CN111766379A - Tissue sample extracting solution treatment method for improving color development of colloidal gold detection card - Google Patents
Tissue sample extracting solution treatment method for improving color development of colloidal gold detection card Download PDFInfo
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Abstract
The invention provides a tissue sample extracting solution treatment method for improving color development of a colloidal gold detection card. The tissue sample extracting solution treatment method for improving the color development of the colloidal gold detection card eliminates the influence of partial interference substances by oxidizing the tissue sample extracting solution, improves the color development of the detection card and improves the detection accuracy.
Description
Technical Field
The invention belongs to the technical field of biochemical detection, and particularly relates to a method for treating a tissue sample extracting solution for improving the color development of a colloidal gold detection card.
Background
In research and production experiments, the following conditions were found when CLE colloidal gold test cards were tested: the color development gray value of the detection card of part of the tissue sample extracting solution is low, so that the detection result is inaccurate. For this portion of the sample, the test card T, C lines were all lighter in color due to the presence of interfering substances in the tissue sample extract. At present, a mode of extracting a tissue extracting solution by using an organic solvent to remove part of interference substances is adopted, but color development cannot be effectively improved, and the organic extraction mode is complex and has low safety. Therefore, there is a need to develop a simple and highly safe method for improving color development, so as to solve the above problems.
Disclosure of Invention
In view of the above, the present invention is directed to a simple, efficient and safe method for processing tissue sample extract to improve the color development of a colloidal gold test card, so as to solve the above problems.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a tissue sample extracting solution treatment method for improving color development of a colloidal gold detection card comprises the step of carrying out oxidation treatment on a tissue sample extracting solution.
Further, the oxidation treatment method is to adopt an oxidant for treatment.
Further, the oxidant is treated in a manner that the oxidant is added to the tissue sample extract or to the sample pad of the colloidal gold test card.
Further, the oxidizing agent is hydrogen peroxide or carbamide peroxide.
Further, the oxidant is hydrogen peroxide, and the treatment mode of the oxidant is to add the hydrogen peroxide solution into the tissue sample extracting solution; the oxidant is carbamide peroxide, and the mode of treating the oxidant is to add the carbamide peroxide solution into the tissue sample extracting solution or on the sample pad of the colloidal gold detecting card.
Further, the mass fraction of the hydrogen peroxide added into the tissue sample extracting solution is 0.03-3%; the mass fraction of the carbamide peroxide added into the tissue sample extracting solution is 0.1-3.2%, and the mass fraction of the carbamide peroxide added onto the sample pad of the colloidal gold detecting card is 0.4-6.4%.
Further, the mass fraction of the hydrogen peroxide added into the tissue sample extracting solution is 0.1-2%; the mass fraction of the carbamide peroxide added into the tissue sample extracting solution is 0.3 percent, and the mass fraction of the carbamide peroxide added on the sample pad of the colloidal gold detecting card is 0.6 percent.
Further, the reaction time of adding the hydrogen peroxide into the tissue sample extracting solution is 5-20 min; the reaction time of adding the carbamide peroxide to the tissue sample extracting solution is 5-30 s.
Further, the tissue sample is an animal tissue sample.
Further, the tissue sample is extracted by a water boiling method or a clamping baking method.
The water boiling method comprises the steps of putting a finely cut tissue sample into a sealed vessel, heating until the tissue sample is well cooked, and cooling to room temperature to obtain liquid in the sealed vessel, namely the tissue sample extracting solution.
The clamping baking method comprises the steps of wrapping a sliced tissue sample with aluminum foil paper, clamping the tissue sample by an extractor, heating for roasting, and collecting liquid flowing out of the aluminum foil paper to obtain tissue sample extracting solution.
The action mechanism is as follows: the tissue sample extracting solution contains some reducing substances such as fat and the like, the substances can interfere the combination of the antigen and the antibody, and the detection target object and the antibody, so that the color development and the accuracy of the detection card are influenced.
Compared with the prior art, the method for treating the tissue sample extracting solution for improving the color development of the colloidal gold detection card has the following advantages:
(1) according to the tissue sample extracting solution treatment method for improving the color development of the colloidal gold detection card, the tissue sample extracting solution is oxidized, so that the influence of partial interference substances is eliminated, the color development of the detection card is improved, and the detection accuracy of the tissue sample is improved;
(2) the method for treating the tissue sample extracting solution for improving the color development of the colloidal gold detection card is simple to operate, consumes less time, does not introduce new interference substances, can convert hydrogen peroxide and carbamide peroxide into water after consumption, does not bring secondary interference, and does not influence the detection accuracy.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
The following preparation methods of the tissue sample extracting solution are both a water boiling method and/or a clamping baking method, and the specific processes of the water boiling method and the clamping baking method are as follows:
take a lean pork sample stored in a refrigerator at-20 c as an example.
A water boiling method: taking a sliced pig lean meat sample, filling the sliced pig lean meat sample into a centrifuge tube (preferably three quarters of the tube is filled), covering a tube cover, heating the tube cover in a water bath at the temperature of more than 90 ℃ until the sample is well cooked, taking out the centrifuge tube, and cooling the centrifuge tube to room temperature to obtain liquid in the tube, namely tissue sample extracting solution.
A clamping and baking method: cutting a pork lean sample into square meat slices with the thickness of 0.5cm and the length of 3cm, wrapping the meat slices with aluminum foil paper, clamping the meat slices in a clamping plate of an extractor, maintaining the temperature of the extractor at 140 ℃ for about 30-40 s, allowing liquid to flow out of the aluminum foil paper, discarding 5-6 drops of liquid separated out at the earliest, collecting subsequent liquid, and cooling to room temperature to obtain a tissue sample extracting solution.
1. Color development influence of standing tissue sample extracting solution on colloidal gold detection card
Respectively adopting 4ml of pig lean meat sample extracting solution obtained by a water boiling method and a clamping baking method to be put in a centrifuge tube, marking as sample 1 and sample 2, standing, respectively sampling sample 1 and sample 2 at 0min, 60min, 180min and 270min, wherein 3 samples are parallel, detecting by using a CLE colloidal gold detection card, dropwise adding the samples on a CLE colloidal gold detection card sample pad, timing, scanning once at 5min, scanning once again at 20min, and calculating and recording a gray value. The results are shown in Table 1.
TABLE 1 influence of the standing extractive solution on the color development of the colloidal gold test card
As can be seen from table 1, after the sample solution is sampled and added dropwise to the CLE colloidal gold test card at different standing times, the gray scale values of the C line and the T line of the test card do not change very obviously, that is, in the standing process, the color development of the C line and the T line after the tissue extract solution is added to the test card cannot be effectively improved by the oxidation of oxygen in the air, and the length of the standing time of the tissue sample extract solution cannot have an obvious influence on the color development of the test card.
2. Effect of Hydrogen peroxide treatment on color development of colloidal gold test cards
Experimental groups:
example 1: taking 1.35ml of lean meat sample extracting solution of pig obtained by water boiling method, putting into 10ml of centrifuge tube, adding 0.15ml of hydrogen peroxide with mass fraction of 30% (the mass fraction of hydrogen peroxide added into the tissue sample extracting solution is 3%), mixing well, and standing for 10 min.
Example 2: taking 1.35ml of the lean meat sample extract obtained by the clamp baking method, putting into a 10ml centrifuge tube, adding 0.15ml of hydrogen peroxide with the mass fraction of 30% (the mass fraction of the hydrogen peroxide added into the tissue sample extract is 3%), mixing uniformly, and standing for 10 min.
Example 3: adding 1.35ml of PBS into a 10ml centrifuge tube, adding 0.15ml of hydrogen peroxide with the mass fraction of 30% (the mass fraction of the hydrogen peroxide added into the PBS is 3%), mixing uniformly and standing for 10 min.
Control group:
comparative example 1: taking 1.35ml of lean meat sample extract obtained by water boiling method, adding 0.15ml of distilled water into a 10ml centrifuge tube, mixing uniformly, and standing for 10 min.
Comparative example 2: taking 1.35ml of lean meat sample extract obtained by a clamping baking method, adding 0.15ml of distilled water into a 10ml centrifuge tube, uniformly mixing, and standing for 10 min.
Comparative example 3: and (3) putting 1.35ml of PBS into a 10ml centrifuge tube, adding 0.15ml of distilled water, uniformly mixing, and standing for 10 min.
Detecting with a CLE colloidal gold detection card:
sampling and detecting the samples of the embodiment 1, the embodiment 2, the embodiment 3, the comparison example 1, the comparison example 2 and the comparison example 3, wherein 3 samples are parallel, the samples are dripped on a sample pad of a CLE colloidal gold detection card and then are timed, the samples are scanned once at 5min and scanned once again at 20min, and the gray value is calculated and recorded. The results are shown in Table 2.
TABLE 2 Experimental results of the effect of hydrogen peroxide treatment on the gray level
The data in table 2 were processed to calculate the relative increase ratio of the gray scale values of the C-line and T-line with and without hydrogen peroxide addition: the relative increase in gray scale value for C or T lines is (hydrogen peroxide added-no hydrogen peroxide added) ÷ no hydrogen peroxide added. The results are shown in Table 3
TABLE 3 relative increase ratio of Gray values of Hydrogen peroxide treated tissue extracts
By analyzing tables 2 and 3, it can be seen that:
as can be seen from the data related to example 1 and comparative example 1 and the data related to example 2 and comparative example 2, the gray values of the C line and the T line are obviously improved after the treatment of adding hydrogen peroxide, especially after the color development is carried out for 20min, compared with the comparative example without adding hydrogen peroxide, the ratio of the increase of the gray values of the color development reaches more than 40%, and it can be seen that the oxidizing agent represented by hydrogen peroxide can effectively improve the color development of the detection card after the tissue sample extraction liquid is added to the detection card;
from example 3(PBS + H)2O2) As can be seen from the data concerning comparative example 3(PBS), the changes in the gray levels of the C line and the T line were not large when hydrogen peroxide was added or when hydrogen peroxide was not added, i.e., the color development of the colloidal gold test card by hydrogen peroxide was substantially observedThe method has the advantages that the method is free from inhibition, hydrogen peroxide mainly oxidizes reducing substances in the tissue sample extracting solution, interference substances in the tissue sample extracting solution are eliminated, and the self color development of the detection card cannot be inhibited;
comparing the data obtained in the detection of 5min and 20min with and without hydrogen peroxide, it can be seen that the release rate of the chromogenic material is not affected by the addition of hydrogen peroxide.
3. Determination of the amount of Hydrogen peroxide added
6 parts of lean pork sample extract obtained by the boiling method were taken, and hydrogen peroxide was added to the sample extract in such amounts that the mass fractions of hydrogen peroxide added to the sample extract were 3%, 1%, 0.3%, 0.1%, 0.03%, and 0%, respectively, and the mixture was mixed and allowed to stand for 10min, which were designated as example 4, example 5, example 6, example 7, example 8, and a blank example in this order. And simultaneously preparing sample standard adding liquid corresponding to the 6 parts of liquid to be detected to obtain 6 sample standard adding examples.
Detecting with a CLE colloidal gold detection card: respectively carrying out sampling detection on 4 to 8 samples, the blank sample and the 6 samples in a standard adding way, wherein 3 samples are parallel, the samples are dripped on a CLE colloidal gold detection card sample pad and then are timed, the samples are scanned once in 5min and then scanned once in 20min, the gray value is calculated and recorded, and the value of the sample in the standard adding way is marked as CSign boardAnd TSign board. The results are shown in Table 4.
TABLE 4 influence of different hydrogen peroxide addition amounts on the color development of the colloidal gold assay cards
As can be seen from Table 4, when H2O2Is 1%, and T is observed after developing for 20min0Color development gray value vs. H2O2The mass fraction of (2%) increased by 52.7% ((113-74) ÷ 74 × 100%), reached essentially the highest value, and followed by H2O2Increase in mass fraction, T0The developed gradation value is substantially stable, and therefore, H is determined2O2The weight percentage of the extract added into the tissue sample extract is 0.03 to 3 percent, preferably 0.1 to 3 percent。
At the same time, T of the standard solution is added from the sample in Table 4Sign boardThe gray scale value is known to increase by H2O2In the course of dosage, TSign boardThe gray values are all 0, the color can not be seen by naked eyes, and the color can be judged to be positive, namely H2O2When the dosage is 0-3%, the sensitivity of the detection card is not affected.
4. Determination of the treatment time of hydrogen peroxide
4 parts of lean meat sample extract obtained by the boiling method were taken, 2.7ml of each extract was added with 0.1ml of 28% hydrogen peroxide (the mass fraction of hydrogen peroxide added to the tissue sample extract was 1%), mixed and left standing for 0min, 5min, 10min and 15min, and the mixture was recorded as example 9, example 10, example 11 and example 12 in this order.
Detecting with a CLE colloidal gold detection card: the samples of examples 9 to 12 were sampled and tested, 3 samples were each tested in parallel, the samples were dropped on the sample pad of the CLE colloidal gold test card and then timed, and the samples were scanned once at 5min and again at 20min, and the gray value was calculated and recorded. The results are shown in Table 5.
TABLE 5 Effect of Hydrogen peroxide treatment time on color development of colloidal gold assay cards
As can be seen from Table 5, the weight fraction of hydrogen peroxide added to the tissue sample extract was 1%, and the reaction time was 5min, T0The gray value of the line remains substantially stable, with increasing resting time T0The grey value of the line is not significantly increased, so it is determined that H is added2O2And then reacting for 5 min.
5. Validation of Hydrogen peroxide-treated tissue sample extract-multiple samples
Experimental groups: 2.7ml of each of the extract solution of the No. 1 pork lean sample, the extract solution of the No. 2 pork lean sample, the extract solution of the No. 3 pork lean sample, the extract solution of the beef sample and the extract solution of the mutton sample obtained by the boiling method was added with 0.1ml of hydrogen peroxide having a mass fraction of 28% (the mass fraction of hydrogen peroxide added to the extract solution of the tissue sample was 1%), mixed and then left stand for 5 minutes, and the mixture was recorded as example 13, example 14, example 15, example 16 and example 17 in this order.
Control group: taking 2.7ml of each of the No. 1 pork lean sample extracting solution, the No. 2 pork lean sample extracting solution, the No. 3 pork lean sample extracting solution, the beef sample extracting solution and the mutton sample extracting solution obtained by the water boiling method, adding 0.1ml of distilled water respectively, mixing uniformly, standing for 5min, and marking as a comparative example 13, a comparative example 14, a comparative example 15, a comparative example 16 and a comparative example 17 in sequence.
Sample labeling group: preparing each to-be-detected liquid in an experimental group and a control group, respectively adding 3ppb standard detection liquid, uniformly mixing, and standing for 5min to obtain 10 sample standard adding examples.
Detecting with a CLE colloidal gold detection card: respectively adding standard samples to the samples of examples 13 to 17, comparative examples 13 to 17 and 10, sampling and detecting, wherein 3 samples are parallel, dropwise adding the samples on a sample pad of a CLE colloidal gold detection card, timing, scanning once at 5min, 8min, 10min, 15min and 20min respectively, calculating and recording gray values, wherein the value of the sample standard adding example is marked as CSign boardAnd TSign board. The results are shown in Table 6.
TABLE 6 hydrogen peroxide treatment of tissue sample extract-multiple samples validation test results
As can be seen from Table 6, T in the examples0The color development is obviously improved and TSign boardAll color development is 0, namely, all positive color development can be judged, and H is not added2O2T of comparative example 17 in the comparative group0Comparative example 17 corresponds to T although color development is observedSign boardA value of greater thanIf the 0 reaches more than 50, the obvious color development can not determine whether the interference substances appear, so that the false negative judgment can be obtained.
6. Influence of hydrogen peroxide treatment on accuracy of colloidal gold detection card
Experimental groups: taking 2.7ml of each of the No. 1 pork lean sample extract, the No. 2 pork lean sample extract, the No. 3 pork lean sample extract, the No. 1 beef sample extract, the No. 2 beef sample extract, the No. 3 beef sample extract, the No. 1 mutton sample extract, the No. 2 mutton sample extract and the No. 3 mutton sample extract obtained by the boiling method, adding 0.1ml of hydrogen peroxide with a mass fraction of 28% (the mass fraction of hydrogen peroxide added to the tissue sample extract is 1%), mixing and standing for 5min, which are described as example 18, example 19, example 20, example 21, example 22, example 23, example 24, example 25 and example 26 in this order.
Control group: taking 2.7ml of each of the No. 1 pork lean sample extracting solution, the No. 2 pork lean sample extracting solution, the No. 3 pork lean sample extracting solution, the No. 1 beef sample extracting solution, the No. 2 beef sample extracting solution, the No. 3 beef sample extracting solution, the No. 1 mutton sample extracting solution, the No. 2 mutton sample extracting solution and the No. 3 mutton sample extracting solution obtained by a water boiling method, respectively adding 0.1ml of distilled water, uniformly mixing, standing for 5min, and sequentially marking as a comparative example 18, a comparative example 19, a comparative example 20, a comparative example 21, a comparative example 22, a comparative example 23, a comparative example 24, a comparative example 25 and a comparative example 26.
Sample labeling group: preparing each to-be-detected liquid in an experimental group and a control group, respectively adding 3ppb standard detection liquid, uniformly mixing, and standing for 5min to obtain 18 sample standard adding examples.
Detecting with a CLE colloidal gold detection card: respectively adding standard samples to the samples of 18 to 26 examples, 18 to 26 comparative examples and 18 samples, sampling and detecting, wherein 3 samples are parallel, dropwise adding the samples on a sample pad of a CLE colloidal gold detection card, timing, scanning once at 10min, calculating and recording gray values, wherein the value of the sample standard adding example is marked as CSign boardAnd TSign board. The results are shown in Table 7.
TABLE 7 influence of hydrogen peroxide treatment on the accuracy of colloidal gold test card
As is clear from Table 7, in the examples, when 1% hydrogen peroxide was added to the sample extract and the mixture was dropped into the colloidal gold test card, the C, T-line color tone values were all improved and the corresponding T values were all improvedSign boardAll color development was 0, and all positive conclusions were drawn, whereas no H was added2O2Of comparative examples 23, 24 and 25 in the control group0Although there is color development, its corresponding TSign boardThe value of (2) is greater than 0, and the determination of false negative is not determined because whether the interference substance appears or not can not be determined, so that the method can improve the detection accuracy of the sample.
7. Effect of Urea peroxide treatment on color development of colloidal gold test cards
Experimental groups:
example 27: taking 1.4ml of lean meat sample extract of pig obtained by water boiling method, putting into 10ml centrifuge tube, adding 0.045g carbamide peroxide (the mass fraction of carbamide peroxide added into the tissue sample extract is 3.2%), mixing well, and standing for 10 min.
Example 28: 1.4ml of PBS is taken to be put into a 10ml centrifuge tube, 0.045g of carbamide peroxide (the mass fraction of carbamide peroxide added into the tissue sample extract is 3.2 percent) is added, and the mixture is evenly mixed and then is kept stand for 10 min.
Control group:
comparative example 27: taking 1.4ml of lean meat sample extract obtained by water boiling method, placing in 10ml centrifuge tube, and standing for 10 min.
Comparative example 28: 1.4ml of PBS was put into a 10ml centrifuge tube and left to stand for 10 min.
Detecting with a CLE colloidal gold detection card:
and (3) sampling and detecting the samples of the example 27, the example 28, the comparative example 27 and the comparative example 28 respectively, wherein 3 samples are parallel, the samples are dripped on a sample pad of a CLE colloidal gold detection card, timing is carried out, scanning is carried out once at the time of 5min, scanning is carried out once again at the time of 20min, and the gray value is calculated and recorded. The results are shown in Table 8.
TABLE 8 Experimental results of the effect of hydrogen peroxide treatment on the gray-scale values
The data in table 8 were processed to calculate the relative increase ratio of the gray scale values of the C-line and T-line with and without urea peroxide addition: the relative increase in gray scale value for the C or T line (urea peroxide added-urea peroxide not added)/urea peroxide not added. The results are shown in Table 9
TABLE 9 relative increase ratio of gray scale values for urea peroxide treated tissue extracts
By analyzing tables 8 and 9, it can be seen that: as can be seen from the data related to example 28 and comparative example 28, the gray values of the C line and the T line are not substantially changed when the carbamide peroxide is added into the PBS compared with that when the carbamide peroxide is not added, which indicates that the addition of the carbamide peroxide has no inhibition effect on the color development of the detection card, however, it is understood from example 27 and comparative example 27 that the gray values of the C line and T line of the test card after the dropwise addition of the sample liquid are significantly improved after the treatment of adding urea peroxide, namely, the color development of the detection card after the sample liquid is dripped is not inhibited, but the color development is actively improved, particularly after the color development is carried out for 20min, compared with the comparative example without the carbamide peroxide, the ratio of the increase of the color development gray value reaches more than 50 percent, and the related data of the example 27 and the comparative example 27 show that the oxidant represented by carbamide peroxide can effectively improve the color development of the detection card after the tissue sample extraction liquid is dripped into the detection card.
8. Color development influence of urea peroxide treatment tissue sample extracting solution with different addition amounts on colloidal gold detection card
6 parts of a lean meat sample extract obtained by a boiling method were taken, urea peroxide was added to the sample extract in such amounts that the urea peroxide was added to the sample extract in the amounts of 0%, 0.1%, 0.3%, 0.9%, 1.8% and 3.2% by mass, and the mixture was uniformly mixed, and then the mixture was designated as a blank example, example 29, example 30, example 31, example 32 and example 33 in this order.
Detecting with a CLE colloidal gold detection card: and (3) sampling and detecting the examples 29 to 33 and the blank example respectively, wherein 3 samples are parallel, the samples are dripped on a sample pad of a CLE colloidal gold detection card and then timed, the samples are scanned once at 5min and scanned once again at 20min, and the gray value is calculated and recorded. The results are shown in Table 10.
TABLE 10 color development of urea peroxide treated tissue sample extracts on colloidal gold test cards at different addition levels
Influence the experimental results
As is clear from Table 10, when the urea peroxide was 0.3% by mass and developed for 20min, T was observed0The developed grey value increased by 46.6% ((88-60) ÷ 60 × 100%) relative to the value of the blank, reaching essentially the highest value, and with increasing mass fraction of carbamide peroxide, T0The color development gray value is basically stable, therefore, the mass fraction of carbamide peroxide added into the tissue sample extracting solution is determined to be 0.1-3.2%, and preferably 0.3%.
9. Color development influence of carbamide peroxide treatment tissue sample extracting solution time on colloidal gold detection card
Weighing 0.32g of carbamide peroxide, adding the carbamide peroxide into 1ml of purified water, and oscillating the carbamide peroxide to be completely dissolved to obtain carbamide peroxide solution;
0.27ml of each of 4 parts of lean meat sample extract solutions obtained by a boiling method was added with 30. mu.l of urea peroxide solution (the mass fraction of urea peroxide added to the tissue sample extract solution was 3.2%), mixed and left standing for 5s, 10s, 20s and 30s, respectively, which were described as example 34, example 35, example 36 and example 37 in this order.
Detecting with a CLE colloidal gold detection card: samples of examples 34 to 37 were taken and tested, 3 samples were taken and tested, and the samples were dropped on the sample pad of the CLE colloidal gold test card and counted, and scanned once at 5min and once again at 20min, and the gray value was calculated and recorded. The results are shown in Table 11.
TABLE 11 Effect of Urea peroxide treatment time on color development of colloidal gold assay cards
As can be seen from Table 11, T is the reaction time of 5s after urea peroxide is added to the tissue sample extract0The grey value of the line remains substantially constant, with increasing resting time T0The grey value of the line did not increase significantly, so it was determined that 5s after the urea peroxide addition.
10. Carbamide peroxide with different addition amounts is added to a sample pad for treating the influence of tissue extract on the color development of a colloidal gold test card
And (3) preparing the CLE colloidal gold detection card with different amounts of carbamide peroxide added on the sample pad, wherein the specific preparation method comprises the steps of adding the prepared carbamide peroxide solution on the sample pad, and drying to obtain the CLE colloidal gold detection card with carbamide peroxide added on the sample pad.
The lean meat sample extract of a pig obtained by a boiling method is dripped on a CLE colloidal gold detection card added with urea peroxide solution with the mass fractions of 0%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.6%, 3.20% and 6.40% on a sample pad, and the samples are sequentially marked as a blank example, an example 38, an example 39, an example 40, an example 41, an example 42, an example 43, an example 44 and an example 45, and 3 samples are detected in parallel by the colloidal gold detection card with the same addition amount of urea peroxide. And (3) dropwise adding the sample on a sample pad of a CLE colloidal gold detection card, timing, scanning once at 5min, scanning once again at 20min, and calculating and recording a gray value. The results are shown in Table 12.
TABLE 12 Effect of urea peroxide in different amounts added to the sample pad on color development of the colloidal gold assay cards
As can be seen from table 12, the sample pad treated with carbamide peroxide can also play the same role as that of carbamide peroxide added into the sample extract after the sample extract is dripped, and the color development of the C line and the T line of the detection card treated with carbamide peroxide is obviously improved compared with that of the detection card not treated with carbamide peroxide after the sample extract is dripped, especially in example 41 with the addition amount of 0.6%, compared with the blank example, the gray value of the T line is raised by up to 46% after the color development is carried out for 20min, so that the same effect of improving the color development of the detection card can be achieved by adding the oxidant represented by carbamide peroxide into the sample pad of the colloidal gold detection card in advance when the oxidant represented by carbamide peroxide is added into the sample pad of the tissue sample extract;
as can be seen from table 12, when the mass fraction of the carbamide peroxide is 0.6%, when the carbamide peroxide is added to the sample pad, the color development gray value of the line C and the line T reaches the highest value after the sample extract is added, and the color development is stable with the increase of the mass fraction of the carbamide peroxide, and has no obvious change, therefore, the mass fraction of the carbamide peroxide solution added to the sample pad of the colloidal gold detection card is determined to be 0.4% -6.4%, preferably 0.6%.
11. For multiple samples, carbamide peroxide is added to the sample pad for treating the color development influence of the tissue extract on the colloidal gold detection card
Experimental groups: the extract solutions of the No. 1 pig lean meat sample, the No. 2 pig lean meat sample, the No. 3 pig lean meat sample, the No. 4 pig lean meat sample, the No. 5 pig lean meat sample, the No. 6 pig lean meat sample and the No. 7 pig lean meat sample obtained by the boiling method were dropped on a CLE colloidal gold test card having a sample pad to which a urea peroxide solution was added at a mass fraction of 0.6%, and they were recorded as example 46, example 47, example 48, example 49, example 50, example 51 and example 52 in this order.
Control group: taking No. 1 pig lean meat sample extracting solution, No. 2 pig lean meat sample extracting solution, No. 3 pig lean meat sample extracting solution, No. 4 pig lean meat sample extracting solution, No. 5 pig lean meat sample extracting solution, No. 6 pig lean meat sample extracting solution and No. 7 pig lean meat sample extracting solution obtained by a boiling method, dropwise adding the extracting solutions onto a CLE colloidal gold detection card without carbamide peroxide on a sample pad, and sequentially marking as comparative example 46, comparative example 47, comparative example 48, comparative example 49, comparative example 50, comparative example 51 and comparative example 52.
And 3 experiments are performed in parallel, the samples are dripped on a CLE colloidal gold detection card sample pad and then timed, scanning is performed once at 5min, scanning is performed again at 20min, and the gray value is calculated and recorded. The results are shown in Table 13.
Table 13 various samples-urea peroxide added to sample pad for treatment of tissue extract versus colloidal gold assay
Color development effect of test card
The data in table 13 were processed to calculate the relative increase ratio of the C-line and T-line gray scale values for the test cards with urea peroxide added to the C-line and T-line gray scale values for the test cards without urea peroxide added. The results are shown in Table 14
TABLE 14 percent Gray value improvement after addition of a 0.6% Urea peroxide sample pad
As can be seen from tables 13 and 14, the addition of urea peroxide in an amount of 0.6% by mass to the sample pad relatively increased the T-ray gray values of seven different samples, with the percentage increase of 40% to 60% for most samples at 5min of development and 30% to 50% for most samples at 20min of development. Namely, the addition of 0.6% of carbamide peroxide has a promoting effect on the color development of CLE colloidal gold test cards (tissues).
12. Urea peroxide added to sample pad for processing influence of tissue extract on detection accuracy of colloidal gold detection card
Experimental groups: a No. 1 pork lean sample extract, a No. 2 pork lean sample extract, a No. 3 pork lean sample extract, a No. 1 beef sample extract, a No. 2 beef sample extract, a No. 3 beef sample extract, a No. 1 mutton sample extract, a No. 2 mutton sample extract, and a No. 3 mutton sample extract obtained by a water boiling method were added dropwise to a CLE colloidal gold test card having a sample pad to which a urea peroxide solution was added at a mass fraction of 0.6%, and the samples were recorded in this order as example 53, example 54, example 55, example 56, example 57, example 58, example 59, example 60, and example 61.
Control group: taking No. 1 pork lean sample extracting solution, No. 2 pork lean sample extracting solution, No. 3 pork lean sample extracting solution, No. 1 beef sample extracting solution, No. 2 beef sample extracting solution, No. 3 beef sample extracting solution, No. 1 mutton sample extracting solution, No. 2 mutton sample extracting solution and No. 3 mutton sample extracting solution obtained by a water boiling method, dropwise adding the extracting solutions on a CLE colloidal gold detection card without carbamide peroxide on a sample pad, and sequentially marking as a comparative example 53, a comparative example 54, a comparative example 55, a comparative example 56, a comparative example 57, a comparative example 58, a comparative example 59, a comparative example 60 and a comparative example 61.
Sample labeling group: preparing sample adding examples of 3ppb standard detection solution added corresponding to an experimental group and a control group, and dropwise adding the sample adding examples corresponding to the experimental group onto a CLE colloidal gold detection card added with a carbamide peroxide solution with the mass fraction of 0.6% on a sample pad; dripping the sample adding standard solution corresponding to the control group on a CLE colloidal gold detection card of which the sample pad does not contain carbamide peroxide;
3 experiments are performed in parallel, the samples are dripped on a CLE colloidal gold detection card sample pad for timing, scanning is performed once in 10min, the gray value is calculated and recorded, wherein the value of the sample added with a standard case is recorded as CSign boardAnd TSign board. The results are shown in Table 15.
Table 15 effect of urea peroxide addition to sample pad for treatment of tissue extract on accuracy of colloidal gold assay cards experimental results
| Name (R) | C0 | T0 | CSign board | CSign board | Judgment of yin and yang after adding mark |
| Comparative example 53 | 129 | 62 | 152 | 0.0 | Yang (Yang) |
| Comparative example 54 | 136 | 78 | 158 | 0.0 | Yang (Yang) |
| Comparative example 55 | 117 | 51 | 152 | 0.0 | Yang (Yang) |
| Comparative example 56 | 128 | 72 | 120 | 0.0 | Yang (Yang) |
| Comparative example 57 | 143 | 62 | 145 | 0.0 | Yang (Yang) |
| Comparative example 58 | 117 | 63 | 154 | 67.5 | Yin (kidney) |
| Comparative example 59 | 129 | 75 | 133 | 16.5 | Yin (kidney) |
| Comparative example 60 | 92 | 163 | 173 | 46.5 | Yin (kidney) |
| Comparative example 61 | 75 | 155 | 156 | 0.0 | Yang (Yang) |
| Example 53 | 175 | 84 | 193 | 0.0 | Yang (Yang) |
| Example 54 | 156 | 112 | 172 | 0.0 | Yang (Yang) |
| Example 55 | 140 | 68 | 154 | 0.0 | Yang (Yang) |
| Example 56 | 161 | 99 | 177 | 0.0 | Yang (Yang) |
| Example 57 | 179 | 80 | 197 | 0.0 | Yang (Yang) |
| Example 58 | 152 | 84 | 167 | 0.0 | Yang (Yang) |
| Example 59 | 142 | 116 | 156 | 0.0 | Yang (Yang) |
| Example 60 | 101 | 179 | 111 | 0.0 | Yang (Yang) |
| Example 61 | 99 | 166 | 109 | 0.0 | Yang (Yang) |
As can be seen from Table 15, in the examples, after the sample extract was dropped on the gold colloidal test card with the sample pad treated with urea peroxide, the C, T-line color tone value was improved, and the corresponding TSign boardAll the color development were 0, and all the results were positive, while in the comparative examples, the T values in comparative examples 58, 59 and 60 were determined after dropping the sample extract on the colloidal gold test card whose sample pad had not been treated with urea peroxide0Although there is color development, its corresponding TSign boardThe value of (A) shows a color development value larger than 0, and it cannot be determined whether or not an interfering substance is present, resulting inThe judgment of false negative is carried out, so that the method can improve the detection accuracy of the sample.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A tissue sample extracting solution processing method for improving color development of a colloidal gold detection card is characterized by comprising the following steps: the treatment method is to carry out oxidation treatment on the tissue sample extracting solution.
2. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 1, wherein the method comprises the following steps: the oxidation treatment method is to adopt an oxidant for treatment.
3. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 2, wherein the method comprises the following steps: the oxidant treatment mode is to add the oxidant into the tissue sample extracting solution or on the sample pad of the colloidal gold detection card.
4. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 2, wherein the method comprises the following steps: the oxidant is hydrogen peroxide or carbamide peroxide.
5. The method for treating the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 4, wherein the method comprises the following steps: the oxidant is hydrogen peroxide, and the treatment mode of the oxidant is to add the hydrogen peroxide solution into the tissue sample extracting solution; the oxidant is carbamide peroxide, and the mode of treating the oxidant is to add the carbamide peroxide solution into the tissue sample extracting solution or on the sample pad of the colloidal gold detecting card.
6. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 5, wherein the method comprises the following steps: the mass fraction of the hydrogen peroxide added into the tissue sample extracting solution is 0.03-3%; the mass fraction of the carbamide peroxide added into the tissue sample extracting solution is 0.1-3.2%, and the mass fraction of the carbamide peroxide added onto the sample pad of the colloidal gold detecting card is 0.4-6.4%.
7. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 6, wherein the method comprises the following steps: the mass fraction of the hydrogen peroxide added into the tissue sample extracting solution is 0.1-2%; the mass fraction of the carbamide peroxide added into the tissue sample extracting solution is 0.3 percent, and the mass fraction of the carbamide peroxide added on the sample pad of the colloidal gold detecting card is 0.6 percent.
8. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 5, wherein the method comprises the following steps: the reaction time of adding the hydrogen peroxide into the tissue sample extracting solution is 5-20 min; the reaction time of adding the carbamide peroxide to the tissue sample extracting solution is 5-30 s.
9. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 1, wherein the method comprises the following steps: the tissue sample is an animal tissue sample.
10. The method for processing the tissue sample extract solution for improving the color development of the colloidal gold test card according to claim 9, wherein: the tissue sample is extracted by water boiling or clamping baking.
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