Ferric hydroxide modified diatomite, preparation method and application in ASFV detection
By using ferric hydroxide-modified diatomaceous earth to concentrate ASFV samples, the problem of negative detection caused by low ASFV pathogen content was solved, and the sensitivity and accuracy of detection were improved.
Patent Information
- Application Number
- CN202511357306.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2025-12-26
AI Technical Summary
In existing technologies, when the ASFV pathogen content is extremely low, qPCR testing is prone to producing negative results, leading to inaccurate detection and insufficient sensitivity.
Ferric hydroxide-modified diatomaceous earth was used as the chromatography material. Suspected ASFV samples were concentrated using an affinity chromatography column, and the supernatant was collected after elution for detection.
It improved the sensitivity and accuracy of ASFV detection, reduced the CT value of fluorescent PCR detection, and enhanced sample amplification efficiency.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of virus detection, in particular to iron hydroxide modified diatomite, a preparation method and application thereof in ASFV detection. BACKGROUND
[0002] African swine fever virus (ASFV) is a virus with high pathogenicity to pigs, and African swine fever (ASF) caused by the virus is one of the most devastating infectious diseases in the global pig industry. Due to the high pathogenicity, complex transmission mechanism, lack of effective vaccine and wide range of epidemic, ASFV has become a key and difficult point of the global pig industry prevention and control, and poses a major challenge to the economy of the animal husbandry, food safety and public health (indirect influence), and causes a serious blow to the pig industry.
[0003] qPCR is the core technology for ASFV diagnosis at present, and is widely used in the diagnosis of suspected cases in clinic, the normalization monitoring of breeding farms / slaughterhouses, the inspection of pork and products at entry and exit ports, the auxiliary verification of virus isolation and culture, etc. The high sensitivity can detect the virus in the early stage (latent period) of infection, and provides a key basis for rapid response and prevention and control of the epidemic. However, for samples with extremely low content of ASFV pathogens, qPCR detection may be negative, and the negative detection does not mean absolutely no virus. After the concentration of the ASFV in the sample, the sensitivity and accuracy of the detection can be improved.
[0004] Based on this, the application is proposed. SUMMARY
[0005] The application aims to provide iron hydroxide modified diatomite, a preparation method and application thereof in ASFV detection, so as to solve the problem of negative qPCR detection in the case of extremely low content of ASFV pathogens in samples in the prior art.
[0006] In order to achieve the above application purposes, the application provides the following technical solutions.
[0007] The application provides a preparation method of iron hydroxide modified diatomite, comprising the following steps:
[0008] (1) Fe(OH)3 colloid is obtained by dropping a saturated FeCl3 solution into boiling water and stirring;
[0009] (2) Fe(OH)3 colloid obtained in step (1) is mixed with diatomite, and iron hydroxide modified diatomite is obtained by drying;
[0010] In step (2), the mass ratio of Fe(OH)3 colloid to diatomite is 1-2:1-2.
[0011] Preferably, the temperature of the water in step (1) is 95-105℃.
[0012] Preferably, the temperature of the drying in step (2) is 40-60℃.
[0013] The application provides the iron hydroxide modified diatomite prepared by the preparation method.
[0014] The application provides application of the iron hydroxide modified diatomite in preparation of concentrated African swine fever virus.
[0015] The application provides a method for concentrating African swine fever virus (ASFV) for non-disease diagnosis purposes, wherein iron hydroxide modified diatomite is filled into an affinity chromatography column as a chromatography material to obtain a chromatography column, a suspected ASFV sample is added to the chromatography column for chromatography, and an eluent is obtained by eluting the chromatography column after the chromatography, and concentrated ASFV is obtained by taking supernatant after standing.
[0016] The iron hydroxide modified diatomite is the iron hydroxide modified diatomite.
[0017] The application has the following technical effects and advantages:
[0018] The application provides an iron hydroxide modified diatomite, a preparation method and application in ASFV detection. The iron hydroxide modified diatomite prepared by the method has good concentration effect on ASFV, and the concentration of ASFV pathogen in a sample with low content and negative in conventional qPCR detection can be improved, the amplification efficiency of the sample is improved, the CT value of the sample is reduced by 1.5-3, and the sensitivity and accuracy of sample detection are improved. DETAILED DESCRIPTION
[0019] The application provides a preparation method of iron hydroxide modified diatomite, comprising the following steps:
[0020] (1) Fe(OH)3 colloid is obtained by adding saturated FeCl3 solution into boiling water and stirring;
[0021] (2) Fe(OH)3 colloid obtained in step (1) is mixed with diatomite, and the mixture is dried to obtain iron hydroxide modified diatomite;
[0022] The mass ratio of Fe(OH)3 colloid to diatomite in step (2) is 1-2:1-2, preferably 1:1.
[0023] In the application, the temperature of the water in step (1) is 95-105℃, preferably 100℃.
[0024] In the present application, the temperature of the drying in step (2) is 40-60 DEG C, preferably 50 DEG C.
[0025] The present application provides the iron hydroxide modified diatomite prepared by the preparation method.
[0026] The present application provides the application of the iron hydroxide modified diatomite in the preparation of concentrated African swine fever virus.
[0027] The present application provides a method for concentrating African swine fever virus for non-disease diagnosis purposes, iron hydroxide modified diatomite is filled into an affinity chromatography column as a chromatography material to obtain a chromatography column, a suspected ASFV sample is added into the chromatography column for chromatography, and an eluent is obtained by eluting the chromatography column after the chromatography, and concentrated ASFV is obtained by standing and taking supernatant.
[0028] The iron hydroxide modified diatomite is the iron hydroxide modified diatomite.
[0029] The technical solutions provided by the present application are described in detail below in combination with embodiments, but they should not be understood as limitations to the protection scope of the present application.
[0030] The configuration method of the saturated FeCl3 solution in the embodiment of the present application is as follows: 1.6 g of ferric chloride hexahydrate is dissolved in 1 ml of distilled water, and is fully dissolved to obtain a saturated FeCl3 solution.
[0031] Example 1
[0032] Preparation of iron hydroxide modified diatomite
[0033] In a 250 mL beaker, 100 mL of distilled water is added and heated to 100 DEG C to obtain boiling water, and the saturated FeCl3 solution is slowly added drop by drop into the boiling water until the solution is reddish brown, and Fe(OH)3 colloid is obtained, and the Fe(OH)3 colloid is mixed with diatomite at a mass ratio of 1:1, and is dried in a drying oven at 50 DEG C to obtain iron hydroxide modified diatomite.
[0034] Experimental Example 1
[0035] 1. Extraction of African swine fever virus sample and nucleic acid
[0036] Suspected ASF serum samples from a harmless treatment plant in Liaocheng, Shandong are obtained, and are subjected to high-temperature inactivation treatment.
[0037] 2. Detection of African swine fever virus sample
[0038] The sample suspected to contain African swine fever virus is diluted at a volume ratio of 1:10 to obtain diluent 1 and diluent 2, respectively.
[0039] Two groups of experiments were set up. Group 1: The diluted solution 1 was extracted using a viral DNA & RNA extraction kit to obtain African swine fever virus nucleic acid DNA. The African swine fever virus nucleic acid DNA was used as a template for fluorescent PCR detection, and the experiment was repeated twice. Group 2: The diluted solution 2 was concentrated using the iron hydroxide modified diatomite prepared in Example 1 to obtain a concentrated solution. The African swine fever virus nucleic acid DNA in the concentrated solution was extracted, and the extracted African swine fever virus nucleic acid DNA was used as a template for fluorescent PCR detection. Two independent fluorescent PCR detections were performed, and each detection was repeated twice.
[0040] The concentration method of the diluted solution 2 in group 2 was as follows:
[0041] (1) 0.5 g of the iron hydroxide modified diatomite prepared in Example 1 was loaded into a 6 mL capacity affinity chromatography column empty column (purchased from Shenzhen Comma Biotechnology Co., Ltd., shelf number: 004203-L, containing 2 pieces of 1.6 mm thick sieve plates, blue upper cover, and lower cover). The affinity chromatography sieve plate was preloaded into the affinity chromatography column empty column, and the upper and lower covers were closed to obtain the loaded chromatography column.
[0042] (2) The loaded chromatography column was fixed on a support and placed on top of a 500 mL beaker (the beaker was used to collect waste liquid).
[0043] (3) The upper and lower covers of the loaded chromatography column were opened, and the diluted solution 2 was added to the loaded chromatography column in sequence. The liquid was allowed to flow naturally until it was exhausted.
[0044] (4) Preparation of 10×NB: 1.9 g of nucleic acid binding buffer (NB) was weighed into a 15 mL centrifuge tube, 10 mL of sterile pure water was added, and it was dissolved and mixed evenly.
[0045] (5) After the chromatography was completed, 1 mL of 10×NB was added to the chromatography column, the upper cover of the chromatography column was tightened, and it was placed on a horizontal shaker for elution for 30 min.
[0046] (6) After elution for 30 min, the eluate was collected, and after standing for 5 min, the supernatant was obtained to obtain the concentrated solution. The CT values of the PCR fluorescent detection of the two groups were determined, and the results are shown in Table 1.
[0047] Table 1 PCR fluorescent detection CT value results
[0048]
[0049] CT value (cycle threshold) is an important indicator in fluorescent quantitative PCR, which represents the cycle number when the fluorescence signal of the amplification product reaches the set threshold. CT value reflects the cycle number when the amplification of the starting template reaches a certain product amount. CT value is negatively correlated with the starting copy number of the target gene in the sample: the higher the starting template amount, the smaller the CT value; the lower the starting template amount, the larger the CT value. According to Table 1, the average CT value of the first group of PCR fluorescence detection is 39.04, and the average CT value of the second group of PCR fluorescence detection is 36.1. It can be seen that the CT value of the second group of PCR fluorescence detection is lower than that of the first group of PCR fluorescence detection, which indicates that the iron hydroxide modified diatomite prepared by the scheme of the application can enrich African swine fever virus and improve the sensitivity of PCR fluorescence detection when used as a concentration reagent for African swine fever virus.
[0050] Experimental Example 2
[0051] A suspected ASF whole blood sample from a harmless treatment plant in Liaocheng, Shandong was obtained and subjected to high temperature inactivation treatment to obtain an inactivated detection sample.
[0052] The experiment was divided into 5 groups, namely control group, group 1, group 2, group 3 and group 4. The control group: extract the African swine fever virus nucleic acid DNA of the detection sample, and perform fluorescent PCR detection with the African swine fever virus nucleic acid DNA as the template; dilute the detection sample according to the ratio of 1:40 to obtain dilution 1; dilute the detection sample according to the ratio of 1:80 to obtain virus dilution 2. Group 1 extracts the nucleic acid of dilution 1, and performs fluorescent PCR detection with the nucleic acid as the template; group 2 concentrates 40 mL of dilution 1 according to the concentration method in experimental example 1 to obtain concentrated solution 1, extracts the nucleic acid of the concentrated solution, and performs fluorescent PCR detection with the nucleic acid as the template; group 3 extracts the nucleic acid of dilution 2, and performs fluorescent PCR detection with the nucleic acid as the template; group 4 concentrates 40 mL of dilution 2 according to the concentration method in experimental example 1 to obtain concentrated solution 2, extracts the nucleic acid of the concentrated solution, and performs fluorescent PCR detection with the nucleic acid as the template.
[0053] The CT values of the control group and groups 1-4 were determined by fluorescent PCR detection, the control group was repeated 3 times, and groups 1-4 were independently detected 2 times, each time with 3 repeats, and the results are shown in Table 2.
[0054] Table 2 CT values of fluorescent PCR detection of different treatment groups
[0055]
[0056] According to Table 2, the detection ratio of the control group is 1, and African swine fever virus can be detected in each of the 3 detections, and the CT value is between 34.43 and 35.98. The detection sample in the first group is diluted at a ratio of 1:40 to obtain diluent 1. Only 1 of the 6 detections of diluent 1 can detect African swine fever virus, and the CT value is 38.56. The second group is concentrated compared with the first group, and diluent 1 is obtained. The 6 detections of concentrated liquid 1 can all detect African swine fever virus, and the CT value range is between 35.77 and 37.05. The detection sample in the third group is diluted at a ratio of 1:80 to obtain diluent 2. Only 1 of the 6 detections of diluent 2 can detect African swine fever virus, and the CT value is 38.97. The fourth group is concentrated compared with the third group, and concentrated liquid 2 is obtained. The 6 detections of concentrated liquid 2 can all detect African swine fever virus, and the CT value range is between 37 and 38.89. It is shown that after the African swine fever virus in the detection sample is concentrated by the iron hydroxide modified diatomite prepared by the scheme of the application as the concentration reagent, the African swine fever virus in the sample can be enriched, the CT value of the fluorescence PCR detection can be reduced when the detection is performed, and the sensitivity and accuracy of the detection can be improved.
[0057] Experimental Example 3
[0058] A soil sample near a pig suspected of African swine fever virus in a harmless treatment plant in Liaocheng area of Shandong was obtained, and high temperature inactivation treatment was performed to obtain a detection sample.
[0059] The detection sample was diluted with sterile water at a mass-volume ratio of 1g:100mL to obtain a diluent. The diluent was concentrated by the concentration method in experimental example 1 to obtain a concentrated liquid.
[0060] The viral DNA&RNA extraction kit was used to extract the African swine fever virus nucleic acid DNA in the diluent and the concentrated liquid, respectively. The African swine fever virus fluorescence PCR nucleic acid detection kit (purchased from Zhengzhou Zhongdao Biotechnology Co., Ltd., shelf number: P1721) was used to perform fluorescence PCR detection on the African swine fever virus nucleic acid DNA of the diluent and the concentrated liquid according to the content of the instruction manual, 3 groups of repetitions were performed, the CT value of the fluorescence PCR detection of the detection sample before and after concentration was determined, and the results are shown in Table 3.
[0061] Table 3 CT value of fluorescence PCR detection of detection sample before and after concentration
[0062] Repeat Pre-concentration CT value Post-concentration CT value 1 NA 34.65 2 37.53 35.41 3 37.66 35.41
[0063] According to Table 3, the soil sample contains African swine fever virus, and false negatives can easily occur in the conventional detection method. For the African swine fever environmental samples that are detected as negative or have a high CT value, the African swine fever virus can be detected after the sample is concentrated by using the iron hydroxide modified diatomite prepared by the scheme of the application as a concentration reagent, and the CT value of the detection can be reduced.
[0064] Experimental Example 4
[0065] The ASFV positive pig whole blood and lymph tissue samples were obtained from a certain agricultural market, and were subjected to high-temperature inactivation treatment to obtain inactivated detection samples. The detection samples were diluted according to a ratio of 1:10 to obtain a diluent.
[0066] 1. Effect of changing the filtration speed on the concentration effect
[0067] The experiment was divided into three groups, namely a control group, a first group and a second group. The African swine fever virus nucleic acid DNA in the diluent was extracted in the control group, and fluorescence PCR detection was performed using the African swine fever virus nucleic acid DNA as a template. The diluent was concentrated according to the concentration method in Example 1 to obtain a concentrated solution in the first group, and the African swine fever virus nucleic acid DNA in the concentrated solution was extracted, and fluorescence PCR detection was performed using the African swine fever virus nucleic acid DNA as a template. The diluent was concentrated according to the concentration method in Example 1 in the second group, and the filtration speed was accelerated by using a syringe pressurization method in step (3) to obtain a concentrated solution, and the African swine fever virus nucleic acid DNA in the concentrated solution was extracted, and fluorescence PCR detection was performed using the African swine fever virus nucleic acid DNA as a template. The control group was repeated in three groups, and the first group and the second group were each independently detected twice, each time with three groups repeated. The CT values of the fluorescence PCR detection of the control group, the first group and the second group were determined, and the results are shown in Table 4.
[0068] Table 4 CT values of fluorescence PCR detection of different treatment groups
[0069]
[0070] According to Table 4, the average CT values of the PCR detection of the first group and the second group are not much different, and are each more than 1.5 lower than that of the control group, indicating that the filtration speed does not affect the concentration effect.
[0071] 2. Effect of different chromatographic materials on the concentration effect
[0072] The effects of different chromatographic materials on the concentration of African swine fever virus were compared. Four groups were set, i.e. a control group, a first group, a second group and a third group. The African swine fever virus nucleic acid DNA in the diluent was extracted, and fluorescence PCR detection was performed with the African swine fever virus nucleic acid DNA as a template in the control group. The diluent was concentrated by the method in Example 1 to obtain a concentrated solution in the first to third groups, and the African swine fever virus nucleic acid DNA in the concentrated solution was extracted, and fluorescence PCR detection was performed with the African swine fever virus nucleic acid DNA as a template. The difference lies in that the first group uses silica gel powder (sodium silicate reacts with acid to generate, and high-purity amorphous silicon dioxide is prepared by dehydration activation) as the packing material of the chromatographic column, the second group uses diatomite (naturally formed, mainly composed of SiO2) containing clay, organic matter and other impurities as the packing material of the chromatographic column, and the third group uses the iron hydroxide modified diatomite prepared in Example 1 as the packing material of the chromatographic column.
[0073] The control group and the first to third groups were independently detected twice at the time of detection, and each time 2 groups were repeated. The CT values of the fluorescence PCR detection of the control group, the first group, the second group and the third group were determined. The CT values of the fluorescence PCR detection of different treatment groups are shown in Table 5.
[0074] Table 5 CT values of fluorescence PCR detection of different treatment groups
[0075]
[0076] According to Table 5, the CT values of the fluorescence PCR detection of the first group, the second group and the third group are 36.29, 36.67 and 35.87 respectively, which are all lower than 38.64 of the control group, indicating that different chromatographic materials have a concentration effect on African swine fever virus. The CT of the fluorescence PCR detection of the third group is different from that of the first group and the second group, indicating that the concentration effect of different materials is not the same. The CT of the fluorescence PCR detection of the third group is lower than that of the first group and the second group, indicating that the iron hydroxide modified diatomite prepared by the scheme has the best concentration effect on African swine fever virus.
[0077] From the above examples, the present application provides a kind of iron hydroxide modified diatomite, preparation method and application in ASFV detection. The iron hydroxide modified diatomite prepared according to the method of the present application has good concentration effect on ASFV. The iron hydroxide modified diatomite is used to concentrate the sample with low content of ASFV pathogen and negative result by conventional qPCR detection, which can improve the amplification efficiency of the detection sample, reduce the sample CT value by 1.5-3, and improve the sensitivity and accuracy of sample detection.
[0078] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.
Claims
1. A method for producing iron hydroxide-modified diatomite, characterized by, It comprises the following steps: (1) Fe(OH)3 colloid is obtained by dropping saturated FeCl3 solution into water and stirring; (2) Fe(OH)3 colloid obtained in step (1) is mixed with diatomite, and dried to obtain iron hydroxide modified diatomite; The mass ratio of Fe(OH)3 colloid to diatomite in step (2) is 1-2:1-2.
2. The production method according to claim 1, characterized by, The temperature of water in step (1) is 95-105℃.
3. The production method according to claim 1, characterized by, The temperature of drying in step (2) is 40-60℃.
4. The iron hydroxide modified diatomite prepared by the preparation method in any one of claims 1-3.
5. The application of the iron hydroxide modified diatomite in claim 4 in the preparation of concentrated African swine fever virus.
6. A method of concentrating African swine fever virus for non-disease diagnostic purposes, characterized by, The iron hydroxide modified diatomite is packed into an affinity chromatography column empty column as chromatography material to obtain a chromatography column, a sample suspected to contain ASFV is added to the chromatography column for chromatography, and after the chromatography is completed, the chromatography column is eluted to obtain an eluate, and the concentrated ASFV is obtained by standing and taking the supernatant; The iron hydroxide modified diatomite is the iron hydroxide modified diatomite in claim 4.