CN112980922B - Detection method and kit for fecal lactose - Google Patents
Detection method and kit for fecal lactose Download PDFInfo
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- CN112980922B CN112980922B CN202110220366.XA CN202110220366A CN112980922B CN 112980922 B CN112980922 B CN 112980922B CN 202110220366 A CN202110220366 A CN 202110220366A CN 112980922 B CN112980922 B CN 112980922B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a detection method for fecal lactose, which comprises the following steps: preparing a sample treatment solution, a first reagent composition and a second reagent composition, wherein the first reagent composition comprises glucose oxidase, peroxidase and an indicator, the second reagent composition comprises lactase besides each component of the first reagent composition, then pretreating a sample to obtain a sample clear solution, taking two equal sample clear solutions, respectively mixing the sample clear solution with the first reagent composition and the second reagent composition for reaction, observing and comparing the color shades of two groups of reaction systems, and judging whether lactose is contained in the sample. The invention also discloses a kit based on the method. The invention has the beneficial effects that: the glucose detection result generated by lactose decomposition is creatively corrected by measuring glucose originally existing in a sample, interference is eliminated, the accuracy is high, the detection method is simple and easy to operate, and the requirements on experimental conditions and expertise of testers are low.
Description
Technical Field
The invention belongs to the field of in-vitro detection of biological samples, and particularly relates to a detection method and a kit for fecal lactose.
Background
Lactose is a disaccharide, mainly derived from mammalian milk. After normal people ingest milk products containing lactose, lactose is decomposed into a part of glucose and a part of galactose by lactase in the small intestine, and can be directly absorbed by intestinal cells and utilized and decomposed by the body. Lactose intolerance (lactose intolerance, LI) refers to the phenomenon of diarrhea-predominant symptoms of the associated digestive tract caused by the relative or absolute deficiency of lactase. The main reason is that the small intestine has small lactase secretion or insufficient activity, so that ingested lactose cannot be completely digested and decomposed, and part of lactose is fermented by intestinal flora to generate lactic acid, methane, carbon dioxide and the like, and the fermented products stimulate the intestinal tract to cause dysfunction. Due to genetic polymorphism, the incidence rates of different families of people are different, chinese people are high incidence people of LI, and the incidence rate of LI in Chinese infants is extremely high and can reach 46.9% -70.0%. Because infants use dairy products as main food, if LI of infants is not important, long-term harm such as chronic diarrhea, malnutrition, anemia and osteoporosis can be caused.
There are several methods of diagnosing LI, including jejunal biopsy to determine lactase activity and histopathological observation, expiration tests, lactose tolerance tests and stool reducing sugar assays. Jejunal biopsy, although accurate, is an invasive method of detection and requires high skill on the person being examined. The expiration test and lactose tolerance test have long detection period and high requirement on compliance of patients. The stool reducing sugar assay is susceptible to other sugars and the detection reagent contains heavy metals. Patent document CN109557088A, CN106645132a discloses a test paper/reagent card for detecting lactose content in feces, respectively, which judges lactose content in a feces sample by detecting galactose generated by lactose decomposition, has a specific effect, however, the detection thereof requires heating to accelerate the reaction, and the raw material reagent, galactose, has a high price. Patent document CN 108070635A discloses a fecal lactose detection kit which semi-quantitatively determines the lactose content in feces by detecting glucose after lactose decomposition, however, ignores the influence of glucose originally present in feces and is prone to false positive results.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for detecting fecal lactose. The technical scheme is as follows:
the detection method for fecal lactose is characterized by comprising the following steps:
step one, preparing a reagent: a sample processing fluid, a first reagent composition, and a second reagent composition;
the first reagent composition comprises glucose oxidase, peroxidase, and an indicator;
the second reagent composition comprises lactase, glucose oxidase, peroxidase, and an indicator;
step two, sample pretreatment: adding a fresh fecal sample into the sample treatment liquid, dispersing and mixing uniformly, and sampling the clear liquid after solid-liquid separation;
step three, color reaction: taking two equal sample clear liquid parts, and respectively mixing and reacting with the first reagent composition and the second reagent composition to form a first reaction system and a second reaction system;
step four, color comparison: observing and comparing the color shades of the first reaction system and the second reaction system, and judging as follows:
if the first reaction system and the second reaction system are both developed and the colors of the first reaction system and the second reaction system are relatively dark and light and cannot be distinguished, the sample is considered to contain no lactose;
if only the second reaction system develops color or both the first reaction system and the second reaction system develop color, and the color of the first reaction system is lighter than that of the second reaction system, the sample is considered to contain lactose;
if neither the first reaction system nor the second reaction system develops color, the sample is considered to contain no glucose or lactose;
and when other color phenomena appear, repeating the second to fourth steps for rechecking, or diluting the sample clear liquid for rechecking.
As a preferable technical scheme, the indicator is 4-aminoantipyrine or one of the following substances:
4-chlorophenol, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 3, 5-dichloro-2-hydroxybenzenesulfonic acid sodium salt, N, N-dialkylaniline, N, N-dialkyl-m-toluidine.
As a preferred embodiment, the first reagent composition and the second reagent composition further comprise a pH buffer.
As a preferred embodiment, the first reagent composition and the second reagent composition further contain a surfactant, a dispersant, and a preservative.
The second object of the invention is to provide a kit based on the method. The technical proposal is as follows:
the kit is characterized by comprising any one of the sample treatment fluid, a first reagent composition and a second reagent composition.
As a preferred embodiment, the first reagent composition is supported on a porous carrier to form a glucose reaction pad;
the second reagent composition is supported on another porous carrier to form a lactose-glucose reaction pad.
As a preferable technical scheme, the glucose reaction pad and the lactose-glucose reaction pad have the same structure, and comprise the porous carrier and a sample filter pad attached to the porous carrier.
As a preferable technical scheme, the glucose reaction pad and the lactose-glucose reaction pad are arranged on the same detection card at intervals in parallel, wherein the porous carrier is arranged on the detection card, the sample filter pad is stuck to one surface of the porous carrier, which is opposite to the detection card, the sample filter pad partially shields the porous carrier, and the exposed part of the porous carrier forms a color development observation area.
As a preferred embodiment, the first reagent composition comprises glucose oxidase, peroxidase, an indicator, potassium dihydrogen phosphate, tween-20, PEG8000 and proclin300;
the second reagent composition contains lactase in addition to all components of the first reagent composition.
Preferably, the sample processing liquid is a solution containing potassium dihydrogen phosphate, sodium chloride and proclin300.
Drawings
FIG. 1 is a schematic diagram of the reaction of the detection method of the present invention;
FIG. 2 is a schematic diagram of a test strip;
fig. 3 is a physical picture of the test strip, wherein: (a) a test strip finished product; (b) both reaction pad color reaction results are negative; (c) The glucose reaction pad detection result is negative, and the lactose-glucose reaction pad detection result is positive.
Detailed Description
The invention is further described below with reference to examples and figures.
The detection method for fecal lactose is characterized by comprising the following steps:
step one, preparing a reagent: a sample processing fluid, a first reagent composition, and a second reagent composition;
the first reagent composition comprises glucose oxidase, peroxidase, and an indicator;
the second reagent composition comprises lactase, glucose oxidase, peroxidase, and an indicator;
to improve the stability of the reaction system and ensure the enzyme activity, the first reagent composition and the second reagent composition further comprise a pH buffer;
in addition, the first reagent composition and the second reagent composition further contain a surfactant, a dispersant and a preservative in order to enhance the reaction effect.
Wherein the indicator comprises 4-aminoantipyrine and further comprises one of the following:
4-chlorophenol, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 3, 5-dichloro-2-hydroxybenzenesulfonic acid sodium salt, N, N-dialkylaniline, N, N-dialkyl-m-toluidine.
Step two, sample pretreatment: and (3) adding a fresh fecal sample into the sample treatment liquid, dispersing and mixing uniformly, and sampling the clear liquid after solid-liquid separation. The separation may be carried out by conventional separation methods such as filtration, centrifugation, etc.
Step three, color reaction: taking two equal sample clear liquid portions, respectively mixing the two equal sample clear liquid portions with the first reagent composition and the second reagent composition, and reacting for 2-10 min at room temperature to form a first reaction system and a second reaction system.
Step four, color comparison: observing and comparing the color shades of the first reaction system and the second reaction system, and judging as follows:
if the first reaction system and the second reaction system are both developed and the colors of the first reaction system and the second reaction system are relatively dark and light and cannot be distinguished, the sample is considered to contain no lactose;
if only the second reaction system develops color or both the first reaction system and the second reaction system develop color, and the color of the first reaction system is lighter than that of the second reaction system, the sample is considered to contain lactose;
if neither the first reaction system nor the second reaction system develops color, the sample is considered to contain no glucose or lactose;
and when other color phenomena appear, repeating the second to fourth steps for rechecking, or diluting the sample clear liquid for rechecking. Specifically, if the color of the first reaction system is darker than that of the second reaction system, the detection is invalid and re-detection is needed; if the color of the first reaction system is darker than that of the second reaction system and cannot be distinguished, the sample clear liquid is diluted 5 times and then re-detected.
The method is based on the following principle: as shown in figure 1, glucose oxidase specifically oxidizes glucose in a sample to generate products, namely glucuronic acid and hydrogen peroxide, wherein the reaction formula (2) shows that the hydrogen peroxide oxidizes an indicator to change color under the action of peroxidase, and the indicator turns purple, and the reaction formula (3) and the reaction formula (4) show that the indicator turns purple; lactase specifically breaks down lactose in a sample to generate a molecule of glucose and a molecule of galactose, the former is shown in a reaction formula (1), glucuronic acid and hydrogen peroxide are generated under the action of glucose oxidase, and the hydrogen peroxide oxidizes an indicator to generate color change under the action of peroxidase. The above determination is made based on the result of the color reaction. The relationship between the color change and whether lactose was contained in the stool sample is summarized in Table 1.
The method is used for detecting lactose in the fecal sample, and presents a qualitative or semi-quantitative detection result. Whether the fecal sample contains lactose can be judged according to whether the color reaction is positive; according to the color depth of the color reaction, the lactose content range in the sample can be semi-quantitatively judged.
Table 1 whether the samples contain glucose and lactose in relation to color change
# Note that: does not contain glucose or lactose, or has the content below the detection limit.
Example two
A kit is based on the method principle of the first embodiment and is used for detecting lactose in excrement. Including the sample processing fluid, the first reagent composition, and the second reagent composition described above.
The first reagent composition includes glucose oxidase, peroxidase, an indicator, potassium dihydrogen phosphate, tween-20, PEG8000, and proclin300. The second reagent composition contains lactase in addition to the first reagent composition.
To facilitate storage and transportation of the kit, the first reagent composition is supported on the porous carrier 2 to form a glucose reaction pad; the second reagent composition is supported on another porous carrier 2 to form a lactose-glucose reaction pad.
For further convenient use, the glucose reaction pad and the lactose-glucose reaction pad have the same structure, and comprise the porous carrier 2 and a sample filter pad 3 attached to the porous carrier 2. The porous support 2 and the sample filter pad 3 may each be filter paper.
For the sake of observation, a glucose reaction pad and a lactose-glucose reaction pad were provided on the test card 1 to form a test strip. The test strip has the structure that a glucose reaction pad and a lactose-glucose reaction pad are arranged on the same detection card 1 at parallel intervals, wherein the porous carrier 2 is arranged on the detection card 1, one surface of the porous carrier 2, which faces away from the detection card 1, is stuck with the sample filter pad 3, the sample filter pad 3 partially shields the porous carrier 2, and the exposed part of the porous carrier 2 forms a color development observation area 2a. A dropper can be arranged in the kit and used for sampling. The sample processing liquid is contained in the sample processing tube.
When in use, the detection card 1 is horizontally placed, and one surface provided with the reaction pad faces upwards. Taking a fresh fecal sample, avoiding pollution by other substances, adding a sample treatment tube, fully and uniformly mixing to form a dispersion liquid, sucking the dispersion liquid, discarding head drops, respectively dripping one drop of the dispersion liquid on a glucose reaction pad and a sample filter pad 3 of a lactose-glucose reaction pad, penetrating the dispersion liquid onto a porous carrier 2 through the sample filter pad 3, standing for 2-5 min at room temperature, and observing the color change of a color development observation area 2a.
In another test strip structure, as shown in fig. 2, a porous carrier 2 carrying a first reagent composition and a porous carrier 2 carrying a second reagent composition are arranged on the same surface of a detection card 1 at intervals, a same sample filter pad 3 is arranged above the two porous carriers, and a pair of opposite sides of the sample filter pad 3 respectively fall on the two porous carriers 2 and have the same area as the overlapped part of the two porous carriers 2. During sample addition, 1 to 2 drops of dispersion liquid are added to the middle part of the sample filter pad 3.
TABLE 2 sample processing fluid composition
The reaction pad is prepared by adopting an impregnation method, and the brief process is as follows: the first reagent composition and the second reagent composition are respectively dissolved in water to form a first solution and a second solution, then filter paper is respectively immersed in the first solution and the second solution to adsorb solute, and finally the filter paper is dried to form the reaction pad.
One specific solution composition used to prepare the reaction pad is shown in tables 3 and 4. Wherein, the N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt can also be replaced by 4-chlorophenol, or 3, 5-dichloro-2-hydroxybenzenesulfonic acid sodium salt, or N, N-dialkylaniline, or N, N-dialkyl-m-toluidine. Tween-20 as a surfactant, PEG8000 as a dispersant and proclin300 as a preservative.
TABLE 3 composition of first solution used to prepare reaction pad
TABLE 4 composition of the second solution used to prepare the reaction pad
Sample testing was performed using the reaction pads prepared as in the formulations in tables 3 and 4.
(1) Standard solution testing
And dissolving a certain amount of lactose in a sample treatment solution to prepare a 1g/L lactose solution, and respectively dripping the sample treatment solution serving as a blank solution onto two detection cards for testing. The detection results are shown in FIG. 3. The blank reaction pad (3 a) is white. After the glucose solution was added dropwise, both the glucose reaction pad and the lactose-glucose reaction pad were wetted and appeared light purple (3 b). After the mixed solution of glucose and lactose was added dropwise, both the glucose reaction pad and the lactose-glucose reaction pad were wetted, the glucose reaction pad appeared light purple, and the lactose-glucose reaction pad appeared dark purple, i.e., the color reaction was positive (3 c).
Preparing a series of lactose solution with gradient concentration, dripping on lactose-glucose reaction pad, observing color change of filter paper, according to color development depth, positive color development results were determined as "-", "+", "+," +++ "," +++ ", as shown in Table 5, standard colorimetric cards can be manufactured accordingly as a basis for detection judgment.
TABLE 5 lactose gradient solution concentration and indicator color intensity control relationship
When galactose solution, fructose solution, maltose solution and sucrose solution are used as reference substances, the detection results are negative, namely the reference substances and the reaction pad do not react, which indicates that the detection of the test strip has specificity, and the common galactose, fructose, maltose, sucrose and the like in food do not influence the detection results.
Different amounts of ascorbic acid or bilirubin were added to lactose solutions and tested. Experiments show that when the concentration of the ascorbic acid is lower than 0.25mg/dL, the detection result is not affected; when the bilirubin concentration is less than 6mg/dL, the detection result is not affected.
(2) Clinical sample detection
86 cases of stool samples of patients aged 0-2 are collected in a clinical laboratory in a period from 21 in 2020 to 31 in 2020, and the kit of the second example is used for detection, and a Ban Shi reagent method commonly used in clinic is used as a reference. In the case where the detection result of the kit is inconsistent with the detection result of the Ban Shi reagent method, the sample is further detected and confirmed by high performance liquid chromatography to determine whether glucose and lactose are contained. The statistics of the detection results are shown in Table 6. It can be seen that the kit of the invention has high consistency with the Ban Shi reagent test results for most samples; however, when the detection results of the two methods are inconsistent, the high performance liquid detection results are consistent with the detection results of the kit provided by the invention, so that the reliability of the detection results of the kit is good.
TABLE 6 results of lactose detection on fecal samples
Compared with the prior art, the invention has the beneficial effects that: (1) The detection method is simple and easy to operate, the sample does not need complex pretreatment, the dry chemical method is adopted, the enzymatic reaction and the chromogenic reaction can be completed at room temperature, the requirements on experimental conditions and experimenters are low, and the method is very suitable for primary hospitals; (2) The glucose detection result generated by lactose decomposition is creatively corrected by measuring the glucose originally existing in the sample, so that interference is eliminated, and the accuracy is high; (3) The kit is easy to use, the test strip has ingenious structure, and the sample almost does not need pretreatment; (4) The color reaction product has large color difference with the sample, and is convenient for observation.
The detection of whether the excrement contains lactose and the content thereof can provide a basis for disease diagnosis. For example diagnosis of lactose intolerance.
Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (3)
1. The detection method for fecal lactose is characterized by comprising the following steps:
step one, preparing a reagent: a sample processing fluid, a first reagent composition, and a second reagent composition;
the first reagent composition comprises glucose oxidase, peroxidase, an indicator, potassium dihydrogen phosphate, tween-20, PEG8000, and proclin300;
the second reagent composition contains lactase in addition to all components of the first reagent composition;
the sample treatment liquid is a solution containing potassium dihydrogen phosphate, sodium chloride and proclin300;
the indicator is 4-aminoantipyrine or one of the following substances: 4-chlorophenol, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 3, 5-dichloro-2-hydroxybenzenesulfonic acid sodium salt, N-dialkylaniline, N-dialkyl-m-toluidine;
step two, sample pretreatment: adding a fresh fecal sample into the sample treatment liquid, dispersing and mixing uniformly, and sampling the clear liquid after solid-liquid separation;
step three, color reaction: taking two equal sample clear liquid parts, and respectively mixing and reacting with the first reagent composition and the second reagent composition to form a first reaction system and a second reaction system;
step four, color comparison: observing and comparing the color shades of the first reaction system and the second reaction system, and judging as follows:
if the first reaction system and the second reaction system are both developed and the colors of the first reaction system and the second reaction system are relatively dark and light and cannot be distinguished, the sample is considered to contain no lactose;
if only the second reaction system develops color or both the first reaction system and the second reaction system develop color, and the color of the first reaction system is lighter than that of the second reaction system, the sample is considered to contain lactose;
if neither the first reaction system nor the second reaction system develops color, the sample is considered to contain no glucose or lactose;
and when other color phenomena appear, repeating the second to fourth steps for rechecking, or diluting the sample clear liquid for rechecking.
2. A kit, characterized in that: a sample processing fluid comprising the method of claim 1, a first reagent composition, and a second reagent composition.
3. The kit of claim 2, wherein: the first reagent composition is supported on a porous carrier (2) to form a glucose reaction pad;
the second reagent composition is supported on another porous carrier (2) to form a lactose-glucose reaction pad;
the glucose reaction pad and the lactose-glucose reaction pad have the same structure and comprise the porous carrier (2) and a sample filter pad (3) attached to the porous carrier (2);
the glucose reaction pad and the lactose-glucose reaction pad are arranged on the same detection card (1) at parallel intervals, the porous carrier (2) is arranged on the detection card (1), the sample filter pad (3) is stuck to one surface of the porous carrier (2) opposite to the detection card (1), the sample filter pad (3) partially shields the porous carrier (2), and the exposed part of the porous carrier (2) forms a color development observation area (2 a).
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