Method for detecting parasite killing capability of fiber fabric

Abstract

The invention discloses a method for detecting parasite killing capability of a fiber fabric, which comprises the following steps: (1) pretreating the fiber fabric to remove biological interference factors; (2) preparing a plurality of groups of standard parasite liquid by a gradient dilution method, drawing a standard curve, and determining the optimal parasite liquid concentration; (3) respectively soaking the fiber fabric and the contrast fabric in the parasite liquid with the optimal parasite liquid concentration, and carrying out shaking culture; (3) after culture is finished, taking an insect liquid sample, diluting, preparing a qPCR reaction system, and carrying out PCR amplification; and (4) calculating the parasite load value in the parasite liquid sample by adopting a delta delta CT method, including the parasite liquid sample for soaking the fiber fabric and the parasite liquid sample for soaking the contrast fabric, and calculating the killing rate. According to the method, the oscillation method and the qPCR method are creatively combined, the parasite killing rate of the fiber fabric is visually calculated according to the load difference value of the calibration genes of the experiment sample and the control sample, and the parasite killing effect of the fiber fabric is more accurately judged from the perspective of the genes.

Description

Technical Field

[0001] This invention belongs to the field of testing technology for the parasite-killing ability of fiber fabrics, specifically relating to a method for testing the parasite-killing ability of fiber fabrics. Background Technology

[0002] With societal development, people are increasingly in contact with their environment. Whether working outdoors, traveling, or keeping pets at home, we are constantly in contact with various invisible parasites. These close encounters can lead to potential risks—parasitic infections. We may come into contact with unknown substances containing parasites in the external environment, or at home, through contact with sofas, beds, or pets. People consider wearing certain fabrics to protect themselves from these risks. However, are these fabrics, such as clothing and sofa upholstery, truly effective against parasites? How can we test whether these fibers kill parasites? Currently, there aren't many relevant testing methods available on the market for insect repellency. Some methods test the repellency properties of the material, while others use a dissecting microscope to directly observe the number of live insects remaining after the actual fiber fabric comes into contact with the insect. However, these methods are only suitable for fabrics with odors or containing leaching insecticides, and there will be errors in counting. Some methods use vat dyes to stain the parasite's metabolic products, and then use a spectrophotometer or ELISA reader to measure the absorbance to roughly determine the number of live insects. These methods are also subject to unstable factors such as the toxicity of reaction additives to cells, the limitations of light on detection conditions, and the easy loss of cells during separation after culture in the culture medium, which will more or less cause inaccurate test calculations. Summary of the Invention

[0003] Purpose of the invention: In order to solve the problems existing in the prior art, the purpose of this invention is to provide a method for detecting the parasite-killing ability of fiber fabrics, which can significantly improve the accuracy of the detection method for the parasite-killing ability of fiber fabrics.

[0004] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0005] A method for detecting the parasite-killing ability of fiber fabrics includes the following steps:

[0006] (1) Pretreatment of fiber fabric to remove biological interference factors;

[0007] (2) Prepare several groups of parasite solutions using the gradient dilution method, plot a standard curve, and determine the optimal concentration of the solution;

[0008] (3) The fiber fabric and the control fabric were respectively immersed in the parasite solution with the optimal concentration of the insect solution and cultured by shaking;

[0009] (4) After the culture is completed, take insect liquid samples, dilute them, prepare qPCR reaction system, and perform PCR amplification;

[0010] (5) The parasite load in the insect fluid samples was calculated using the ΔΔCT method, including insect fluid samples soaked in fiber fabric and insect fluid samples soaked in control fabric, and the kill rate was calculated.

[0011] As a specific implementation plan, in step (1), the pretreatment includes high-pressure steam sterilization or ultraviolet irradiation.

[0012] Preferably, the conditions for the high-pressure steam sterilization treatment are: 121±5℃, 0.1±0.02MPa, 30±2min; and the ultraviolet sterilization irradiation time is 1.5-2.5h.

[0013] As a specific implementation plan, in step (2), the method for selecting the concentration of the parasite fluid includes: preparing a standard parasite fluid, diluting it 5 to 6 times in a 10-fold gradient, creating a standard curve, determining that the activity of the parasite fluid meets the requirements by the amplification factor and the slope of the curve, and selecting the concentration value closest to the standard according to the standard curve.

[0014] As a specific implementation plan, in step (3), the parasite is selected from Toxoplasma gondii, and the concentration of the parasite fluid is 10. 5 pcs / ML.

[0015] As a specific implementation plan, in step (3), the control fabric is selected from fiber fabrics that have no ability to kill parasites.

[0016] As a specific implementation plan, in step (3), the conditions for the shaking culture are: temperature 30±37℃, 100~120rpm, shaking reaction for 10-14h.

[0017] As a specific implementation plan, in step (4), the qPCR reaction system includes insect fluid sample, upstream primer, downstream primer, buffer solution and enzyme-free water.

[0018] As a specific implementation plan, the PCR amplification conditions in step (4) are as follows:

[0019] Stage 1: 95℃, 5min;

[0020] Stage 2: 95℃, 30s; 55℃, 30s; 72℃, 60s; (40 cycles)

[0021] Stage 3: 72℃, 5min;

[0022] Stage 4: 25℃, 5min.

[0023] As a specific implementation plan, in step (5), the parasite load of the insect liquid sample soaked in the fiber fabric is set as the experimental group load, and the parasite load of the insect liquid sample soaked in the control fabric is set as the control group load. The killing rate is (control group load - experimental group load) / control group load * 100%.

[0024] Beneficial effects: Compared with existing technologies, this invention is based on the specific properties of fiber fabrics that kill parasite growth. It innovatively combines the shaking method with the qPCR method, selects the optimal concentration of insect solution and shakes the fiber fabric for a certain period of time, and then uses the qPCR amplification reaction to intuitively calculate the killing rate of the fiber fabric against parasites from the difference in the labeled gene load between the experimental sample and the control sample. This allows for a more accurate assessment of the killing effect of the fiber fabric on parasite growth from a genetic perspective. Attached Figure Description

[0025] Figure 1 This is the standard curve for Toxoplasma gondii worm fluid. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example

[0028] 1. Testing the ability of fiber fabrics to kill Toxoplasma gondii:

[0029] 1.1 Experimental Materials:

[0030] 1.1.1 Equipment: Vortex mixer, handheld centrifuge, pipettes, sealing film maker, microplate centrifuge, PCR reaction plate, qPCR sealing film, centrifuge tubes, filter cartridges and pipette tips, qPCR instrument, clean bench;

[0031] 1.1.2 Reagents: 2X Universal SYBR Green Fast qPCR Mix, Water Nuclease-Free, Anhydrous Ethanol, Gene Primers (Upstream + Downstream), Single-Stranded DNA Fragments, TaqMan Universal Premix;

[0032] 1.1.3 Experimental samples: Toxoplasma gondii protozoan solution of known concentration, experimental fiber fabric, and control fiber fabric;

[0033] 1.2 Experimental Procedure:

[0034] 1.2.1. Take a weight of 200~250g / cm³. 2 The experimental cotton insect-resistant fabric has a weight of 200-250 g / cm³. 2 The pure cotton control fabric was subjected to UV sterilization for 2 hours and then turned over after 30 minutes.

[0035] 1.2.2 qPCR treatment (operated in a clean bench):

[0036] 1.2.2.1, with 10 1 ~10 6 A standard curve was established using six 10-fold dilution gradients (copy / μL) to determine the optimal concentration (ideal slope of 100%, corresponding to a slope of -3.32; acceptable range of 90%–110%, corresponding to a slope of -3.58–-3.10). Within this range, the standard curve is considered efficient and reliable, with R0. 2 Ideally, the value should be ≥0.99. The closer this value is to 1, the more reliable the standard curve and the more accurate the gene expression. Figure 1 As shown: These six values ​​are basically on a straight line. Substituting the corresponding concentrations into the standard curve formula confirms the unique optimal concentration value. The slope in this case is -3.4117, R0. 2 The value was 0.9982, and the optimal concentration of the Toxoplasma gondii worm solution was 10. 5 pcs / ML;

[0037] 1.2.2.2, Prepare 10 5 5 mL of Toxoplasma gondii solution per mL, and experimental groups (weight 200-250 g / cm³) were added. 2 Pure cotton insect-resistant fabric (1cm*1cm) compared with control group (weight 200~250g / cm) 2 A 1cm*1cm pure cotton fabric was soaked in a Toxoplasma gondii solution at a temperature of 30±37℃ and a shaking reaction of 100~120rpm for 12 hours.

[0038] 1.2.2.3. Dilute the sample in test buffer solution and prepare qPCR reaction in a 96-well optical plate using TaqMan universal premix. The reaction system is shown in the table below:

[0039]

[0040] The sequences of the above gene primers are shown below:

[0041] Upstream primer: GGAGACGGAGCCAGAT;

[0042] Downstream primer: GCACCCATACCAACAGC.

[0043] 1.2.2.4. Perform PCR amplification on a qPCR instrument:

[0044] Stage 1: 95℃, 5min;

[0045] Stage 2: 95℃, 30s; 55℃, 30s; 72℃, 60s; (40 cycles)

[0046] Stage 3: 72℃, 5min;

[0047] Stage 4: 25℃, 5min;

[0048] 1.3 Result Processing:

[0049] 1.3.1 ΔΔCT method:

[0050] A = CT(target gene, experimental sample) - CT(internal control gene, experimental sample);

[0051] B = CT (target gene, control sample) - CT (internal standard gene, control sample).

[0052] K=AB

[0053] Expression multiple = 2 -K

[0054] 2. Results

[0055] The test results are shown in the table below:

[0056]

[0057] The results showed that the CT values ​​of the internal reference genes in the experimental and control samples were within the ideal range (around 20-25), the PCR amplification efficiency was very good, and the accuracy of the killing rate of Toxoplasma gondii calculated by the difference in load values ​​was very high. The pure cotton insect-resistant fabric in this experiment had a very good killing effect on Toxoplasma gondii.

[0058] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method of testing the ability of a fibrous fabric to kill parasites, characterized in that, The method comprises the following steps: (1) pretreatment of the fiber fabric to remove biological interference factors; (2) preparing a plurality of groups of parasite solutions by gradient dilution method, drawing a standard curve, and determining the optimal solution concentration; (3) immersing the fiber fabric and the control fabric in the parasite solution with the optimal solution concentration respectively, and oscillating and culturing; (4) after the culture, taking the solution sample, diluting, preparing a qPCR reaction system, and performing PCR amplification; (5) calculating the parasite load value in the solution sample by using the ΔΔCT method, including the parasite load value in the solution sample immersed in the fiber fabric and the parasite load value in the solution sample immersed in the control fabric, and calculating the killing rate.

2. The method of testing the ability of a fibrous fabric to kill parasites according to claim 1, characterized in that, In step (1), the pretreatment comprises high-pressure steam sterilization treatment or ultraviolet sterilization irradiation.

3. The method of testing the ability of a fibrous fabric to kill parasites according to claim 2, wherein, The high-pressure steam sterilization treatment is performed at 121±5℃, 0.1±0.02MPa, and for 30±2min; and the ultraviolet sterilization irradiation is performed for 1.5-2.5h.

4. The method of testing the ability of a fibrous fabric to kill parasites according to claim 1, wherein, In step (2), the method for determining the optimal solution concentration comprises the following steps: preparing a standard parasite solution, diluting 5-6 gradients by 10 times, drawing a standard curve, determining the activity of the parasite solution to meet the requirements through the amplification multiple and the curve slope, and selecting the concentration value closest to the standard according to the standard curve.

5. The method of testing the ability of a fibrous fabric to kill parasites according to claim 1, wherein, In step (3), the parasite is selected from Toxoplasma gondii and the concentration of the parasite is 10 5 organisms / ML.

6. The method of testing the ability of a fibrous fabric to kill parasites of claim 1, wherein, In step (3), the control fabric is selected from a fiber fabric without parasite killing ability.

7. The method of testing the ability of a fibrous fabric to kill parasites according to claim 1, wherein, In step (3), the oscillating culture is performed at 30±37℃, 100-120rpm, and for 10-14h.

8. The method of testing the ability of a fibrous fabric to kill parasites of claim 1, wherein, In step (4), the qPCR reaction system comprises the solution sample, an upstream primer, a downstream primer, a buffer solution, and enzyme-free water.

9. The method of testing the ability of a fibrous fabric to kill parasites of claim 1, wherein, In step (4), the PCR amplification is performed under the following conditions: Stage 1: 95℃, 5min; Stage 2: 95℃, 30s; 55℃, 30s; 72℃, 60s; (40 cycles) Stage 3: 72℃, 5min; Stage 4: 25℃, 5min.

10. The method of testing the ability of a fibrous fabric to kill parasites according to claim 1, wherein, In step (5), the parasite load of the solution sample immersed in the fiber fabric is set as the experimental group load, the parasite load of the solution sample immersed in the control fabric is set as the control group load, and the killing rate=(control group load-experimental group load) / control group load*100%.

Images