Method for detecting hemozoin content in plasmodium falciparum and use thereof
Patent Information
- Application Number
- PCT/CN2025/147163
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-10-16
- Filing Date
- 2025-12-30
- Publication Date
- 2026-09-03
Smart Images
Figure CN2025147163_03092026_PF_FP_ABST
Abstract
Description
A method for detecting the pigment content of Plasmodium falciparum and its application Technical Field
[0001] This invention belongs to the field of Plasmodium falciparum pigment detection technology, specifically relating to a method for detecting the content of Plasmodium falciparum pigment and its application. Background Technology
[0002] With the widespread use of antimalarial drugs, their effectiveness against malaria is diminishing, making the development of new drugs and vaccines to address the health risks posed by Plasmodium parasites an urgent priority. The development of new drugs and vaccines requires screening suitable Plasmodium strains and candidate strains.
[0003] Existing methods for detecting Plasmodium pigment include Raman spectroscopy, absorption spectroscopy, polarization microscopy, and immunochromatography. Raman spectroscopy results in a weak Raman signal and relatively low detection sensitivity, potentially requiring longer detection times and higher laser power. The equipment is expensive, and the operation demands skilled operators. Absorption spectroscopy is relatively simple and inexpensive, suitable for preliminary screening of large-scale samples. However, its accuracy is significantly affected by interference from other substances in the sample, potentially requiring sample pretreatment to remove impurities. Polarization microscopy is relatively simple and easy to operate, but requires specialized microscope equipment and operators, and demands high standards for sample preparation and staining. Its detection speed is relatively slow, making it unsuitable for rapid screening of large-scale samples. Dark-field microscopy is relatively simple and easy to operate, but its sensitivity and accuracy are relatively low, and it requires high standards for sample preparation and staining. Flow cytometry can rapidly and accurately quantify Plasmodium pigment content, exhibiting high sensitivity and specificity, and can distinguish between different types of Plasmodium pigment. However, it requires specialized flow cytometers and operators, and the equipment is expensive. Immunochromatography is simple and rapid, suitable for rapid on-site screening. It has a lower cost and is suitable for preliminary testing of large-scale samples. However, its sensitivity and accuracy are relatively low, and false positive or false negative results may occur. Specific antibodies are required, and the quality and stability of the antibodies have a significant impact on the test results.
[0004] Current methods for detecting malaria pigment cannot simultaneously achieve high detection sensitivity, ease of operation, and simple equipment. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a method for detecting the pigment content of Plasmodium falciparum and its application.
[0006] The purpose of this invention is to provide a method for detecting the pigment content of Plasmodium falciparum, comprising the following steps:
[0007] Red blood cells infected with Plasmodium falciparum were resuspended in a solvent, saponin was added, mixed, allowed to stand, centrifuged, and the precipitate was collected to obtain a crude precipitate of Plasmodium falciparum. The crude precipitate of Plasmodium falciparum was centrifuged and the precipitate was collected to obtain a Plasmodium falciparum precipitate. The Plasmodium falciparum precipitate was resuspended in an SDS solution with a pH of 7.6 and a mass fraction of 1.5%–2.5%, and centrifuged repeatedly 2–3 times. The precipitate was collected until the absorbance at 400 nm in the supernatant was zero. The precipitate was collected to obtain a clean Plasmodium falciparum pigment precipitate. The clean Plasmodium falciparum pigment precipitate was dissolved in an SDS solution with a pH of 7.6 and a mass fraction of 1.5%–2.5% and a NaOH solution to obtain a Plasmodium falciparum pigment solution. The absorbance of the Plasmodium falciparum pigment solution at a wavelength of 400 nm was measured, a standard curve was prepared using heme chloride, and the content of Plasmodium falciparum pigment was calculated based on the standard curve.
[0008] Preferably, the solvent is PBS or physiological saline at 4°C to 10°C. The PBS is a phosphate buffer solution with a pH of 7.2 to 7.4.
[0009] Preferably, the volume ratio of the malaria parasite to the solvent is 1:5 to 10.
[0010] Preferably, the final concentration of the saponin is 0.14% to 0.16% by mass.
[0011] Preferably, the settling temperature is 4℃~10℃ and the settling time is 10min~20min.
[0012] Preferably, the conditions for high-speed centrifugation are 10000g to 12000g and centrifugation for 5min to 10min.
[0013] Preferably, the SDS solution contains 2% SDS by mass, and the SDS solution is prepared by dissolving SDS in a Tris-HCl buffer solution with a pH of 7.6 or less and a concentration of 0.5 mol / L to 1.5 mol / L to obtain the SDS solution.
[0014] Preferably, the number of repeated centrifugations is 2.
[0015] Preferably, the volume ratio of SDS solution to NaOH solution is 7-9:1, wherein the concentration of NaOH solution is 1.8 mol / L-2.2 mol / L. Too high or too low a concentration of the Plasmodium solution will lead to inaccurate detection results; the concentration of the Plasmodium solution should be adjusted as needed. Reducing the amount of SDS and NaOH solution can increase the concentration of the Plasmodium solution. Diluting with SDS solution can reduce the concentration of the Plasmodium solution.
[0016] Preferably, the absorbance value of the malignant plasmodium pigment solution at wavelength 400nm is adjusted to 0.5-0.8 by controlling the amount of SDS solution or NaOH solution. When the absorbance value of the malignant plasmodium pigment solution at wavelength 400nm is 0.5-0.8, the measurement result is accurate. If the absorbance value is greater than 1, the mother liquor should be diluted for measurement again so that the OD value is between 0.5 and 0.8.
[0017] Preferably, the calculation method is to bring the absorbance value of the malignant plasmodium pigment solution at wavelength 400nm into a standard curve equation to calculate the corresponding concentration of the plasmodium pigment solution.
[0018] Preferably, the standard curve is made according to the following method: 5mg of hematin chloride powder is dissolved in 1mmol / L NaOH solution, diluted with SDS solution with pH 7.6 and mass fraction 2% to obtain gradient concentrations, the absorbance value at 400nm is measured respectively, and the standard curve and the equation of the standard curve are obtained according to the absorbance value and the concentration of the SDS solution.
[0019] The second object of the present application is the application of the reagent for the detection method of the content of malignant plasmodium pigment in the detection of the content of malignant plasmodium pigment.
[0020] Compared with the prior art, the present application has the following beneficial effects:
[0021] 1. The method for detecting the content of Plasmodium falciparum pigment according to the present application, the red blood cells infected by Plasmodium falciparum are resuspended by a solvent, and saponin is added, mixed, and left to stand, and then centrifuged to collect the precipitate to obtain a crude Plasmodium falciparum precipitate. Saponin is added to ensure sufficient hemolysis, and when the supernatant is discarded, attention should be paid to retaining the precipitate, because the pigment molecules are small and easy to be lost. It is necessary to centrifuge sufficiently, and carefully discard the supernatant, and prevent sample loss. The Plasmodium falciparum precipitate is centrifuged at high speed, and the precipitate is collected to obtain a Plasmodium falciparum precipitate. The Plasmodium falciparum pigment precipitate is resuspended by a SDS solution with a mass fraction of 1.5% to 2.5% and a pH less than or equal to 7.6, and centrifuged repeatedly for 2 to 3 times, and the precipitate is collected until the absorbance value at 400 nm of the supernatant after centrifugation is zero, and the precipitate is collected to obtain a clean Plasmodium falciparum precipitate. The SDS solution can wash away the residual hemoglobin in the red blood cells in the solution, and remove the red blood cell residues, which can avoid the interference of the hemoglobin in the red blood cells. The pH of the SDS solution should not be greater than 7.6 when resuspending, otherwise the pigment will increase the dissolution and cause the measured value to be low. The attention should be paid to the number of times of washing by the SDS solution, too few times of washing by the SDS solution will cause the measured value to be high due to the residual hemoglobin. Too many times of washing by the SDS solution will cause the pigment in the sample to be lost and cause the measured result to be low. Therefore, the absorbance value at 400 nm of the supernatant should be detected after each centrifugation, and the washing is stopped when the absorbance value is zero. The clean Plasmodium falciparum precipitate is dissolved by adding a SDS solution and a NaOH solution to obtain a Plasmodium falciparum pigment solution. The absorbance value at 400 nm of the Plasmodium falciparum pigment solution is detected, and the content of the Plasmodium falciparum pigment is calculated. If the concentration of the Plasmodium falciparum pigment solution is too high, the solution can be diluted for measurement, and if the concentration is too low, the total amount of the system solution can be reduced in proportion.
[0022] The method for detecting the content of Plasmodium falciparum pigment according to the presentapplication can quickly detect the content of Plasmodium falciparum pigment, the result is accurate, and large and valuable equipment is not needed, the operation method is simple and easy to be accepted. The present application provides a method for detecting the content of pigment of a constructed or screened Plasmodium falciparum strain, which is used to determine the influence of Plasmodium falciparum strains with different pigment contents on the effectiveness and safety of vaccines.
[0023] 2. The method for detecting the content of Plasmodium falciparum pigment according tothe present application does not need Raman spectrum and magnetic separation technology, and most ordinary laboratories can screen and determine the content and pigment formation ability of Plasmodium falciparum by using conventional equipment. The method for detecting the content of Plasmodium falciparum pigment according to present application can be detected by conventional instruments such as Nano Drop or conventional spectrophotometer. It is particularly noted that the absorbance value at 400 nm is used for comparison in the present system, and the result is accurate. The method provided by the present application is accurate, sensitive, easy to operate, and has a wide range of applications. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 is a comparison of the morphology of wild-type P. falciparum and K13 mutant strains according to the present application. Wherein, A is wild-type P. falciparum. B is K13 mutant strain.
[0025] Figure 2 is a comparison of the pigment content of wild-type P. falciparum and K13 mutant strains according to the present application. Wherein, A is a comparison of the pigment content of wild-type P. falciparum and K13 mutant strains in alkaline SDS solution. B is a comparison of the pigment content of wild-type P. falciparum and K13 mutant strains.
[0026] Figure 3 is a comparison of the number of schizonts of wild-type P. falciparum and K13 mutant strains according to the present application.
[0027] Figure 4 is a graph of the absorbance of the pigment of P. falciparum at different wavelengths detected by spectrophotometry according to the present application.
[0028] Figure 5 is a graph of the effect of different pH SDS solutions on the measured values according to the present application.
[0029] Figure 6 is a graph of the effect of the number of washes on the results according to the present application.
[0030] Figure 7 is a graph of the analysis of the supernatant after washing according to the present application. DETAILED DESCRIPTION
[0031] In order to enable those skilled in the art to better understand the technical solutions of the present application and to implement them, the technical solutions in the embodiments of the present application are described clearly and completely below in conjunction with the preferred embodiments and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all of them. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts fall within the scope of protection of the present application.
[0032] It should be noted that all the professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the scope of protection of the present application. Unless otherwise specified, all the materials, reagents, instruments and equipment used in the following embodiments of the present application can be purchased from the market or prepared by existing methods.
[0033] Example 1
[0034] A method for detecting the pigment content of P. falciparum, comprising the following steps:
[0035] One mL of red blood cells infected with Plasmodium falciparum were resuspended in 5 mL of physiological saline to obtain a cell suspension. 3% saponin was added to bring the saponin content in the cell suspension to 0.15%. The mixture was stirred, allowed to stand at 4°C for 15 min, and then centrifuged at 12000g for 5 min. The precipitate was collected to obtain a crude precipitate of Plasmodium falciparum. The crude precipitate was centrifuged at 12000g for 5 min, and the precipitate was collected to obtain a Plasmodium falciparum precipitate. The precipitate was resuspended in a 2% SDS solution (pH 7.6), centrifuged at 12000g for 5 min, and this centrifugation was repeated twice. The absorbance at 400 nm in the supernatant was zero. The precipitate was collected to obtain a clean Plasmodium falciparum pigment precipitate. A 2% SDS solution and a 2 mol / L NaOH solution were added to the clean Plasmodium falciparum pigment precipitate at a volume ratio of 9:1 to dissolve the precipitate, obtaining a Plasmodium falciparum pigment solution. The absorbance of the Plasmodium falciparum pigment solution was diluted to 0.8% using a 2 mol / L NaOH solution at a wavelength of 400 nm, and the content of Plasmodium falciparum pigment was calculated based on the standard curve.
[0036] Example 2
[0037] A method for detecting the pigment content of Plasmodium falciparum includes the following steps:
[0038] One mL of red blood cells infected with Plasmodium falciparum were resuspended in 7 mL of physiological saline to obtain a cell suspension. 3% saponin was added to bring the saponin content in the cell suspension to 0.14%. The mixture was stirred, allowed to stand at 7°C for 10 min, and then centrifuged at 11000g for 7 min. The precipitate was collected to obtain a crude precipitate of Plasmodium falciparum. The crude precipitate was centrifuged at 11000g for 7 min, and the precipitate was collected to obtain a Plasmodium falciparum precipitate. The precipitate was resuspended in a 1.5% SDS solution (pH 7.4), centrifuged at 11000g for 7 min, and this centrifugation was repeated three times. The absorbance at 400 nm in the supernatant was zero. The precipitate was collected to obtain a clean Plasmodium falciparum pigment precipitate. A 2% SDS solution and a 1.8 mol / L NaOH solution were added to the clean Plasmodium falciparum pigment precipitate at a volume ratio of 8:1 to dissolve the precipitate, obtaining a Plasmodium falciparum pigment solution. The Plasmodium falciparum pigment solution was diluted with 2% SDS to an absorbance value of 0.6 at a wavelength of 400 nm, and the content of Plasmodium falciparum pigment was calculated according to the standard curve.
[0039] Example 3
[0040] A method for detecting the pigment content of Plasmodium falciparum includes the following steps:
[0041] One mL of red blood cells infected with Plasmodium falciparum were resuspended in 10 mL of physiological saline to obtain a cell suspension. 3% saponin was added to bring the saponin content in the cell suspension to 0.16%. The mixture was stirred, allowed to stand at 10°C for 20 min, and then centrifuged at 10000g for 10 min. The precipitate was collected to obtain a crude precipitate of Plasmodium falciparum. The crude precipitate was centrifuged at 10000g for 10 min, and the precipitate was collected to obtain a Plasmodium falciparum precipitate. The precipitate was resuspended in a 2.5% SDS solution (pH 7), centrifuged at 10000g for 10 min, and this centrifugation was repeated three times. The absorbance at 400 nm in the supernatant was zero. The precipitate was collected to obtain a clean Plasmodium falciparum pigment precipitate. A 2% SDS solution and a 2.2 mol / L NaOH solution were added to the clean Plasmodium falciparum pigment precipitate at a volume ratio of 7:1 to dissolve the precipitate, obtaining a Plasmodium falciparum pigment solution. The Plasmodium falciparum pigment solution was diluted with 2.2 mol / L NaOH to a absorbance value of 0.5 at a wavelength of 400 nm, and the content of Plasmodium falciparum pigment was calculated according to the standard curve.
[0042] Comparative Example 1
[0043] A method for detecting the pigment content of Plasmodium falciparum includes the following steps:
[0044] One mL of red blood cells infected with Plasmodium falciparum were resuspended in 5 mL of physiological saline to obtain a cell suspension. 3% (w / w) saponin was added to bring the saponin content in the cell suspension to 0.15%. The mixture was stirred, allowed to stand at 4°C for 5 min, and then centrifuged at 12000g for 5 min. The precipitate was collected to obtain a crude precipitate of Plasmodium falciparum. The crude precipitate was centrifuged at 12000g for 5 min, and the precipitate was collected to obtain a Plasmodium falciparum precipitate. The precipitate was resuspended in a 2% (w / w) SDS solution (pH 9), centrifuged at 12000g for 5 min, and this centrifugation was repeated three times. The absorbance at 400 nm in the supernatant was zero. The precipitate was collected to obtain a clean Plasmodium falciparum precipitate. A 2% (w / w) SDS solution and a 2 mol / L NaOH solution were added to the clean Plasmodium falciparum precipitate at a volume ratio of 9:1 to dissolve the precipitate, yielding a Plasmodium falciparum pigment solution. The absorbance of the Plasmodium falciparum pigment solution was diluted with NaOH solution to 0.5 at a wavelength of 400 nm, and the content of Plasmodium falciparum pigment was calculated according to the standard curve.
[0045] To illustrate the beneficial effects of the present invention, the following experiments were also conducted.
[0046] The pigment in Plasmodium falciparum is pathogenic; the higher the pigment content, the stronger the pathogenicity of the parasite. A wild-type Plasmodium falciparum strain is designated 3D7. WTIt is related to the K13 mutant strain C580Y, denoted as 3D7. C580Y The morphology is shown in Figure 1, the pigment content in Figure 2, and the number of merozoites in Figure 3. Wild-type Plasmodium falciparum strains have higher pigment content and contain more merozoites. The C580Y mutation in the K13 mutant strain leads to a reduction in the number of merozoites and weakened pathogenicity.
[0047] I. Materials and Equipment
[0048] 1. Experimental Materials
[0049] The main material used in this invention is saponin, with a purity of ≥95%, CAS number 8047-15-2, purchased from Sinopharm Group Pharmaceutical Co., Ltd.
[0050] II. Experimental Methods
[0051] 1. Wavelength scanning of Plasmodium falciparum pigment
[0052] To accurately detect the content of Plasmodium falciparum pigment, the absorbance of the pigment at different wavelengths was scanned to obtain the characteristic absorption wavelengths of the pigment. The experimental method is as follows: Plasmodium falciparum pigment was scanned at wavelengths of 350 nm to 450 nm using a UV-Vis spectrophotometer.
[0053] 2. Effects of SDS solutions with different pH values on the measurement values of Plasmodium falciparum pigment.
[0054] The Plasmodium falciparum precipitate was washed with 2% w / v SDS solutions at pH 7.6 and pH 9.0 respectively until the absorbance at 400 nm in the supernatant was zero. The precipitate was collected to obtain a clean Plasmodium falciparum precipitate. A 2% (w / v) SDS solution and a 2 mol / L NaOH solution were added to the clean Plasmodium falciparum precipitate at a volume ratio of 9:1 to dissolve the precipitate, yielding a Plasmodium falciparum pigment solution. The absorbance of the Plasmodium falciparum pigment solution at 400 nm was measured. If the OD value was greater than 0.8, further dilution was performed to a value between 0.5 and 0.8. The Plasmodium pigment content was calculated based on the absorbance value and a standard curve, and then multiplied back by the dilution factor.
[0055] 3. Effect of washing frequency on the pigment of Plasmodium falciparum
[0056] The Plasmodium falciparum precipitate was washed with 2% w / v SDS solution at pH 7.6 and pH 9.0, respectively, and centrifuged at 12000g for 5 min, repeated three times. After each centrifugation, the supernatant was collected and discarded. 100 μL of the precipitate from each wash was mixed with 100 μL of 2% w / v SDS solution and 10 μL of 2 mol / L NaOH solution to dissolve the precipitate, obtaining a Plasmodium pigment solution. The absorbance of the Plasmodium solution at 400 nm was measured. If the OD value was greater than 0.8, it was further diluted with NaOH solution to a value between 0.5 and 0.8. The Plasmodium pigment content was calculated based on the absorbance value and the standard curve, and then multiplied back by the dilution factor.
[0057] III. Experimental Results
[0058] 1. Wavelength scanning of Plasmodium falciparum pigment
[0059] Figure 4 shows the absorbance scan results of Plasmodium falciparum pigment at different wavelengths. The results indicate that the absorbance value of Plasmodium falciparum pigment reaches its maximum value within the scanning range of 350 nm to 450 nm, with the maximum value occurring at a wavelength of 400 nm. Therefore, the characteristic absorption wavelength of Plasmodium falciparum pigment is 400 nm. Thus, detecting the content of Plasmodium falciparum pigment at a wavelength of 400 nm yields the most accurate results and the most sensitive method.
[0060] 2. Effects of SDS solutions with different pH values on the measurement values of Plasmodium falciparum pigment.
[0061] Figure 5 shows the effect of SDS solutions with different pH values on the measurement values of Plasmodium falciparum pigment. The results show that after washing the Plasmodium falciparum precipitate with a 2% w / v SDS solution at pH 9.0, the absorbance of the Plasmodium falciparum pigment solution decreased. This indicates that an excessively high pH will wash away the Plasmodium falciparum pigment, resulting in a lower detection value. Therefore, the pH value of the SDS solution should be less than or equal to 7.6.
[0062] 3. Effect of washing frequency on the pigment of Plasmodium falciparum
[0063] The effect of washing frequency on the pigment of Plasmodium falciparum is shown in Figure 6. The absorbance of the supernatant after each washing and centrifugation is shown in Figure 7. The results show that with increasing washing frequency, the absorbance of the supernatant after centrifugation decreases, indicating that the Plasmodium falciparum precipitate is gradually washed away. However, the absorbance of the Plasmodium falciparum pigment solution also decreases with increasing washing frequency, indicating that the pigment in Plasmodium falciparum is lost during washing. Therefore, the number of washings should not be too high. The number of washings should be controlled within 3 times to effectively remove the Plasmodium falciparum precipitate while minimizing the loss of the pigment.
[0064] IV. Discussion of Results
[0065] In this invention's method for detecting Plasmodium falciparum pigment content, saponin is added to ensure sufficient hemolysis. When discarding the supernatant, care should be taken to retain the precipitate, as the pigment molecules are small and easily lost. Thorough centrifugation is necessary, and the supernatant should be carefully discarded to prevent loss of the Plasmodium falciparum pigment. When resuspending the precipitate in a 2% SDS solution with a pH less than or equal to 7.6, this serves two purposes: first, to wash away residual heme from red blood cells; second, to remove any remaining red blood cell residue. Otherwise, the results will be affected. Third, the pH should not exceed 7.6, otherwise the pigment will dissolve. This process should be repeated, ideally until the absorbance of the supernatant at 400 nm is zero according to the spectrophotometer. However, excessive repetition should be avoided, as it can dissolve the pigment and lead to lower measurement results. The Plasmodium falciparum pigment precipitate is completely dissolved in 2% SDS and 2 mol / L NaOH. The solution is then diluted with 2% SDS solution according to the concentration. It is important to note that high concentrations will result in inaccurate results; therefore, the concentration of the Plasmodium falciparum pigment solution needs to be adjusted.
[0066] This method can be used with conventional instruments, such as Nano Drop or a common spectrophotometer. It is particularly important to note that in this system, the absorbance value at 400 nm should be used for comparison to ensure accurate results. This method is simple to learn and operate, and is suitable for analysis and research in most ordinary laboratories.
[0067] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.
[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.
Claims
1. A method for detecting the pigment content of Plasmodium falciparum, characterized in that, Includes the following steps: Resuspend the erythrocytes infected with Plasmodium falciparum in a solvent, add saponin, mix well, let stand, centrifuge, and collect the precipitate to obtain a crude precipitate of Plasmodium falciparum. The coarse precipitate of Plasmodium falciparum was centrifuged and the precipitate was collected to obtain the Plasmodium falciparum precipitate. The Plasmodium falciparum precipitate was resuspended in an SDS solution with a pH of 7.6 or less and a mass fraction of 1.5% to 2.5%. The precipitate was centrifuged 2 to 3 times and collected. The precipitate was collected when the absorbance at 400 nm in the supernatant was zero. The precipitate was then collected to obtain clean Plasmodium falciparum pigment precipitate. Add SDS solution with a pH of ≤7.6 and a mass fraction of 1.5%–2.5% and NaOH solution to dissolve the clean Plasmodium falciparum pigment precipitate, and obtain Plasmodium falciparum pigment solution; The absorbance of the Plasmodium falciparum pigment solution at a wavelength of 400 nm was measured, a standard curve was prepared using heme chloride, and the content of Plasmodium falciparum pigment was calculated based on the standard curve.
2. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The volume ratio of the malaria parasite to the solvent is 1:5 to 10; wherein the solvent is physiological saline or phosphate buffer.
3. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The final concentration of the saponin is 0.14% to 0.16% by mass.
4. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The settling temperature is 4℃~10℃, and the time is 10min~20min.
5. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The centrifugation conditions were all 10000g~12000g, centrifuged for 5min~10min.
6. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The SDS solution has a mass fraction of 2%.
7. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The volume ratio of the SDS solution to the NaOH solution is 7–9:1, wherein the concentration of the NaOH solution is 1.8 mol / L–2.2 mol / L.
8. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The number of times the centrifugation was repeated was 2.
9. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The absorbance value of the Plasmodium falciparum pigment solution at a wavelength of 400 nm is adjusted to 0.5–0.8 by controlling the amount of SDS solution or NaOH solution used.
10. The application of the reagent used in the method for detecting the pigment content of Plasmodium falciparum according to claim 1 in the detection of the pigment content of Plasmodium falciparum.