WO2014082439A1 - Method for improving detection sensitivity of colloidal gold immunochromatographic test strip - Google Patents
Method for improving detection sensitivity of colloidal gold immunochromatographic test strip Download PDFInfo
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- WO2014082439A1 WO2014082439A1 PCT/CN2013/077201 CN2013077201W WO2014082439A1 WO 2014082439 A1 WO2014082439 A1 WO 2014082439A1 CN 2013077201 W CN2013077201 W CN 2013077201W WO 2014082439 A1 WO2014082439 A1 WO 2014082439A1
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- test strip
- colloidal gold
- gold immunochromatographic
- immunochromatographic test
- detection
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
Definitions
- the invention belongs to the field of colloidal gold detection, and particularly relates to a method for improving the sensitivity of detection of colloidal gold immunochromatographic test strips.
- the colloidal gold immunochromatographic test strip reader can improve the detection sensitivity, the detection sensitivity is limited, and the combined optical detection technique is easily interfered by the background of the test strip itself.
- the researchers optimized from the aspects of signal enhancement and data processing.
- the sensitizing reagent is mainly used to sensitize the test strip to improve the sensitivity of the test, but chloroauric acid and ascorbic acid are commonly used as sensitizing reagents, and the cost is high, which is not suitable for wide application. Therefore, based on these studies, it is important to study a treatment method that can reduce the background value of the test strip itself, which is of great significance for further improving the sensitivity of the colloidal gold immunochromatographic test strip.
- the technical problem to be solved by the present invention is to provide a method for improving the detection sensitivity of colloidal gold immunochromatographic test strips in view of the deficiencies of the above prior art.
- the utility model can improve the detection sensitivity of the colloidal gold immunochromatographic test strip, increase the linear range of detection, and is simple and convenient to operate.
- a method for improving the sensitivity of detection of colloidal gold immunochromatographic test strips the specific steps are as follows:
- test strip processing the colloidal gold immunochromatographic test strip is tested for the sample to be tested, and after the test is completed, the transparent reagent prepared by the step (1) is added dropwise in the detection area of the colloidal gold immunochromatographic test strip. After the test strip of the test strip is placed from white to transparent, the reader can take readings.
- the amount of the clearing agent used in the step (2) is 50 to 100 ⁇ L.
- the placement time of the step (2) is 3 to 5 minutes.
- the colloidal gold immunochromatographic test strip is a mycotoxin colloidal gold immunochromatographic test strip or a virus colloidal gold immunochromatographic test strip.
- the invention pre-treats the colloidal gold immunochromatographic test strip before the detection, so that the nitrocellulose membrane in the detection zone on the colloidal gold immunochromatographic test strip can be transformed from a white non-transparent fragile structure into a transparent and A certain tough film structure (see Figure 1), which reduces the background interference caused by light reflection; at the same time, it can prevent the coloration area from gradually changing due to the self-oxidation of colloidal gold, and the colloidal gold immunochromatographic test strip
- the color of the color area is fixed to avoid the adverse effect of the detection of the test strip by the oxidation of the colloidal gold.
- the reading of the reading instrument can be achieved to reduce the influence of the background value and improve the colloidal gold immunochromatography.
- the purpose of the detection sensitivity of the test strip is provided.
- the processing time is short, and it can be completed in 3 ⁇ 5 minutes after the completion of the detection.
- Example 1 is a longitudinal cross-sectional view of a total amount of aflatoxin colloidal gold immunochromatographic test strip before and after treatment according to Example 1 of the present invention, wherein the T detection line and the C quality control line are shown.
- test group
- Test strip processing preparation of a series of concentrations of aflatoxin (AFT) standard solution (0 ng / mL, 0.01 ng / mL, 0.03 ng/mL, 0.06 ng/mL, 0.125 ng/mL, 0.25 ng/mL, 0.5 Ng/mL, 1 ng/mL), 100 ⁇ L of each of the above concentrations of the standard solution was added dropwise to the sample pad of the colloidal gold test strip of the total amount of aflatoxin, and the test was carried out. After 10 minutes of detection, the gel was immunized in colloidal gold. 2 drops (about 100 ⁇ L) of clearing reagent were added to the test area of the chromatographic test strip.
- AFT aflatoxin
- the above-mentioned colloidal gold test strip of the total amount of aflatoxin is prepared according to the method in the patent document "High-sensitivity immunochromatographic test strip for rapid detection of aflatoxin total amount and preparation method thereof" (Patent No. 201010245801.6).
- Control group The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
- Control group The effective detection range is 0.03ng/mL ⁇ 0.5ng/mL, that is, when the total concentration of aflatoxin in the system to be tested reaches 0.03ng/mL, the reader reaches its highest test threshold, higher than 0.5ng/mL. When the reader reaches its minimum test threshold.
- the effective detection range is 0.01 ng/mL ⁇ 1 ng/mL, that is, when the total concentration of aflatoxin in the system to be tested reaches 0.01 ng/mL, the reader reaches its highest test threshold, higher than 1 ng/mL. The reader reaches its minimum test threshold.
- the detection sensitivity of the aflatoxin total colloidal gold immunochromatographic test strip is improved by treating the aflatoxin total colloidal gold immunochromatographic test strip by the method of the invention. It detects the linear range.
- test group
- Benzyl alcohol and methanol are selected as the formulation components, and the mixture is uniformly mixed with 100 ⁇ L of benzyl alcohol according to 1 mL of methanol, and sealed and stored in the dark;
- Test strip processing prepare a series of standard aflatoxin M1 (AFM1) standard solution (0 ng / mL, 0.05 ng / mL, 0.1 ng/mL, 0.25 ng/mL, 0.50 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL , 4ng/mL), respectively, 100 ⁇ L of each of the above concentrations of the standard solution was added dropwise to the sample pad of the aflatoxin M1 (AFM1) colloidal gold test strip, after 10 minutes of testing, in the test area of the test strip 2 drops (about 100 ⁇ L) of clearing reagent, place the test strip of the test strip to change from white to transparent, immediately read the reading instrument and record the test value.
- AFM1 aflatoxin M1
- Control group The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
- Control group The effective detection range is 0.1 ng/mL ⁇ 3 ng/mL, that is, when the aflatoxin M1 concentration in the system to be tested is lower than 0.03 ng/mL, the reader reaches its highest test threshold, which is higher than the 3 ng/mL test value. When the reader reaches its minimum test threshold.
- the effective detection range is 0.05 ng/mL ⁇ 4 ng/mL, that is, when the aflatoxin M1 concentration in the system to be tested reaches 0.05 ng/mL, the reader reaches its highest test threshold, which is higher than the 4 ng/mL test value. The reader has reached its minimum test threshold.
- the aflatoxin M1 colloidal gold immunochromatographic test strip is treated by the method of the invention, and the detection sensitivity of the aflatoxin M1 colloidal gold immunochromatographic test strip is improved, and the sensitivity thereof is increased. Detect linear range.
- test group
- Benzyl alcohol and methanol are selected as the formulation components, and the mixture is uniformly mixed with 105 ⁇ L of benzyl alcohol according to 1 mL of methanol, and sealed and stored in the dark;
- Test strip processing and testing preparation of a series of concentrations of zearalenone standard solution (0 ng / mL, 0.05 ng / mL, 0.1 Ng/mL, 0.25 ng/mL, 0.50 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL 4ng/mL), 100 ⁇ L of each of the above concentrations of the standard solution was added dropwise to the sample pad of the zearalenone colloidal gold test strip. After 10 minutes of testing, the clearing reagent was added to the test strip detection area. After 4 minutes, the test strip detection area changes from white to transparent, and the reading of the reader is immediately performed, and the test value is recorded.
- Control group The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
- Control group The effective detection range is 0.1 ng/mL ⁇ 2 ng/mL, that is, when the concentration of zearalenone in the system reaches 0.1 ng/mL, the reader reaches its highest test threshold, and when it is higher than 2 ng/mL, the reading is performed. The instrument reaches its minimum test threshold.
- the effective detection range is 0.05 ng/mL ⁇ 3 ng/mL, that is, when the concentration of zearalenone in the system reaches 0.05 ng/mL, the reader reaches its highest test threshold, and when it is higher than 3 ng/mL, the reading strip The instrument reaches its minimum test threshold.
- the detection sensitivity of the zearalenone colloidal gold immunochromatographic test strip is improved by using the method of the invention to treat the zearalenone colloidal gold immunochromatographic test strip. It detects the linear range.
- test group
- Test strip treatment inactivate live H5 avian influenza virus positive allantoic fluid, using 0.01M PBS 1:2 times dilution, then separately take different dilution sample solution (1:2 ⁇ 1:214) and add it to the sample pad of H5 avian influenza test strip purchased on the market for H5 avian influenza detection, 10 After the minute test is completed, 100 ⁇ L of the clearing reagent is added dropwise to the test area of the test strip. After 3 minutes, the test strip detection area is changed from white to transparent, and the reading of the reader is immediately performed, and the test value is recorded.
- Control group The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
- Control group The minimum detection dilution was 2 10 , that is, when the dilution ratio was higher than 2 10 , the reader reached its minimum test threshold and could not detect positive.
- the minimum detection dilution was 2 12 , that is, when the dilution ratio was higher than 2 12 , the reader reached its minimum test threshold and could not detect positive.
Abstract
Disclosed is a method for improving the detection sensitivity of a colloidal gold immunochromatographic test strip. The method comprises the following specific steps: phenylmethanol and methanol, at a volume ratio of (9 : 1)-(10 : 1), are selected to be fully and uniformly mixed, so as to obtain a transparent reagent, after a sample to be detected is detected by the colloidal gold immunochromatographic test strip, the transparent reagent is added dropwise into a detection area of the test strip, the test strip is stood up, and after the white detection area of the test strip becomes transparent, the test strip is read by a test strip reading instrument. The above-mentioned operation can increase the detection sensitivity and linear detection range of the colloidal gold immunochromatographic test strip and has the advantages of simple operation and a short processing time.
Description
本发明属于胶体金检测领域，具体涉及一种提高胶体金免疫层析试纸条检测灵敏度的方法。 The invention belongs to the field of colloidal gold detection, and particularly relates to a method for improving the sensitivity of detection of colloidal gold immunochromatographic test strips.
食品是人类赖以生存和发展的物质基础，食品安全则直接关系到人类健康。然而，近年来恶性食品安全事故接连不断出现，如病原微生物超标、三聚氰胺牛奶、瘦肉精猪肉、黄曲霉毒素奶等。针对食品中微生物、真菌毒素、药物残留、重金属等污染问题，目前，免疫层析试纸条技术已广泛应用到食品，农产品等领域。其中最为常见的免疫层析试纸条为胶体金免疫层析试纸条。胶体金免疫层析试纸条适用于现场快速检测，最初主要用于蛋白质、微生物及多种化合物的定性检测，仅需肉眼即可完成判断。随着食品安全学科领域发展，各国对于食品中的污染物都有明确的限量要求，单纯的胶体金免疫层析试纸条定性检测对于很多种污染物检测已经难以满足消费者对污染物的定量要求。胶体金免疫层析试纸条读条仪的出现使得胶体金试纸条由定性检测逐渐转变到定量检测水平。Food is the material basis for human survival and development, and food safety is directly related to human health. However, in recent years, malignant food safety accidents have appeared continuously, such as pathogens exceeding the standard, melamine milk, lean meat pork, aflatoxin milk and so on. In view of the pollution problems of microorganisms, mycotoxins, drug residues and heavy metals in food, immunochromatographic test strip technology has been widely applied to food, agricultural products and other fields. The most common immunochromatographic test strips are colloidal gold immunochromatographic test strips. The colloidal gold immunochromatographic test strip is suitable for rapid detection on the spot. It is mainly used for the qualitative detection of proteins, microorganisms and various compounds, and can be judged only by the naked eye. With the development of food safety disciplines, countries have clear limits on the pollutants in food. The qualitative detection of pure colloidal gold immunochromatographic test strips has been difficult for consumers to quantify pollutants. Claim. The appearance of the colloidal gold immunochromatographic test strip reader has gradually changed the colloidal gold test strip from qualitative detection to quantitative detection.
然而，胶体金免疫层析试纸条读条仪虽然能提高检测灵敏度，但是检测灵敏度提高有限，且这种结合光学的检测技术容易受试纸条本身背景的干扰。为进一步提高读条仪的读数灵敏度，研究者分别从信号增强、数据处理等方面进行优化。现有技术中，主要采用增敏试剂对检测试纸条进行增敏处理，来提高检测的灵敏度，但氯金酸和抗坏血酸作为常用的增敏试剂，成本较高，不适于广泛应用。因此在这些研究基础上，研究一种能降低试纸条自身背景值的处理方法，对于进一步改善胶体金免疫层析试纸条的读数灵敏度具有重要意义。However, although the colloidal gold immunochromatographic test strip reader can improve the detection sensitivity, the detection sensitivity is limited, and the combined optical detection technique is easily interfered by the background of the test strip itself. In order to further improve the reading sensitivity of the reader, the researchers optimized from the aspects of signal enhancement and data processing. In the prior art, the sensitizing reagent is mainly used to sensitize the test strip to improve the sensitivity of the test, but chloroauric acid and ascorbic acid are commonly used as sensitizing reagents, and the cost is high, which is not suitable for wide application. Therefore, based on these studies, it is important to study a treatment method that can reduce the background value of the test strip itself, which is of great significance for further improving the sensitivity of the colloidal gold immunochromatographic test strip.
本发明所要解决的技术问题是针对上述现有技术存在的不足而提供一种提高胶体金免疫层析试纸条检测灵敏度的方法。其可提高胶体金免疫层析试纸条的检测灵敏度，增大检测线性范围，操作简单方便。
The technical problem to be solved by the present invention is to provide a method for improving the detection sensitivity of colloidal gold immunochromatographic test strips in view of the deficiencies of the above prior art. The utility model can improve the detection sensitivity of the colloidal gold immunochromatographic test strip, increase the linear range of detection, and is simple and convenient to operate.
本发明为解决上述提出的问题所采用的技术方案为：The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:
一种提高胶体金免疫层析试纸条检测灵敏度的方法，具体步骤如下：A method for improving the sensitivity of detection of colloidal gold immunochromatographic test strips, the specific steps are as follows:
（1）透明化试剂的配制：选取苯甲醇与甲醇作为配方组分，将两者以体积为9:1～10:1充分混匀，制得透明化试剂，避光密封保存；(1) Preparation of the transparent reagent: Benzyl alcohol and methanol are selected as the formulation components, and the two are thoroughly mixed in a volume of 9:1 to 10:1 to obtain a transparent reagent, which is sealed and protected from light;
（2）试纸条处理：取胶体金免疫层析试纸条对待测样品进行检测，检测完成后在胶体金免疫层析试纸条的检测区滴加步骤（1）配制得到的透明化试剂，放置待试纸条检测区由白色变成透明后，读条仪即可进行读数。(2) Test strip processing: the colloidal gold immunochromatographic test strip is tested for the sample to be tested, and after the test is completed, the transparent reagent prepared by the step (1) is added dropwise in the detection area of the colloidal gold immunochromatographic test strip. After the test strip of the test strip is placed from white to transparent, the reader can take readings.
按上述方案，所述步骤（2）中透明化试剂的使用量为50~100μL。According to the above scheme, the amount of the clearing agent used in the step (2) is 50 to 100 μL.
按上述方案，所述步骤（2）的放置时间为3~5min。According to the above scheme, the placement time of the step (2) is 3 to 5 minutes.
按上述方案，所述的胶体金免疫层析试纸条为真菌毒素胶体金免疫层析试纸条或病毒胶体金免疫层析试纸条。According to the above scheme, the colloidal gold immunochromatographic test strip is a mycotoxin colloidal gold immunochromatographic test strip or a virus colloidal gold immunochromatographic test strip.
本发明在检测前通过对胶体金免疫层析试纸条进行预处理，可使胶体金免疫层析试纸条上检测区的硝酸纤维素膜由白色非透明的易碎结构转变成透明且具备一定韧性的薄膜结构（见图1），而降低光反射作用造成的背景干扰；同时还可防止因胶体金自身氧化而使显色区域逐渐变色，而通过对胶体金免疫层析试纸条显色区域的颜色固定，达到避免因胶体金氧化而对试纸条的读数检测造成不良影响的作用，预处理完毕后再进行读条仪读数，可达到降低背景值影响，提高胶体金免疫层析试纸条的检测灵敏度的目的。The invention pre-treats the colloidal gold immunochromatographic test strip before the detection, so that the nitrocellulose membrane in the detection zone on the colloidal gold immunochromatographic test strip can be transformed from a white non-transparent fragile structure into a transparent and A certain tough film structure (see Figure 1), which reduces the background interference caused by light reflection; at the same time, it can prevent the coloration area from gradually changing due to the self-oxidation of colloidal gold, and the colloidal gold immunochromatographic test strip The color of the color area is fixed to avoid the adverse effect of the detection of the test strip by the oxidation of the colloidal gold. After the pre-treatment is completed, the reading of the reading instrument can be achieved to reduce the influence of the background value and improve the colloidal gold immunochromatography. The purpose of the detection sensitivity of the test strip.
与现有技术相比，本发明的有益效果在于：Compared with the prior art, the beneficial effects of the present invention are:
（l）提高检测灵敏度，增大检测线性范围；采用本发明方法对胶体金免疫层析试纸条进行处理后可使读条仪进行检测时的最小有效读数减小，从而可提高胶体金免疫层析试纸条的检测灵敏度，增大其检测线性范围；(l) Increasing the detection sensitivity and increasing the linear range of detection; using the method of the present invention to treat the colloidal gold immunochromatographic test strip can reduce the minimum effective reading when the reader is tested, thereby improving colloidal gold immunity The detection sensitivity of the chromatographic test strip increases the linear range of detection;
（2）操作简单方便，无需专业人员； (2) Simple and convenient operation, no need for professionals;
（3）处理时间短，检测完成后3~5分钟即可完成。(3) The processing time is short, and it can be completed in 3~5 minutes after the completion of the detection.
图1为本发明实施例1处理前后的黄曲霉毒素总量胶体金免疫层析试纸条的纵切面剖视图，图中T检测线，C质控线。
1 is a longitudinal cross-sectional view of a total amount of aflatoxin colloidal gold immunochromatographic test strip before and after treatment according to Example 1 of the present invention, wherein the T detection line and the C quality control line are shown.
实施例1：Example 1:
实验组： test group:
（1）透明化试剂的配制：选取苯甲醇与甲醇作为配方组分，按照1mL甲醇加入110μL苯甲醇比例充分混匀，避光密封保存；(1) Preparation of the transparent reagent: Benzyl alcohol and methanol are selected as the formulation components, and the ratio of 110 μL of benzyl alcohol is added to 1 mL of methanol to mix well, and sealed in the dark;
（2）试纸条处理：配制系列浓度黄曲霉毒素（AFT）标准溶液（0 ng/mL、0.01 ng/mL、
0.03 ng/mL、0.06 ng/mL、0.125 ng/mL、0.25 ng/mL、0.5
ng/mL、1ng/mL），分别取100μL上述各浓度的标准溶液滴加到黄曲霉毒素总量的胶体金检测试纸条的样品垫上，进行检测，10分钟检测完成后，在胶体金免疫层析试纸条的检测区滴加2滴（约100μL）透明化试剂，放置3分钟后，试纸条检测区由白色变成透明（见图1），立即进行读条仪读数，记录测试值。上述黄曲霉毒素总量的胶体金检测试纸条按专利文献“快速检测黄曲霉毒素总量的高灵敏度免疫层析试纸条及其制备方法”（专利号为201010245801.6）中的方法制备获得。(2) Test strip processing: preparation of a series of concentrations of aflatoxin (AFT) standard solution (0 ng / mL, 0.01 ng / mL,
0.03 ng/mL, 0.06 ng/mL, 0.125 ng/mL, 0.25 ng/mL, 0.5
Ng/mL, 1 ng/mL), 100 μL of each of the above concentrations of the standard solution was added dropwise to the sample pad of the colloidal gold test strip of the total amount of aflatoxin, and the test was carried out. After 10 minutes of detection, the gel was immunized in colloidal gold. 2 drops (about 100 μL) of clearing reagent were added to the test area of the chromatographic test strip. After standing for 3 minutes, the test strip detection area was changed from white to transparent (see Figure 1), and the reading of the reading instrument was immediately performed. value. The above-mentioned colloidal gold test strip of the total amount of aflatoxin is prepared according to the method in the patent document "High-sensitivity immunochromatographic test strip for rapid detection of aflatoxin total amount and preparation method thereof" (Patent No. 201010245801.6).
对照组：对照组与实验组的不同之处在于：检测完成后直接读数不进行透明化试剂处理。Control group: The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
结果分析：Result analysis:
对照组：有效检测范围为0.03ng/mL~0.5ng/mL，即待测体系中黄曲霉毒素总量浓度达0.03ng/mL时，读条仪达到其最高测试阈值，高于0.5ng/mL时，读条仪达到其最低测试阈值。Control group: The effective detection range is 0.03ng/mL~0.5ng/mL, that is, when the total concentration of aflatoxin in the system to be tested reaches 0.03ng/mL, the reader reaches its highest test threshold, higher than 0.5ng/mL. When the reader reaches its minimum test threshold.
实验组：有效检测范围为0.01ng/mL~1ng/mL，即待测体系中黄曲霉毒素总量浓度达到0.01ng/mL时，读条仪达到其最高测试阈值，高于1ng/mL时，读条仪达到其最低测试阈值。Experimental group: The effective detection range is 0.01 ng/mL~1 ng/mL, that is, when the total concentration of aflatoxin in the system to be tested reaches 0.01 ng/mL, the reader reaches its highest test threshold, higher than 1 ng/mL. The reader reaches its minimum test threshold.
由此对比结果可以看出：采用本发明方法对黄曲霉毒素总量胶体金免疫层析试纸条进行处理后提高了黄曲霉毒素总量胶体金免疫层析试纸条的检测灵敏度，增大了其检测线性范围。From the comparison results, it can be seen that the detection sensitivity of the aflatoxin total colloidal gold immunochromatographic test strip is improved by treating the aflatoxin total colloidal gold immunochromatographic test strip by the method of the invention. It detects the linear range.
实施例2：Example 2:
实验组：test group:
（1）透明化试剂的配制：选取苯甲醇与甲醇作为配方组分，按照1mL甲醇加入100μL苯甲醇比例充分混匀，避光密封保存； (1) Preparation of the transparent reagent: Benzyl alcohol and methanol are selected as the formulation components, and the mixture is uniformly mixed with 100 μL of benzyl alcohol according to 1 mL of methanol, and sealed and stored in the dark;
（2）试纸条处理：配制系列浓度黄曲霉毒素M1（AFM1）标准溶液（0 ng/mL、0.05 ng/mL、
0.1 ng/mL、0.25 ng/mL、0. 50 ng/mL、1 ng/mL、2 ng/mL、3 ng/mL
、4ng/mL），分别取100μL上述各浓度的标准溶液滴加到黄曲霉毒素M1（AFM1）胶体金检测试纸条的样品垫上，10分钟检测完成后，在试纸条的检测区滴加2滴（约100μL）透明化试剂，放置待试纸条检测区由白色变成透明，立即进行读条仪读数，记录测试值。(2) Test strip processing: prepare a series of standard aflatoxin M1 (AFM1) standard solution (0 ng / mL, 0.05 ng / mL,
0.1 ng/mL, 0.25 ng/mL, 0.50 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL
, 4ng/mL), respectively, 100μL of each of the above concentrations of the standard solution was added dropwise to the sample pad of the aflatoxin M1 (AFM1) colloidal gold test strip, after 10 minutes of testing, in the test area of the test strip 2 drops (about 100 μL) of clearing reagent, place the test strip of the test strip to change from white to transparent, immediately read the reading instrument and record the test value.
对照组：对照组与实验组的不同之处在于：检测完成后直接读数不进行透明化试剂处理。Control group: The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
结果分析：Result analysis:
对照组：有效检测范围为0.1ng/mL~3ng/mL，即待测体系中黄曲霉毒素M1浓度低于0.03ng/mL时，读条仪达到其最高测试阈值，高于3ng/mL测试值时，读条仪达到其最低测试阈值。Control group: The effective detection range is 0.1 ng/mL~3 ng/mL, that is, when the aflatoxin M1 concentration in the system to be tested is lower than 0.03 ng/mL, the reader reaches its highest test threshold, which is higher than the 3 ng/mL test value. When the reader reaches its minimum test threshold.
实验组：有效检测范围为0.05ng/mL~4ng/mL，即待测体系中黄曲霉毒素M1浓度达0.05ng/mL时，读条仪达到其最高测试阈值，高于4ng/mL测试值时，读条仪达到其最低测试阈值。Experimental group: The effective detection range is 0.05 ng/mL~4 ng/mL, that is, when the aflatoxin M1 concentration in the system to be tested reaches 0.05 ng/mL, the reader reaches its highest test threshold, which is higher than the 4 ng/mL test value. The reader has reached its minimum test threshold.
由此对比结果可以看出：采用本发明方法对黄曲霉毒素M1胶体金免疫层析试纸条进行处理后提高了黄曲霉毒素M1胶体金免疫层析试纸条的检测灵敏度，增大了其检测线性范围。From the comparison results, it can be seen that the aflatoxin M1 colloidal gold immunochromatographic test strip is treated by the method of the invention, and the detection sensitivity of the aflatoxin M1 colloidal gold immunochromatographic test strip is improved, and the sensitivity thereof is increased. Detect linear range.
实施例3：Example 3:
实验组：test group:
（1）透明化试剂的配制：选取苯甲醇与甲醇作为配方组分，按照1mL甲醇加入105μL苯甲醇比例充分混匀，避光密封保存；(1) Preparation of the transparent reagent: Benzyl alcohol and methanol are selected as the formulation components, and the mixture is uniformly mixed with 105 μL of benzyl alcohol according to 1 mL of methanol, and sealed and stored in the dark;
（2）试纸条处理及检测：配制系列浓度玉米赤霉烯酮标准溶液（0 ng/mL、0.05 ng/mL、 0.1
ng/mL、0.25 ng/mL、0. 50 ng/mL、1 ng/mL、2 ng/mL、3 ng/mL
、4ng/mL），分别取100μL上述各浓度的标准溶液滴加到玉米赤霉烯酮胶体金检测试纸条的样品垫上，10分钟检测完成后，在试纸条检测区滴加透明化试剂，4分钟后试纸条检测区由白色变成透明，立即进行读条仪读数，记录测试值。(2) Test strip processing and testing: preparation of a series of concentrations of zearalenone standard solution (0 ng / mL, 0.05 ng / mL, 0.1
Ng/mL, 0.25 ng/mL, 0.50 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL
4ng/mL), 100μL of each of the above concentrations of the standard solution was added dropwise to the sample pad of the zearalenone colloidal gold test strip. After 10 minutes of testing, the clearing reagent was added to the test strip detection area. After 4 minutes, the test strip detection area changes from white to transparent, and the reading of the reader is immediately performed, and the test value is recorded.
对照组：对照组与实验组的不同之处在于：检测完成后直接读数不进行透明化试剂处理。Control group: The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
结果分析：Result analysis:
对照组：有效检测范围为0.1ng/mL~2ng/mL，即体系中玉米赤霉烯酮浓度达0.1ng/mL时，读条仪达到其最高测试阈值，高于2ng/mL时，读条仪达到其最低测试阈值。Control group: The effective detection range is 0.1 ng/mL~2 ng/mL, that is, when the concentration of zearalenone in the system reaches 0.1 ng/mL, the reader reaches its highest test threshold, and when it is higher than 2 ng/mL, the reading is performed. The instrument reaches its minimum test threshold.
实验组：有效检测范围为0.05ng/mL~3ng/mL，即体系中玉米赤霉烯酮浓度达到0.05ng/mL时，读条仪达到其最高测试阈值，高于3ng/mL时，读条仪达到其最低测试阈值。Experimental group: The effective detection range is 0.05 ng/mL~3 ng/mL, that is, when the concentration of zearalenone in the system reaches 0.05 ng/mL, the reader reaches its highest test threshold, and when it is higher than 3 ng/mL, the reading strip The instrument reaches its minimum test threshold.
由此对比结果可以看出：采用本发明方法对玉米赤霉烯酮胶体金免疫层析试纸条进行处理后提高了玉米赤霉烯酮胶体金免疫层析试纸条的检测灵敏度，增大了其检测线性范围。From the comparison results, it can be seen that the detection sensitivity of the zearalenone colloidal gold immunochromatographic test strip is improved by using the method of the invention to treat the zearalenone colloidal gold immunochromatographic test strip. It detects the linear range.
实施例4Example 4
实验组： test group:
（1）透明化试剂的配制：选取苯甲醇与甲醇作为配方组分，按照1mL甲醇加入110μL苯甲醇比例充分混匀，避光密封保存；(1) Preparation of the transparent reagent: Benzyl alcohol and methanol are selected as the formulation components, and the ratio of 110 μL of benzyl alcohol is added to 1 mL of methanol to mix well, and sealed in the dark;
（2）试纸条处理：取灭活H5型禽流感病毒阳性尿囊液，用0.01M PBS
1:2倍比稀释，然后分别取不同稀释度样品溶液（1:2~1:214）滴加到市场上购买的H5型禽流感检测试纸条的样品垫上进行H5型禽流感检测，10分钟检测完成后，在试纸条的检测区滴加100μL透明化试剂，3分钟后试纸条检测区由白色变成透明，立即进行读条仪读数，记录测试值。(2) Test strip treatment: inactivate live H5 avian influenza virus positive allantoic fluid, using 0.01M PBS
1:2 times dilution, then separately take different dilution sample solution (1:2~1:214) and add it to the sample pad of H5 avian influenza test strip purchased on the market for H5 avian influenza detection, 10 After the minute test is completed, 100 μL of the clearing reagent is added dropwise to the test area of the test strip. After 3 minutes, the test strip detection area is changed from white to transparent, and the reading of the reader is immediately performed, and the test value is recorded.
对照组：对照组与实验组的不同之处在于：检测完成后直接读数不进行透明化试剂处理。Control group: The difference between the control group and the experimental group was that the direct reading was not processed by the clearing reagent after the test was completed.
结果分析：Result analysis:
对照组：最低检测稀释度为210，即稀释倍数高于210时，读条仪达到其最低测试阈值，不能检测到阳性。Control group: The minimum detection dilution was 2 10 , that is, when the dilution ratio was higher than 2 10 , the reader reached its minimum test threshold and could not detect positive.
实验组：最低检测稀释度为212，即稀释倍数高于212时，读条仪达到其最低测试阈值，不能检测到阳性。Experimental group: The minimum detection dilution was 2 12 , that is, when the dilution ratio was higher than 2 12 , the reader reached its minimum test threshold and could not detect positive.
Claims (4)
1. 一种提高胶体金免疫层析试纸条检测灵敏度的方法，其特征在于：具体步骤如下：A method for improving the sensitivity of colloidal gold immunochromatographic test strips, characterized in that the specific steps are as follows:
（1）透明化试剂的配制：选取苯甲醇与甲醇作为配方组分，将两者以体积为9:1～10:1充分混匀，制得透明化试剂，避光密封保存；(1) Preparation of the transparent reagent: Benzyl alcohol and methanol are selected as the formulation components, and the two are thoroughly mixed in a volume of 9:1 to 10:1 to obtain a transparent reagent, which is sealed and protected from light;
（2）试纸条处理：取胶体金免疫层析试纸条对待测样品进行检测，检测完成后在胶体金免疫层析试纸条的检测区滴加步骤（1）配制得到的透明化试剂，放置待试纸条检测区由白色变成透明后，读条仪即可进行读数。(2) Test strip processing: the colloidal gold immunochromatographic test strip is tested for the sample to be tested, and after the test is completed, the transparent reagent prepared by the step (1) is added dropwise in the detection area of the colloidal gold immunochromatographic test strip. After the test strip of the test strip is placed from white to transparent, the reader can take readings.
2.
根据权利要求1所述的提高胶体金免疫层析试纸条检测灵敏度的方法，其特征在于：所述步骤（2）中透明化试剂的使用量为50～100μL。2.
The method for improving the detection sensitivity of a colloidal gold immunochromatographic test strip according to claim 1, wherein the amount of the clearing reagent used in the step (2) is 50 to 100 μL.
3.
根据权利要求1所述的提高胶体金免疫层析试纸条检测灵敏度的方法，其特征在于：所述步骤（2）的放置时间为3-5min。3.
The method for improving the detection sensitivity of a colloidal gold immunochromatographic test strip according to claim 1, wherein the step (2) has a standing time of 3-5 min.
4.
根据权利要求1所述的提高胶体金免疫层析试纸条检测灵敏度的方法，其特征在于：所述的胶体金免疫层析试纸条为真菌毒素胶体金免疫层析试纸条或病毒胶体金免疫层析试纸条。4.
The method for improving the detection sensitivity of a colloidal gold immunochromatographic test strip according to claim 1, wherein the colloidal gold immunochromatographic test strip is a mycotoxin colloidal gold immunochromatographic test strip or a viral colloid. Gold immunochromatographic test strips.
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