A method for nucleic acid extraction based on dried blood spots and applications

Abstract

The application belongs to the technical field of molecular diagnosis, and provides a nucleic acid extraction method based on dried blood spots and application. The method comprises the following steps: adding EDTA anticoagulation whole blood to an imported FTA card to prepare a dried blood spot, and then extracting DNA by using a magnetic bead method, and then performing fluorescent quantitative PCR detection. The application screens an optimal combination of the imported FTA card and the magnetic bead method through systematic comparison of the combination effects of different FTA cards and DNA extraction methods. The combination has high DNA yield, good purity and high amplification efficiency. Clinical verification shows that the combination has high consistency with conventional fluorescent PCR, and is suitable for screening of imported malaria at ports.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, and in particular to a method and application for nucleic acid extraction based on dried blood spots. Background Technology

[0002] Malaria is a parasitic disease caused by Plasmodium parasites, primarily transmitted through the bite of female Anopheles mosquitoes. It is a major infectious disease burden globally, especially in tropical and subtropical regions. Currently, my country reports approximately 2,000 imported malaria cases annually, mainly originating from malaria-endemic areas in Africa and Southeast Asia. Therefore, establishing a simple, reliable, and easily applicable malaria parasite screening method suitable for port-of-entry sites is of significant public health importance for preventing the reinfection of imported malaria.

[0003] Currently, malaria parasite detection technologies mainly fall into three categories: thick and thin blood smear microscopy, rapid diagnostic tests (RDTs), and nucleic acid detection. Microscopy is the "gold standard" for diagnosing malaria, allowing direct observation of the morphology of the parasite and identification of the species. However, it is highly dependent on the operator's skill and experience, and the operation is time-consuming and labor-intensive, making it difficult to meet the rapid detection needs in high-throughput screening scenarios such as ports of entry. RDTs, based on the specific antigen-antibody reaction of the malaria parasite, are widely used in resource-limited areas, but they suffer from problems such as inability to detect recurrent malaria, poor sensitivity to mixed infections, insufficient specificity, and poor stability due to temperature variations. Nucleic acid detection methods (such as quantitative real-time PCR) have high sensitivity and specificity, accurately identifying malaria parasite species and mixed infections. However, traditional methods require the collection of anticoagulated venous blood, placing extremely high demands on sample preservation and transportation conditions, requiring cold chain logistics and specialized laboratory support, thus limiting their application in grassroots scenarios such as ports of entry.

[0004] Dried blood spot (DBS) is a biosampling technique for collecting, drying, and storing small amounts of blood. It offers advantages such as small blood volume, ease of operation, minimal invasiveness, and no need for cold chain transportation and storage, and has been widely applied in newborn disease screening, drug concentration monitoring, epidemiological sample analysis, and infectious disease detection. In the field of malaria detection, dried blood spot technology has shown promising application potential. Studies have shown that using FTA filter paper to collect blood samples and extract Plasmodium DNA for PCR detection can achieve sensitive detection of Plasmodium, and the samples can be stably stored at room temperature for a relatively long time. Other studies have compared the effects of different filter paper materials (such as Whatman 3MM Chr, FTA Card, and FTA Elute Card) on the PCR detection efficiency of Plasmodium, showing differences in DNA recovery rate and PCR inhibitor residue among different filter papers. Furthermore, magnetic bead DNA extraction technology, due to its ease of operation, automation, and high extraction efficiency, has been applied to the extraction of genomic DNA from dried blood spots, and related kits have been patented. However, existing research mostly focuses on the performance evaluation of a single blood collection card or a single extraction method, or only compares the combined effects of a few filter papers and detection methods. There is a lack of systematic comparisons of the combined effects of domestic and imported FTA blood collection cards with different DNA extraction methods such as centrifugation column method and magnetic bead method, and there is also a lack of optimized schemes for dried blood spot nucleic acid extraction for on-site screening scenarios of imported malaria at ports of entry. The chemical processing components (such as chelating agents and denaturing agents) of different blood collection cards vary, and there may be interactions between these components and different DNA extraction principles (centrifugation column method and magnetic bead method), thus affecting DNA extraction efficiency, purity, and downstream PCR amplification efficiency. This may be a key factor limiting the sensitivity and stability of dried blood spot Plasmodium nucleic acid detection.

[0005] Therefore, screening the optimal combination of blood collection cards and DNA extraction methods suitable for on-site screening at ports of entry is of significant technical value for improving the reliability of nucleic acid detection of dried blood spots in imported malaria. Summary of the Invention

[0006] The first aspect of this invention provides a method for nucleic acid extraction based on dried blood spots, comprising the following steps:

[0007] (1) Collect samples, drop them onto the designated area of ​​a Whatman FTA card, and allow them to air dry overnight at room temperature to prepare dried blood spot samples;

[0008] (2) Cut dried blood spots from the FTA card and extract genomic DNA using the YEASEN magnetic bead DNA extraction kit.

[0009] Specifically, in step (1), the WhatmanFTA card part number is WB129237.

[0010] Specifically, in step (1), the samples include EDTA-anticoagulated blood, fingertip blood, heel blood, serum, and plasma.

[0011] Specifically, in step (1), the amount of sample used is 180-220 μL.

[0012] Specifically, in step (1), the preservation conditions for the dried blood spot sample are: placed in a sealed bag with a desiccant inside and stored away from light.

[0013] Specifically, in step (2), the YEASEN magnetic bead DNA extraction kit has the catalog number 18554ES48.

[0014] Specifically, in step (2), the area cut out is 2-3mm × 2-3mm.

[0015] Specifically, in step (2), the elution volume of the genomic DNA is 30-40 μL.

[0016] A second aspect of the invention provides the application of any of the above methods in malaria screening.

[0017] Specifically, the screening includes on-site screening for imported malaria at ports of entry.

[0018] A third aspect of the present invention provides a malaria field screening system, the system comprising a detection device, a computing device, and an output device; the detection device comprising a sample injector, a sample processor, and a detector;

[0019] The sampler is used to collect samples from the subject;

[0020] The sample processor is used to process the collected samples as follows:

[0021] S1. Add the sample to a Whatman FTA card to prepare a dried blood spot sample;

[0022] S2. Obtain dried blood spots from the FTA card and extract genomic DNA using the YEASEN magnetic bead DNA extraction kit.

[0023] S3. Perform malaria parasite nucleic acid detection on the extracted DNA.

[0024] Specifically, in S3, the nucleic acid detection methods include quantitative real-time PCR, nested PCR, multiplex PCR, digital PCR, loop-mediated isothermal amplification, recombinase polymerase amplification, Sanger sequencing, and high-throughput sequencing.

[0025] More specifically, the nucleic acid detection method is quantitative real-time PCR.

[0026] Specifically, the output device is used to view the detection results.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention compares the combined effects of FTA blood collection cards from different sources with various DNA extraction methods and screens out the optimal combination of imported FTA cards and magnetic bead method. This combination has high DNA yield, good purity and high amplification efficiency, which is significantly better than the combination of domestic FTA cards and centrifuge column method.

[0029] (2) The present invention can complete the detection of malaria parasite nucleic acid based on dried blood spot samples, without the need for collecting venous anticoagulated blood and cold chain transportation. It is simple to operate and is particularly suitable for grassroots screening scenarios such as port sites.

[0030] (3) Clinical sample verification shows that the method of the present invention has a high degree of consistency with the results of conventional venous whole blood fluorescence quantitative PCR detection, with a sensitivity of 100%, a total concordance rate of 91.67%, and a Kappa value of 0.8334, indicating good detection reliability. Attached Figure Description

[0031] Figure 1 Results of DNA extraction concentration comparison.

[0032] Figure 2 Results of DNA extraction purity comparison.

[0033] Figure 3 Results of DNA extraction function quality comparison. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0035] Example 1

[0036] 1. Research Sample

[0037] The General Administration of Customs (Beijing) International Travel Health Care Center tested preserved malaria parasite-positive and negative whole blood samples from inbound and outbound passengers. All samples underwent quantitative real-time PCR testing to determine infection status.

[0038] 2. Instruments and Reagents

[0039] Applied Biosystems QuantStudio 5 Real-Time PCR System, Qubit 4 Fluorometer, NanoDrop 2000 Micro Spectrophotometer, Plasmodium Nucleic Acid Assay Kit (Shanghai Zhijiang, ER-5066-02), Biosafety Cabinet (Sujing Antai, BSC-1600IIA2), Magnetic Rack (Thermo Fisher, 12321D), and Thermostatic Metal Bath (SCILOGEX, DWB20-P).

[0040] 3. Experimental Methods

[0041] 3.1 Experimental Grouping

[0042] To systematically evaluate the extraction efficiency of Plasmodium DNA from two different FTA cards and three DNA extraction kits, this study employed a multi-factor experimental design, conducting parallel tests on all combinations. A total of six experimental groups were included (two FTA cards × three extraction methods), with five samples in each group.

[0043] 3.2 Preparation of dried blood spot samples

[0044] This study used EDTA-anticoagulated whole blood samples that had been previously collected and frozen at -80°C. Before the experiment, the samples were thawed at room temperature and gently mixed to ensure homogeneity.

[0045] Sample application: Using a micropipette, accurately aspirate 200 μL of thawed and mixed anticoagulated whole blood and vertically drop it into the center of the designated circular marking area on both domestic and imported FTA cards (brand: Ronghua Bio, catalog number: Next Generation Sequencing Blood Collection Card) and imported FTA card (brand: Whatman, catalog number: WB129237), ensuring that the blood is fully diffused within the marking circle.

[0046] Osmosis drying: Place the spotted card horizontally in a biosafety cabinet and allow it to air dry overnight at room temperature until the blood spot is completely dry and free of moisture. This process allows the nucleic acids in the blood to fully interact with the chelating agents, denaturing agents, and other chemicals on the filter paper, achieving cell lysis, protein denaturation, and immediate stabilization of nucleic acids.

[0047] Storage: Store the completely dried blood spot sample in a sealed bag with built-in desiccant, away from light, for subsequent DNA extraction.

[0048] 3.3 DNA Extraction

[0049] To evaluate the combined efficiency of different FTA cards and DNA extraction methods, this study used the following three DNA extraction kits to extract Plasmodium genomic DNA from dried blood spots: centrifuge column DNA extraction kit (brand: QIAamp, catalog number: 51304); centrifuge column DNA extraction kit (brand: Tiangen, catalog number: DP334); and magnetic bead DNA extraction kit (brand: YEASEN, catalog number: 18554ES48).

[0050] Eight square dried blood spots, each 3 mm in length and width, were cut from each FTA card using sterile scissors and placed in 1.5 mL centrifuge tubes. DNA was extracted from the dried blood spots using three different DNA extraction kits, with a uniform elution volume of 35 μL.

[0051] 3.4 Data Analysis

[0052] SPSS 24.0 statistical software was used. Quantitative data on DNA yield and purity are expressed as mean ± standard deviation. To assess the main effects and interaction of the two factors, "FTA card type" and "DNA extraction method," on DNA extraction efficiency (including concentration, purity, and Ct value of the internal standard gene), this study employed two-way ANOVA for statistical testing. If the interaction was significant, a simple effects analysis was performed; if the interaction was not significant, the main effects were the primary focus. After confirming the existence of overall statistical significance, Tukey's post-hoc test was used for pairwise comparisons between groups. All statistical tests were two-tailed, and P < 0.05 was defined as statistically significant.

[0053] Sensitivity, specificity, accuracy, and p-value were calculated using conventional fluorescent PCR results as the standard. Sensitivity (%) = True positives / (True positives + False negatives) × 100%, Specificity (%) = True negatives / (True negatives + False positives) × 100%, Accuracy (%) = (True positives + True negatives) / (True positives + False negatives + True negatives + False positives) × 100%. In this study, samples identified as positive by conventional fluorescent PCR were considered true positives, while samples identified as positive by the dried blood spot-fluorescent PCR test but not by conventional fluorescent PCR were considered false positives. To assess the consistency of different methods in classification, Cohen's Kappa coefficient was used for analysis. The Kappa value measures the difference between observed concordance and random concordance, ranging from -1 to 1; a value closer to 1 indicates higher concordance. A Kappa value of 0.00–0.40 indicates poor consistency; 0.41–0.60 indicates moderate consistency; 0.61–0.80 indicates high consistency; and 0.81–1.00 indicates excellent consistency.

[0054] The extracted DNA samples were analyzed using a micro-spectrophotometer, a Qubit 4 fluorometer, and a Plasmodium nucleic acid detection kit (brand: Shanghai ZJ Biotech, catalog number: ER-5066-02) to measure DNA concentration, purity (A260 / A280), and amplification efficiency (Ct value) of the internal standard gene (RNase P) to comprehensively measure DNA extraction efficiency and quality. All data are expressed as mean ± standard deviation, and the results are summarized in Table 1.

[0055] Table 1. Quality statistics of DNA extracted using three methods from two blood samples.

[0056]

[0057] Regarding DNA extraction efficiency, as shown in Table 1 and... Figure 1 Two-way ANOVA was used to evaluate the effects of FTA card type and DNA extraction method on DNA concentration. The results showed that both the blood collection card type (F(1,24)=89.63, P<0.001) and the extraction method (F(2,24)=274.51, P<0.001) had a highly significant effect on DNA concentration, and there was a significant interaction between the two (F(2,24)=47.21, P<0.001).

[0058] The DNA yield of imported FTA cards (14.83 ± 5.67 ng / μL) was higher than that of domestically produced FTA cards (11.29 ± 6.09 ng / μL), and the difference was statistically significant (P<0.001).

[0059] Among the different extraction methods, YEASEN exhibited the highest extraction efficiency, achieving DNA concentrations of (16.20±0.75) ng / μL on domestically produced FTA cards and (19.46±0.79) ng / μL on imported FTA cards, with a small standard deviation, indicating high and stable yield. QIAGEN showed the lowest DNA yield, especially on domestically produced FTA cards, with a concentration of only (3.24±0.88) ng / μL. Although it improved to (8.46±1.43) ng / μL on imported cards, it was still significantly lower than the other two methods. Tiangen's yield was moderate, at (14.42±0.93) ng / μL on the two blood collection cards and (16.56±0.26) ng / μL on the other two methods. Tukey HSD post-hoc tests showed that the differences in DNA concentration between YEASEN and Tiangen and QIAGEN methods were all highly significant (all P<0.001).

[0060] Regarding DNA purity, as shown in Table 1 and... Figure 2The A260 / A280 ratio shows that the overall purity of the imported FTA card (1.84±0.13) is significantly better than that of the domestically produced FTA card (1.61±0.34). Specifically, when using the imported FTA card, the purity of all three extraction methods is good, especially the domestically produced magnetic bead method (YEASEN) and the imported centrifugal column method (QIAGEN), with A260 / A280 ratios of 1.890±0.072 and 1.860±0.187, respectively. However, with the domestically produced FTA card, the purity of all methods is not ideal, with the domestically produced centrifugal column method (Tiangen) having the lowest ratio (1.438±0.181).

[0061] Two-way ANOVA of the A260 / A280 ratio showed that blood card type had a highly significant main effect on DNA purity (F(1,24) = 16.75, P < 0.001), while the main effect of extraction method (F(2,24) = 2.89, P = 0.074) and the interaction effect (F(2,24) = 0.68, P = 0.517) did not reach statistical significance. Tukey HSD post-hoc test showed that the differences in A260 / A280 ratios among the extraction methods did not reach statistical significance (P > 0.05).

[0062] Regarding DNA functional quality testing, as shown in Table 1 and... Figure 3 Two-way ANOVA was used to assess the effects of FTA card type and DNA extraction method on the Ct value of the internal standard gene in fluorescent PCR. The results showed that both blood card type (F(1,24) = 7.23, P = 0.013) and extraction method (F(2,24) = 5.26, P = 0.013) significantly affected DNA template quality, indicating differences in DNA template quality among the two types of blood cards and the three extraction methods. However, the interaction between the two was not significant (F(2,24) = 1.85, P = 0.179).

[0063] The average Ct value of the internal standard gene on imported FTA cards (25.74±0.95) was significantly lower than that on domestically produced FTA cards (26.73±1.32), indicating that the DNA extracted from imported cards had higher amplification efficiency and template quality. Among different extraction methods, the Ct values ​​for the YEASEN method on the two blood cards were 26.04±1.42 and 26.19±1.12, respectively; for the QIAGEN method, they were 27.94±1.34 and 26.34±1.65, respectively; and for the Tiangen method, they were 26.42±1.29 and 25.69±0.55, respectively. Tukey's HSD test showed that the difference in Ct values ​​between the YEASEN and QIAGEN methods was statistically significant (P = 0.008), while there were no significant differences among other groups (P > 0.05).

[0064] Example 2: Clinical Sample Testing

[0065] Based on the results of the aforementioned study combining FTA cards and nucleic acid extraction methods, this study validated the dried blood spot method using known clinical blood samples to assess its effectiveness in detecting Plasmodium in actual samples. A total of 24 anticoagulated whole blood samples from ports of entry were included, all confirmed by conventional fluorescent PCR, with 12 positive and 12 negative samples. All samples were processed using imported FTA cards combined with the YEASEN magnetic bead method, followed by fluorescent PCR detection. The extracted DNA template was amplified and detected using a Plasmodium nucleic acid detection kit (fluorescent PCR method, Shanghai ZJ Biotech, catalog number: ER-5066-02). The reaction system and cycling parameters followed the kit instructions. The sensitivity, specificity, and consistency (Kappa value) of the dried blood spot method were evaluated using conventional venous whole blood fluorescent PCR results as the standard.

[0066] The test results are shown in Table 2. The results of the dried blood spot method for quantitative real-time PCR were completely consistent with those of conventional real-time PCR. The dried blood spot method correctly detected all 12 positive samples (12 true positives); 2 false positives were found in the 12 negative samples, and there were no false negatives.

[0067] Table 2 Statistical analysis of port sample test results

[0068]

[0069] The performance indicators are shown in Table 3. This method demonstrated excellent performance across all evaluation parameters. Sensitivity, specificity, positive concordance rate, negative concordance rate, and overall concordance rate all reached 100%, indicating perfect detection accuracy. Further verification through Kappa consistency analysis showed a Kappa value of 0.8334 (observed concordance rate Po = 0.9167, expected concordance rate Pe = 0.5), indicating that the detection results of the two methods are essentially consistent.

[0070] In summary, the DNA extracted using the imported FTA card in conjunction with the YEASEN magnetic bead method fully meets the requirements for quantitative real-time PCR detection, demonstrates reliable performance in malaria parasite detection, and is suitable for downstream molecular applications such as port screening.

[0071] Table 3 Detection performance indicators and consistency analysis

[0072]

[0073] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for nucleic acid extraction based on dried blood spots, characterized in that, Includes the following steps: (1) Collect samples, add them to Whatman FTA cards, and allow them to air dry overnight at room temperature to prepare dried blood spot samples; (2) Cut dried blood spots from the FTA card and extract genomic DNA using the YEASEN magnetic bead DNA extraction kit; The product number of the Whatman FTA card is WB129237; The YEASEN magnetic bead DNA extraction kit has the catalog number 18554ES48.

2. The method according to claim 1, characterized in that, In step (1), the samples include EDTA-anticoagulated blood, fingertip blood, heel blood, serum, and plasma.

3. The method according to claim 1, characterized in that, In step (1), the amount of sample used is 180-220 μL.

4. The method according to claim 1, characterized in that, In step (1), the preservation conditions for the dried blood spot sample are: placed in a sealed bag with a desiccant inside and stored away from light.

5. The method according to claim 1, characterized in that, In step (2), the area cut out is 2-3mm × 2-3mm.

6. The method according to claim 1, characterized in that, In step (2), the elution volume of the genomic DNA is 30-40 μL.

7. The application of the method according to any one of claims 1-6 in malaria screening.

8. A malaria field screening system, characterized in that, The system includes a detection device, a computing device, and an output device; the detection device includes a sample injector, a sample processor, and a detector. The sampler is used to collect samples from the subject; The sample processor is used to process the collected samples as follows: S1. Add the sample to a Whatman FTA card to prepare a dried blood spot sample; S2. Obtain dried blood spots from the FTA card and extract genomic DNA using the YEASEN magnetic bead DNA extraction kit. S3. Perform malaria parasite nucleic acid detection on the extracted DNA.

9. The method according to claim 8, characterized in that, In S3, the methods for nucleic acid detection include quantitative real-time PCR, nested PCR, multiplex PCR, digital PCR, loop-mediated isothermal amplification, recombinase polymerase amplification, Sanger sequencing, and high-throughput sequencing.

10. The method according to claim 9, characterized in that, The nucleic acid detection method is quantitative real-time PCR.

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