection system for helicobacter pylori is characterized in that: comprises a primer for detecting a strain identification gene 16S rRNA and a primer for detecting genes ureC and beta-globin which respectively carry out quantitative analysis on the copy number of helicobacter pylori;
the 5 'end of the forward primer of each primer is provided with a forward universal primer sequence, and the 5' end of the reverse primer of each primer is provided with a reverse universal primer sequence;
the nucleotide sequence of the forward primer aiming at the 16S rRNA gene is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer aiming at the 16S rRNA gene is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer aiming at the ureC gene is shown as SEQ ID No.33, and the nucleotide sequence of the reverse primer aiming at the ureC gene is shown as SEQ ID No. 34;
the nucleotide sequence of a forward primer aiming at the beta-globin gene is shown as SEQ ID No.35, and the nucleotide sequence of a reverse primer aiming at the beta-globin gene is shown as SEQ ID No. 36;
the nucleotide sequence of the forward universal primer is shown as SEQ ID No. 37;
the nucleotide sequence of the reverse universal primer is shown as SEQ ID No. 38.
2. The qualitative and quantitative multiple gene detection system for helicobacter pylori according to claim 1, characterized in that: the final concentration of the forward primers aiming at the 16S rRNA and the ureC gene in the detection system is 200 nM; the final concentration of reverse primers aiming at 16S rRNA, ureC and beta-globin genes in a detection system is 100 nM; the final concentration of the forward primer against the beta-globin gene in the detection system was 100 nM.
3. The qualitative and quantitative multiple gene detection system for helicobacter pylori according to claim 1 or 2, characterized in that: also includes PCR buffer solution, MgCl2Solution, dNTPs, hot start DNA polymerase, a fluorescent universal label mixture and a DNA template; the fluorescent universal label mixture comprises a reverse universal primer and a forward universal primer with a fluorescent label.
4. The qualitative and quantitative multiple gene detection system for helicobacter pylori according to claim 3, characterized in that: the fluorescent label is CY5 or CY3 or FAM.
5. The qualitative and quantitative multiple gene detection system for helicobacter pylori according to claim 4, characterized in that: also comprises a positive control solution and a negative control solution; the positive control solution is a plasmid mixture comprising all target gene targets; the negative control solution was ultrapure water without nuclease.
6. The qualitative and quantitative multiple gene detection system for helicobacter pylori according to claim 4, characterized in that: the components in the reaction system are 10X PCR buffer solution 1 volume, fluorescent universal label mixture and dNTPs 2mmol/L in total 1 volume, and MgCl 25mmol/L22 volumes of solution, 1 volume of primer mixture, 1 volume of 5U/. mu.L hot start DNA polymerase, 2 volumes of DNA template and 2 volumes of pure water; the use amount of the DNA template is 5-50 ng/system.
7. A qualitative and quantitative multiple gene detection kit for helicobacter pylori using the detection system according to claim 1.
8. Use of the test system according to claim 1 for the preparation of a test and diagnostic product for helicobacter pylori.
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