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Journal of Insect Science logoLink to Journal of Insect Science
. 2023 Oct 7;23(5):15. doi: 10.1093/jisesa/iead080

Comparison of 3 DNA extraction methods for extracting DNA from an adult Culex quinquefasciatus (Diptera: Culicidae)

Nurhadi Eko Firmansyah 1, Thaksaporn Thongseesuksai 2, Thidarut Boonmars 3,4, Saleha Sungkar 5, Porntip Laummaunwai 6,7,✉
Editor: Amr Mohamed
PMCID: PMC10560001  PMID: 37804500

Abstract

The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ± 3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ± 4.57 ng/µl) and a microwave-based method (119.26 ± 3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ± 0.02, 1.79 ± 0.01, and 1.87 ± 0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.

Keywords: mosquito, cost- and time-effective, polymerase chain reaction, microwave irradiation, DNAzol Direct reagent

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Introduction

Mosquitoes are regarded as one of the most important arthropods in the transmission of several vector-borne pathogens to humans, companion animals, and livestock (Silbermayr et al. 2014, WHO 2014). The primary mosquito genera responsible for the spread of human pathogens are Anopheles, Aedes, Mansonia, and Culex (Bamou et al. 2021, Sriwichai et al. 2016). Anopheles species play a role in transmitting malaria and lymphatic filariasis. Aedes aegypti can transmit dengue virus to humans and some Aedes species are vectors of chikungunya and lymphatic filariasis (Dahmana and Mediannikov 2020). Culex species transmit lymphatic filariasis and Japanese encephalitis. Mansonia species are the vectors of chikungunya and lymphatic filariasis (WHO 2014, Lee et al. 2018). Vector control programs in endemic areas for mosquito-borne pathogens require data on feeding behavior, biting habits, vector competence, diversity, and distribution of mosquito species. Accurate information on the species complexes and sibling species present in any area is especially important (Tainchum et al. 2015, Weeraratne et al. 2017). Identification of mosquitoes by morphology requires an experienced taxonomist. Additionally, distinguishing members of a species complex is difficult. Therefore, molecular techniques have been developed and applied to identify mosquitoes and complement morphological identification, including ecological, phylogenetic, and genetic variations (Chan et al. 2014, Poolphol et al. 2017). DNA extraction is one of the important steps for molecular analysis. In general, commercial DNA extraction kits are considered the conventional method (Psifidi et al. 2015). However, commercial kits can be expensive and time-consuming, which may not be suitable for large-scale identification, particularly in low-resource settings. Therefore, different protocols for mosquito DNA extraction have been established in search of simple, inexpensive, and accurate results (Vasuki et al. 2012, Musapa et al. 2013, Chen et al. 2021).

There are rapid and efficient reagents available for DNA extraction from biological samples, such as DNAzol reagent and DNAzol Direct reagent. DNAzol is a guanidine-detergent lysing solution that hydrolyzes RNA and permits DNA precipitation from a cell lysate with ethanol (Molecular Research Center 2015a). The DNAzol method had been reported for extracting DNA from mosquito larvae and adults to study mosquito-borne malaria (Gupta and Preet 2012, Rider et al. 2012). DNAzol Direct is an alkaline solution containing a high concentration of polyethylene glycol (PEG) which has the properties of lysing biological samples and yielding DNA into the lysate (Chomczynski et al. 2006, García-Romero et al. 2019). The DNAzol Direct had been used for extracting DNA from Ae. aegypti to identify a new endogenous viral element (Suzuki et al. 2020).

Microwave-assisted extraction has been used in various studies for DNA extractions from blood, plants, and microorganisms (Rasmussen et al. 2008, Port et al. 2014, Melendez et al. 2016, Altemimi et al. 2017). Heating can be achieved by microwave irradiation (Port et al. 2014, Melendez et al. 2016). However, there have been no reports of using microwave irradiation for DNA extraction from adult mosquitoes. The aim of this study was to compare 3 DNA extraction methods: PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method for extracting DNA from an adult Cx. quinquefasciatus for subsequent polymerase chain reaction (PCR) assays in terms of DNA concentration, absorbance ratios, cost-effectiveness, and time required.

Materials and Methods

Mosquito Collection

Adult female Cx. quinquefasciatus were collected around Khon Kaen University using an electric aspirator. Mosquitoes were caught from 6:00 PM to 9:00 PM, the peak biting period according to some studies (Chen et al. 2017, Poolphol et al. 2017, St Laurent et al. 2017). Identification of all female mosquitoes was based on the Thailand keys to species level (Rattanarithikul et al. 2005). All mosquitoes were kept in sterile microcentrifuge tubes and stored at −20 °C.

Design of DNA Extraction

Three methods were used to extract DNA: a commercial DNA extraction kit (PureLink Genomic DNA Kit, Invitrogen, USA), DNAzol Direct reagent (Molecular Research Center, Inc., Catalog # DN131, Cincinnati, OH, USA), and the microwave-based method. DNA extraction was carried out in a microwave oven (Samsung, Model Q019, 900W, Samsung Co. Ltd., Japan) and operated at high power levels (800 W). The heating time was recorded accurately using a stopwatch. In this study, each method was optimized by varying the incubation time to achieve optimal DNA isolation. The cuticle of mosquitoes is composed of chitin, protein, and lipid. Chitin, being an insoluble fiber, is difficult to break down (Kouřimská and Adámková 2016). Therefore, grinding the mosquito is a crucial step that must be taken before following the protocol of each method. The PureLink Genomic Kit contains 2 main reagents: cell lysis and protein precipitation solutions. After griding with a sterile micro pestle, the lysates were incubated for 1 and 2 h at 55 °C. For DNAzol Direct, the mosquitoes were incubated at 80 °C for 10 and 20 min after griding, and for a microwave-based method, the mosquitoes were optimized by heating at 800 W for 5 and 10 min after griding. For optimization, each condition of each method was performed 3 times. After optimization, 10 mosquitoes were individually used for DNA extraction by each optimized method.

DNA Amplification

A standard PCR was used to amplify DNA samples using primers for the coding region locus CQ11 of Cx. quinquefasciatus. The primer sequences are forward (5ʹ-CCT TCT TGA ATG GCT GTG GCA-3ʹ) and reverse (5ʹ-TGG AGC CTC CTC TTC ACG G-3ʹ), yielding a PCR product of 274 bp (Smith and Fonseca 2004). The PCR mixture (20 μl volume) contained 10 mM KCl buffer, 5 mM Tris-HCl, 2.5 mM MgCl2, 200 mM dNTP, 0.5 μM of each primer, and 0.08 units of RBC Taq DNA polymerase (RBC Bioscience, Taiwan). The PCR cycle consisted of 1 cycle at 94 °C for 2 min, followed by 40 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min, and then an additional extension at 72 °C for 10 min. The PCR products were visualized by electrophoresis on 1.5% agarose gel stained with ethidium bromide.

DNA Quality and Quantity, Estimated Cost, and Time Required

DNA concentrations and purity of 3 DNA extraction methods were measured at 260/280 ratio nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). Generally, the expected absorbance ratios for extracted DNA samples should range from 1.7 to 2.0 (Linton et al. 2001, Miller et al. 2009). The estimated cost was based on the price of chemicals and disposable items, including pipette tips and microcentrifuge tubes. The estimated cost of a sample for each method was calculated in US dollars (USD). The time required by each method to finish 1 extraction from an adult single mosquito was estimated based on the procedures used in this study.

Statistical Analysis

Statistical analysis was performed using SPSS software (IBM SPSS Statistics version: 28.0.1.0 [142]). The mean and standard error (SE) of DNA yield rate and absorbance ratio (A260/A280) were performed by descriptive analysis. For statistical comparisons, the mean of DNA yield from different DNA isolation methods was analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The value of P < 0.05 was considered to be statistically significant.

Results

For the optimization results, it was observed that using the PureLink Genomic DNA kit method, the solution from incubation for 1 h did not produce any PCR product after electrophoresis. On the other hand, when the sample was incubated for 2 h, a PCR product of approximately 274 bp size was obtained. The DNA concentration of 2-h incubation ranged from 98 to 112 ng/µl, and the 260/280 ratio was 1.8. The result of the ANOVA analysis revealed a significant increase in DNA yield when incubating for 2 h compared to 1 h (F = 9.5, df = 4, P < 0.001), and the purity of the DNA obtained after 2 h of incubation was significantly higher than that obtained after 1 h (F = 9.143, df = 4, P < 0.001). For the optimization of DNAzol Direct method, lysates from 10 and 20 min incubation were amplified with specific primers, resulting in a PCR product with approximately 274 bp in size. The DNA concentration for the 10-min lysate incubation was in the range from 43 to 67 ng/µl, while that for the 20-min incubation was in the range from 143 to 172 ng/µl. The A260/A280 ratio for the 10 min incubation was 1.5–1.6, while that for the 20 min incubation was 1.7–1.8. The statistical analysis showed a significant increase in DNA yield when incubating for 20 min compared to 10 min (F = 0.081, df = 4, P < 0.001), and the purity increased significantly for 20-min incubation compared to 10 min (F = 0.663, df = 4, P = 0.013). After optimizing the microwave-based method, it was observed that the lysate heated for both 5 and 10 min presented a PCR product of approximately 274 bp when using specific primers. The DNA concentration for the lysate heated for 5 min ranged from 96 to 115 ng/µl, whereas that for the lysate heated for 10 min ranged from 59 to 73 ng/µl. Additionally, the A260/A280 ratio for both the 5- and 10-min heating was 1.8. The statistical analysis showed a significant increase in DNA yield when incubating for 5 min compared with 10 min (F = 0.514, df = 4, P < 0.004), and the purity was not significant for 20-min incubation compared with 10-min incubation (F = 0.681, df = 4, P = 0.74).

Based on the optimization results, it was determined that the optimal conditions for the PureLink Genomic DNA kit were incubation at 55 °C for 2 h, while for the DNAzol Direct method, it was incubation at 80 °C for 20 min. The microwave-based method showed optimal results with heating at 800 W for 5 min. The workflow of the 3 optimized methods for DNA extraction is presented in Table 1. The results presented in Fig. 1, showed the PCR product of DNA extracted from individual mosquitoes by 3 optimized methods after amplification with specific primers, yielding fragments of approximately 274 bp. There were no PCR products from the negative control which used distilled water as a template (Fig.1, Lane 1).

Table 1.

DNA extraction protocols of 3 optimized methods

Commercial DNA extraction (PureLink Genomic DNA kits) DNAzol Direct extraction (Molecular Research Center, Catalog # DN131) Microwave-based method

  • One female mosquito in a sterile 1.5-ml micro-centrifuge tube

  • Add 20 μl of proteinase-K and 180 μl of lysis buffer, grind with micro-pestle for 1 min then vortex vigorously for 30 s

  • Incubate at 55 °C, 2 h

  • Centrifuge at 12,000 g for 3 min, transfer supernatant to a new sterile tube

  • Add 20 µl RNase and incubate at room temperature for 2 min

  • Add 200 μl of binding buffer and vortex FOR HOW LONG?

  • Add 200 μl of absolute ethanol to lysate and mix gently for 5 s

  • Transfer lysate to spin column in a collection tube and centrifuge at 12,000 g for 3 min

  • Discard flow-through and then add 500 µl of washing buffer to the column tube

  • Centrifuge at 12,000 g for 3 min

  • Place the spin column into a new micro-centrifuge tube, then add 30 µl of elution buffer

  • Centrifuge at 12,000 g for 5 min, then DNA keep at −20 °C

  • One female mosquito in a sterile 1.5-ml micro-centrifuge tube

  • Add 150 µl of DNAzol Direct, grind with a sterile micro-pestle for 1 min then vortex vigorously for 30 s

  • Incubate at 80 °C for 20 min

  • Centrifuge at 12,000 g for 5 min, transfer supernatant to a new sterile tube

  • Store supernatant at −20 °C

  • One female mosquito in a sterile 1.5-ml micro-centrifuge tube

  • Add 200-μl sterile distilled water, grind with sterile micro-pestle for 1 min, then vortex vigorously for 30 s

  • Heat in microwave at 800 W for 5 min

  • Centrifuge at 12,000 g for 5 min, transfer supernatant to a new sterile tube

  • Store supernatant at −20 °C
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Fig. 1.

Fig. 1.

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Culex quinquefasciatus gene products. Lane M, DNA ladder; Lane 1, negative control; Lane 2, PCR product of DNA extracted from commercial DNA extraction kit; Lane 3, PCR product of DNA extracted from DNAzol Direct method; Lane 4, PCR product of DNA extracted by the microwave-based method. The arrow indicates a PCR product of about 274 bp.

Figure 2 shows the PCR products of DNA extracted from 10 Cx. quinquefasciatus individuals using the optimization conditions of 3 methods: the PureLink Genomic DNA kit (Fig. 2A), the DNAzol Direct reagent (Fig. 2B), and the microwave-based method (Fig. 2C). The DNA yields, absorbance ratios, time required, and cost per sample for the 3 methods of DNA extraction are listed in Table 2. The results of the ANOVA analysis indicated that the yield rates of the DNAzol Direct method show a significantly greater DNA yield (F = 84.869, df = 2, 27, P < 0.001) when compared with the yields of the PureLink Genomic DNA kit and microwave-based method. The yield rates between the commercial kit and microwave-based method are not significantly different (F = 84.869, df = 2, 27, P = 0.820). The mean absorbance ratios for a PureLink Genomic DNA kit method and microwave-based methods were 1.92 ± 0.02 and 1.87 ± 0.01, respectively, while the mean absorbance ratio for DNAzol Direct method was 1.79 ± 0.01. After ANOVA analysis, the purity of DNA from the microwave method is not significantly different (F = 11.936, df = 2,27, P = 0.126) when compared with the PureLink Genomic DNA kit and DNAzol Direct. Whereas the DNA purity from DNAzol Direct is significantly different when compared with the PureLink Genomic DNA kit (F = 11.936, df = 2,27, P < 0.001). The estimated time required for each DNA extraction method was 2.30 h for the PureLink Genomic DNA extraction kit, 30 min for the DNAzol Direct method, and 15 min for the microwave-based method. The estimated cost of each method was as follows: the PureLink Genomic DNA kit cost 16 USD, the DNAzol Direct cost 2 USD, and the microwave-based method cost 0.2 USD.

Fig. 2.

Fig. 2.

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Culex quinquefasciatus gene products. Lane M, DNA ladder; Lane 1, negative control. A, the PCR products of DNA extracted by the PureLink Genomic DNA kit; B, PCR product of DNA extracted by the DNAzol Direct reagent, C; PCR product of DNA extracted by the microwave-based method. The arrow indicates a PCR product of about 274 bp.

Table 2.

Comparison of DNA yields, absorbance ratios, estimated cost, and time required for 3 DNA extraction methods

Evaluation PureLink Genomic DNA Kits (n = 10) DNAzol Direct (n  = 10) Microwave-based method (n  = 10)
DNA yield rate (mean ± SE) (ng/µl) 115.98 ± 4.57 179.08 ± 3.77 119.26 ± 3.06
Absorbance ratio (mean ± SE) and range 1.92 ± 0.02
1.81–2.06		 1.79 ± 0.01
1.78–1.8		 1.87 ± 0.01
1.8–1.94
Estimated cost (USD) per sample 16 2 0.2
Time consuming (h, min) 2 h and 30 min 30 min 15 min
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Discussion

As commercial DNA extraction kits can be costly and time consuming, alternative DNA extraction methods have been developed and evaluated the reliability of various DNA extraction methods from mosquitoes (Vasuki et al. 2012, Musapa et al. 2013, Chen et al. 2021). When designing an optimal DNA extraction method, certain requirements such as optimized performance, removal of PCR inhibitors, simple laboratory procedures, low expense, good reproducibility, and safety must be achieved (Rantakokko-jalava and Jalava 2002). This study aimed to compare 3 DNA extraction methods for adult Cx. quinquefasciatus: PureLink Genomic DNA kit, DNAzol Direct, and the microwave-based method. The comparison was focused on the quantity and purity of genomic DNA, as well as cost-efficiency and time consuming. The mosquito cuticle is made up of the polysaccharide chitin, proteins, and lipids, which form an extracellular layer that covers the external surface and acts as a protective barrier between the mosquito and its environment (Moussian 2010). Chitin acts as an insoluble fiber, meaning it does not dissolve in water, and therefore it does not easily break down (Kouřimská and Adámková 2016). Therefore, we needed to grind the mosquito prior to processing and each method had to optimize the conditions to achieve a high DNA yield in a time-efficient manner. The temperature and duration of cell lysis incubation have an impact on both the quality and quantity of extracted DNA. In this study, the duration of incubation time for each method was optimized. The optimal conditions for the PureLink Genomic DNA kit were found to involve incubation at 55 °C for 2 h, as evidenced by the presence of a PCR product of approximately 274 bp following electrophoresis analysis. Whereas no specific PCR product was observed in the solution that underwent 1 h of incubation. These results suggest that 2-h incubation time is required for complete cell lysis in this method, as evidenced by the presence of cell debris after centrifugation following the 1 h incubation. Regarding the DNAzol Direct method, the DNA yield and quality were improved with incubation at 80 °C for 20 min compared to the 10 min incubation, as evaluated using a nanodrop spectrophotometer. This can reduce the risk of contamination from incomplete cell lysis. The microwave-based method revealed that heating at 800 W for 5 min resulted in a higher DNA yield compared to a 10-min incubation. This observation suggests that DNA degradation occurs during the 10-min incubation period. After obtaining the optimal conditions for each method, the quality and quantity of DNA yield, cost, and time consumption were compared among the 3 methods.

PureLink Genomic DNA kit involves multiple steps, including cell lysis using a specialized buffer system, DNA binding to a spin column, and elution of purified DNA. Following the manufacturer’s instructions, this may result in loss of DNA throughout the process. Comparing with the PureLink Genomic DNA kit, the DNA concentration rates of the DNAzol Direct method are significantly higher than those of the PureLink Genomic DNA kit and a microwave-based method. This suggests that the DNA yield from the PureLink Genomic DNA kit may be loss of DNA yield during the extraction process, as mentioned above. The DNA purity from DNAzol Direct (1.79 ± 0.01) is significantly different when compared with the PureLink Genomic DNA (1.92 ± 0.02), whereas the purity of DNA from the microwave method is not significantly different (1.87 ± 0.01). However, the absorbance ratio (A260/A280) between 1.8 and 2.0 is generally accepted as pure for DNA (Kasem et al. 2008). The DNAzol Direct reagent is an alkaline solution containing PEG. It quickly and effectively lyses biological samples, releasing DNA into the lysate. The alkaline pH and chaotropic properties of DNAzol Direct can inactivate PCR inhibitors, including proteases and nucleic acid degradation enzymes, and the lysate does not require neutralization before its use in PCR (Molecular Research Center 2015b). The DNAzol Direct reagent can be utilized for a wide range of samples, and the process is both simple and time effective (Chomczynski et al. 2006, García-Romero et al. 2019). According to the manufacturer’s instructions, DNAzol Direct reagent can be incubated at either room temperature or 80 °C. Suzuki et al. (2020) utilized DNAzol Direct reagent to extract DNA from Ae. aegypti in order to identify the viral genome of Ae. aegypti by homogenizing mosquitoes with beads prior to incubation at room temperature. Whereas, our study incubated the sample at 80 °C after grinding with a sterile micro pestle. However, Suzuki et al. did not report the DNA concentration and purity, making it difficult to compare the results.

The microwave-based method employs microwave irradiation to disrupt cells and release DNA into the solution, enabling the use of the resulting supernatant as a PCR template. Microwave-based methods have been applied for extracting DNA from various samples. For example, Sato et al. conducted a study comparing 6 different DNA extraction methods from formalin-fixed and paraffin-embedded tissue, which included a microwave-based approach. Their findings indicated that the microwave-based method was simpler, had a lower contamination risk, and resulted in a higher yield of DNA than the chemical reagent-based extraction method employed in their study (Sato et al. 2001). Port et al. (2014) found that microwave irradiation quickly and simply yielded DNA from blood samples suitable for nested PCR, with a detection limit of 5 Plasmodium parasites/μl. Melendez et al. (2016) employed a microwave-based approach for the rapid extraction and detection of Neisseria gonorrhoeae DNA, the causative agent of the sexually transmitted infection gonorrhea. According to their findings, the utilization of this microwave-based method for DNA release represents a notable advancement in the detection of gonorrhea infections (Melendez et al. 2016).

We also compared the cost of extracting DNA and the time required to complete the process of 3 methods, as shown in Table 2. In developing countries, low cost is an important factor. Since commercial DNA extraction kits can be expensive and time-consuming, the use of an inexpensive but efficient DNA extraction method is preferred. As noted above, analyzing diversity within a species complex and distinguishing sibling species requires an experienced taxonomist. Molecular methods are increasingly used to overcome this problem. Regarding the DNA quality for molecular applications, Genomic DNA isolated by microwave-based method can provide sufficient DNA for PCR as demonstrated by successful PCR amplification as shown in Fig. 1.

Our use of the combination of microwave irradiation for heating and sterile distilled water appears to be the first for a mosquito sample. As shown in Table 1, our protocol has clear advantages over the others, such as simplicity, speed, and cost-effectiveness as shown in Table 2. Many variables, such as organisms of choice, extraction and preservation methods, and precipitation procedure, affect DNA yield. Additionally, incubation temperature also affects the concentration of DNA extracted (Chen et al. 2010). In the present study, the microwave-based method shows good results without using proteinase-K, although there are several inhibiting factors such as exoskeletal chitin and parasitic microorganisms in the body of the mosquito (Kouřimská and Adámková 2016, Cansado-Utrilla et al. 2021).

The DNA extracted from an adult Cx. quinquefasciatus using these 3 methods exhibited good quality and quantity, as revealed by our study. To the best of our knowledge, this is the first report of DNA extraction from a single mosquito using microwave irradiation. The simplified microwave-based method enables efficient extraction of high-quality DNA from an adult Cx. quinquefasciatus for PCR-based assays. This method can be used for molecular analysis of other mosquito species in large-scale studies, particularly in low-resource settings.

Acknowledgments

We want to thank the Faculty of Medicine, Khon Kaen University, for supporting this study, in part, by grants and the Invitation Research Fund, Faculty of Medicine, Khon Kaen University (no. IN63361). We want to thank Mr. Kevin McCracken, under the aegis of the KKU Publication Clinic, for assistance with the English presentation of this manuscript.

Contributor Information

Nurhadi Eko Firmansyah, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

Thaksaporn Thongseesuksai, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

Thidarut Boonmars, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Neglected, Zoonosis and Vector-Borne Disease Research Group, Khon Kaen University, Khon Kaen, Thailand.

Saleha Sungkar, Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia.

Porntip Laummaunwai, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Neglected, Zoonosis and Vector-Borne Disease Research Group, Khon Kaen University, Khon Kaen, Thailand.

Funding

This research was funded with the support of the Faculty of Medicine, Khon Kaen University, for supporting this study, in part, by grants and the Invitation Research Fund, Faculty of Medicine, Khon Kaen University (no. IN63361).

Author Contributions

Porntip Laummaunwai (Conceptualization [Equal], Formal analysis [Equal], Funding acquisition [Equal], Investigation [Equal], Methodology [Equal], Project administration [Equal], Writing—original draft [Equal], Writing—review & editing [Equal]), Nurhadi Firmansyah (Conceptualization [Equal], Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing—original draft [Equal], Writing—review & editing [Equal]), Thaksaporn Thongseesuksai (Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing—review & editing [Equal]), Saleha Sungkar (Investigation [Equal], Methodology [Equal], Writing—review & editing [Equal]), and Thidarut Boonmars (Conceptualization [Equal], Investigation [Equal], Methodology [Equal], Writing—review & editing [Equal])

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