ABSTRACT.
This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.
INTRODUCTION
Malaria remains a significant health concern, with an estimated 247 million cases reported globally.1 Within 87 developing countries, a substantial portion of the population resides in tropical and subtropical areas, which are high-risk malaria transmission regions.1 In Thailand, the regions where malaria is endemic are located along the border, particularly along the Thai–Myanmar border, where the prevalence is highest.2 Malaria control faces a significant challenge in providing widespread access to accurate and effective diagnosis of malaria parasites. For malaria diagnosis, microscopic examination, and rapid diagnosis tests exhibit low sensitivity, with the detection limit as low as 100 parasites/µL.3–5 Furthermore, the microscopy technique requires skilled technicians to accurately distinguish malaria, especially when there are morphological similarities that lead to potential confusion.6–8 To overcome these problems, molecular techniques have been used as a solution. DNA extraction is one of the important steps of molecular diagnosis. Commercial DNA extraction kits are frequently used to reduce inhibitors such as hemoglobin in blood samples.9–12 However, the high cost of these kits limits their use in low-resource settings. As a result, several uncomplicated and cost-effective methods have been used as alternative approaches for extracting Plasmodium DNA.13–15 Traditionally, polymerase chain reaction (PCR) has been conducted using DNA extracted directly from whole blood. Nevertheless, an increasingly used method in low-resource settings involves PCR amplification of Plasmodium DNA extracted from dried blood spots (DBSs) on filter paper, as opposed to using whole blood samples.11,16 The use of DBSs offers the advantage of blood collection in remote rural areas with the benefit of room-temperature storage, so there is no necessity for a cold chain.11 In addition, DBSs require a smaller blood volume by finger prick compared with whole blood collected by venipuncture, making DBS favorable for collection, transportation, and storage.9,11 In this study, the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit; Vivantis Technologies Sdn. Bhd., Shah Alam, Malaysia) was used as the commercial DNA extraction kit. This commercial kit was used for extracting DNA from whole blood when studying the prevalence of Plasmodium vivax in Pakistan and extracting DNA from fighting cocks in Thailand for epidemiology studies.17,18 However, there have been no reports about the limit of detection (LOD) and use of DBS extraction with the GF-1 BD Kit. The DNAzol Direct reagent (DN 131) is an alkaline solution containing a high concentration of polyethylene glycol, which has the properties of lysing biological samples and yielding DNA into the lysate.19,20 The alkaline pH and chaotropic properties of DN 131 can inactivate PCR inhibitors, including proteases and nucleic acid degradation. Nevertheless, no studies of DN 131 for extracting Plasmodium DNA from whole blood and dried blood. The tris-ethylenediaminetetraacetic acid and proteinase K (TE-PK) method has been used to compare commercial kits and the limit of Plasmodium detection; however, this method has not been studied for DBS extraction.21
The objective of this study was to compare the efficiency of three DNA extraction methods, namely, the GF-1 BD Kit, DN 131, and TE-pK methods for extracting Plasmodium falciparum DNA from both whole blood and DBSs. We evaluated these methods based on various criteria, including DNA concentration, purity, time requirements, and cost-effectiveness. In addition, the limit of parasite detection was evaluated for each extraction method.
MATERIALS AND METHODS
Whole blood and dried blood preparation.
An EDTA blood sample containing P. falciparum was obtained from a hospital in Chaing Mai Province, Thailand, in 2016 and stored at −80°C. The presence of the parasite was examined by thick blood film microscopy, and the parasite density in the sample was quantified as 5,000 parasites/µL. After routine diagnosis, the samples were transported to the laboratory in a cooler and then stored at −20°C until used. Normal blood samples were taken from healthy persons with no history of malaria infection. A volume of 100 µL of positive whole blood was used for each experiment. For DBSs, 20 µL of the blood sample was spotted onto Whatman filter paper No. 4 (St Neots, United Kingdom), left to dry overnight, and subsequently stored in plastic bags at a temperature of −20°C until used. The study protocol was approved by the Khon Kaen University Ethics Committee for Human Research (HE651337). Informed consent was obtained from the study subjects using a standard approved procedure.
Determination of the limitation of three DNA extraction methods.
To prepare whole blood samples, a positive blood sample was diluted with uninfected human whole blood to generate mock P. falciparum whole blood mixtures of 1,000, 200, 40, 8, 1.6, 0.32, 0.06, 0.012, and 0.002 parasites/µL. A volume of 100 µL was used for each dilution of the whole blood samples. For the DBSs, 20 μL of fresh mock whole blood mixtures was spotted onto Whatman filter paper No. 4, dried overnight, and then stored in plastic bags at −20°C until used.
DNA extraction methods.
Plasmodium falciparum DNA was extracted from whole blood and DBS samples using three methods: the GF-1 BD Kit; DNAzol Direct reagent (DN 131; Molecular Research Center, Cincinnati, OH); and TE-pK, a previously published DNA extraction method.21 In this process, 100 µL of whole blood and 20 µL of DBSs were used. The GF-1 BD Kit was used following the manufacturer’s instructions, which encompassed a series of steps. These steps included cell lysis facilitated by a lysis buffer and incubation at 65°C, followed by two consecutive washing steps, and finally, a DNA elution step using a spin column in conjunction with an elution buffer. In the DNAzol Direct protocol, we made slight modifications to the procedure for extracting DNA from both whole blood and DBSs. The process began with mixing the blood sample with 1 mL of sterile distilled water (DW) to reduce inhibitors such as hemoglobin, followed by the addition of 100 µL of DN 131 reagent. After thorough vortexing, the samples were incubated at 80°C for 10 minutes during the whole blood extraction process. In the case of DBS extraction, incubation periods at 80°C for 10 minutes and 20 minutes were used for optimization. After centrifugation, the genomic DNA (gDNA) was used directly in the PCR reaction. In the Tris (Vivantis Technologies Snd. Bhd.)-EDTA (AppliChem, GmbH, Darmstadt, Germany) and proteinase K (Vivantis Technologies Snd. Bhd.) method, the sample was initially combined with 1 mL of sterile DW. After vigorous vortexing and centrifugation, the supernatant was discarded. The resulting lysate was then mixed with TE and proteinase K, followed by incubation at 56°C for 30 minutes for whole blood extraction and for 40 minutes and 1 hour for DBS extraction. Subsequently, the samples were further incubated at 80°C for 10 minutes before undergoing centrifugation, and the supernatant was collected for DNA template. The experiments were performed using microcentrifuge tubes and were conducted in duplicate and repeated three times. The protocols of the three difference DNA extraction methods from whole blood and DBSs are given in Tables 1 and 2, respectively.
Table 1.
The protocol of three different methods for extracting Plasmodium falciparum DNA from whole blood
| GF-1 BD Kit | TE-pK method | DN 131 |
|---|---|---|
| Add 100 µL of BB to 100 µL of whole blood in a 1.5-mL centrifuge tube and vortex vigorously. Add 10 µL of proteinase K; mix immediately. Incubate at 65°C for 10 minutes. Add 10 µL of RNase A, incubate at 37°C for 5 minutes, and then mix gently. Add 100 µL of absolute ethanol to the tube, mix vigorously, and load into a column with a collection tube. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard flow through. Add 250 µL of washing buffer 1 into a column with a collection tube. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard flow through. Add 250 µL of washing buffer 2 into a column with a collection tube. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard flow through. Centrifuge again at 12,000 × g, 2 minutes at 4°C for removing residual alcohol. Transfer the column to a new 1.5-mL centrifuge tube, add 30 µL of sterile DI to column tubes, and let it stand for 2 minutes at room temperature. Centrifuge at 12,000 × g, 5 minutes at 4°C; supernatant is collected and kept at −80°C. |
Add 1 mL of sterile DW to 100 µL of whole blood in a 1.5-mL centrifuge tube and vortex vigorously. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard supernatant. Add 40 µL of TE buffer and 20 µL of proteinase K (20 mg/mL) and mix gently. Incubate at 56°C for 30 minutes and then further incubate at 80°C for 10 minutes to inactivate proteinase K. Centrifuge at 12,000 × g, 5 minutes at 4°C; supernatant is collected and kept at −80°C until use. |
Add 1 mL of sterile DW to 100 µL of whole blood and vortex vigorously. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard supernatant. Add 100 µL of DNAzol Direct reagent into the tube and vortex. Incubate at 80°C, 10 minutes. Centrifuge at 12,000 × g, 5 minutes at 4°C; supernatant is collected and kept at −80°C. |
BB = blood lysis buffer; DI = deionized water; DN 131 = DNAzol Direct; DW = distilled water; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; RNase = ribonuclease; TE-pK = Tris-EDTA and proteinase K.
Table 2.
The protocol of three different methods for extracting Plasmodium falciparum DNA from DBSs by optimizing lysis time
| GF-1 BD Kit | TE-pK method | DN 131 |
|---|---|---|
| Add 100 µL of BB to a 1.5-mL centrifuge tube containing DBS, vortex to dissolve blood into buffer, and then discard filter paper (5 minutes). Add 10 µL of proteinase K and mix gently. Incubate at 65°C for 30 minutes and 1 hour, separate sample tube. Add 10 µL of RNase A, incubate at 37°C for 5 minutes, and then mix gently. Add 100 µL of absolute ethanol to the tube, mix vigorously, and load into a column with a collection tube. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard flow through. Add 250 µL of washing buffer 1 into a column with a collection tube. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard flow through. Add 250 µL of washing buffer 2 into a column with a collection tube. Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard flow through. Centrifuge again at 12,000 × g, 2 minutes at 4°C for removing residual alcohol. Transfer the column to a new 1.5-mL centrifuge tube, add 30 µL of sterile DI into column tubes, and let it stand for 2 minutes at room temperature. Centrifuge at 12,000 × g, 5 minutes at 4°C; supernatant is collected and kept at −80°C. |
Add 1 mL of sterile DW to a 1.5-mL centrifuge tube containing DBS, vortex to dissolve blood into sterile DW, and then discard filter paper (5 minutes). Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard supernatant. Add 40 µL of TE buffer and 20 µL of proteinase K (20 mg/mL) and mix gently. Incubate at 56°C, 40 minutes and 1 hour, separate sample tube, and then further incubate at 80°C for 10 minutes to inactivate proteinase K. Centrifuge at 12,000 × g, 5 minutes at 4°C; supernatant is collected and kept at −80°C. |
Add 1 mL of sterile DW to a 1.5-mL centrifuge tube containing DBS, vortex to dissolve blood into sterile DW, and then discard filter paper (5 minutes). Centrifuge at 12,000 × g, 5 minutes at 4°C, and then discard supernatant. Add 100 µL of DNAzol Direct reagent into the tube and vortex. Incubate at 80°C for 10 minutes and 20 minutes, separate sample tube. Centrifuge at 12,000 × g, 5 minutes at 4°C; supernatant is collected and kept at −80°C. |
BB = blood lysis buffer; DBS = dried blood spot; DI = deionized water; DN 131 = DNAzol Direct; DW = distilled water; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; RNase = ribonuclease; TE-pK = Tris-EDTA and proteinase K.
Polymerase chain reaction amplification.
For PCR, P. falciparum DNA was performed using specific primers designed from 18S rRNA, as previously described.22 The oligonucleotide primers were as follows: PLF, 5′-AGTGTGTATCAATCGAGTTTC-3′; FAR, 5′-AGTTCCCCTAGAATAGTTACA-3′. The PCR tube contained a total volume of 20 μL, consisting of 10 mM KCl buffer, 5 mM Tris–HCl, 2.5 mM MgCl2, 200 mM deoxyribonucleotide triphosphate, 0.5 μM of each primer, and 0.1 unit of Taq DNA polymerase (Vivantis Technologies). Reactions were performed in a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific, Waltham, MA). The cycling conditions consisted of an initial denaturation phase at 94°C for 2 minutes, 40 repetitions at 94°C for 30 seconds, annealing of each primer pair at 42°C for 1 minute, and extension at 72°C for 1 minute, followed by a final extension for 10 minutes at 72°C. The PCR products were visualized by electrophoresis on 1.5% agarose gel stained with ethidium bromide. The target size of P. falciparum is a 395-bp product.
DNA quality and quantity, estimated cost, and time required.
DNA concentrations and purity of the three DNA extraction methods were measured at A260/A280 nm ratio using a NanoDrop™ One spectrophotometer (Thermo Fisher Scientific). The absorbance ratio (A260/A280) of approximately 1.8 is generally considered for DNA purity. If the ratio notably drops to < 1.8, it could indicate the presence of proteins, phenol, or other contaminants that absorb strongly around 280 nm.23 The estimated cost was based on the price of the chemical and disposable items, including pipette tips and microcentrifuge tube. The estimated cost of a sample for each method was calculated in U.S. dollars (USD). The time required for each method to finish one extraction from whole blood and DBSs was estimated according to the procedures used in this study.
Data analysis.
The DNA yields and purities for both whole blood and DBS samples were determined as the mean ± SD through descriptive analysis. For statistical comparisons, the means of DNA yield and purity obtained from different DNA isolation methods were subjected to one-way analysis of variance (ANOVA), followed by the Tukey post hoc test. In addition, the independent t-test was used to analyze the optimization of DNA extraction from DBSs and for comparison between whole blood and DBSs in each DNA extraction method. A P value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (IBM SPSS Statistics version 28.0.1.0. [142], Armonk, NY).
RESULTS
Quantity and quality of gDNA from whole blood extraction.
A comparative analysis of the quantity and quality of gDNA was conducted using three different methods: the GF-1 BD Kit, DN 131, and TE-pK methods. The mean concentrations ±SD of DNA obtained with these methods were as follow: 67.1 ± 0.81 ng/µL for the GF-1 BD Kit, 80.8 ± 2.48 ng/µL for DN 131, and 52.8 ± 1.86 ng/µL for the TE-pK method. An ANOVA analysis revealed a significant difference in DNA concentrations, with the DN 131 method yielding significantly higher DNA concentrations than both the GF-1 BD Kit and the TE-pk method (P < 0.001). Conversely, the TE-pK method presented significantly lower DNA quantities than both the DN 131 and GF-1 BD Kit (P < 0.001).
The DNA purity, as indicated by the A260/A280 ratio, was 1.9 ± 0.02 for the GF-1 BD Kit, 1.7 ± 0.02 for DN 131, and 1.7 ± 0.02 for the TE-pK method. An ANOVA analysis revealed a significant difference in DNA purity with use of the GF-1 BD Kit compared with both the DN 131 and TE-pK methods (P < 0.001). However, there was no significant difference in DNA purity observed between the DN 131 and TE-pK methods (P = 0.104). The quantity and quality of DNA extracted from 100 µL of whole blood are presented in Table 3.
Table 3.
The quantity and quality of gDNA extracted from whole blood using three different DNA extraction methods
| DNA extraction method | GF-1 BD Kit | DN 131 | TE-pK method |
|---|---|---|---|
| Quantity of gDNA (ng/µL) | 67.1 ± 0.81 | 80.8 ± 2.48 | 52.8 ± 1.86 |
| A260/A280 | 1.9 ± 0.02 | 1.7 ± 0.02 | 1.7 ± 0.02 |
| Estimated time | 40 minutes | 30 minutes | 60 minutes |
| Estimated cost (USD) | 20 | 1 | 0.15 |
DN 131 = DNAzol Direct; EDTA = ethylenediaminetetraacetic acid; gDNA = genomic DNA; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; TE-pK = Tris-EDTA and proteinase K; USD = U.S. dollars.
Quantity and quality of gDNA from DBS extraction.
Dried blood spot extraction was optimized because of a lack of previous studies employing the techniques used in this study. In this study, we compared the GF-1 BD Kit at two different incubation times, 30 minutes and 1 hour. The results revealed that DNA extracted with a 30-minute lysis time (31.2 ± 1.09 ng/µL) showed significantly higher yields than the 1-hour lysis time (28.5 ± 1.05 ng/µL) (P = 0.002). However, there was no significant difference in DNA purity between the 30-minute (1.9 ± 0.04) and 1-hour (1.9 ± 0.05) lysis times (P = 0.105). Regarding the DN 131 method, the means of DNA yields were 33.3 ± 2.13 ng/µL and 42 ± 3.56 ng/µL when utilizing lysis times of 10 minutes and 20 minutes, respectively. The DNA concentration was significantly higher when a 20-minute lysis time was used compared with a 10-minute lysis time (P < 0.001). As for DNA purity, the A260/A280 ratio was 1.7 ± 0.03 for 10-minute lysis and 1.7 ± 0.01 for 20-minute lysis, and there was no significant difference in A260/A280 between these two lysis times when using the DN 131 method (P = 0.055).
In the case of the TE-pK method, a comparison was conducted between lysis times of 40 minutes and 1 hour. The mean DNA yields were 18.3 ± 0.62 ng/µL for 40-minute lysis time and 15.8 ± 0.4 ng/µL for 1-hour lysis time. Significantly higher DNA yields were obtained with a 40-minute lysis time compared with a 1-hour lysis time (P < 0.001). The DNA purity, as indicated by the A260/A280 ratio, was 1.7 ± 0.03 for 40 minutes of lysis and 1.7 ± 0.03 for 1 hour of lysis when using the TE-pK method. However, there was no significant difference in DNA purity observed between the 40-minute and 1-hour lysis times with the TE-pK method (P = 0.19). Detailed results on the optimization of DNA extractions from DBSs, including varying lysis times within each DNA extraction method, are shown in Table 4.
Table 4.
Quantity and quality of DNA extracted from DBSs with different lysis times
| DNA extraction method | Lysis time | DNA yield (ng/µL) (mean ± SD) | P value | A260/A280 (mean ± SD) | P value |
|---|---|---|---|---|---|
| GF-1 BD Kit | 30 minutes | 31.2 ± 1.09 | 0.002 | 1.9 ± 0.04 | 0.105 |
| 1 hour | 28.5 ± 1.05 | 1.9 ± 0.05 | |||
| DN 131 | 10 minutes | 33.3 ± 2.13 | < 0.001 | 1.7 ± 0.03 | 0.055 |
| 20 minutes | 42 ± 3.56 | 1.7 ± 0.01 | |||
| TE-pK method | 40 minutes | 18.3 ± 0.62 | < 0.001 | 1.7 ± 0.03 | 0.19 |
| 1 hour | 15.8 ± 0.40 | 1.7 ± 0.03 |
DN 131 = DNAzol Direct; EDTA = ethylenediaminetetraacetic acid; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; TE-pK = Tris-EDTA and proteinase K.
After optimization, optimal conditions for DBSs when considering the DNA yields and purity of each DNA extraction method were determined as follows: 30 minutes of lysis time for the GF-1 BD Kit, 20 minutes of lysis time for DN 131, and 40 minutes of lysis time for the TE-pK method. Among three DNA extraction methods, the DN 131 method yielded the highest DNA quantity (42 ± 3.56 ng/µL), followed by the GF-1 BD Kit (31.2 ± 1.09 ng/µL) and the TE-pK method (18.3 ± 0.62 ng/µL). From the ANOVA analysis, the DNA yields obtained with the DN 131 method were significantly higher than those with the GF-1 BD Kit and TE-pK methods (P < 0.001), and the GF-1 BD Kit also yielded significantly higher DNA yields than the TE-pK method (P < 0.001).
The purity of DNA extracted using the GF-1 BD Kit was 1.9 ± 0.04, and this method showed a significant difference in purity compared with both the DN 131 method and the TE-pK method (P < 0.001). However, there was no significant difference in DNA purity between the DN 131 method (1.7 ± 0.01) and the TE-pK method (1.7 ± 0.03) (P = 0.31). A comprehensive overview of the quantity and quality of DNA extracted from DBSs using the optimal conditions for each method is shown in Table 5.
Table 5.
Comparison of quantity and quality of DNA extracted from DBSs by using optimal conditions for each method
| DNA extraction method | Optimal lysis time | DNA yield (ng/µL) (mean ± SD) | A260/A280 (mean ± SD) | Estimated time | Estimated cost per sample (USD) |
|---|---|---|---|---|---|
| GF-1 BD Kit | 30 minutes | 31.2 ± 1.09 | 1.9 ± 0.04 | 1 hour | 20 |
| DN 131 | 20 minutes | 42 ± 3.56 | 1.7 ± 0.01 | 45 minutes | 1 |
| TE-pK method | 40 minutes | 18.3 ± 0.62 | 1.7 ± 0.03 | 1.2 hours | 0.15 |
DBS = dried blood spot; DN 131 = DNAzol Direct; EDTA = ethylenediaminetetraacetic acid; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; TE-pK = Tris-EDTA and proteinase K; USD = U.S. dollars.
Limitation of detection from whole blood and DBS extractions.
The LOD from whole blood extraction was 0.012 parasites/µL for the GF-1 BD Kit and DN 131 methods and 1.6 parasites/µL for the TE-Pk method (Figure 1). In DBS extraction, the LOD was 1.6 parasites/µL for the DN 131 method, 8 parasites/µL for the GF-1 BD Kit, and 200 parasites/µL for the TE-pK method (Figure 2).
Figure 1.
Limit of detection of Plasmodium falciparum from whole blood using the GF-1 BD Kit, DN 131, and TE-pK methods. The numbers above each lane indicate the number of parasites present per microliter of blood. Infected whole blood was serially diluted with heathy whole blood. The initial parasite density was 5,000 parasites/µL. Lane M is a DNA marker (100 bp). (A) The DN 131 method showed a limit of detection of 0.012 parasites/µL. (B) The GF-1 BD Kit showed a limit of detection of 0.012 parasites/µL. (C) The TE-pK method showed a limit of detection of 1.6 parasites/µL. The arrows indicate a PCR product of about 395 bp. DN 131 = DNAzol Direct; EDTA = ethylenediaminetetraacetic acid; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; PCR = polymerase chain reaction; TE-pK = Tris-EDTA and proteinase K.
Figure 2.
Limit of detection of Plasmodium falciparum from DBSs using the GF-1 BD Kit, DN 131, and TE-pK methods. The numbers above each lane indicate the number of parasites present per microliter of blood. Infected whole blood was serially diluted with heathy whole blood. The initial parasite density was 5,000 parasites/µL. Lane M is a DNA marker. (A) The DN 131 method showed a limit of detection of 1.6 parasites/µL. (B) The GF-1 BD Kit showed a limit of detection of 8 parasites/µL. (C) The TE-pK method showed a limit of detection of 200 parasites/µL. The arrows indicate a PCR product of about 395 bp. DBS = dried blood spot; DN 131 = DNAzol Direct; EDTA = ethylenediaminetetraacetic acid; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; PCR = polymerase chain reaction; TE-pK = Tris-EDTA and proteinase K.
Comparison of quantity and quality between whole blood and DBSs in each DNA extraction method.
The quantity of DNA extracted from whole blood (67.1 ± 0.81 ng/µL) using the GF-1 BD Kit was significantly higher than the quantity obtained from DBSs (31.2 ± 1.09 ng/µL) (P < 0.001). However, there was no significant difference in purity between whole blood (1.9 ± 0.02) and DBSs (1.9 ± 0.04) when using the GF-1 BD Kit (P = 0.27). In the case of the DN 131 method, the DNA yields extracted from whole blood (80.8 ± 2.48 ng/µL) were significantly higher than those obtained from DBSs (42 ± 3.56 ng/µL) (P < 0.001). However, there was no significant difference in purity between whole blood (1.7 ± 0.02) and DBSs (1.7 ± 0.01) when using DN 131 (P = 0.24). Similarly with the TE-pK method, the DNA yields extracted from whole blood (52.8 ± 1.86 ng/µL) were significantly higher than those obtained from DBSs (18.3 ± 0.62 ng/µL) (P < 0.001). Although the quality of DNA from whole blood (1.7 ± 0.02) was higher than that from DBSs (1.7 ± 0.03), there was no significant difference in purity of DNA between whole blood and DBSs using the TE-pK method (P = 0.33). A comparative analysis of the quantity and quality of DNA extracted from whole blood and DBSs is presented in Table 6.
Table 6.
Comparison between quantity and quality of DNA extractions from whole blood and DBSs
| DNA extraction method | Type of blood sample | DNA yields (ng/µL) (mean ± SD) A260/A280 | P value | A260/A280 | P value |
|---|---|---|---|---|---|
| GF-1 BD Kit | Whole blood | 67.1 ± 0.81 | < 0.001 | 1.9 ± 0.02 | 0.27 |
| DBS | 31.2 ± 1.09 | 1.9 ± 0.04 | |||
| DN 131 | Whole blood | 80.8 ± 2.48 | < 0.001 | 1.7 ± 0.02 | 0.24 |
| DBS | 42 ± 3.56 | 1.7 ± 0.01 | |||
| TE-pK method | Whole blood | 52.8 ± 1.86 | < 0.001 | 1.7 ± 0.02 | 0.33 |
| DBS | 18.3 ± 0.62 | 1.7 ± 0.03 |
DBS = dried blood spot; DN 131 = DNAzol Direct; EDTA = ethylenediaminetetraacetic acid; GF-1 BD Kit = GF-1 Blood DNA Extraction Kit; TE-pK = Tris-EDTA and proteinase K.
DISCUSSION
This study reports a comparative analysis of three DNA extraction methods used on blood infected with P. falciparum. These methods were applied to both whole blood and DBSs, with a focus on evaluating DNA concentration, purity, cost-effectiveness, and time efficiency. In addition, the study examined the limitations of parasite detection under optimal conditions for each extraction method, considering both whole blood and DBS samples. The temperature and duration of cell lysis incubation have an impact on both the quality and quantity of extracted DNA.24,25 For DNA extraction from whole blood, 100 µL of the blood sample was used, and the protocols provided by each method’s manufacturer were followed. Nevertheless, because no prior research has been reported on DNA extraction from P. falciparum–infected blood on DBSs using the three methods in this study, a comprehensive optimization of the incubation period duration for each method was carried out.
Regarding whole blood DNA extraction, the DNA concentration rates achieved through the DN 131 reagent were significantly higher than those of the GF-1 BD Kit and the TE-pK method, whereas the DNA concentration obtained via the GF-1 BD Kit was significantly higher than that of the TE-pK method. Notably, the GF-1 BD Kit involves a multistep process that includes cell lysis utilizing a specialized buffer system, DNA binding to a spin column, and the subsequent elution of purified DNA. This multistep procedure may lead to a loss of DNA quantity during the process. The DNA purity extracted by the DN 131 reagent was significantly different than that of the GF-1 BD Kit, whereas the purity of DNA from the TE-pK method was not significantly different. The GF-1 BD Kit involves the step of adding RNase, which can enhance the purity of the DNA template, distinguishing it from the other two methods used in this study.
The DNA concentration yields achieved through the DN 131 reagent exhibited a notable superiority over those yielded by the GF-1 BD Kit, primarily because of the simplicity of the DN 131 protocol. In this protocol, a blood sample was incubated at 80°C followed by centrifugation, and the resulting supernatant was directly used as a DNA template. In contrast, the standard procedure for commercial DNA extraction kits, including the GF-1 BD Kit, involves multiple steps, such as cell lysis with a specialized buffer system, DNA binding to a spin column, and subsequent elution of purified DNA as mentioned above. This complex process can potentially result in a loss of DNA quantity. These findings align with the research of Tomasek et al.26 in 2008, who reported higher DNA yields from avian blood samples using the DN 131 method (ranging from 620 to 3,370 ng/µL) compared with the commercial NucleoSpin® Kit (ranging from 7.2 to 11.8 ng/µL).
For DBS extraction results, the optimal conditions for the GF-1 BD Kit were found to involve incubation at 65°C for 30 minutes, as evidenced by the significantly higher DNA yield observed during the 30-minute incubation compared with the 1-hour incubation, potentially indicating that DNA might degrade with prolonged incubation times. Under optimal conditions for the DN 131 method, the DNA yield improved with incubation at 80°C for 20 minutes compared with incubation for 10 minutes. This observation was determined through use of a nanodrop spectrophotometer, revealing a higher yield from the 20-minute incubation. This outcome suggests that the shorter 10-minute incubation duration did not adequately facilitate cell lysis. In the case of the TE-pK method, a significant increase in DNA yield was observed with a 40-minute incubation compared with a 1-hour incubation. This indicates that the longer time of incubation can degrade DNA yield, as evidenced by the presence of a lower DNA concentration.
The sensitivity of DNA extracted from whole blood and DBSs using three different methods in this study was also examined. The findings demonstrated that the LODs achieved with the GF-1 BD Kit and the DN 131 method were higher than that obtained with the TE-pK method. This difference could be attributed to the beneficial properties of the cell lysis buffers in both the GF-1 BD Kit and the DN 131 method. These buffers contain potent components that effectively protect against nuclease activity and even inhibit potential PCR inhibitors, thus facilitating a substantial yield of gDNA.27,28
In the comparative analysis between DNA extraction from whole blood and DBSs, the DNA yield from the whole blood extraction method was statistically significantly greater than the yields obtained from each method when applied to DBS samples. Although the use of DNA extracted from DBSs results in lower DNA yield, the technique of blood collection on filter paper retains several practical advantages: 1) less invasive blood collection with a finger prick, 2) convenient storage and transportation of samples at room temperature, and 3) minor risk of biohazard exposure.9 Moreover, DBSs offer cost-effectiveness and simplicity and have previously been used in malaria research.10,14,29 Furthermore, we conducted a comparison of the costs associated with DNA extraction, as well as the time required for the completion of the procedures for all three methods. Particularly in developing countries, where cost-effectiveness is of utmost importance, commercial DNA extraction kits can be financially burdensome, and there is a preference for employing a DNA extraction approach that is both economical and efficient.12
A limitation of the study was the lack of known quantities of Plasmodium DNA at each dilution as controls. To address this issue, we duplicated each dilution and repeated the dilution process three times for both whole blood and DBSs. Another limitation of this study was that we used only one sample for comparing three DNA extraction methods. In future research, we intend to apply these findings in the field of malaria control in endemic areas, particularly along border areas such as Thailand–Myanmar. The ongoing challenge of malaria along the Thailand–Myanmar border is frequently attributed to a spillover effect, with the movement of people from Myanmar playing a significant role in the continued prevalence of malaria in the region.30,31 Previous studies had reported molecular techniques for malaria control in this region.32,33
The GF-1 BD Kit, priced at approximately $20 USD per sample, is the most expensive among the three methods owing to its multistep process involving various reagents, including those for cell lysis and the spin column for DNA washing and elution, whereas the DN 131 method cost only $1 USD per sample, as it uses a single reagent for extraction. The TE-pK method is the most cost-effective, with a cost of approximately $0.15 USD per sample, as it allowed for preparation in larger volumes and used only 40 µL of TE buffer and 20 µL of proteinase K per sample under the conditions of the current study. The results indicate that the TE-pK method is the most economically viable option, whereas the DN 131 method stands out for its simplicity.
CONCLUSION
To the best of our knowledge, this study represents an initial investigation into the use of DBS conditions with the GF-1 BD Kit, DN 131, and TE-pK method. The results unequivocally indicate that the DNAzol Direct reagent stands out as the optimal choice owing to its cost-effectiveness, time efficiency, and superior LOD in comparison to both the GF-1 BD Kit and the TE-pK method for extracting DNA from both whole blood and DBS samples. Nevertheless, the TE-pK method could be a suitable, more economical alternative for whole blood extraction compared with use of a commercial kit. These findings offer great promise for the application of these approaches in large-scale studies in resource-limited regions, facilitating malaria control efforts. In future studies, these findings will be implemented in the field to enhance malaria surveillance in endemic areas in Thailand, promoting its effectiveness.
ACKNOWLEDGMENTS
We thank the Department of Parasitology, Faculty of Medicine, Khon Kaen University, for providing research space. In addition, we extend our thanks to Chiangmai Hospital for its contribution of the positive blood sample used in this research.
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