CN116027035A - Kit for improving detection accuracy of HIV1/2 urine colloidal gold immunochromatography and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection and a preparation method thereof. The kit comprises: the test strip with the chromatographic membrane is provided with a detection line and a quality control line from left to right in sequence, the detection line is coated with protein II, and the left side of the detection line is provided with a filtering area coated with protein I. According to the invention, the filtering area is arranged at the left side of the detection line, substances which possibly interfere with the detection result in the sample to be detected are intercepted by utilizing the protein I on the filtering area, so that the false positive rate caused by the bridging action of the interfering substances is reduced, the antigens or antibodies coated on the detection line are not interfered by steric hindrance, the specific reaction capacity of the antigens or antibodies on the detection line is improved, the false negative rate is reduced, and the method has important significance for improving the accuracy of the human immunodeficiency virus HIV1/2 urine colloidal gold immunochromatography kit.

Description

Kit for improving detection accuracy of HIV1/2 urine colloidal gold immunochromatography and preparation method thereof

Technical Field

The invention belongs to the technical field of medicine detection, and particularly relates to a kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection and a preparation method thereof.

Background

The colloidal gold immunochromatography test strip detection technology is a detection technology which is widely applied at present, and the mechanism of action is as follows: the method comprises the steps of dripping a sample to be detected on a sample pad of a detection test strip, automatically moving a liquid sample to the front of the test strip by capillary chromatography, dissolving a colloidal gold-labeled reagent on the binding pad and reacting with each other in the moving process, and further moving to a region coated with a specific antigen or antibody, wherein if a target detection object exists in the sample, the conjugate of the target detection object and the gold-labeled reagent is trapped due to specific binding with the specific antigen or antibody, is accumulated on a detection line (T line), and can observe a color development result by naked eyes.

The colloidal gold immunochromatographic test strip detection technology has the advantages of low cost, convenient operation, no need of depending on special equipment and professionals in the operation process, and the like, and can be suitable for on-site rapid detection, so that the colloidal gold immunochromatographic test strip detection technology is also widely applied to various detection fields such as food, agricultural products, epidemic prevention and the like.

However, in the actual detection process, the specific antigen or antibody on the T-line may also bind to substances other than the target analyte in the sample to be detected and having a structure similar to the target analyte, and these are mainly based on the coincidence similarity and homology of amino acid sequences, which are difficult to avoid, so that when the colloidal gold immunochromatographic test strip is used for detection, interference of non-specific substances is unavoidable, and phenomena of false positive and false negative occur, resulting in inaccurate detection results, and many researches are conducted in order to avoid occurrence of non-specific reactions in the detection process in the prior art.

As in patent CN106290880a, a reagent composition for immunochromatography for canine coronavirus is provided, in which components such as a diluent, a chelating agent, a surfactant and the like are added to a treatment solution of a sample to dilute the sample, so as to reduce the concentration of an interfering antibody protein contained in the sample, and avoid the occurrence of false positive phenomenon caused by too high binding property of the antibody protein with T-wire protein due to too high concentration of the antibody protein. However, the method is characterized in that components such as a chelating agent, a surfactant, a diluent and the like are artificially added, so that the virus antibodies in the to-be-detected object are synchronously diluted while the to-be-detected object is diluted, so that the possible virus concentration in the to-be-detected object is low, the binding force with protein at a T line is poor, or the color development is not obvious due to the too low concentration, and the occurrence of false negative phenomenon is difficult to avoid.

In addition, patent CN107907679a discloses an immunochromatographic test strip with an independent quality control system, which adds an inert protein with a special label on a label compound, a monoclonal antibody capable of specifically binding with the special label carried on the inert protein is contained on a quality control line (C line), and the sensitivity is improved by improving the specificity of C line reaction, but in the preparation process of the test strip, not only the monoclonal antibody required by the test strip is required to be prepared, but also the coupling of the inert protein and the special label is required, and the operations increase the raw material cost of the kit and improve the complexity of the preparation of the kit.

In addition, since the human immunodeficiency virus HIV1/2 is detected by taking serum, urine, saliva of a human body as a sample, substances such as protein, creatinine, non-target antibody and the like contained in the sample may compete with the virus antibody and act on the T-line specific protein, so that the detection result is inaccurate, and particularly as shown in fig. 3, when the capture protein coated on the chromatographic membrane acts on the impurity proteins such as inert proteins and the like, the binding sensitivity of the capture protein on the target protein is reduced, so-called false negative phenomenon is caused; if the impurity protein interacts with the labeled complex and the capture protein on the T-line by bridging, as shown in fig. 4, it will still show a strong signal, i.e. a false positive, even if no target molecule is present.

Therefore, if a simple and practical mode can be adopted, nonspecific substances possibly combined with the T line antibody are removed when the sample to be detected reaches the left side of the T line, so that interference of the nonspecific substances on HIV1/2 detection by the HIV is avoided, and the method has important significance for improving the detection accuracy of an HIV1/2 (human immunodeficiency virus) urine colloidal gold immunochromatography detection test strip.

Disclosure of Invention

In order to solve the technical problems, the invention provides a kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection and a preparation method thereof.

The invention provides a kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection, which comprises the following components: the test strip with the chromatographic membrane is provided with a detection line and a quality control line from left to right in sequence, the detection line is coated with protein II, and the left side of the detection line is provided with a filtering area coated with protein I. "left to right" is defined as: the direction of flow of the sample to be tested.

The term "filtration zone" as used herein refers to a filtration zone constructed by the present invention, which essentially comprises a filtration zone constructed by the present invention, wherein specific proteins are coated thereon, and the purpose of adsorbing and trapping these impurity proteins is achieved by weak adsorption and binding of specific proteins to certain nonspecific substances in the sample to be tested, and thus, the filtration zone is named as "filtration zone" for the convenience of better understanding of the meaning of the present invention, and further, the filtration zone may be named as "filter strip", "filter belt", "filter line", etc. depending on the specific shape of the filtration zone, such as strip, line, etc.

For ease of understanding, the antigen or antibody protein/capture protein/specific antigen or antibody protein and the like coated on the T line (detection line) will hereinafter be simply referred to as protein ii, and the protein coated on the filtration zone will hereinafter be referred to as protein i.

The protein I is inactivated protein II; alternatively, the protein I is a protein which has similar physicochemical properties to the protein II but has no immunological specificity to the detection target; alternatively, the protein I is an inert protein which does not have the ability to specifically react with the substance to be detected.

The inert protein is at least one of bovine serum albumin, chicken egg albumin and casein.

The concentration of the protein I is less than or equal to the concentration of the protein II.

The filtering area is a filtering line which is identical to the detecting line in shape and size.

At least 1 filter line is arranged.

The distance from the filtering line to the detection line is 4 mm-6 mm, if the distance from the filtering line to the T line is too close or too far, impurity proteins in the sample are easy to reach the T line, and the impurity proteins are not adsorbed timely with protein I loaded by the filtering line, so that the purification is not thorough enough, and the filtering effect is influenced.

Preferably, when the number of the filter lines is 2, the types of the proteins I coated on the two filter lines are different.

Preferably, when the number of the filter lines is 2, the protein I coated on one filter line is inert protein or protein which has similar physical and chemical properties with the protein II coated on the T line and has no immunological specificity to the detection target; the other filter line is coated with protein I, which is T-line protein II inactivated and loses the ability to specifically react with the target substance.

Preferably, when the number of the filter lines is 2, the protein I coated on one filter line far away from the T line is inert protein or is similar to the protein II coated on the T line in physicochemical property, but has no immune specificity to the detection target; the protein I coated on the filter line which is nearer to the T line is the T line protein II which loses the ability of specifically reacting with the detection target substance after being inactivated.

The invention also provides a preparation method of the kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection, which comprises the steps of sequentially arranging a filtering area, a detection line and a quality control line on a chromatographic membrane of a test strip in the kit from left to right, coating protein II on the detection line, coating protein I on the filtering area, and drying the chromatographic membrane coated with the protein I; the protein I is inactivated protein II; alternatively, the protein I is a protein which has similar physicochemical properties to the protein II but has no immunological specificity to the detection target; alternatively, the protein I is an inert protein which does not have the ability to specifically react with the substance to be detected.

More specifically, the preparation method of the kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection is prepared by the following steps:

step S1, construction of a filtering area: the filtering area is a filtering line with the same shape and size as the detecting line, phosphate buffer solution is adopted to dilute the filtering line coated protein I, so that the concentration of the coated protein I on the filtering line is 1.0-3.0 mg/mL, then a scribing instrument is adopted to scribe a line at the position 4-6 mm in front of the detecting line of the chromatographic membrane, the line width is the same as the detecting line width, the scribing quantity is at least 1, and the construction of the filtering line is completed; the filter line, the detection line and the quality control line are sequentially distributed on the chromatographic membrane from left to right; preferably, the number of the filtering lines is 2;

s2, placing the chromatographic membrane subjected to membrane dividing in the step S1 in a vacuum drying oven, and drying at 40-60 ℃;

step S3, assembling a kit: and (3) assembling the chromatographic membrane dried in the step (S2) into a test strip, and further assembling into a kit.

In the above steps, the concentration of the phosphate buffer solution is 10-25 mM.

Preferably, the phosphate buffer solution has a concentration of 20 mM.

Further, the method provided by the invention has the following action principle: adding a filtering area in front of the T line and coating specific proteins on the filtering area, wherein the specific proteins coated on the filtering area entrap interfering substances in a sample to be detected before the interfering substances are not in non-specific binding with the protein II of the T line, so that the interfering substances are prevented from bridging between a signal marker and the protein II of the T line to cause false positive signals of the T line; meanwhile, the protein II coated on the T line is not interfered by steric hindrance, and can smoothly react with a substance to be detected, so that the possibility of false negative results is reduced to a great extent, and the detection accuracy and sensitivity are improved.

The invention has the beneficial effects that:

(1) According to the kit provided by the invention, the filtering area is arranged at the left side of the T line, namely 1-2 filtering lines are constructed, and the interference substances in the sample to be detected are retained before the interference substances are not contacted with the T line antigen/antibody by combining the protein I coated by the filtering lines with the non-target substances in the sample to be detected; meanwhile, the method plays a certain role in purifying the sample to be detected, on one hand, avoids false positive caused by non-specific impurity bridging signal markers and T-line capture proteins in the sample to be detected, and reduces the occurrence probability of false positive phenomena; on the other hand, the non-specific reaction of the interfering substance and the capture protein on the T line is avoided, so that the capture protein is not affected by steric hindrance, the capability of carrying out specific reaction with a detection target is improved, the probability of false negative results is reduced, and the detection accuracy and sensitivity are improved.

(2) According to the method provided by the invention, only the filtering area is arranged on the left side of the T line, namely 1-2 filtering lines are constructed, the proteins coated on the filtering lines are all common proteins, the preparation operation of the kit is simple, the manufacturing cost is low, and the popularization and the application of the kit are facilitated.

Drawings

FIG. 1 is a schematic diagram of a test strip chromatographic membrane prepared by the method of the invention; (a) is a schematic view of a chromatographic membrane provided with a filter line; (b) is a schematic view of a chromatographic membrane provided with two filter lines;

in fig. 2, the outer shape photographs of the kits in examples 1 to 4 are shown from left to right;

FIG. 3 is a simulated graph of the case of a false negative;

FIG. 4 is a simulated view of a false positive condition;

FIG. 5 is a schematic diagram of the kit of example 1 of the present invention.

Detailed Description

The present invention will now be further described in connection with specific embodiments in order to enable those skilled in the art to better understand the invention.

Example 1

Step S1, construction of a filtering area: f1 filter line, F2 filter line, detection line and quality control line are sequentially arranged on chromatographic membrane of test paper in the kit from left to right, protein II is coated on the detection line, and protein I is coated on the F1 filter line and the F2 filter line. The positional relationship among the F1 filter line, the F2 filter line, the T line, and the C line is shown in fig. 1 (b).

Wherein the distance from the F1 filter line to the T line is 6 mm, and the coating protein is heart-type fatty acid binding protein; the distance from the F2 filter line to the T line is 4 mm, the coated protein is an antigen protein with the immune activity removed, and the widths of the F1 filter line and the F2 filter line are the same as the width of the T line.

In this example, the core fatty acid binding protein and the immunocompetent HIV1/2 antigen protein were diluted with 20 mM phosphate buffer so that the concentration of the protein coated on both filter lines was the same as that of the protein coated on the T line, and then the protein was coated on both filter lines on the left side of the T line of the chromatographic membrane using a membrane-dividing instrument.

S2, placing the chromatographic membrane subjected to membrane dividing in the step S1 in a vacuum drying oven, and drying at the temperature of 55 ℃;

step S3, assembling a kit: assembling the chromatographic membrane dried in the step S2 into a test strip, and further assembling into a kit;

and S4, detecting 300 samples, and simultaneously, corroborating by an elisa method, and calculating the false positive rate, the false negative rate and the accuracy rate. The detection results are shown in the following table 1.

The principle of the kit obtained in this example in use is shown in FIG. 5. The appearance of the kit in example 1 is shown as 64 in FIG. 2.

Table 1 results of testing 300 samples using the example 1 kit

According to the detection results in table 1, the false positive rate, false negative rate and accuracy rate conditions of the method are calculated, and the calculation results are as follows:

false positive rate=0/(291+9) ×100% =0;

false negative rate=0/(291+9) ×100% =0;

accuracy= (291+9)/(291+9) ×100% =100%.

According to the embodiment, two filter wires are constructed on the left side of the T wire, impurity components and non-specific interfering substances in a sample to be detected are intercepted by the two filter wires, and when the finally prepared kit is applied to detection of HIV1/2 urine samples, the accuracy is extremely high, no false negative or false positive condition occurs, and the accuracy reaches 100%.

Example 2

Step S1, construction of a filtering area: constructing 1 filter line;

diluting Deoxyribonucleoprotein (DNP) with 20 mM phosphate buffer to make the concentration of DNP protein be 1.8 mg/mL, and coating the protein on a filter line on the left side of a T line of a chromatographic membrane by using a membrane dividing instrument, wherein the distance from the filter line to the T line is 5 mm, and the width of the filter line is the same as that of the T line;

the operations of steps S2-S4 are the same as in example 1.

The positional relationship among the filter line, T line and C line is shown in (a) of FIG. 1. The appearance of the kit in example 2 is shown as number 65 in FIG. 2.

The detection results of the kit prepared in this example applied to HIV1/2 urine samples are shown in Table 2 below.

TABLE 2 results of detection of 300 samples using the example 2 kit

According to the detection results in Table 2, the false positive rate, false negative rate and accuracy rate conditions of the method are calculated, and the calculation results are as follows:

false positive rate=1/(20+280) ×100% =0.33%;

false negative rate=0/(20+280) ×100% =0;

accuracy = (280+19)/(280+20) ×100% = 99.67%.

The main reason why the false positive rate can be obtained is that the left filtering line of the T line is constructed, DNP protein loaded on the filtering line is a protein in a special form commonly existing in various biological cell nuclei, and the existence of the protein can be combined with most of proteins in a detection sample, especially the proteins in human urine, which possibly interfere with the detection result, so that a good filtering effect is achieved, the interference of the non-specific substances on the detection is avoided, the sample is purer after the filtering, the effect of the sample and the T line is more obvious after the detection sample reaches the T line, and the detection sensitivity and accuracy are synchronously improved.

Example 3

Step S1, construction of a filtering area: the filtering area is a filtering line with the same shape and size as the detection line, phosphate buffer solution of 20 mM is adopted to dilute the yellow meal worm fibrinolytic active protein (TFP) to ensure that the concentration of the TFP protein is 1.5 mg/mL, then a film dividing instrument is adopted to coat the protein on the filtering line before the T line of the chromatographic film, wherein the distance from the filtering line to the T line is 5 mm, the width of the filtering line is the same as the width of the T line, the number of the filtering lines is 1, and the construction of the filtering area is completed;

wherein, the TFP protein adopted in the steps is given by Nanjinouzan biotechnology limited company;

otherwise, the operations of steps S2-S4 are the same as in example 1.

The appearance of the kit in example 3 is shown as 66 in FIG. 2.

The detection results of the prepared kit applied to human immunodeficiency virus HIV1/2 urine samples are shown in the following table 3.

TABLE 3 results of detection of 300 samples using the example 3 kit

According to the detection results in Table 3, the false positive rate, false negative rate and accuracy rate conditions of the method are calculated, and the calculation results are as follows:

false positive rate=0/(27+273) ×100% =0;

false negative rate=1/(272+28) ×100% =0.33%;

accuracy = (272+27)/(273+27) ×100% = 99.67%.

From the detection results of the embodiment, it can be seen that the filter line is constructed by adopting TFP protein, so that a better effect can be achieved, and the prepared kit is applied to the detection of HIV1/2 urine samples, and the detection accuracy is up to 99.67%.

Comparative example 1

And S1, coating T lines and C lines on the chromatographic membrane according to a conventional process, and constructing no filtering area.

The operations of steps S2-S4 are the same as in example 1, and the detection results are shown in Table 4 below.

Table 4 results of testing 300 samples using the comparative example 1 kit

The false positive rate, false negative rate and accuracy rate of the method of the present invention were calculated from the detection results in table 4, and the calculation results are as follows:

false positive rate=5/(268+32) ×100% =1.67%;

false negative rate=3/(268+32) ×100% =1%;

accuracy= (265+27)/(273+27) ×100% = 97.33%.

For ease of understanding, the invention is briefly described herein:

in this comparative example, 300 urine samples provided by public health departments were selected for detection, wherein 30 positive samples were found and 270 negative samples were found.

When the kit in comparative example 1, namely the traditional kit, is used for detection, 32 positive samples are detected, wherein 3 false positives are detected; simultaneously measuring 268 parts of negative samples and 1 part of false negative;

i.e. the actual number of positive samples is: 32 (positive) -3 (false positive) +1 (false negative) =30 (positive); the actual number of negative samples is: 268 (negative) -1 (false negative) +3 (false positive) =270 (negative).

It is apparent that in the comparative example 1, the conventional kit is adopted to detect the sample, the left side of the T line is not provided with a filter line, under the same test conditions, the false positive rate and the false negative rate are both increased, even the false positive rate is up to 1.66%, the detection accuracy is only 97.33%, the detection accuracy of the kit is greatly reduced, and once the number of the detected samples is increased, the number of misdiagnosis caused by the conventional kit is greatly increased, so that the detection of HIV1/2 urine samples is not facilitated.

Comparative example 2

The experimental protocol was the same as that of comparative example 1, and only the sample to be tested was changed, and the test results were as shown in Table 5 below.

Table 5 results of testing 300 samples using the comparative example 2 kit

The false positive rate, false negative rate and accuracy rate of the method of the invention are calculated according to the detection results in table 5, and the calculation results are as follows:

false positive rate=3/(288+12) ×100% =1%;

false negative rate=2/(288+12) ×100% =0.67%;

accuracy= (286+9)/(288+12) ×100% =98.33%.

As can be seen from the detection data of the above examples 1-3 and comparative examples 1-2, the colloidal gold immunochromatography detection kit for HIV1/2 urine prepared by the method has extremely high accuracy in detecting samples, the range of false positive rate and false negative rate is only 0.33%, and the detection accuracy is as high as 99.67%.

If the detection test strip is not processed by the method of the invention, as described in comparative examples 1-2, the values of the false positive rate and the false negative rate are increased and even reach 1.66%, and the accuracy of detection of HIV1/2 urine samples of human immunodeficiency virus is lower than 99%, even the lowest is only 97.33%.

The experiments prove that the method has good effect on improving the accuracy of the human immunodeficiency virus HIV1/2 urine colloidal gold immunochromatography detection kit.

In addition, in order to further verify the method provided by the invention, the inventor also performs experiments in which the experimental results of examples 4-5 are good, and the false positive rate and the false negative rate can be kept at a low level within 0.5%.

Example 4

The distinction is only made in the construction process of the filtering area in step S1, specifically:

diluting DNP protein by using 10 mM phosphate buffer solution to make the concentration of DNP protein be 1.0 mg/mL;

the subsequent operations of step S1 and steps S2-S4 are the same as those of example 1.

The appearance of the kit in example 4 is shown as 67 in FIG. 2.

When the improved kit of the embodiment is used for detecting HIV1/2 urine samples, the false positive rate and the false negative rate can be kept at lower levels and still are smaller than 0.5%, but the color depth of individual samples is insufficient, the color of some samples is lighter, the detection sensitivity is not as high as that of the kit of the embodiment 1, and the main reason is presumed to be that: the concentration of the coating protein on the filter line was too low and its trapping ability for nonspecific substances contained in the sample was limited, so that the sensitivity was slightly different from that of the kit of example 1.

Example 5

The distinction is only made in the construction process of the filtering area in step S1, specifically:

diluting DNP protein by using a phosphate buffer solution of 20 mM to ensure that the concentration of DNP protein is 3.0 mg/mL;

the subsequent operations of step S1 and steps S2-S4 are the same as those of example 1.

The kit prepared in this example has almost the same effect as that achieved by the kit of example 1, but the preparation cost of the kit is definitely greatly increased on the basis of the excessively high concentration of the coating protein I, and the kit is unfavorable for the industrialized production of the kit under the same conditions, so that the filtering area is not coated with the protein I with such high concentration.

Claims (9)

1. A kit for improving the accuracy of an HIV1/2 urine colloidal gold immunochromatographic assay, comprising: the test strip with chromatographic membrane, there are detection line and quality control line sequentially from left to right on the chromatographic membrane, there are protein II on the detection line, characterized by that, there are filtering areas coated with protein I on the left side of the detection line; the protein I is inactivated protein II; alternatively, the protein I is a protein which has similar physicochemical properties to the protein II but has no immunological specificity to the detection target; alternatively, the protein I is an inert protein which does not have the ability to specifically react with the substance to be detected.

2. The kit for improving the accuracy of colloidal gold immunochromatographic assay of HIV1/2 urine according to claim 1, wherein the inert protein is at least one of bovine serum albumin, chicken ovalbumin and casein.

3. The kit for improving the accuracy of colloidal gold immunochromatographic assay of HIV1/2 urine according to claim 1, wherein the concentration of protein I is less than or equal to the concentration of protein II.

4. The kit for improving the accuracy of the colloidal gold immunochromatographic assay for HIV1/2 urine according to claim 1, wherein the filtering area is a filtering line which has the same shape and size as the detecting line.

5. The kit for improving the accuracy of a colloidal gold immunochromatographic assay for HIV1/2 urine according to claim 4, wherein the number of the filter lines is at least 1.

6. The kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatographic assay according to claim 4, wherein the distance from the filter line to the detection line is 4 mm-6 mm.

7. A preparation method of a kit for improving the accuracy of HIV1/2 urine colloidal gold immunochromatography detection is characterized in that a filtering area, a detection line and a quality control line are sequentially arranged on a chromatographic membrane of a test strip in the kit from left to right, a protein II is coated on the detection line, a protein I is coated on the filtering area, and the chromatographic membrane coated with the protein I is dried; the protein I is inactivated protein II; alternatively, the protein I is a protein which has similar physicochemical properties to the protein II but has no immunological specificity to the detection target; alternatively, the protein I is an inert protein which does not have the ability to specifically react with the substance to be detected.

8. The method for preparing the kit for improving the accuracy of the colloidal gold immunochromatographic assay of the HIV1/2 urine according to claim 7, which is prepared by the following steps:

step S1, construction of a filtering area: the filtering area is a filtering line which is identical to the shape and the size of the detection line, and the filtering line, the detection line and the quality control line are sequentially distributed on the chromatographic membrane from left to right; diluting the coating protein I of the filter line by using a phosphate buffer solution to ensure that the concentration of the coating protein I on the filter line is 1.0-3.0 mg/mL, and then scribing at the position 4-6 mm in front of a chromatographic membrane detection line by using a scribing instrument, wherein the line width is the same as the detection line width, the scribing quantity is at least 1, and the construction of a filter region is completed;

s2, placing the chromatographic membrane subjected to membrane dividing in the step S1 in a vacuum drying oven, and drying at 40-60 ℃;

step S3, assembling a kit: and (3) assembling the chromatographic membrane dried in the step (S2) into a test strip, and further assembling into a kit.

9. The method for preparing the kit for improving the accuracy of the colloidal gold immunochromatographic assay of HIV1/2 urine according to claim 8, wherein the number of the filter lines is 2.

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