Method and kit for detecting helicobacter pylori virulence gene type by using loop-mediated isothermal amplification method
阅读说明：本技术 一种使用环介导等温扩增法检测幽门螺杆菌毒力基因类型的方法及试剂盒 (Method and kit for detecting helicobacter pylori virulence gene type by using loop-mediated isothermal amplification method ) 是由 郗日沫 王倍 甘奇 孟萌 佟悦 乔岩旗 于 2020-12-30 设计创作，主要内容包括：本发明创造提供了一种用环介导等温扩增检测幽门螺杆菌毒力基因类型的方法和试剂盒。通过查阅文献筛选了7种毒力基因类型进行检测,其中针对cagA、vacA s2、vacA m1、vacA m2、iceA1、iceA2、babA2、oipA、dupA、luxS设计了5条引物,针对vacA s1设计了6条引物进行检测。这种检测方法具有特异性强,操作简便,成本低,反应时间短的优点,不需要昂贵的反应仪器,可以在水浴锅或金属浴中完成反应,并且反应完成后无需电泳等后续检测手段,直接通过肉眼便能观察反应结果。本发明首次将幽门螺杆菌的毒力基因进行一系列完整的检测,针对不同的毒力基因设计出相应的LAMP引物并进行筛选。(The invention provides a method and a kit for detecting the helicobacter pylori virulence gene type by loop-mediated isothermal amplification. 7 virulence gene types are screened and detected by consulting the literature, wherein 5 primers are designed for cagA, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA2, oipA, dupA and luxS, and 6 primers are designed for vacA s 1. The detection method has the advantages of strong specificity, simple and convenient operation, low cost and short reaction time, does not need expensive reaction instruments, can finish the reaction in a water bath or a metal bath, does not need subsequent detection means such as electrophoresis and the like after the reaction is finished, and can directly observe the reaction result by naked eyes. The invention carries out a series of complete detections on the virulence genes of the helicobacter pylori for the first time, designs corresponding LAMP primers aiming at different virulence genes and carries out screening.)
1. A detection method for detecting the type of a helicobacter pylori virulence gene is characterized in that a loop-mediated isothermal amplification method is used, so that the detection efficiency is improved, the time is short, and the efficiency is high.
2. The method of claim 1, wherein the detection primer comprises:
primers for detecting cagA
cagA-F3:5’-TCTGCCAAACAATCTTTTGC-3’
cagA-B3:5’-TGGTTCTTGATTGATTGCTTC-3’
cagA-FIP:5’-TTCTGCTTCTTGCCTTTCTTTCAAGAATCATTATAGGGAATCAAATCCG-3’
cagA-BIP:5’-ATGGAGAGCCTACTGGTGGGTTTGACATCAGAAGATTGTTCT-3’
cagA-LF:5’-GCCCATGAACTTTTGATCCGT-3’；
Primers for detecting vacAs1
vacAs1-F3:5’-CCTCTGGTTTCTCTTGCTT-3’
vacAs1-B3:5’-TCTGGGGTTTTATTGGCT-3’
vavAs1-FIP:5’-CACGGTTGTGAAAAAGGCGGTAGTAGGAGCGTTAGTCAGC-3’
vacAs1-BIP:5’-CATTGTTGGGGGGATCGCTACTCTTCGGCTTGTTTGAGC-3’
vacAs1-LB:5’-GGTCTCAGGGCTTCTTAGCT-3’
vacAs1-LF:5’-ATGACTTTGTTGCGGTGT-3’；
Primers for detecting vacAs2
vacAs2-F3:5’-CCTTAATCGTAAATGCAACAGAA-3’
vacAs2-B3:5’-GACTCTCGCTGTGTATGG-3’
vacAs2-FIP:5’-CGGTGTGTTTGTTGTATTTCCATTTATTTTCTAGTCTAAAGTCGCAC-3’
vacAs2-BIP:5’-CAATCGCCCTATTATTTCTCTCGCTGATCATTTGGCGTGTTAGC-3’
vacAs2-LB:5’-GGTGTTAATGGGCACCGAAC-3’；
Primers for detecting vacAm1
vacAm1-F3:5’-AACTATCTGGTCCGAGGC-3’
vacAm1-B3:5’-CCATTGGTACCTGTAGAAACA-3’
vacAm1-FIP:5’-GGTCGCGCTGTCTATATCATTATTAAGTGGCAACCTTAAATGTAGG-3’
vacAm1-BIP:5’-TACAAACCGCTCATCAAGATTAACAACCAATGATTTTCGCTTTCA-3’
vacAm1-LB:5’-CAAGATCTCATTAAAAATACAG-3’；
Primers for detecting vacAm2
vacAm2-F3:5’-TAGTGGATAGCGCGACTG-3’
vacAm2-B3:5’-GTTGTTGTTATAAAGGGCTAGG-3’
vavAm2-FIP:5’-CGCCCTTTCACTAAAACATGTTCTTTTACAAACCACTCATTAAGATCA-3’
vacAm2-BIP:5’-GGAGTGCAAGGCGCTAGTTATTTGAATTGCTCTTGCAGAT-3’
vacAm2-LB:5’-ATATTTCTGCAAGCAACACC-3’；
Primers for detecting iceA1
iceA1-F3:5’-CTTGAATGGCTATAATACCGAAT-3’
iceA1-B3:5’-ATCATCAAAAGTCTGTGTGTT-3’
iceA1-FIP:5’-CGCGCAACATTGTTGCTTATAGGTGTTTTTAACCAAAGTATCTGT-3’
iceA1-BIP:5’-GTGTGCGTGGCAACTCTGAAAGAAACTCTTGAATCATCCTTG-3’
iceA1-LB:5’-TAGAAGTGGATCATAAAGACGGCC-3’；
Primers for detecting iceA2
iceA2-F3:5’-GAGAATGGTATCCATAAGAGAAC-3’
iceA2-B3:5’-ACTGCTCATTAAAACACCG-3’
iceA2-FIP:5’-CTTTGATGTGGTTACAGCCACTACTTATGGTAGTAACGCTATTAATGTG-3’
iceA2-BIP:5’-AAGGACCTACTAGAAAATAGGGCACTAGAAAAACAGCGACACC-3’
iceA2-LB:5’-GCAAAAGCTTTGGCGCATT-3’；
Primers for detecting babA2
babA2-F3:5’-CGGAACGGTGAATGTAAGCT-3’
babA2-B3:5’-AGGCACACCGTCTAATTGG-3’
babA2-FIP:5’-GTCGTGGTTCCGCCATGTTGGTACACATGCTCAGGGGAAGG-3’
babA2-BIP:5’-CGGCAAAAGCGTAACCACCACAGACACGTTGGGTGTTACCT-3’
babA2-LF:5’-GTGGCTTTTTCCGAGCAGTT-3’；
Primers for detection of oipA
oipA-F3:5’-AAAAAAAGCATCAAGGCATG-3’
oipA-B3:5’-TCATCGCCATAGCGATCA-3’
oipA-FIP:5’-ACTTGCAAACCAAGTGCTACCTTAGAAAATCTTCAGGGCTTGT-3’
oipA-BIP:5’-ACAATCTCACCCCTTTCAATCAAGTTCATCATTATTCCAACGAACG-3’
oipA-LB:5’-CAAGAGTCGCACGATTTTTCAGT-3’；
Primers for detecting dupA
dupA-F3:5’-CAAAAGAACACAACAAACCTT-3’
dupA-B3:5’-GTAGATAATCACTTGAGAAAGGT-3’
dupA-FIP:5’-CGAGCGCGTTAGCGATATAGGGATGAAACTAAAGACTACATTATGC-3’
dupA-BIP:5’-CAAGCTAGAAAGATCAACGGAACACTCTTTGCTTTATCAATGCCTAA-3’
dupA-LB:5’-TTTGCATGGCGTTTCA-3’；
Primers for detecting luxS
luxS-F3:5’-CAACCCATAGGCGACCAATC-3’
luxS-B3:5’-ATGTGCGTATCGCTGATCG-3’
luxS-FIP:5’-GGACATGCCAAGCTTGCACTCTATTAGCATGGTTGCGGATGA-3’
luxS-BIP:5’-CTTTGTTGGGCTGCTTGAAGCGAAAAGGGCGTTAATGGGGAT-3’
luxS-LF：5’-TAGAGCATTTAGTCGCTGAGA-3’。
3. The use of the primer set according to claim 2 for detecting the type of helicobacter pylori virulence gene based on loop-mediated isothermal amplification.
4. The detection method of claim 1, wherein the detection method comprises:
preparing an LAMP system: 0.1. mu.l each of 100 μm outer primers F3 and B3; 0.4. mu.l each of 100 μm inner primers FIP and BIP; 0.25. mu.l each of 100 μm loop primers LF and LB; 2x reaction buffer 12.5 μ l; bst DNase 1. mu.l; 2. mu.l of template DNA; fluorescent dye 1. mu.l, make up to 25. mu.l with deionized water.
5. The detection method of claim 1, wherein the LAMP reaction conditions are 63 ℃ for 60min, followed by inactivation at 85 ℃ for 5 min.
6. The assay of claim 1 wherein the presence of the virulence gene is determined by visual observation of a color change or fluorescence.
7. A kit for detecting the type of helicobacter pylori virulence gene, comprising: the primer set of claim 2, 2x reaction buffer, Bst dnase, fluorescent dye, deionized water.
8. The kit of claim 7, wherein the 2x buffer comprises the following components: a final concentration of 40mM Tris-HCl (pH 8.8);
kcl at a final concentration of 20 Mm; MgSO 2 to a final concentration of 16mM4(ii) a To a final concentration of 20mM (NH)4)2SO4(ii) a 0.2% Tween 20; betaine at a final concentration of 1.6M; dNTPs at a final concentration of 2.8 mM.
9. The kit according to claim 7, wherein the fluorescent dye is a calcein-manganese chloride mixture. The preparation method comprises the following steps:
first, a 1M sodium hydroxide solution (10 mL) was prepared: 0.4g of solid sodium hydroxide was weighed, dissolved in 10ml of sterilized ultrapure water and mixed well.
Next, 6.5mM calcein (50ml) was prepared: weighing 0.162g of calcein, putting the calcein into a 2ml centrifuge tube, adding 1.2ml of 1M sodium hydroxide for assisting dissolution, centrifuging at 25000rpm for 2 minutes, transferring into a 50ml centrifuge tube, adding 38.8ml of sterilized ultrapure water, mixing uniformly, wherein the pH of the obtained solution is 8.0-8.5, performing filtration sterilization by using a 0.22 mu M filter membrane in a super clean bench, and preparing the calcein with the concentration of 6.5 mM.
Finally, 1.0ml of fluorescent dye was prepared: 12 ml centrifuge tube was filled with 500. mu.l of sterilized ultrapure water, 200. mu.l of the above-described calcein solution, 13. mu.l of manganese dichloride, and 287. mu.l of ultrapure water to make up to 1.0 ml.
10. The kit of claim 7, wherein the visual interpretation of the color results is specifically: if the reaction product is orange transparent liquid, the strain does not contain the virulence gene; if the reaction product is a green turbid liquid, the strain is considered to contain the virulence genes.
11. The kit of claim 7, wherein the color is determined by fluorescence, wherein a bright green fluorescence under 365nm UV illumination is positive to indicate that the strain contains the virulence gene, and a non-fluorescence is negative to indicate that the strain does not contain the virulence gene.
12. The kit of any one of claims 7 to 11 for use in the detection of H.pylori virulence gene type.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for detecting a helicobacter pylori virulence gene type by using a loop-mediated isothermal amplification method.
Background
Helicobacter pylori (h. pylori) is a gram-negative, campylotrophic bacterium, first discovered in gastric biopsies by Warren and Marshall in the early 80 s of the 20 th century, and it is mainly distributed in gastric mucosal tissues, the only microbial species currently known to be able to survive in the human stomach. According to statistics, the worldwide prevalence rate reaches 20-80%, wherein the prevalence rate in developed countries is 34.7%, and the prevalence rate in developing countries is 50.8%. The infection of Hp is closely related to digestive tract diseases such as chronic gastritis, upper gastrointestinal ulcer, gastric mucosal metaplasia and the like, and is listed as a class I carcinogenic factor by WHO.
China is a high-incidence country of helicobacter pylori infection (59 percent of infection rate), and the research aiming at the helicobacter pylori is very important. Helicobacter pylori is highly infected, and the infected person can develop serious gastrointestinal diseases including gastritis, gastric ulcer, duodenal ulcer, lymphoma, even gastric cancer. The pathogenesis of the disease outcome is thought to be mediated by complex interactions between host, environmental and bacterial virulence factors. The first step in the entry of H.pylori into the human body is colonization. Helicobacter pylori improves the pH value of the surrounding microenvironment through the expression of urease, and the tail flagella of the helicobacter pylori can provide power, so that the helicobacter pylori can rapidly penetrate through the gastric acid environment and submerge into the deep mucous membrane layer of the stomach wall with a neutral environment. Then, helicobacter pylori establishes permanent colonization on the gastric mucosa through outer membrane proteins and adhesins, and becomes a habitat of the helicobacter pylori by using mucosal protection of a host, so that chronic inflammation and tissue damage of the host are caused, and finally peptic ulcer and gastric cancer of a human body may be caused. At present, Hp strains are divided into two types according to the expression of the Hp strains cagA and vacA and corresponding proteins thereof, wherein the type I contains a cagA gene and can express a VacA toxin and a CagA protein, and the type is a high-toxicity strain; type II, which does not contain the cagA gene and does not express VacA toxin and CagA protein, is a low-toxicity strain. Held and other researches find that the risk of gastric adenocarcinoma of a patient with positive CagA is 7.4 times of that of CagA negative, which indicates that CagA infection is one of risk factors of gastric cancer, and the expression of genes of iceA1, iceA2, babA2, oipA, dupA and luxS can cause certain damage to the gastric mucosa of the human body, and increase the probability of gastritis, gastric ulcer and the like.
Notomi of Japan, 2000, discloses Loop-mediated isothermal amplification (LAMP) in the journal of Nucleic Acids Res, which has been successfully applied to the detection of SARS, avian influenza, HIV and other diseases, and designs 4 specific primers and Loop primers for 6 regions of a target gene, and under the action of a strand displacement DNA polymerase (Bst DNA polymerase), the isothermal amplification is carried out at 60-65 ℃ for about 15-60 minutes, and the 10^ 9-10 times of Nucleic acid amplification can be realized. Most importantly, the LAMP result is simple to detect, does not need gel electrophoresis like PCR, is judged only by observing whether color is changed or whether green fluorescence is generated or not by naked eyes, is simple, convenient and quick, and is suitable for rapid diagnosis of a basic layer.
Disclosure of Invention
The invention provides a visual detection method and a kit for specifically detecting main virulence genes of helicobacter pylori.
To detect the major virulence gene type of H.pylori. The invention detects genes of helicobacter pylori such as cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA2, oipA, dupA, luxS and the like. The primers used were as follows:
1. primers for detecting cagA
cagA-F3:5’-TCTGCCAAACAATCTTTTGC-3’
cagA-B3:5’-TGGTTCTTGATTGATTGCTTC-3’
cagA-FIP:5’-TTCTGCTTCTTGCCTTTCTTTCAAGAATCATTATAGGGAATCAAATCCG-3’
cagA-BIP:5’-ATGGAGAGCCTACTGGTGGGTTTGACATCAGAAGATTGTTCT-3’
cagA-LF:5’-GCCCATGAACTTTTGATCCGT-3’
2. Primers for detecting vacA s1
vacA-S1-F3:5’-CCTCTGGTTTCTCTTGCTT-3’
vacA-S1-B3:5’-TCTGGGGTTTTATTGGCT-3’
vavA-S1-FIP:5’-CACGGTTGTGAAAAAGGCGGTAGTAGGAGCGTTAGTCAGC-3’
vacA-S1-BIP:5’-CATTGTTGGGGGGATCGCTACTCTTCGGCTTGTTTGAGC-3’
vacA-S1-LB:5’-GGTCTCAGGGCTTCTTAGCT-3’
vacA-S1-LF:5’-ATGACTTTGTTGCGGTGT-3’
3. Primers for detecting vacA s2
vacA-S2-F3:5’-CCTTAATCGTAAATGCAACAGAA-3’
vacA-S2-B3:5’-GACTCTCGCTGTGTATGG-3’
vacA-S2-FIP:5’-CGGTGTGTTTGTTGTATTTCCATTTATTTTCTAGTCTAAAGTCGCAC-3’
vacA-S2-BIP:5’-CAATCGCCCTATTATTTCTCTCGCTGATCATTTGGCGTGTTAGC-3’
vacA-S2-LB:5’-GGTGTTAATGGGCACCGAAC-3’
4. Primers for detecting vacA m1
vacA-M1-F3:5’-AACTATCTGGTCCGAGGC-3’
vacA-M1-B3:5’-CCATTGGTACCTGTAGAAACA-3’
vacA-M1-FIP:5’-GGTCGCGCTGTCTATATCATTATTAAGTGGCAACCTTAAATGTAGG-3’
vacA-M1-BIP:5’-TACAAACCGCTCATCAAGATTAACAACCAATGATTTTCGCTTTCA-3’
vacA-M1-LB:5’-CAAGATCTCATTAAAAATACAG-3’
5. Primers for detecting vacA m2
vacA-M2-F3:5’-TAGTGGATAGCGCGACTG-3’
vacA-M2-B3:5’-GTTGTTGTTATAAAGGGCTAGG-3’
vavA-M2-FIP:5’-CGCCCTTTCACTAAAACATGTTCTTTTACAAACCACTCATTAAGATCA-3’
vacA-M2-BIP:5’-GGAGTGCAAGGCGCTAGTTATTTGAATTGCTCTTGCAGAT-3’
vacA-M2-LB:5’-ATATTTCTGCAAGCAACACC-3’
6. Primers for detecting iceA1
iceA1-F3:5’-CTTGAATGGCTATAATACCGAAT-3’
iceA1-B3:5’-ATCATCAAAAGTCTGTGTGTT-3’
iceA1-FIP:5’-CGCGCAACATTGTTGCTTATAGGTGTTTTTAACCAAAGTATCTGT-3’
iceA1-BIP:5’-GTGTGCGTGGCAACTCTGAAAGAAACTCTTGAATCATCCTTG-3’
iceA1-LB:5’-TAGAAGTGGATCATAAAGACGGCC-3’
7. Primers for detecting iceA2
iceA2-F3:5’-GAGAATGGTATCCATAAGAGAAC-3’
iceA2-B3:5’-ACTGCTCATTAAAACACCG-3’
iceA2-FIP:5’-CTTTGATGTGGTTACAGCCACTACTTATGGTAGTAACGCTATTAATGTG-3’
iceA2-BIP:5’-AAGGACCTACTAGAAAATAGGGCACTAGAAAAACAGCGACACC-3’
iceA2-LB:5’-GCAAAAGCTTTGGCGCATT-3’
8. Primers for detecting babA2
babA2-F3:5’-CGGAACGGTGAATGTAAGCT-3’
babA2-B3:5’-AGGCACACCGTCTAATTGG-3’
babA2-FIP:5’-GTCGTGGTTCCGCCATGTTGGTACACATGCTCAGGGGAAGG-3’
babA2-BIP:5’-CGGCAAAAGCGTAACCACCACAGACACGTTGGGTGTTACCT-3’
babA2-LF:5’-GTGGCTTTTTCCGAGCAGTT-3’
9. Primers for detection of oipA
oipA-F3:5’-AAAAAAAGCATCAAGGCATG-3’
oipA-B3:5’-TCATCGCCATAGCGATCA-3’
oipA-FIP:5’-ACTTGCAAACCAAGTGCTACCTTAGAAAATCTTCAGGGCTTGT-3’
oipA-BIP:5’-ACAATCTCACCCCTTTCAATCAAGTTCATCATTATTCCAACGAACG-3’
oipA-LB:5’-CAAGAGTCGCACGATTTTTCAGT-3’
10. Primers for detecting dupA
dupA-F3:5’-CAAAAGAACACAACAAACCTT-3’
dupA-B3:5’-GTAGATAATCACTTGAGAAAGGT-3’
dupA-FIP:5’-CGAGCGCGTTAGCGATATAGGGATGAAACTAAAGACTACATTATGC-3’
dupA-BIP:5’-CAAGCTAGAAAGATCAACGGAACACTCTTTGCTTTATCAATGCCTAA-3’
dupA-LB:5’-TTTGCATGGCGTTTCA-3’
11. Primers for detecting luxS
luxS-F3:5’-CAACCCATAGGCGACCAATC-3’
luxS-B3:5’-ATGTGCGTATCGCTGATCG-3’
luxS-FIP:5’-GGACATGCCAAGCTTGCACTCTATTAGCATGGTTGCGGATGA-3’
luxS-BIP:5’-CTTTGTTGGGCTGCTTGAAGCGAAAAGGGCGTTAATGGGGAT-3’
luxS-LF：5’-TAGAGCATTTAGTCGCTGAGA-3’
Specifically, the loop-mediated isothermal amplification method is further applied to detect the virulence gene type of the helicobacter pylori by utilizing the primer group, and whether the detected strain is a high-virulence strain or not is determined.
The detection steps of the amplification product are as follows:
(1) preparing an LAMP system: 0.1. mu.l each of 100 μm outer primers F3 and B3; 0.4. mu.l each of 100 μm inner primers FIP and BIP; 0.25. mu.l each of 100 μm loop primers LF and LB; 2x reaction buffer 12.5 μ l; bst DNase 1. mu.l; 2. mu.l of template DNA; fluorescent dye 1. mu.l, make up to 25. mu.l with deionized water.
(2) Carrying out an amplification reaction: the LAMP reaction condition is that the reaction is carried out for 60min at 63 ℃ and then the inactivation is carried out for 5min at 85 ℃.
(3) And (4) judging the result: whether the virulence gene is contained or not is determined by visual observation of a color change or by fluorescence.
Specifically, the 2 × buffer solution in (1) comprises the following components: a final concentration of 40mM Tris-HCl (pH 8.8); kcl at a final concentration of 20 Mm; MgSO 2 to a final concentration of 16mM4(ii) a To a final concentration of 20mM (NH)4)2SO4(ii) a 0.2% Tween 20; betaine at a final concentration of 1.6M; dNTPs at a final concentration of 2.8 mM.
Specifically, the fluorescent dye in (1) is a mixed solution of calcein and manganese chloride. The preparation method comprises the following steps:
1.1 preparation of 1M sodium hydroxide solution (10 mL):
0.4g of solid sodium hydroxide was weighed, dissolved in 10ml of sterilized ultrapure water and mixed well.
Preparation of 2.6.5mM calcein (50 ml):
weighing 0.162g of calcein, putting the calcein into a 2ml centrifuge tube, adding 1.2ml of 1M sodium hydroxide for assisting dissolution, centrifuging at 25000rpm for 2 minutes, transferring into a 50ml centrifuge tube, adding 38.8ml of sterilized ultrapure water, mixing uniformly, wherein the pH of the obtained solution is 8.0-8.5, performing filtration sterilization by using a 0.22 mu M filter membrane in a super clean bench, and preparing the calcein with the concentration of 6.5 mM.
3.1.0ml of fluorescent dye preparation:
12 ml centrifuge tube was filled with 500. mu.l of sterilized ultrapure water, 200. mu.l of the above-described calcein solution, 13. mu.l of manganese dichloride, and 287. mu.l of ultrapure water to make up to 1.0 ml.
Specifically, the result of interpreting the color with naked eyes in the step (3) is as follows: if the reaction product is orange transparent liquid, the strain does not contain the virulence gene; if the reaction product is a green turbid liquid, the strain is considered to contain the virulence genes.
The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.
The invention provides a method for rapidly detecting the type of a helicobacter pylori virulence gene in the field of biotechnology.
The invention provides a kit for visually detecting a helicobacter pylori virulence gene, which comprises: the primer set, the 2x reaction buffer solution, the Bst DNA enzyme, the fluorescent dye and the deionized water.
The beneficial effects of the invention include:
(1) the detection method can detect the main virulence genes of the helicobacter pylori and has important guiding significance clinically.
(2) Compared with other detection systems, the method has the advantages that the fluorescent dye is added before the reaction, the reaction is completed in one step, the cover opening is not needed after the reaction is finished, and the aerosol pollution caused by the cover opening is avoided.
(3) The fluorescent dye contained in the kit is a mixed solution of calcein and manganese dichloride. Loop-mediated isothermal amplification produces a large amount of pyrophosphate ions as a by-product while synthesizing a large amount of nucleic acids. The calcein contained in the fluorescent dye is initially in a fluorescence quenching state due to the binding with manganese ions, but as the loop-mediated isothermal amplification reaction proceeds, the bound manganese ions are deprived by pyrophosphate ions which are reaction by-products, and the calcein is released and emits fluorescence, and further binds with magnesium ions in the reaction solution, so that the fluorescence is further enhanced. Because a plurality of primers are involved in the loop-mediated isothermal amplification reaction process, non-specific binding between the primers is easy to generate primer dimers, so that the result is misjudged. The principle of fluorescent dye detection is to combine pyrophosphate ions as a reaction byproduct rather than amplified product DNA, so that the false positive problem caused by primer dimer does not exist.
(4) The kit has the advantages that the reaction result is easy to read, the result can be observed visually, the positive result is green liquid, the negative result is orange liquid, the result can be judged and read through ultraviolet irradiation, under the irradiation of ultraviolet light, the positive result can emit bright green fluorescence, and the negative result does not generate fluorescence.
(5) The method for interpreting the result not only comprises visual observation and fluorescence, but also can collect fluorescence signals per minute by using a real-time quantitative fluorescence PCR instrument, thereby avoiding errors.
(6) The amplification can be completed in a water bath or a metal bath without setting temperature change and circulation, is suitable for popularization in primary hospitals, and has wide application prospect.
Drawings
FIG. 1 shows the annealing sites and amplification directions of LAMP primers for the cagA gene
FIG. 2 shows the annealing sites and amplification directions of LAMP primers for vacA s1 gene
FIG. 3 shows the annealing sites and amplification directions of LAMP primers for vacA s2 gene
FIG. 4 shows the VAcA m1 gene LAMP primer annealing sites and amplification directions
FIG. 5 shows the VAcA m2 gene LAMP primer annealing sites and amplification directions
FIG. 6 shows the LAMP primer annealing sites and amplification directions of the iceA1 gene
FIG. 7 shows the LAMP primer annealing sites and amplification directions of the iceA2 gene
FIG. 8 shows LAMP primer annealing sites and amplification directions of babA2 gene
FIG. 9 shows LAMP primer annealing sites and amplification directions of oipA gene
FIG. 10 shows the annealing sites and amplification directions of LAMP primers for dupA gene
FIG. 11 shows LAMP primer annealing sites and amplification directions of luxS gene
FIG. 12 is a result chart of detecting virulence genes of a standard strain 43504
FIG. 13 is a result chart of detection of virulence genes of a standard strain 26695
FIG. 14 is the result chart of detecting the virulence genes of clinical strains
Detailed Description
Embodiments of the present invention are further described below with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of a portion of the invention and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
firstly, the sequence of the virulence gene is searched on NCBI website, DNAMAN 8 software is used for comparing the conserved region of the target gene, according to the conserved region, online software Primer Explorer v5 is used for designing LAMP Primer, the LAMP Primer is synthesized by Shanghai biological engineering corporation,
the primer sequences obtained are as follows.
Primers for detecting cagA
Forward outer primer F3: 5'-TCTGCCAAACAATCTTTTGC-3'
Reverse outer primer B3: 5'-TGGTTCTTGATTGATTGCTTC-3'
5'-TTCTGCTTCTTGCCTTTCTTTCAAGAATCATTATAGGGAATCAAATCCG-3' forward inner primer FIP
Reverse inner primer BIP 5'-ATGGAGAGCCTACTGGTGGGTTTGACATCAGAAGATTGTTCT-3'
Forward loop primer LF: 5'-GCCCATGAACTTTTGATCCGT-3'
Primers for detecting vacA s1
Forward outer primer F3: 5'-CCTCTGGTTTCTCTTGCTT-3'
Reverse outer primer B3: 5'-TCTGGGGTTTTATTGGCT-3'
5'-CACGGTTGTGAAAAAGGCGGTAGTAGGAGCGTTAGTCAGC-3' forward inner primer FIP
Reverse inner primer BIP 5'-CATTGTTGGGGGGATCGCTACTCTTCGGCTTGTTTGAGC-3'
Forward loop primer LF: 5'-ATGACTTTGTTGCGGTGT-3'
Reverse loop primer LB: 5'-GGTCTCAGGGCTTCTTAGCT-3'
Primers for detecting vacA s2
Forward outer primer F3: 5'-CCTTAATCGTAAATGCAACAGAA-3'
Reverse outer primer B3: 5'-GACTCTCGCTGTGTATGG-3'
5'-CGGTGTGTTTGTTGTATTTCCATTTATTTTCTAGTCTAAAGTCGCAC-3' forward inner primer FIP
Reverse inner primer BIP 5'-CAATCGCCCTATTATTTCTCTCGCTGATCATTTGGCGTGTTAGC-3'
Reverse loop primer LB: 5'-GGTGTTAATGGGCACCGAAC-3'
Primers for detecting vacA m1
Forward outer primer F3: 5'-AACTATCTGGTCCGAGGC-3'
Reverse outer primer B3: 5'-CCATTGGTACCTGTAGAAACA-3'
5'-GGTCGCGCTGTCTATATCATTATTAAGTGGCAACCTTAAATGTAGG-3' forward inner primer FIP
Reverse inner primer BIP 5'-TACAAACCGCTCATCAAGATTAACAACCAATGATTTTCGCTTTCA-3'
Reverse loop primer LB: 5'-CAAGATCTCATTAAAAATACAG-3'
Primers for detecting vacA m2
Forward outer primer F3: 5'-TAGTGGATAGCGCGACTG-3'
Reverse outer primer B3: 5'-GTTGTTGTTATAAAGGGCTAGG-3'
5'-CGCCCTTTCACTAAAACATGTTCTTTTACAAACCACTCATTAAGATCA-3' forward inner primer FIP
Reverse inner primer BIP 5'-GGAGTGCAAGGCGCTAGTTATTTGAATTGCTCTTGCAGAT-3'
Reverse loop primer LB: 5'-ATATTTCTGCAAGCAACACC-3'
Primers for detecting iceA1
Forward outer primer F3: 5'-CTTGAATGGCTATAATACCGAAT-3'
Reverse outer primer B3: 5'-ATCATCAAAAGTCTGTGTGTT-3'
5'-CGCGCAACATTGTTGCTTATAGGTGTTTTTAACCAAAGTATCTGT-3' forward inner primer FIP
Reverse inner primer BIP 5'-GTGTGCGTGGCAACTCTGAAAGAAACTCTTGAATCATCCTTG-3'
Reverse loop primer LB: 5'-TAGAAGTGGATCATAAAGACGGCC-3'
Primers for detecting iceA2
Forward outer primer F3: 5'-GAGAATGGTATCCATAAGAGAAC-3'
Reverse outer primer B3: 5'-ACTGCTCATTAAAACACCG-3'
5'-CTTTGATGTGGTTACAGCCACTACTTATGGTAGTAACGCTATTAATGTG-3' forward inner primer FIP
Reverse inner primer BIP 5'-AAGGACCTACTAGAAAATAGGGCACTAGAAAAACAGCGACACC-3'
Reverse loop primer LB: 5'-GCAAAAGCTTTGGCGCATT-3'
Primers for detecting babA2
Forward outer primer F3: 5'-CGGAACGGTGAATGTAAGCT-3'
Reverse outer primer B3: 5'-AGGCACACCGTCTAATTGG-3'
5'-GTCGTGGTTCCGCCATGTTGGTACACATGCTCAGGGGAAGG-3' forward inner primer FIP
Reverse inner primer BIP 5'-CGGCAAAAGCGTAACCACCACAGACACGTTGGGTGTTACCT-3'
Forward loop primer LF: 5'-GTGGCTTTTTCCGAGCAGTT-3'
Primers for detection of oipA
Forward outer primer F3: 5'-AAAAAAAGCATCAAGGCATG-3'
Reverse outer primer B3: 5'-TCATCGCCATAGCGATCA-3'
5'-ACTTGCAAACCAAGTGCTACCTTAGAAAATCTTCAGGGCTTGT-3' forward inner primer FIP
Reverse inner primer BIP 5'-ACAATCTCACCCCTTTCAATCAAGTTCATCATTATTCCAACGAACG-3'
Reverse loop primer LB: 5'-CAAGAGTCGCACGATTTTTCAGT-3'
Primers for detecting dupA
Forward outer primer F3: 5'-CAAAAGAACACAACAAACCTT-3'
Reverse outer primer B3: 5'-GTAGATAATCACTTGAGAAAGGT-3'
5'-CGAGCGCGTTAGCGATATAGGGATGAAACTAAAGACTACATTATGC-3' forward inner primer FIP
Reverse inner primer BIP 5'-CAAGCTAGAAAGATCAACGGAACACTCTTTGCTTTATCAATGCCTAA-3'
Reverse loop primer LB: 5'-TTTGCATGGCGTTTCA-3'
Primers for detecting luxS
Forward outer primer F3: 5'-CAACCCATAGGCGACCAATC-3'
Reverse outer primer B3: 5'-ATGTGCGTATCGCTGATCG-3'
5'-GGACATGCCAAGCTTGCACTCTATTAGCATGGTTGCGGATGA-3' forward inner primer FIP
Reverse inner primer BIP 5'-CTTTGTTGGGCTGCTTGAAGCGAAAAGGGCGTTAATGGGGAT-3'
Forward loop primer LF: 5'-TAGAGCATTTAGTCGCTGAGA-3'
Reaction System (25. mu.L)
Composition (I)
Stock solution
Dosage of
Final concentration
2 Xbuffer
12.5μL
F3
100μM
0.1μL
0.4μM
B3
100μM
0.1μL
0.4μM
FIP
100μM
0.4μL
1.6μM
BIP
100μM
0.4μL
1.6μM
LF
100μM
0.25μL
1.0μM
LB
100μM
0.25μL
1.0μM
Bst DNA enzyme
8000U/ml
1.0μL
0.32U/μL
Fluorescent dyes
1.0μL
Nucleic acid template
2.0μL
ddH2O
Make up to 25 μ L
2x reaction buffer composition and concentration
The fluorescent dye is a mixed solution of calcein and manganese chloride. The preparation method comprises the following steps:
1.1 preparation of 1M sodium hydroxide solution (10 mL):
0.4g of solid sodium hydroxide was weighed, dissolved in 10ml of sterilized ultrapure water and mixed well.
Preparation of 2.6.5mM calcein (50 ml):
weighing 0.162g of calcein, putting the calcein into a 2ml centrifuge tube, adding 1.2ml of 1M sodium hydroxide for assisting dissolution, centrifuging at 25000rpm for 2 minutes, transferring into a 50ml centrifuge tube, adding 38.8ml of sterilized ultrapure water, mixing uniformly, wherein the pH of the obtained solution is 8.0-8.5, performing filtration sterilization by using a 0.22 mu M filter membrane in a super clean bench, and preparing the calcein with the concentration of 6.5 mM.
3.1.0ml of fluorescent dye preparation:
12 ml centrifuge tube was filled with 500. mu.l of sterilized ultrapure water, 200. mu.l of the above-described calcein solution, 13. mu.l of manganese dichloride, and 287. mu.l of ultrapure water to make up to 1.0 ml.
DNA extraction step:
1. the standard strain and the clinical isolates of H.pylori were inoculated on Columbia medium, streaked, and after two days, scraped off with an inoculating loop and dissolved in PBS solution.
2. The bacteria were dispensed into 1.5 ml centrifuge tubes, centrifuged at 12000rpm for 2min, and the supernatant was discarded.
3. Adding 180 microliter Buffer GL, 20 microliter ProteinaseK and 10 microliter RNase A, fully and uniformly blowing by using a gun head, and carrying out warm bath for 10min in 56 ℃ water bath.
4. 200 microliters of Buffer GB and 200 microliters of absolute ethanol were added and mixed well.
5. The Spin Column was placed on the Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12000rpm for 2min, and the filtrate was discarded.
6. 500 microliters of Buffer WA WAs added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate WAs discarded.
7. 700. mu.l of Buffer WB was added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.
8. 700. mu.l of Buffer WB was added to Spin Column again, centrifuged at 12000rpm for 1min, and the filtrate was discarded.
9. The tube from which the filtrate was discarded was centrifuged again at 12000rpm for 2 min.
10. Spin Column was placed in a new 1.5 ml centrifuge tube, 50 μ l of Elution buffer was added to the center of the Spin Column membrane, and allowed to stand at room temperature for 5 min.
11. The DNA was eluted by centrifugation at 12000rpm for 2 min. The centrifuged solution is the extracted DNA.
The detection steps of the amplification product are as follows:
preparing an LAMP system: 0.1. mu.l each of 100 μm outer primers F3 and B3; 0.4. mu.l each of 100 μm inner primers FIP and BIP; 0.25. mu.l each of 100 μm loop primers LF and LB; 2x reaction buffer 12.5 μ l; bst DNase 1. mu.l; 2. mu.l of template DNA; fluorescent dye 1. mu.l, make up to 25. mu.l with deionized water.
Carrying out an amplification reaction: the LAMP reaction condition is that the reaction is carried out for 60min at 63 ℃ and then the inactivation is carried out for 5min at 85 ℃.
And (4) judging the result: whether the virulence gene is contained or not is determined by visual observation of a color change or fluorescence.
FIG. 12 shows the virulence gene types of the standard strain 43504, which are consistent with the types reported in the literature. Graph A shows the fluorescent signal condition of each gene in each minute, and the result shows that the virulence genotypes of the standard strains are as follows: cagA, vacA 1/m1, iceA2, babA2, oipA, luxS. The B picture is a visual result, and whether the virulence gene is contained or not is determined by visually observing the change of color. From 1 to 12, cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA2, oipA, dupA, luxS, and negative control, respectively. Interpretation criteria of results: green liquid is positive and orange liquid is negative. Panel C shows the results under 365nm UV light. 1-12 are identical to those in the B diagram. Interpretation criteria of results: under the irradiation of ultraviolet light, the fluorescence emitting bright green is positive, and the fluorescence without fluorescence is negative.
FIG. 13 shows the virulence gene types of the standard strain 26695. Panel A shows the results of visual observation of color change to determine whether the virulence gene is contained. From 1 to 12, cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA2, oipA, dupA, luxS, and negative control, respectively. Interpretation criteria of results: green liquid is positive and orange liquid is negative. Panel B shows the results under 365nm uv light. 1-12 are identical to those in Panel A. Interpretation criteria of results: under the irradiation of ultraviolet light, the fluorescence emitting bright green is positive, and the fluorescence without fluorescence is negative. As shown in the figure, the virulence gene types of the standard strain 26695 are cagA, vacA s1/m1, iceA2, babA2, oipA, luxS.
FIG. 14 shows the results of detecting virulence genes in clinical isolates, and panel A is a visual result, in which the presence of virulence genes is determined by visual observation of color changes. From 1 to 12, cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA2, oipA, dupA, luxS, and negative control, respectively. Interpretation criteria of results: green liquid is positive and orange liquid is negative. Panel B shows the results under 365nm uv light. 1-12 are identical to those in Panel A. Interpretation criteria of results: under the irradiation of ultraviolet light, the fluorescence emitting bright green is positive, and the fluorescence without fluorescence is negative. As shown, the clinical isolates had virulence gene types vacA s1/m2, iceA1, oipA, dupA, luxS.
Sequence listing
<110> university of southern kayak
<120> a method and kit for detecting helicobacter pylori virulence gene type by using loop-mediated isothermal amplification method
<160> 56
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 1
tctgccaaac aatcttttgc 20
<210> 2
<211> 21
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 2
tggttcttga ttgattgctt c 21
<210> 3
<211> 49
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 3
ttctgcttct tgcctttctt tcaagaatca ttatagggaa tcaaatccg 49
<210> 4
<211> 42
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 4
atggagagcc tactggtggg tttgacatca gaagattgtt ct 42
<210> 5
<211> 21
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 5
gcccatgaac ttttgatccg t 21
<210> 6
<211> 19
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 6
cctctggttt ctcttgctt 19
<210> 7
<211> 18
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 7
tctggggttt tattggct 18
<210> 8
<211> 40
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 8
cacggttgtg aaaaaggcgg tagtaggagc gttagtcagc 40
<210> 10
<211> 39
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 10
cattgttggg gggatcgcta ctcttcggct tgtttgagc 39
<210> 10
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 10
ggtctcaggg cttcttagct 20
<210> 11
<211> 18
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 11
atgactttgt tgcggtgt 18
<210> 12
<211> 23
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 12
ccttaatcgt aaatgcaaca gaa 23
<210> 13
<211> 18
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 13
gactctcgct gtgtatgg 18
<210> 14
<211> 47
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 14
cggtgtgttt gttgtatttc catttatttt ctagtctaaa gtcgcac 47
<210> 15
<211> 44
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 15
caatcgccct attatttctc tcgctgatca tttggcgtgt tagc 44
<210> 16
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 16
ggtgttaatg ggcaccgaac 20
<210> 17
<211> 18
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 17
aactatctgg tccgaggc 18
<210> 18
<211> 21
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 18
ccattggtac ctgtagaaac a 21
<210> 19
<211> 46
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 19
ggtcgcgctg tctatatcat tattaagtgg caaccttaaa tgtagg 46
<210> 20
<211> 45
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 20
tacaaaccgc tcatcaagat taacaaccaa tgattttcgc tttca 45
<210> 21
<211> 22
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 21
caagatctca ttaaaaatac ag 22
<210> 22
<211> 18
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 22
tagtggatag cgcgactg 18
<210> 23
<211> 22
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 23
gttgttgtta taaagggcta gg 22
<210> 24
<211> 48
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 24
cgccctttca ctaaaacatg ttcttttaca aaccactcat taagatca 48
<210> 26
<211> 40
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 26
ggagtgcaag gcgctagtta tttgaattgc tcttgcagat 40
<210> 26
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 26
atatttctgc aagcaacacc 20
<210> 27
<211> 23
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 27
cttgaatggc tataataccg aat 23
<210> 29
<211> 21
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 29
atcatcaaaa gtctgtgtgt t 21
<210> 29
<211> 45
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 29
cgcgcaacat tgttgcttat aggtgttttt aaccaaagta tctgt 45
<210> 31
<211> 42
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 31
gtgtgcgtgg caactctgaa agaaactctt gaatcatcct tg 42
<210> 32
<211> 24
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 32
tagaagtgga tcataaagac ggcc 24
<210> 33
<211> 23
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 33
gagaatggta tccataagag aac 23
<210> 34
<211> 19
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 34
actgctcatt aaaacaccg 19
<210> 35
<211> 49
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 35
ctttgatgtg gttacagcca ctacttatgg tagtaacgct attaatgtg 49
<210> 36
<211> 43
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 36
aaggacctac tagaaaatag ggcactagaa aaacagcgac acc 43
<210> 37
<211> 19
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 37
gcaaaagctt tggcgcatt 19
<210> 38
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 38
cggaacggtg aatgtaagct 20
<210> 39
<211> 19
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 39
aggcacaccg tctaattgg 19
<210> 40
<211> 41
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 40
gtcgtggttc cgccatgttg gtacacatgc tcaggggaag g 41
<210> 41
<211> 41
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 41
cggcaaaagc gtaaccacca cagacacgtt gggtgttacc t 41
<210> 42
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 42
gtggcttttt ccgagcagtt 20
<210> 43
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 43
aaaaaaagca tcaaggcatg 20
<210> 44
<211> 18
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 44
tcatcgccat agcgatca 18
<210> 45
<211> 43
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 45
acttgcaaac caagtgctac cttagaaaat cttcagggct tgt 43
<210> 46
<211> 46
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 46
acaatctcac ccctttcaat caagttcatc attattccaa cgaacg 46
<210> 47
<211> 23
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 47
caagagtcgc acgatttttc agt 23
<210> 48
<211> 21
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 48
caaaagaaca caacaaacct t 21
<210> 49
<211> 23
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 49
gtagataatc acttgagaaa ggt 23
<210> 50
<211> 46
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 50
cgagcgcgtt agcgatatag ggatgaaact aaagactaca ttatgc 46
<210> 51
<211> 47
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 51
caagctagaa agatcaacgg aacactcttt gctttatcaa tgcctaa 47
<210> 52
<211> 16
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 52
tttgcatggc gtttca 16
<210> 53
<211> 20
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 53
caacccatag gcgaccaatc 20
<210> 54
<211> 19
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 54
atgtgcgtat cgctgatcg 19
<210> 55
<211> 42
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 55
ggacatgcca agcttgcact ctattagcat ggttgcggat ga 42
<210> 56
<211> 42
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 56
ctttgttggg ctgcttgaag cgaaaagggc gttaatgggg at 42
<210> 57
<211> 21
<212> DNA
<213> Helicobacter pylori (Helicobacter pylori)
<400> 57
tagagcattt agtcgctgag a 21