Plasmodium falciparum pigment content detection method and application thereof

Abstract

The invention belongs to the technical field of plasmodium falciparum pigment detection, and particularly relates to a plasmodium falciparum pigment content detection method and application thereof. The plasmodium falciparum pigment content detection method comprises the following steps: resuspending erythrocytes infected by plasmodium falciparum by using a solvent, adding saponin, uniformly mixing, standing, centrifuging, and collecting precipitate to obtain plasmodium falciparum coarse precipitate. And centrifuging the plasmodium falciparum coarse precipitate, and collecting the precipitate to obtain the plasmodium falciparum precipitate. And re-suspending the plasmodium falciparum precipitate by using an SDS solution, repeatedly centrifuging and collecting the precipitate until the absorbance value at 400nm in the centrifuged supernatant is zero, collecting the precipitate, adding the SDS solution and a NaOH solution for dissolving, detecting the absorbance value at the wavelength of 400nm, and calculating to obtain the content of the plasmodium falciparum pigment. The plasmodium falciparum pigment content detection method does not need complex technologies such as Raman spectrum and magnetic separation, and the pigment level and forming ability of plasmodium can be rapidly screened and determined.

Description

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: 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 again, 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.

[0007] 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.

[0008] Preferably, the volume ratio of the malaria parasite to the solvent is 1:5~10.

[0009] Preferably, the final concentration of the saponin is 0.14% to 0.16% by mass.

[0010] Preferably, the settling temperature is 4℃~10℃ and the settling time is 10min~20min.

[0011] Preferably, the high-speed centrifugation conditions are 10000g~12000g, centrifugation for 5min~10min.

[0012] 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.

[0013] Preferably, the number of repeated centrifugations is 2.

[0014] 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 increases the concentration of the Plasmodium solution. Diluting with SDS solution decreases the concentration of the Plasmodium solution.

[0015] Preferably, 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. When the absorbance value of the Plasmodium falciparum pigment solution at a wavelength of 400 nm is 0.5~0.8, the measurement result is accurate. If the absorbance value is greater than 1, the stock solution should be diluted before measurement to bring the OD value between 0.5~0.8.

[0016] Preferably, the calculation method involves substituting the absorbance value of the Plasmodium falciparum pigment solution at a wavelength of 400 nm into the standard curve equation to calculate the corresponding concentration of the Plasmodium falciparum pigment solution.

[0017] Preferably, the standard curve is prepared by the following method: 5 mg of heme chloride powder is dissolved in 1 mmol / L NaOH solution, diluted with 2% SDS solution at pH 7.6 to form a gradient concentration, and the absorbance value is measured at 400 nm. The standard curve and its equation are prepared based on the absorbance value and the concentration of SDS solution.

[0018] The second objective of this invention is the application of a reagent used in a method for detecting the pigment content of Plasmodium falciparum in the detection of the pigment content of Plasmodium falciparum.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The method for detecting the pigment content of Plasmodium falciparum of this invention involves resuspending Plasmodium falciparum-infected red blood cells in a solvent, adding saponin, mixing, allowing to stand, centrifuging, and collecting the precipitate to obtain a crude precipitate of Plasmodium falciparum. Adding saponin ensures sufficient hemolysis. When discarding the supernatant, care should be taken to retain the precipitate, as pigment molecules are small and easily lost. Thorough centrifugation is necessary, and the supernatant should be carefully discarded to prevent sample loss. The crude precipitate of Plasmodium falciparum is centrifuged at high speed, and the precipitate is collected to obtain the Plasmodium falciparum precipitate. The Plasmodium falciparum pigment precipitate is resuspended in an SDS solution with a pH less than or equal to 7.6 and a mass fraction of 1.5% to 2.5%, and centrifuged repeatedly 2 to 3 times. The precipitate is collected until the absorbance at 400 nm in the supernatant is zero, and the precipitate is collected to obtain a clean Plasmodium falciparum precipitate. The SDS solution can wash away residual heme from the red blood cells in the solution and remove red blood cell residues, thus avoiding interference from heme within the red blood cells. When resuspending, the pH of the SDS solution should not exceed 7.6; otherwise, increased pigment dissolution will lead to lower measured values. When washing with SDS solution, it is important to note that too few washes will result in residual heme, leading to higher measured values. Too many washes will result in pigment loss, leading to lower measured results. Therefore, after each centrifugation, the absorbance of the supernatant at 400 nm must be measured; if the absorbance is zero, washing should be stopped. Then, add SDS solution and NaOH solution to dissolve the clean Plasmodium falciparum precipitate, obtaining the Plasmodium falciparum pigment solution. Measure the absorbance of the Plasmodium falciparum pigment solution at 400 nm and calculate the Plasmodium falciparum pigment content. If the concentration of the Plasmodium falciparum pigment solution is too high, it can be diluted before measurement; if the concentration is too low, the total volume of the solution can be reduced proportionally.

[0020] The method for detecting the pigment content of Plasmodium falciparum in this invention can rapidly detect the pigment content of Plasmodium falciparum, providing accurate results. It does not require large, expensive equipment, is simple to operate, and is easily accepted. This invention will provide a method for detecting the pigment content of low-pigment Plasmodium strains that have been constructed or screened, in order to determine the impact of strains with different pigment contents on vaccine efficacy and safety.

[0021] 2. The method for detecting the pigment content of Plasmodium falciparum in this invention does not require Raman spectroscopy or magnetic separation techniques. Most ordinary laboratories can screen and determine the pigment content and pigment-forming ability of Plasmodium using conventional equipment. The method for detecting the pigment content of Plasmodium falciparum in this invention can be performed using conventional instruments, such as Nano Drop or a commonly used spectrophotometer. It is particularly important to note that the absorbance value at 400 nm should be used for comparison in this system for accurate results. The method provided by this invention is accurate, sensitive, easy to operate, and has a wide range of applications for detecting the pigment content of Plasmodium falciparum. Attached Figure Description

[0022] Figure 1This is a morphological comparison diagram of the wild-type Plasmodium falciparum strain and the K13 mutant strain of this invention. In the diagram, A represents the wild-type Plasmodium falciparum strain, and B represents the K13 mutant strain.

[0023] Figure 2 This is a comparison chart of pigment content between the wild-type and K13 mutant strains of Plasmodium falciparum of the present invention. In chart A, the pigment of the wild-type and K13 mutant strains is deeper than that in alkaline SDS solution; in chart B, the pigment of the wild-type and K13 mutant strains is compared.

[0024] Figure 3 This is a graph comparing the number of merozoites between the wild-type and K13 mutant strains of Plasmodium falciparum of the present invention.

[0025] Figure 4 This invention provides a spectrophotometric method for detecting the absorbance of Plasmodium falciparum pigments at different wavelengths.

[0026] Figure 5 This is a graph showing the effect of SDS solutions with different pH values ​​on the measured values ​​according to the present invention.

[0027] Figure 6 This is a graph showing the effect of the number of washes on the results of this invention.

[0028] Figure 7 This is an analysis diagram of the supernatant after washing according to the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0031] Example 1 A method for detecting the pigment content of Plasmodium falciparum includes the following steps: 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 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% (w / w) 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% (w / w) 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.

[0032] Example 2 A method for detecting the pigment content of Plasmodium falciparum includes the following steps: One mL of red blood cells infected with Plasmodium falciparum were resuspended in 7 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 0.14%. 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 Plasmodium falciparum precipitate. 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% (w / w) 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% (w / w) 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 pigment, 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.

[0033] Example 3 A method for detecting the pigment content of Plasmodium falciparum includes the following steps: 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.

[0034] Comparative Example 1 A method for detecting the pigment content of Plasmodium falciparum includes the following steps: 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 to 0.5% with NaOH solution at a wavelength of 400 nm, and the content of Plasmodium falciparum pigment was calculated based on the standard curve.

[0035] To illustrate the beneficial effects of the present invention, the following experiments were also conducted.

[0036] 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. WT It is related to the K13 mutant strain C580Y, denoted as 3D7. C580Y The shape is like Figure 1 As shown, the pigment content is as follows Figure 2 As shown, the number of merozoites is as follows Figure 3 As shown, 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.

[0037] I. Materials and Equipment 1. Experimental Materials 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.

[0038] II. Experimental Methods 1. Wavelength scanning of Plasmodium falciparum pigment 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: the pigment of Plasmodium falciparum was scanned at wavelengths of 350 nm to 450 nm using a UV-Vis spectrophotometer.

[0039] 2. Effects of SDS solutions with different pH values ​​on the measurement values ​​of Plasmodium falciparum pigment. 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 the standard curve, and then multiplied back by the dilution factor.

[0040] 3. Effect of washing frequency on the pigment of Plasmodium falciparum 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.

[0041] III. Experimental Results 1. Wavelength scanning of Plasmodium falciparum pigment The scan results of the absorbance of Plasmodium falciparum pigment at different wavelengths are as follows: Figure 4 As shown in the figure, the absorbance of Plasmodium falciparum pigment reaches its maximum value within the scanning range of 350 nm to 450 nm, with the wavelength at which the maximum value occurs at 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.

[0042] 2. Effects of SDS solutions with different pH values ​​on the measurement values ​​of Plasmodium falciparum pigment. The effect of SDS solutions with different pH values ​​on the measured values ​​of Plasmodium falciparum pigments, as follows: Figure 5 As shown, the results indicate that washing the Plasmodium falciparum precipitate with a 2% w / v SDS solution at pH 9.0 reduced the absorbance of the Plasmodium falciparum pigment solution. This suggests that an excessively high pH will wash away the Plasmodium falciparum pigment, leading to a lower detection result. Therefore, the pH of the SDS solution should be less than or equal to 7.6.

[0043] 3. Effect of washing frequency on the pigment of Plasmodium falciparum The effect of washing frequency on the pigment of Plasmodium falciparum, such as Figure 6 As shown. The absorbance values ​​of the supernatant after each wash and centrifugation are as follows. Figure 7 As shown in the figure, the absorbance of the supernatant after centrifugation decreased with increasing washing cycles, indicating that the Plasmodium falciparum precipitate was gradually washed away. However, the absorbance of the Plasmodium falciparum pigment solution also decreased with increasing washing cycles, indicating that the pigment in the Plasmodium falciparum is lost during washing. Therefore, the number of washing cycles should not be too high. The number of washing cycles should be controlled within 3 times to effectively remove the Plasmodium falciparum precipitate while minimizing the loss of the Plasmodium falciparum pigment.

[0044] IV. Discussion of Results 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.

[0045] 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.

[0046] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two 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.

[0047] 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, The method comprises the following steps: resuspending red blood cells infected with Plasmodium falciparum with a solvent, adding saponin, mixing, standing, centrifuging, collecting the precipitate, and obtaining a crude Plasmodium falciparum precipitate; centrifuging the crude Plasmodium falciparum precipitate, collecting the precipitate, and obtaining a Plasmodium falciparum precipitate; resuspending the Plasmodium falciparum precipitate with a SDS solution with a mass fraction of 1.5% to 2.5% and a pH less than or equal to 7.6, centrifuging repeatedly for 2 to 3 times, collecting the precipitate, until the absorbance value at 400 nm in the supernatant is zero, collecting the precipitate, and obtaining a clean Plasmodium falciparum pigment precipitate; dissolving the clean Plasmodium falciparum pigment precipitate with a SDS solution with a mass fraction of 1.5% to 2.5% and a pH less than or equal to 7.6 and a NaOH solution, and obtaining a Plasmodium falciparum pigment solution; detecting the absorbance value of the Plasmodium falciparum pigment solution at 400 nm, preparing a standard curve with hematin, and calculating the Plasmodium falciparum pigment content according to 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 Plasmodium falciparum to the solvent is 1:5 to 10; wherein the solvent is physiological saline or a phosphate buffer.

3. The method of claim 1, wherein the pigment content of P. falciparum is determined by measuring the amount of hemozoin in the sample. 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 standing temperature is 4°C to 10°C, and the standing time is 10 min to 20 min.

5. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The centrifugation conditions are all 10000g to 12000g for 5 min to 10 min.

6. The method for detecting the pigment content of Plasmodium falciparum according to claim 1, characterized in that, The mass fraction of the SDS solution is 2%.

7. The method of claim 1, wherein the pigment content of P. falciparum is determined by measuring the amount of hemozoin in the sample. The volume ratio of the SDS solution to the NaOH solution is 7 to 9:1, wherein the concentration of the NaOH solution is 1.8 mol / L to 2.2 mol / L.

8. The method of claim 1, wherein the pigment content of P. falciparum is determined by measuring the amount of hemozoin in the sample. The number of repeated centrifugations is 2.

9. The method of claim 1, wherein the pigment content of P. falciparum is determined by measuring the amount of hemozoin in the sample. The absorbance value of the Plasmodium falciparum pigment solution at 400 nm is adjusted to 0.5 to 0.8 by controlling the amount of the SDS solution or the NaOH solution.

10. Application of a reagent used in the Plasmodium falciparum pigment content detection method of claim 1 in detecting the Plasmodium falciparum pigment content.

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