CN106645132B - Test paper for detecting lactose content in sample and preparation method of test paper - Google Patents
Test paper for detecting lactose content in sample and preparation method of test paper Download PDFInfo
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Abstract
A test paper for detecting lactose in a sample and a preparation method thereof are provided, filter paper is selected as a substrate carrier, lactase is added on the filter paper for drying by a drying technology, TMB, horseradish peroxidase and galactose oxidase are sequentially added on another filter paper, and the filter paper is dried step by step; selecting a pvc plate for fixing the dried filter paper, and finally cutting into lactose detection test paper; the test paper can detect whether lactose exists in a sample, and has the advantages of quick reaction, high sensitivity, detection by one-time sample addition, judgment of a result by color change, simplicity and easiness in use.
Description
Technical field:
the invention relates to a test paper, belongs to the technical field of detecting whether lactose is contained in a sample, and particularly relates to a test paper which is particularly suitable for rapidly detecting whether lactose is contained in a fecal sample and a preparation method of the test paper.
The background technology is as follows:
lactose is mainly derived from the milk of mammals, and the lactose content of the milk is different for each mammal, for example, human milk contains 7% lactose, milk contains 4.2% lactose, and goat milk contains 4.6% lactose. Milk is a very nutritious drink, and is an optimal substitute for breast milk for infants. The milk contains protein, carbohydrate, phospholipid, calcium, zinc and other trace elements. The drink milk can supplement nutrients such as protein and microelements such as calcium and zinc; reducing the occurrence rate of rickets, osteoporosis, anemia, cardiovascular diseases and other diseases.
After normal people drink milk products containing lactose, lactose can be decomposed into a molecule of glucose and a molecule of galactose in the small intestine by lactase positioned on brush-shaped edges of villus distal ends of mucous membrane surfaces of the small intestine, the glucose and the galactose are monosaccharides and can be directly absorbed by cells, and the glucose enters a glycolysis path to be continuously decomposed; galactose is converted into galactose-1-phosphate by galactose kinase, then UDP-galactose is formed under the action of galactose-1-phosphate uridyltransferase, UDP-galactose is converted into UDP-glucose by UDP-galactose 4-epimerase under the condition that NAD+ is coenzyme, UPD-glucose is converted into glucose-1-phosphate under the catalysis of UDP-glucose pyrophosphorylase, and then the glucose-1-phosphate enters a glycolytic pathway to be decomposed continuously.
Lactose intolerance refers to the fact that the lactase secretion amount at the villus top of the mucous membrane surface of small intestine, especially jejunum, is reduced or the lactase is not high in activity, and can not completely digest and decompose lactose in milk, and part of lactose is glycolyzed into lactic acid, hydrogen, methane and carbon dioxide by colonic flora. Lactic acid stimulates the intestinal wall, increasing intestinal peristalsis and causing diarrhea. Carbon dioxide produces flatulence and increases intestinal peristalsis in the intestines, causing the child to be dysphoric, and occasionally may induce intestinal cramps to develop intestinal cramps. The increase in lactic acid also lowers the pH of the feces.
It is counted that lactose intolerance exists worldwide, the incidence rate is related to race, asians are 95-100%, blackmen are 60-80%, central European are 2-23% and American white are 6-22%. According to the investigation of Chinese disease prevention and control centers, the incidence rate of lactose intolerance and lactose malabsorption of children aged 3-13 years in Beijing, shanghai, guangzhou and the like in China is about 80 percent. It is currently known in medicine that a reliable method for determining the diagnosis of lactose intolerance symptoms is that jejunal biopsy directly measures lactase activity, but its invasiveness makes it difficult as a routine means. Hydrogen expiration experiments (HBTs) are an accurate non-invasive diagnostic method, but require special instruments, equipment, and time, are not suitable for popularization in basic units, and are also not suitable for large sample size population surveys. The Filin reagent method for measuring reducing sugar is also an accurate in-vitro noninvasive diagnosis, but the reaction process of the Filin reagent method needs to be heated, so that the diagnosis process needs more time and corresponding equipment. Lead acetate is commonly used clinically at present, but lead acetate has certain danger, and the operation process needs to be heated, and the reaction process is relatively complex. The method adopts dry chemical method to measure lactose in excrement, and the whole reaction is carried out at normal temperature, so that rapid qualitative diagnosis can be realized without any equipment.
Currently, a dry chemical method is disclosed in a technical patent 200610056826.5 for detecting lactose intolerance, which judges the basis of lactose intolerance by detecting galactose in urine, and the limitation of the patent is that: 1) The galactose in human urine is detected, a urine purifying device is needed to be added, and the detection steps and the detection cost are increased; 2) The galactose oxidase pad, the peroxidase pad and the color developing agent pad are respectively manufactured and then are overlapped together, and the sample is added and then reacts through the forward chromatographic action of each test paper, so that the display mode is not visual. 3) The above-mentioned technique cannot detect whether lactose is contained in other samples such as feces, and has limitations in detecting samples.
The invention comprises the following steps:
aiming at the defects in the prior art, the invention provides the test paper for directly detecting lactose, which is simple to prepare and convenient to use, and the preparation method of the test paper.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a test strip for detecting lactose content in a sample, comprising: the substrate, lactase adsorption test paper, chromogenic test paper and water absorbing paper; the lactase adsorption test paper, the chromogenic test paper and the absorbent paper are attached to the bottom plate in a mode of tail end superposition for fixation.
Further, the overlapping position of the lactase adsorption test paper and the chromogenic test paper is fixed by an adhesive tape.
Further, the overlapping length of the lactase adsorption test paper and the chromogenic test paper at the tail end of the water absorption paper is 2-3 mm.
Further, the bottom plate is a PVC bottom plate.
A method for preparing test paper for detecting lactose content in a sample comprises the following steps:
step 1: dripping lactase solution onto filter paper until saturation, and drying in an oven to form lactase adsorption test paper;
step 2, cutting the dried lactase adsorption test paper into paper strips with proper widths, and adhering the paper strips to the lactase adsorption test paper positions on the bottom plate;
step 3, additionally taking a piece of filter paper, dripping TMB solution on the filter paper until the filter paper is saturated, and then drying in an oven;
step 4, dripping the solution of horseradish peroxidase and galactose oxidase on the filter paper dried in the step 3 to a saturated state, and drying in a drying oven to form color development test paper;
step 5, cutting the color development test paper dried in the step 4 into paper strips with proper widths, and adhering the paper strips to the position of the color development test paper on the bottom plate, wherein the head part of the color development test paper and the tail part of the lactase adsorption test paper are provided with overlapping sections with proper widths;
step 6, cutting the water absorbing paper into paper strips with proper widths, adhering the paper strips to the water absorbing paper position on the bottom plate, wherein the head part of the water absorbing paper and the tail part of the color development test paper are provided with overlapping sections with proper widths;
and 7, cutting the bottom plate adhered with the lactase adsorption test paper, the chromogenic test paper and the absorbent paper into test paper with proper width by using a cutter, and thus obtaining the test paper for detecting lactose content.
Further, the lactase adsorption test paper and the chromogenic test paper and the absorbent paper are respectively overlapped by 2-3 mm, and the surfaces of the lactase adsorption test paper and the chromogenic test paper are covered by adhesive tapes to ensure firm connection between the lactase adsorption test paper and the chromogenic test paper.
Further, the bottom plate is a PVC bottom plate.
Further, the step 1, the lactase adsorption test paper is prepared by preparing lactase into lactase solution, and dripping the prepared lactase solution onto filter paper until saturation; then putting the saturated filter paper into a baking oven to be baked into lactase test paper; the lactase solution contains phosphate buffer solution, sodium azide, lactase and bovine serum albumin, and the components of the lactase solution are as follows: phosphate buffer, pH7.5, 0.02N containing per liter 1 gram sodium azide, 12000 International units of lactase, 4.5 grams of bovine serum albumin.
Further, the preparation method of the color development test paper comprises the steps of firstly dripping TMB solution onto the filter paper until the filter paper is saturated, and then drying the filter paper in an oven; dripping the solution of horseradish peroxidase and galactose oxidase into the filter paper dried in the step 3 to be in a saturated state, and drying in a drying oven to finally form color development test paper; the chromogenic test paper treatment solution in the step 4 contains phosphate buffer solution, sodium azide, horseradish peroxidase, galactose oxidase and bovine serum albumin, and the components of the chromogenic test paper treatment solution are as follows: phosphate buffer solution of pH7.5 of 0.02N containing 1 g sodium azide, 20000 international unit horseradish peroxidase, 12000 international unit galactose oxidase, and 4.5g bovine serum albumin per liter.
And 3,5 and 6, respectively cutting the lactase test paper, the chromogenic test paper and the water absorbing paper into pieces with proper widths, adhering the pieces on a PVC board, overlapping the lactase test paper and the chromogenic test paper by 2-3 mm, and adhering the overlapped parts by using an adhesive tape to prepare the lactose detection test paper.
By adopting the technical scheme, the test paper provided by the invention can detect whether lactose exists in a sample. The method has the advantages of quick response, high sensitivity, simple and easy use, and can complete detection by one-time sample addition and judge the result by color change due to the use of enzymatic reaction. The preparation process is simple, has lower cost and is easy to popularize, and can be used for rapidly detecting whether the sample contains lactose.
Description of the drawings:
FIG. 1 is an exploded view of an embodiment of the present invention;
FIG. 2 is a schematic diagram of a combination of embodiments of the present invention.
The specific embodiment is as follows:
the invention will be further described with reference to the drawings and examples.
Referring to fig. 1-2, a test paper for detecting lactose content in a sample according to an embodiment of the present invention includes: a bottom plate 1, lactase adsorption test paper 2, chromogenic test paper 3 and absorbent paper 4; the lactase adsorption test paper 1, the chromogenic test paper 2 and the absorbent paper 3 are attached to the bottom plate 1 in a mode of tail end superposition for fixation. The stacking length is generally 2-3 mm, and can be other stacking lengths, so long as the smooth lateral chromatography of the sample can be ensured. The base plate 1 may be a PVC base plate or any other suitable base plate. The overlapping position of the lactase adsorption test paper 2 and the chromogenic test paper 3 is fixed by an adhesive tape 5, so that the sample can be smoothly chromatographed from the lactase adsorption test paper side to the chromogenic test paper.
The method for preparing the test paper for detecting lactose content in the sample comprises the following steps:
and step 1, dripping the lactase solution on the filter paper 1 until the lactase solution is saturated, and then drying the lactase solution in an oven to form the lactase adsorption test paper.
And 2, cutting the dried lactase adsorption test paper into paper strips with proper widths, and adhering the paper strips to the lactase adsorption test paper positions on the PVC bottom plate. As shown in fig. 1.
Step 3, additionally taking a piece of filter paper, dripping TMB (tetramethyl benzidine) solution on the filter paper until the filter paper is saturated, and then drying the filter paper in an oven.
And step 4, dripping the solution of horseradish peroxidase and galactose oxidase into the filter paper dried in the step 3 to be in a saturated state, and drying in a drying oven to finally form the color development test paper.
And 5, cutting the dried color development test paper 3 in the step 4 into paper strips with proper widths, and adhering the paper strips to the color development test paper positions on the PVC bottom plate 6. As shown in fig. 1.
And step 6, cutting the water absorbing paper into paper strips with proper widths, and adhering the paper strips to the water absorbing paper positions on the PVC bottom plate. As shown in fig. 1.
And 7, cutting the PVC bottom plate adhered with the lactase adsorption test paper, the chromogenic test paper and the absorbent paper into test paper with proper width by using a cutter, and thus obtaining the test paper for detecting lactose content. The lactase adsorption test paper and the chromogenic test paper are arranged between the lactase adsorption test paper and the chromogenic test paper; the chromogenic test paper and the absorbent paper are respectively overlapped by 2-3 mm, and the surfaces of the lactase adsorption test paper and the chromogenic test paper are covered by adhesive tapes, so that firm connection between the chromogenic test paper and the absorbent paper is ensured.
The preparation method of the lactase adsorption test paper comprises the steps of preparing lactase into lactase solution, and dripping the prepared lactase solution onto filter paper until saturation; and then the saturated filter paper is put into an oven to be dried into lactase test paper.
The lactase adsorption test paper treatment solution in the step 1 contains phosphate buffer solution, sodium azide, lactase and bovine serum albumin, and the components of the lactase adsorption test paper treatment solution are as follows: 0.02N PB (phosphate buffer) solution pH7.5 containing 1 g sodium azide, 12000 International units of lactase, 4.5g bovine serum albumin per liter.
As described above, the preparation method of the chromogenic test paper comprises the steps of dripping TMB solution onto the filter paper until saturated, and oven drying. And (3) dripping the solution of horseradish peroxidase and galactose oxidase into the filter paper dried in the step (3) to be saturated, and drying in a drying oven to finally form the color development test paper.
The chromogenic test paper treatment solution in the step 4 contains phosphate buffer solution, sodium azide, horseradish peroxidase, galactose oxidase and bovine serum albumin, and the components of the chromogenic test paper treatment solution are as follows: PB solution at pH7.5 of 0.02N contains 1 g sodium azide, 20000 international units of horseradish peroxidase, 12000 international units of galactose oxidase, and 4.5g of bovine serum albumin per liter.
And 3,5 and 6, respectively cutting the lactase test paper, the chromogenic test paper and the water absorbing paper into proper widths, pasting the proper widths on a PVC board, overlapping the lactase test paper and the chromogenic test paper by 2-3 mm, and pasting the overlapped parts by using an adhesive tape to prepare the lactose detection test paper.
As described above, the lactose test paper after being prepared can be used for completing the whole process of decomposing lactose into galactose and glucose and then decomposing galactose into hydrogen peroxide by one sample addition, and the pH value of the chromogenic test paper is changed by the hydrogen peroxide, so that the chromogenic test paper changes from light yellow to blue.
Further, the present invention provides a specific example to further illustrate the present invention, system Cheng Shizhi:
1. the lactase solution is dripped on filter paper until the lactase solution is saturated, and then dried in an oven at 45 ℃ for 1h, so as to form lactase adsorption test paper. The temperature of the oven and the drying time can be adjusted according to the quantity of the products until the products are dried.
2. Cutting the dried lactase adsorption test paper into strips with the width of 1.6cm, and adhering the strips to the positions of the lactase adsorption test paper on a PVC bottom plate.
3. A piece of filter paper was additionally taken, and 1mg/ml TMB solution was added dropwise to the filter paper until saturated, and then dried in an oven at 45℃for 1h. The temperature of the oven and the drying time can be adjusted according to the quantity of the products until the products are dried.
4. And (3) dripping the solution of horseradish peroxidase and galactose oxidase into the filter paper dried in the step (3) to be saturated, and drying the filter paper in an oven at 45 ℃ for 1h. The temperature of the oven and the drying time can be adjusted according to the quantity of the products until the products are dried.
5. Cutting the color development test paper dried in the step 4 into strips with the width of 2.5cm, and sticking the strips on the PVC bottom plate.
6. Cutting the water absorbing paper into strips with the width of 2cm, and adhering the strips to the water absorbing paper on the PVC bottom plate.
7. And cutting the PVC bottom plate stuck with the lactase adsorption test paper, the color development test paper and the absorbent paper into test strips with the width of 4.3mm by using a cutter.
The test paper is used when:
1. sample treatment and sample addition: the feces are diluted by pure water without lactose and galactose according to a proper proportion to prepare a sample solution, and 100 microliters of the prepared sample solution is dripped on lactase adsorption test paper of a lactose detection test strip.
2. Lactose decomposition: if lactose is present in the sample, it breaks down into galactose and glucose under the action of lactase. This process was completed on lactose adsorption paper 1.
3. Color development: the decomposed galactose and sample liquid are attracted to the color development paper 3 by the dried filter paper. Under the action of galactose of the color development test paper, galactose is decomposed into hydrogen peroxide, and the color of the color development test paper is changed from light yellow to blue. This process is completed on a chromogenic test paper.
Compared with the prior art, the invention has the following differences: 1. the test paper is judged by detecting whether the excrement contains lactose or not, and is different from the judgment in the prior art by detecting galactose in urine. 2. The test paper of the invention detects lactose in human fecal samples with different detected substances; whereas the disclosed technique detects galactose in human urine; 3. the test paper of the invention detects that the sample is feces, and the disclosed technology detects that the sample is urine. 4. The sample pretreatment is different, and the test paper only needs to dilute the fecal sample (because the fecal is solid); while the disclosed technology requires the addition of urine purification devices; 5. the test paper has different structures, the test paper consists of three parts of test paper, namely lactase adsorption test paper, chromogenic test paper and absorbent paper, the lactose is decomposed by the lactase adsorption test paper and then is moved to the chromogenic test paper through the lateral chromatography, and the redundant liquid is absorbed by the absorbent paper after the chromogenic reaction; the disclosed technology is that galactose oxidase pad, peroxidase pad and color developing agent pad are respectively manufactured and then are overlapped together, and after sample is added, the reaction is carried out through the forward chromatography of each test paper. 6. The test paper has different manufacturing processes, and the galactose oxidase, the peroxidase and the color developing agent are dried and adsorbed on the same test paper, and the lactase is adsorbed on another filter paper; the technology disclosed is to adsorb galactose oxidase, peroxidase and color-developing agent on 3 pieces of test paper, respectively. 7. The present technology contains lactase to break down lactose and the disclosed technology does not contain lactase. 8. Different color developing agents, the color developing agent of the test paper is TMB, and the disclosed color developing agents are 4-aminoantipyrine and 3, 5-dichloro dihydroxybenzene sulfonic acid. 9. The test paper of the invention has different drying processes.
In conclusion, the method has the characteristics of simple preparation, convenient use, direct lactose detection and the like, and has unique advantages.
While the foregoing description illustrates and describes the preferred embodiments of the present invention, as noted above, it is to be understood that the invention is not limited to the forms disclosed herein but is not to be construed as excluding other embodiments, and that various other combinations, modifications and environments are possible and may be made within the scope of the inventive concepts described herein, either by way of the foregoing teachings or by those of skill or knowledge of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (4)
1. A method for preparing test paper for detecting lactose content in a sample is characterized by comprising the following steps: the test paper comprises: the substrate, lactase adsorption test paper, chromogenic test paper and water absorbing paper; the lactase adsorption test paper, the chromogenic test paper and the absorbent paper are attached to the bottom plate in a mode of overlapping the tail ends and fixed, and the overlapping position of the lactase adsorption test paper and the chromogenic test paper is fixed through an adhesive tape; the manufacturing method comprises the following steps:
step 1: preparing lactase into lactase solution, and dripping the prepared lactase solution onto filter paper until saturation; then putting the saturated filter paper into a baking oven to be baked into lactase test paper; the lactase solution contains phosphate buffer solution, sodium azide, lactase and bovine serum albumin, and the components of the lactase solution are as follows: phosphate buffer of 0.02N pH7.5 containing 1 g sodium azide, 12000 international units of lactase, 4.5g bovine serum albumin per liter;
step 2, cutting the dried lactase adsorption test paper into paper strips with proper widths, and adhering the paper strips to the lactase adsorption test paper positions on the bottom plate;
step 3, additionally taking a piece of filter paper, firstly dripping TMB solution on the filter paper until the filter paper is saturated, and then drying the filter paper in an oven;
step 4, dripping the solution of horseradish peroxidase and galactose oxidase into the filter paper dried in the step 3 to be in a saturated state, and drying in a drying oven to finally form color development test paper; the chromogenic test paper treatment solution contains phosphate buffer solution, sodium azide, horseradish peroxidase, galactose oxidase and bovine serum albumin, and the components of the chromogenic test paper treatment solution are as follows: phosphate buffer solution of 0.02N pH7.5 containing 1 g sodium azide, 20000 international unit horseradish peroxidase, 12000 international unit galactose oxidase, 4.5g bovine serum albumin per liter;
step 5, cutting the color development test paper dried in the step 4 into paper strips with proper widths, and adhering the paper strips to the position of the color development test paper on the bottom plate, wherein the head part of the color development test paper and the tail part of the lactase adsorption test paper are provided with overlapping sections with proper widths;
step 6, cutting the water absorbing paper into paper strips with proper widths, adhering the paper strips to the water absorbing paper position on the bottom plate, wherein the head part of the water absorbing paper and the tail part of the color development test paper are provided with overlapping sections with proper widths;
and 7, cutting the bottom plate adhered with the lactase adsorption test paper, the chromogenic test paper and the absorbent paper into test paper with proper width by using a cutter, and thus obtaining the test paper for detecting lactose content.
2. A method of preparing a test strip for detecting lactose content in a sample as claimed in claim 1, wherein: the lactase adsorption test paper and the chromogenic test paper are respectively overlapped by 2-3 mm, and the surfaces of the lactase adsorption test paper and the chromogenic test paper are covered by adhesive tapes to ensure firm connection between the lactase adsorption test paper and the chromogenic test paper.
3. A method of preparing a test strip for detecting lactose content in a sample as claimed in claim 1, wherein: the bottom plate is a PVC bottom plate.
4. A method of preparing a test strip for detecting lactose content in a sample as claimed in claim 1, wherein: and 3,5 and 6, respectively cutting the lactase test paper, the chromogenic test paper and the water absorbing paper into pieces with proper widths, adhering the pieces on a PVC board, overlapping the lactase test paper and the chromogenic test paper by 2-3 mm, and adhering the overlapped parts by using an adhesive tape to prepare the lactose detection test paper.
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| CN108070635B (en) * | 2017-10-20 | 2021-02-23 | 重庆医科大学附属儿童医院 | Fecal lactose detection kit and application and detection method thereof |
| CN107941799A (en) * | 2017-12-20 | 2018-04-20 | 珠海科域生物工程股份有限公司 | A kind of fecal sample Test paper and preparation method thereof |
| CN108918865A (en) * | 2018-05-21 | 2018-11-30 | 德康润生物科技（北京）有限公司 | Fluorescence immune chromatography test paper bar and reagent card |
| CN109557088A (en) * | 2019-01-11 | 2019-04-02 | 四川沃文特生物技术有限公司 | A kind of reagent card for detecting excrement lactose content and the excrement lactose detection method based on this |
| CN109706069A (en) * | 2019-03-13 | 2019-05-03 | 湖南省天骑医学新技术股份有限公司 | A kind of culture dish and production method applied to drug sensitive test |
| CN112980922B (en) * | 2021-02-26 | 2023-06-16 | 康帕思(重庆)生物技术有限公司 | Detection method and kit for fecal lactose |
| CN113267618B (en) * | 2021-07-20 | 2021-09-28 | 北京华益精点生物技术有限公司 | Biological sensor |
| CN116640655B (en) * | 2023-05-26 | 2024-03-29 | 北京中生金域诊断技术股份有限公司 | Portable urine galactose detection kit stored at normal temperature, reaction liquid and preparation method thereof |
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| WO2000066765A1 (en) * | 1999-04-30 | 2000-11-09 | Biohit Oyj | Method for the determination of disaccharidases and kit therefor |
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