CN112683893A - High-sensitivity helicobacter pylori detection kit - Google Patents
High-sensitivity helicobacter pylori detection kit Download PDFInfo
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- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 52
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 47
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 23
- 239000002250 absorbent Substances 0.000 claims abstract description 17
- 230000002745 absorbent Effects 0.000 claims abstract description 17
- 206010019375 Helicobacter infections Diseases 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 105
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 46
- 239000001963 growth medium Substances 0.000 claims description 45
- 239000004202 carbamide Substances 0.000 claims description 33
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 32
- 239000000758 substrate Substances 0.000 claims description 24
- 239000002696 acid base indicator Substances 0.000 claims description 21
- 210000003296 saliva Anatomy 0.000 claims description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000000337 buffer salt Substances 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- 210000001156 gastric mucosa Anatomy 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 6
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims description 4
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 4
- 239000003086 colorant Substances 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
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- 239000011259 mixed solution Substances 0.000 claims description 4
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000001632 sodium acetate Substances 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
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- 235000011008 sodium phosphates Nutrition 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 2
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 2
- 235000011009 potassium phosphates Nutrition 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000011975 tartaric acid Substances 0.000 claims description 2
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- 238000006243 chemical reaction Methods 0.000 description 4
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- 239000000843 powder Substances 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 4
- 201000011549 stomach cancer Diseases 0.000 description 4
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 3
- 208000007107 Stomach Ulcer Diseases 0.000 description 3
- 208000023652 chronic gastritis Diseases 0.000 description 3
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Inorganic materials Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 3
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 3
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 208000004300 Atrophic Gastritis Diseases 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
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- 239000003153 chemical reaction reagent Substances 0.000 description 2
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- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 description 2
- 229940012189 methyl orange Drugs 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000012134 rapid urease test Methods 0.000 description 2
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 2
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- 235000011004 sodium tartrates Nutrition 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- MBXSHBIQMDKTEW-UHFFFAOYSA-N 3-bromobutanenitrile Chemical compound CC(Br)CC#N MBXSHBIQMDKTEW-UHFFFAOYSA-N 0.000 description 1
- OXDOIOYUIISYNE-UHFFFAOYSA-N 4-methylthiadiazole-5-carbaldehyde Chemical compound CC=1N=NSC=1C=O OXDOIOYUIISYNE-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000060 Abdominal distension Diseases 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
- 206010015137 Eructation Diseases 0.000 description 1
- 208000036495 Gastritis atrophic Diseases 0.000 description 1
- 235000000177 Indigofera tinctoria Nutrition 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
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- 206010046274 Upper gastrointestinal haemorrhage Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
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- 229940088710 antibiotic agent Drugs 0.000 description 1
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- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 229940097275 indigo Drugs 0.000 description 1
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- NMKFVGALBGZKGW-UHFFFAOYSA-M sodium;2-[(3-bromo-4-hydroxy-2-methyl-5-propan-2-ylphenyl)-(3-bromo-2-methyl-4-oxo-5-propan-2-ylcyclohexa-2,5-dien-1-ylidene)methyl]benzenesulfonate Chemical compound [Na+].CC1=C(Br)C(=O)C(C(C)C)=CC1=C(C=1C(=CC=CC=1)S([O-])(=O)=O)C1=CC(C(C)C)=C(O)C(Br)=C1C NMKFVGALBGZKGW-UHFFFAOYSA-M 0.000 description 1
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Abstract
The application provides a helicobacter pylori detection kit with high sensitivity, the kit adopts a rapid culture method to shorten the detection time, and the kit also uses an ammonia absorbent to increase the detection accuracy and reduce the misjudgment rate of clinical medical personnel. The kit can be used for large-scale clinical screening to prevent helicobacter pylori infection and transmission.
Description
Technical Field
The application relates to the field of medical equipment, in particular to a kit for detecting helicobacter pylori, and belongs to the field of medicines.
Background
Helicobacter pylori lives in the pylorus and oral cavity of the human stomach, and is one of the most common bacterial pathogens. Most of the world's population is infected with H.pylori, and in some countries almost 90% of people are infected with this bacterium. People are usually infected at early age, reaching 50% under 5 years of age. This bacterial infection first causes chronic gastritis and leads to gastric ulcers and gastric atrophy, and in severe cases gastric cancer.
According to statistics, the incidence rate of atrophic gastritis and gastric cancer of people with early age of the initial helicobacter pylori infection is high, and the helicobacter pylori infection and the death rate of gastric cancer are in parallel. Helicobacter pylori is parasitic in the tissues of the gastric mucosa, and 67% to 80% of gastric ulcers and 95% of duodenal ulcers are caused by helicobacter pylori. The common symptoms of patients with chronic gastritis and peptic ulcer are: after eating, the upper abdomen is full, uncomfortable or painful, often accompanied by other adverse symptoms such as belching, abdominal distension, acid regurgitation, anorexia, etc. Some patients may also have recurrent severe abdominal pain, minor upper gastrointestinal bleeding, etc. Therefore, experts think that people who find helicobacter pylori infection as soon as possible can kill helicobacter pylori with antibiotics timely and effectively, and the method has great significance for preventing and controlling gastric cancer.
Currently, there are many methods for detecting helicobacter pylori infection, such as biopsy, isolated culture of helicobacter pylori, rapid urease test, urea breath test, urinary ammonia excretion test, serology test, polymerase chain reaction, and the like. In comparison with the above-mentioned results,the most widespread detection methods for clinical applications are mainly the rapid urease test and the urea breath test. The two detection methods are mainly based on that helicobacter pylori generates urease which decomposes urea to generate CO2And NH3. Helicobacter pylori produces urease, and the urease needs a certain time to decompose urea, so that the conventional rapid urease method needs more time for detecting each sample, thereby being not beneficial to large-scale screening development. Detection of helicobacter pylori by pH value saliva urease test[1]The method comprises the steps of mixing 12-15% of urea with ethanol in equal proportion, mixing saliva with a urea solution during detection by using a self-made saliva urease detection kit, measuring the pH value after mixing for 5-20 minutes, taking the pH value of the measured saliva as a negative control, and judging whether helicobacter pylori infection exists or not according to the pH value difference. The method has the following defects: (1) helicobacter pylori produces urease, which takes a certain time to decompose urea, resulting in the inability of a tester to rapidly detect helicobacter pylori. The applicant carries out rechecking by the method, helicobacter pylori is added into the urea solution, the pH value of the urea solution is not changed within 5 minutes, the pH value is slightly increased within 15 minutes, and the change is obvious within 25-30 minutes; (2) when the pH value is judged to change, pH test paper is used, the measured pH value is coarser, and the change of the pH value within 0.5 cannot be judged; (3) NH produced by urease decomposition of urea3Form NH with water4OH, but NH4OH reversibly decomposes to NH3And H2O, although a slight amount of such change affects the result judgment. The test shows that the self-made kit is directly added with urease, the minimum detection amount reaches 8EU, and the urease generated by helicobacter pylori in saliva in a short time can not reach 8EU, so that the detection time is prolonged. CN1266461 discloses a urease kit for endoscope, which is composed of urea and KH2PO4Hypochlorite, phenate solution and phenol red. The main action principle is that urease decomposes urea to produce CO2And NH3Then, the indigo method is used for detecting trace ammonia to judge the helicobacter pylori infection, and in the system, gel made of agar is used as a carrier of the whole system. Since of hypochlorite and phenateThe stability is poor, and the carrier is agar gel, urea can be decomposed in the system in advance, so that the substrate urea is insufficient during detection, and accurate detection cannot be carried out. And the detection result needs 1 to 3 hours.
The patent CN202010184232.2 of our company uses an ultra-rapid culture method to make helicobacter pylori nourished, thereby rapidly producing urease and accelerating the decomposition of the substrate urea to produce substances for identification. CN202010184232.2 also solves a problem that plagues the accuracy of this method: through adding the ammonia absorbent that can absorb ammonia on the kit, lock ammonia on the kit, can strengthen the color reaction like this, make the phenomenon more obvious, have more kind and judge in medical personnel. Through research, the application can be extended to a certain extent on the basis of CN 202010184232.2. When the reagent is used, urea, an ammonia absorbent and a culture medium are dissolved in an ethanol solution to prepare a solution A, an ammonia absorbent and a culture medium with equal proportion are dissolved in an ethanol solution to prepare a negative solution B, the pH values of the solution A and the solution B are adjusted to be consistent, equal volume of saliva is respectively dripped into the solution A and the negative solution B according to the proportion of 2: 1 (if gastric mucosa tissues are used, the gastric mucosa tissues at the same sampling point are respectively placed into about 0.75ml of the solution A and the negative solution B), 1 drop of an acid-base indicator is respectively dripped, the mixture is uniformly mixed, after 1 minute, the color of the solution A and the color of the negative solution B are observed, compared with the color change pH value of the acid-base indicator, the color pH value of the solution A minus the color pH value of the negative solution B is positive, the value is negative when the value is more than 0, and the value is negative when the value is less than or. The present application solves the following problems: (1) the culture medium in the substrate solution can ensure that the helicobacter pylori can be nourished, so that urease is rapidly produced, and the detection time can be greatly shortened; (2) the ammonia absorbent in the substrate solution can be used
Equilibrium of the reaction is shifted to NH4The OH end moves, the change of the pH value of the substrate solution is obvious, and the color change difference of the acid-base indicator is increased after reaction; (3) the general acid-base indicator has a large color change range, such as a color change range of phenol red of 6.6-8.0 and a pH value of 6.6-8.0The color is orange, the pH is yellow when being lower than 6.6, the pH is red when being higher than 8.0, the difference is 1.4, the intermediate excessive color is gradually changed, and the urease decomposes urea to change the pH value of the substrate solution to be less than 0.5 to a great extent, so when an acid-base indicator like phenol red is used for judging whether the helicobacter pylori exists, the judgment cannot be carried out on the critical point, and the misjudgment is caused. The acid-base indicator used in the application is a mixed acid-base indicator, has a short color change range, and can reduce negative or positive misjudgment of helicobacter pylori near critical to a great extent; (4) through tests, the minimum detection amount of the kit prepared by the method is 0.025EU which is far lower than the minimum detection amount of the kit prepared by the surplus token and the like, and the minimum detection amount of the kit prepared by the method is 8 EU. Because the minimum detection amount of the urease is greatly reduced, the sensitivity is greatly improved, and because the sensitivity is improved, only helicobacter pylori exists, trace urease originally secreted by the helicobacter pylori can be well detected, so the detection time is also accelerated to a certain extent; (5) the residual token and the like are used for detecting helicobacter pylori by a pH value method saliva urease test, a 50% ethanol solution is selected, and through the test, the activity of the ethanol solution with the concentration of 50% or more than 50% can be inhibited to a certain extent, and even the helicobacter pylori can die. The method selects 20-40% ethanol solution, preferably 25% ethanol solution, the ethanol with the concentration can inhibit trace urease secreted by other microorganisms in saliva, so that the interference is reduced, and the activity of helicobacter pylori or the secreted urease is not influenced; (6) the hydrolysis of urea is affected by acid and alkali, temperature and other factors, and through experiment, the urea solution with pH value of 6.54 is placed, the pH value is 7.75 measured on the second day, and the pH value is 8.63 measured on the third day. This indicates that the urea solution has poor stability, which can lead to clinical false positives. The substrate is prepared into freeze-dried powder and is prepared into a substrate solution during use. This increases the stability of the substrate. (7) Some medical personnel can not use the kit because of the sensitivity to color change by naked eyes, the kit can use a color difference meter to compare colors, the difference value can show the serious condition that the patient is infected with the helicobacter pylori, a special high-sensitivity pH meter can be matched to measure the pH value difference, and the difference value can also show the serious condition that the patient is infected with the helicobacter pylori
Disclosure of Invention
The application aims to provide a helicobacter pylori detection kit capable of rapidly detecting helicobacter pylori, and the kit can inform medical staff of helicobacter pylori infection of patients in a short time.
The helicobacter pylori detection kit is characterized by comprising a substrate urea, an ammonia absorbent, an acid-base indicator, a culture medium and an ethanol solution
The helicobacter pylori detection kit is characterized in that the ammonia absorbent is one or more of citric acid and salts, tartaric acid and salts, boric acid, nickel chloride, aluminum sulfate, aluminum trichloride and copper sulfate, wherein the nickel chloride, the aluminum sulfate and the aluminum trichloride are preferred.
The helicobacter pylori detection kit is characterized in that the culture medium is one or more of 199 culture medium, BME culture medium, 1640 culture medium, RPMI-1640 culture medium, DMEM-F12 culture medium, DMEM culture medium, F10 culture medium, F12 culture medium, Fischer's culture medium, IMDM culture medium, MEM culture medium and lactic acid.
The helicobacter pylori detection kit is characterized in that the pH value buffer salt is one or more of acetic acid/sodium acetate, ammonia/ammonium chloride, phosphoric acid/sodium phosphate or potassium phosphate, disodium hydrogen phosphate/sodium dihydrogen phosphate, phthalic acid buffer salt and citric acid buffer salt.
The helicobacter pylori detection kit is characterized in that urea, a culture medium, a buffer salt and an ammonia absorbent are dissolved by an ethanol solution to form a substrate solution when in use, the pH value of the substrate solution is 3.0-8.0, the pH value of the substrate solution is lower than the pH value at which an acid-base indicator starts to change color, when the substrate solution is mixed with saliva according to a ratio of 4: 1, the pH value of the mixed solution is not more than 0.15 relative to the pH value of the substrate solution, and the pH value of the mixed solution is still lower than the pH value at which the acid-base indicator starts to change color.
The helicobacter pylori detection kit is characterized in that the acid-base indicator is a mixed indicator or a monochromatic indicator, and when the mixed indicator is used, the color change pH range of the mixed indicator is not more than 0.5.
The helicobacter pylori detection kit is characterized in that the substrates of urea, ammonia absorbent, acid-base indicator, buffer salt and culture medium exist in a solid form, wherein freeze-dried powder is preferred.
The helicobacter pylori detection kit as described above, wherein the detection is carried out in the following steps when detecting whether helicobacter pylori is infected:
(1) dissolving urea, buffer salt, an ammonia absorbent and a culture medium in an ethanol solution to prepare a solution A, dissolving the buffer salt, the ammonia absorbent and the culture medium in the ethanol solution in equal proportion to prepare a negative solution B, and adjusting the pH values of the solution A and the solution B to be consistent;
(1a) respectively taking the solution A and the negative solution B, respectively dripping equal-volume saliva according to the proportion of 4: 1, respectively dripping 1 drop of an acid-base indicator, and uniformly mixing;
(1b) if the gastric mucosa tissue is used, the gastric mucosa tissue at the same sampling point is respectively placed in about 0.75ml of solution A and negative solution B, 1 drop of acid-base indicator is respectively added dropwise, and the mixture is uniformly mixed;
(2) and after 1 minute, observing the colors of the solution A and the negative solution B, comparing the color change pH value of the solution A with the color change pH value of the acid-base indicator, subtracting the color pH value of the negative solution B from the color pH value of the solution A, wherein the value is positive when the value is more than 0, the value is negative when the value is less than or equal to 0, and the larger the value is, the larger the positive value is.
The helicobacter pylori detection kit is characterized in that the difference value between the sample solution A and the negative control solution B can be compared by a color difference meter to obtain an accurate difference value so as to judge the severity of helicobacter pylori infection.
The helicobacter pylori detection kit is characterized in that the difference between the sample solution A and the negative control solution B can be accurately determined by comparing the pH value of the pH value to determine the severity of helicobacter pylori infection.
Detailed Description
Example 1
Dissolving 2 parts of urea, 0.5 part of sodium citrate, 0.02 part of acetic acid/sodium acetate and 0.2 part of 199 culture medium in 100 parts of 25% ethanol respectively to prepare a solution A, dissolving 0.5 part of sodium citrate and 0.2 part of 199 culture medium in 100 parts of 25% ethanol to prepare a negative solution B, and adjusting the pH values of the solution A and the negative solution B to 3.5. Another part of 0.1% bromocresol green sodium salt aqueous solution and another part of 0.2% methyl orange aqueous solution were mixed and the pH was adjusted to 3.5. The indicators were color-corrected at pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, respectively, labeled 0, 1, 2, 3, 4, 5, 6, 7, respectively.
Example 2
Dissolving 3 parts of urea, 0.3 part of sodium tartrate, 0.03 part of phosphoric acid/sodium phosphate and 0.25 part of 1640 culture medium in 100 parts of 30% ethanol respectively to prepare a solution A, dissolving 0.3 part of sodium tartrate and 0.25 part of 1640 culture medium in 100 parts of 30% ethanol to prepare a negative solution B, and adjusting the pH values of the solution A and the negative solution B to 4.5. Another part of the 0.1% chlorophenol red sodium salt aqueous solution and another part of the 0.1% aniline blue aqueous solution are mixed, and the pH is adjusted to 4.5. The indicators were color-corrected at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, respectively, labeled 0, 1, 2, 3, 4, 5, 6, 7, respectively.
Example 3
Respectively dissolving 2.5 parts of urea, 0.3 part of nickel chloride and 0.2 part of DMEM culture medium in 100 parts of 25% ethanol to prepare a solution A, dissolving 0.3 part of nickel chloride and 0.2 part of DMEM culture medium in 100 parts of 25% ethanol to prepare a negative solution B, and adjusting the pH values of the solution A and the negative solution B to 6.0. Another part of 0.1% bromocresol purple sodium salt aqueous solution and another part of 0.1% bromothymol blue sodium salt aqueous solution are mixed, and the pH is adjusted to 6.0. The indicators were color-corrected at pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, respectively, labeled 0, 1, 2, 3, 4, 5, 6, respectively.
Test example 1
167 patients with gastritis, chronic gastritis or gastric ulcer as clinical preliminary diagnosis were selected and tested using the reagent prepared in example 1, the test method: 1.0ml of each of the solution A and the negative solution B prepared in example 1 was added to 0.5ml of saliva of the same patient, and 1 drop of bromocresol green/methyl orange indicator was added dropwiseAnd mixing uniformly. Observing the color of the solution A, comparing the color with that of the negative solution B, simultaneously subtracting the corresponding numerical values of the solution A, the negative solution B and the corrected standard color from the pH value of the color of the solution A, wherein the numerical value is positive when the numerical value is more than 0, and the numerical value is negative when the numerical value is less than or equal to 0, and the larger the numerical value of the determination result is, the larger the positive value is. Subjects who collected saliva were validated on a 14 carbon insufflation test. The air blowing value is more than or equal to 100dpm/mmol, the positive result (Hp infection) is obtained, and the measurement result is less than 100dpm/mmol, the negative result (no Hp infection) is obtained. The results of the two methods are compared: saliva method (HPS) and 14 carbon blow test (A)14C-UBT) assay results 167 cases, corresponding to HPS positive specimens, were evaluated by Bayesian theorem14The C-UBT positive specimen has 16 cases of false positives; and 167 cases of HPS negative samples corresponded to14The C-UBT negative specimen has 13 cases of false negatives; HPS to14C-UBT was used as the standard, the positive predictive value of HPS was 93.35%, the negative predictive value was 96.38%, the accuracy was 96.84%, and the variability was 4.62%.
Test example 2
Take the recipe of example 1: dissolving 2 parts of urea, 0.5 part of sodium citrate, 0.02 part of acetic acid/sodium acetate and 0.2 part of 199 culture medium in 100 parts of purified water respectively to prepare a solution A, dissolving 0.5 part of sodium citrate and 0.2 part of 199 culture medium in 100 parts of purified water to prepare a negative solution B, adding an indicator into the solution A and the solution B respectively, freeze-drying the solution into freeze-dried powder, and storing the solution with 25% ethanol as a reaction solution independently. Placing the lyophilized powder with different water contents in 40 deg.C environment, and measuring the contents in 0, 1, 2, 3, and 6 months respectively.
TABLE 1 stability of lyophilized powders of different water contents
As can be seen from Table 1, the higher the water content of urea, the poorer the stability thereof, so that the substrate is made into lyophilized powder, i.e. the product stability can be increased; secondly, the freeze-dried powder is dissolved more rapidly, the product dissolving time can be shortened, and the workload of medical staff is reduced.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the spirit of the present application, which should be considered as belonging to the protection scope of the present application.
Claims (10)
1. A helicobacter pylori detection kit for detecting helicobacter pylori comprises a substrate urea, an ammonia absorbent, an acid-base indicator, buffer salt, a culture medium solution and an ethanol solution.
2. The helicobacter pylori detection kit according to claim 1, wherein the ammonia absorbent is one or more of citric acid and salts, tartaric acid and salts, boric acid, nickel chloride, aluminum sulfate, aluminum trichloride and copper sulfate, and preferably nickel chloride, aluminum sulfate and aluminum trichloride.
3. The helicobacter pylori detection kit according to claim 1, wherein the culture medium is one or more of 199 culture medium, BME culture medium, 1640 culture medium, RPMI-1640 culture medium, DMEM-F12 culture medium, DMEM culture medium, F10 culture medium, F12 culture medium, Fischer's culture medium, IMDM culture medium, MEM culture medium, and lactic acid.
4. The helicobacter pylori detection kit according to claim 1, wherein the pH buffer salt is one or more selected from the group consisting of acetic acid/sodium acetate, ammonia/ammonium chloride, phosphoric acid/sodium phosphate or potassium phosphate, disodium hydrogen phosphate/sodium dihydrogen phosphate, phthalic acid buffer salt, and citric acid buffer salt.
5. The helicobacter pylori detection kit according to claim 1, wherein urea, a culture medium, a buffer salt and an ammonia absorbent are dissolved in an ethanol solution to form a substrate solution, the pH of the substrate solution is 3.0 to 8.0, the pH of the substrate solution is lower than the pH at which the pH indicator starts to change color, when the substrate solution is mixed with saliva in a ratio of 4: 1, the pH of the mixed solution is not higher than 0.15 relative to the pH of the substrate solution, and the pH of the mixed solution is lower than the pH at which the pH indicator starts to change color.
6. The helicobacter pylori detection kit according to claim 1, wherein the acid-base indicator is a mixed indicator or a monochromatic indicator, and the pH range of the color change of the mixed indicator is not greater than 0.5.
7. Helicobacter pylori detection kit according to claim 1, wherein the substrates urea, ammonia absorbent, acid-base indicator, buffer salt, culture medium are present in solid form, preferably as lyophilized powder.
8. A helicobacter pylori detection kit according to claim 1, wherein the detection is carried out in the following steps in the case of detecting the infection with helicobacter pylori:
(1) dissolving urea, buffer salt, an ammonia absorbent and a culture medium in an ethanol solution to prepare a solution A, dissolving the buffer salt, the ammonia absorbent and the culture medium in the ethanol solution in equal proportion to prepare a negative solution B, and adjusting the pH values of the solution A and the solution B to be consistent;
(1a) respectively taking the solution A and the negative solution B, respectively dripping equal-volume saliva according to the proportion of 4: 1, respectively dripping 1 drop of an acid-base indicator, and uniformly mixing;
(1b) if the gastric mucosa tissue is used, the gastric mucosa tissue at the same sampling point is respectively placed in about 0.75ml of solution A and negative solution B, 1 drop of acid-base indicator is respectively added dropwise, and the mixture is uniformly mixed;
(2) and after 1 minute, observing the colors of the solution A and the negative solution B, comparing the color change pH value of the solution A with the color change pH value of the acid-base indicator, subtracting the color pH value of the negative solution B from the color pH value of the solution A, wherein the value is positive when the value is more than 0, the value is negative when the value is less than or equal to 0, and the larger the value is, the larger the positive value is.
9. The helicobacter pylori detection kit according to claim 8, wherein the difference between the sample solution A and the negative control solution B is determined by comparing the difference between the colors of the sample solution A and the negative control solution B with a color difference meter to obtain an accurate difference value, thereby determining the severity of helicobacter pylori infection.
10. The helicobacter pylori detection kit according to claim 8, wherein the difference between the sample solution A and the negative control solution B is determined by comparing the pH value of the pH value with a correct difference to determine the severity of helicobacter pylori infection.
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| CN113341142A (en) * | 2021-06-07 | 2021-09-03 | 北京捷乐生物科技有限公司 | Detection method and kit for helicobacter pylori antibody |
| CN114277086A (en) * | 2021-12-28 | 2022-04-05 | 南京康容健康科技有限公司 | Helicobacter pylori rapid detection reagent |
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