Development of Nested PCR-Heteroduplex Mobility Assay for Determination of Genetic Diversity in the Block 2 Region of the Plasmodium falciparum Merozoite Surface Protein 1 Gene.

Genetic diversity of Plasmodium parasite has significantly related to malaria control and vaccine development. The P. falciparum merozoite surface protein 1 (Pfmsp1) gene is a commonly used molecular marker to differentiate genetic diversity. This study is aimed at developing a nested PCR-Heteroduplex Mobility Assay (nPCR-HMA) for determination of the block 2 of the Pfmsp1 gene. The MAD20 family allele of P. falciparum was used as a control for optimization of the annealing and polyacrylamide gel electrophoresis conditions. In order to evaluate the developed nPCR-HMA, 8 clinical P. falciparum isolates were examined for allelic variants. The results revealed 9 allelic variants. Our study indicated that the successful nPCR-HMA with good precision and accuracy offers a more rapid, efficient, and cheap method for large-scale molecular epidemiological studies as compared to nucleotide sequencing.


Background
Malaria is a severe infectious disease, caused by the parasites which belongs to the Plasmodium genus and transmits to humans by the female anopheles mosquito vector. It is a significant public health problem worldwide, especially in tropical and subtropical regions. Regardless of widespread efforts to eradicate or control malaria, this disease continues as the leading cause of morbidity and mortality [1]. In 2017, around 219 million people are at risk of malaria and the estimated number of malaria deaths accounted for 435,000 [1].
Human malaria is caused by five species including P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. Among these, P. falciparum is the most widespread and the most dangerous. P. falciparum responsible for 99.7% of estimated malaria cases in the WHO African region, as well as in the majority of cases in the WHO regions of South-East Asia (62.8%), the Eastern Mediterranean (69%), and the Western Pacific (71.9%) [1].
The spread and mortality rate of malaria infection associate to several features. The increasing of the immigration of foreign workers from endemic areas into countries can lead to the spread of malaria between regions [2][3][4][5]. In addition, the genetic variation has been reported as a cause of the mechanisms that escape from the immune response of the host, and the increasing resistance to the antimalarial drugs [6][7][8][9] also is a cause of malaria spreading.
The genotyping of malaria parasite populations is used to investigate the genetic diversity of malaria infections with consideration of several factors, including transmission intensity, drug-resistant parasite, precise treatment, host immunity, and epidemiologic study. Thus, simple and accurate genetic diversity detection has developed. Several molecular techniques have been used for genetic diversity detection, including nested PCR, Restriction Fragment Length Polymorphism (RFLP), Single-Strand Conformation Polymorphism (SSCP), Heteroduplex Tracking Assay (HTA), Heteroduplex Mobility Assay (HMA), and DNA sequencing. The HMA has been used for subtyping, identification, and classification of human immunodeficiency virus (HIV) and other viruses [10][11][12][13][14]. The HMA is a simple, rapid, sensitive, and cost-effective technique compared to DNA sequencing [10].
The principle of HMA is based on a reference DNA and a sample DNA that will be amplified separately, mixed, denatured, and slowly cooled to permit the formation of heteroduplex from incomplete base matching between reference DNA and sample DNA. Then, the heteroduplex is determined by using polyacrylamide gel electrophoresis. The differences in the migration of heteroduplex form homoduplex indicate the difference in nucleotide sequence between reference DNA and a sample DNA. These are due to mismatched regions which present single-stranded configuration and slower migration than completed match dsDNA. In this system, if the sample DNA is not a perfect match to the reference DNA, multiple bands will be observed. The fastest migrating band is the homoduplex reference DNA and/or the homoduplex DNA sample. A heteroduplex band with mismatches migrates more slowly than a homoduplex band. HMA is easily applied to large numbers of samples and is particularly suited to the analysis of PCR products, because both the sample DNA and the reference DNA must be of the same size. In PCR, this would correspond to the use of the same amplification primers.
In principle, HMA shows the characteristics of 3 bands: the first band is a fast DNA moving as a homoduplex band caused by a complete sequence matching self DNA or a complete sequence matching in the sample DNA. The second band was the slowest-moving band caused by an incomplete match between 2 DNA sequences. The last band is a single-stranded DNA that occurs in the annealing step that has a matching DNA with the reference DNA independent of the sample; reducing the temperature to minimize single partial DNA matching thus forms a single-stranded DNA band.
The P. falciparum merozoite surface protein 1 (Pfmsp1) gene is typically used as a genetic marker to determine the genetic diversity and multiplicity of infection (MOI) of P. falciparum [15][16][17]. PfMSP1 located on chromosome 9 contains 17 blocks, of which block 2 shows prevalent allelic polymorphism worldwide [16,17]. The block 2 allele is mainly represented by three families, namely, K1, MAD20, and RO33 in the field isolates. These families' allelic sequences show a highly diverse and specific geographical distribution [18]. In addition, the pattern and fragment size polymorphism of the block 2 allele serve as molecular indicators, host immunity, and malaria transmission dynamics [19].
However, data on the HMA to investigate the genetic diversity of the Pfmsp1 gene have not been evaluated. In this study, we developed and evaluated the Nested PCR-Heteroduplex Mobility Assay (nPCR-HMA) to determine the genetic diversity of the Pfmsp1 gene in the block 2 region. The technique could be used for the screening of P. falciparum genetic diversity, reinfection, recrudescence, and molecular epidemiology study in a malaria-endemic area.

Material and Methods
2.1. Sample Collection. The study was approved and reviewed by the Ethics Committee of Rangsit University, Thailand (RSUERB2019-061). The samples tested composed of 8 P. falciparum-infected blood detected by microscopic examination. Blood samples were collected from malaria-endemic areas, Thailand, including Mae Hong Son and Kanchanaburi provinces. P. falciparum-positive blood specimens were collected in an EDTA tube, and approximately 100 μl of blood samples was dropped on a piece of Whatman 3M filter paper and dried (dried blood spot; DBS). Each DBS was individually stored in small plastic bags at room temperature before transporting to laboratory until the next step of the investigation.
P. falciparum MAD20, RO33, and K1 families reference DNA, which were recombinant plasmid, contained block 2 of Pfmsp1 described by Ngrenngarmlert et al., [20]. The known sequence reference DNA (recombinant clones) is showed in Figure 1. The P. falciparum K1 culture that was kindly provided by Dr. Sumali Kamchonwongphaisan, Office of the National Science and Technology Development, Thailand, was used as monoclone P. falciparum control.

DNA Extraction.
Parasite DNA was extracted from DBS using the Chelex method with some modifications [21]. Pieces of DBS were put in a microcentrifuge tube, and 300 μl of sterile distilled water was added. After 10 min room temperature incubation, 300 μl of supernatant was transferred to a microcentrifuge tube, and then, 1 ml of sterile distilled water was added and incubated at room temperature for 30 min. After centrifugation for 3 min at 12,000 rpm, removed the supernatant, and 6% Chelex-100 Resin (Bio-Rad Laboratories, Hercules, CA), was added and then, the tube was incubated for 30 min at 56°C and followed by incubation at 100°C for 8 min. After centrifugation for 3 min at 12,000 rpm, the supernatant of extracted DNA was kept at -20°C for long-term storage.

Block 2 Pfmsp1 Gene Amplification by Nested PCR.
Nested PCR amplification of P. falciparum DNA was carried out using the Techne TC-3000 Thermal cycler (Chemoscience, USA). The primary PCR was performed according to Snounou et al. [22]; primer pairs corresponding to blocks 1-4 regions of Pfmsp1 gene consisted of forward primer M1-OF: 5 ′ -CTAGAAGCTTTAGAAGATGCAGTA TTG-3′ and reverse primer M1-OR: 5′-CTTAAATAGTA TTCTAATTCAAGTGGATCA-3′. The PCR reaction was set up in a final volume of 20 μl containing 1 μl of DNA extraction, 250 nM of each primers, 10x PCR Buffer containing 1.5 mM MgCl2 (Invitrogen, USA), 125 nM of dNTPs, and 1 unit of Taq DNA Polymerase (Intron, Korea). The PCR amplification conditions were as follows: initial denaturation at 95°C for 15 min, 25 cycles of 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min with the final extension at 72°C, 2 minutes. The secondary PCR was done with specific primers to the block 2 region, previously designed by Ngrenngarmlert et al., [20], and the forward and reverse primers were C1F: 5 ′ -GAAGATGCAGTATTGACAGG-3 ′ and C3R: 5 ′ -TGATTGGTTAAATCAAAGAG-3 ′ , respectively. The conditions and PCR reaction used for the second amplification were identical to those used for the first PCR, except that amplification was conducted for 24 cycles. Then, the PCR products were determined on 1.8% agarose gel electrophoresis, visualized under a UV transilluminator after staining with ethidium bromide. MAD20, and RO33) were the DNA sequences cloned in recombinant plasmids. The control DNA was amplified from P. falciparum K1 culture.
The HMA assay consisted of 5 μL of PCR amplicons containing the block 2 of Pfmsp1 from the second reaction by nPCR, 1 μl of 10x annealing buffer (1 M NaCl, 100 mM Tris-HCl, pH 7.5, 20 mM EDTA), 1 μl of 6x DNA loading buffer, and 5 μl of reference DNA. The HMA conditions were as follows: denature dsDNA at 98°C for 3 minutes; after denaturing, the DNA fragments were hybridized at 4°C for 15 minutes. The heteroduplex products were analyzed on 8% polyacrylamide gel electrophoresis at 10 mA for 1.30 h. Homoduplex and heteroduplex DNA bands were stained with ethidium bromide and visualized under a UV transilluminator. The selection of one reference DNA is performed by observing the most clear heteroduplex band of the reference DNA that occurred with each of the three samples.

The Optimum Conditions to Perform Heteroduplex
Mobility Assay (HMA). The HMA conditions were tested with the reference DNA selected from the above step. The selected reference DNA and K1 DNA with proportions were 2 μl/2 μl and 5 μl/5 μl following denaturation at 98°C for 3 minutes; after denaturing, the DNA fragments were hybridized at 4°C for 15 minutes. The heteroduplex products were analyzed on a 6%, 8%, and 12% polyacrylamide gel electrophoresis at 10 mA for 1.30 h. After polyacrylamide gel elec-trophoresis, the heteroduplex bands were observed. The most clear of the heteroduplex band condition was selected for the next analysis.

The Genetic Diversity of Pfmsp1
Was Analyzed by HMA. The optimum conditions as selected by previously described studies were used to investigate for Pfmsp1 diversity in 8 clinical samples.

The
Interpretation of HMA. The measurement of heteroduplex mobility was presented as a mobility index (MI) as shown in the equation below. In this study, we used the P. falciparum K1 strain from a culture as a control to determine the heteroduplex pattern between the references DNA.

Validation of Established HMA.
In this experiment, we used the optimum conditions of HMA for validating the genetic diversity and reliability of HMA. In addition, the samples were repeatably tested, by within run and between run for precision evaluation of the test.  Journal of Parasitology Research selected three samples each (S1, S2, and S3). In Figure 3, we found that there were clear heteroduplex bands in the lane that annealing with MAD20 DNA in all samples tested. So we selected MAD20 as the reference DNA for the next step of HMA. The percentages of polyacrylamide gel and DNA volume were also validated. The optimum condition of the HMA which revealed a respectable heteroduplex band was the use of 12% polyacrylamide gel with 5 μl of each DNA by static electricity 10 mA for 1 h and 30 min. Then, the MAD20 reference DNA was tested with K1 culture control DNA. In Figure 4, the results showed a homoduplex 1 band in lane K1+K1 DNA and lane MAD20+MAD20 DNA with the single-stranded DNA band in each lane. Only lane MAD20 +K1 DNA presented one heteroduplex band.
In Figure 5, the results showed the representative gel image of homoduplex and heteroduplex bands in samples H18 and H24 using MAD20 reference DNA.

Genetic Diversity of Pfmsp1 Gene Block 2 by nPCR-
Heteroduplex Mobility Assay (nPCR-HMA) Using MAD20 as Reference DNA. The optimum HMA conditions as mentioned above were used to validate the genetic diversity of the Pfmsp1 block 2 gene from 8 clinical blood samples infected with P. falciparum. The experiment was accomplished by mixing the nPCR amplicon of Pfmsp1 block 2 DNA from each sample with MAD20 reference DNA before denaturing and annealing. As shown in Figure 6, we found 2 heteroduplex bands that occurred in samples H14 and H18 which referred to MOI. Samples H21, H24, G3, and G11 obtained 1 heteroduplex band (single strain infection). There were two samples that did not have a heteroduplex band (H22, G6). This presented the allelic diversity of 8 allelic variants observed in different movement bands on the same PAGE. Including the 2 samples that have no heteroduplex band, we found in total 9 allele variants from 8 samples.

Genetic
Diversity by nPCR-HMA Using MI. Next, we validated the heteroduplex band with an MI value. The movement of each band was measured to calculate MI. The results are shown in Table 1. The result revealed that the differences in MI vary from 5.32 to 17.39%.
The allelic variants were analyzed by the variation of the MI value. The result of MI values from 8 samples showed different MI accounts for 8 variants as shown in Table 1.
The 10 repeatable tested MAD20+K1 culture control heteroduplexes by HMA were calculated for the standard deviation (SD) of MI values as shown in Table 2. Then, this SD value (0.8) was used as the interval value to set up the interval scale starting from MI 5 (the round value of the lowest MI) as shown in Table 3. The results demonstrated that allelic type no.5 was the most prevalent (n = 5) (MI range = 8:6 − 9:4) followed by no. 1 (n = 3). Reproducibility. The product of nPCR for Pfmsp1 block2 gene of K1 and MAD20 DNA was used to validate the HMA for 10 repeats. The MI was calculated for heteroduplex DNA. The results found that MI has a variance at an acceptable coefficient of variation (CV%) that was less than 10% (CV = 5:59%) as shown in Table 4. The sampling selected 4 samples, H14, H18, G3, and G11, that were classified gene variance by triplicate MI each. The results showed the same allelic variant no. of 5/6 allelic variants (83.33%). But all samples presented the same amount of heteroduplex bands in each repeat (100%). This indicated a good reproducibility of the established nPCR-MHA as shown in Table 4.

Accuracy
Validation. This experiment was carried to validate the accuracy of HMA by using P. falciparum (K1) which have similarities with 77% of MAD20 DNA by nucleotide sequencing and performed the HMA between them. The results found only one heteroduplex band and one homoduplex band. The single homoduplex band found in MAD20 DNA matched to MAD20 DNA or K1 DNA match to K1 DNA. This indicates that HMA performance as theory presents the accuracy of the MHA.

Discussion
Widespread genetic diversity of P. falciparum in field isolates is believed to be a major complication for the development of  Journal of Parasitology Research an effective vaccine, with the increasing drug resistant and MOI detection against P. falciparum malaria. Therefore, a survey on genetic polymorphism of malaria parasite in clinical isolates from diverse endemic areas is required to control the disease and vaccine development. One of the most commonly used markers for genotyping of P. falciparum is PfMSP-1 [20,22,23].
In this study, we developed nPCR-HMA using P. falciparum MAD20 as the reference DNA and validated it to screen Pfmsp1 allelic variations, since HMA has been shown to have a high efficiency for genetic variation detection as previously reported [10,24,25]. In addition, it is simple and more cost-efficient versus nucleotide sequencing.
The nPCR for amplifying the Pfmsp1 gene which has a product size of 300-400 bp succeed for all of 8 samples tested (100%). This indicates that the method has high sensitivity (100%) to amplify the Pfmsp1 target gene.
We have established the optimum conditions using MAD20 P. falciparum DNA as reference DNA and P. falciparum strain K1 from culture as control DNA-the use of 5 μl of each nPCR product, with 98°C denature for 3 min and annealing for 15 min and then run on 12% polyacrylamide gel electrophoresis using a constant current supply of 10 mA of 1 h and 30 min. It was able to present obvious heteroduplex and homoduplex bands. This indicated that this technique can be used to separate variants of P. falciparum infection with convenience and rapid for a large number of samples simultaneously.
In addition, the established nPCR-HMA can discriminate the P. falciparum allelic variants to be 9 allelic variants from 8 clinical isolates. This clearly confirmed because each sample tested was on the same run of polyacrylamide gel electrophoresis. The results indicated that nPCR-HMA of Pfmsp1 has good efficiency to detect the genetic diversity of P. falciparum.
Compared to previous researches, this study showed more rapid than the report by Kuroiwa (Kuroiwa et al.,  2004), which can be excluded species of respiratory syncytial virus A for 31 alleles by using 150 V at 3 hours. In addition, Wang & Hiruki (Wang & Hiruki, 2001) differentiated the aster yellows phytoplasma group and the clover proliferation which uses voltage of 200 to 250 V, 3 to 4 hours. Nevertheless, there is a report that presented that HMA are less timeconsuming than this study, such as the study of species differentiation of enteric adenoviruses by using 110 V, 75 minutes (Soares, et al., 2004). However, these depend on different DNA sizes, concentration of polyacrylamide gel, and the voltage of polyacrylamide gel electrophoresis.
This study accessible that the established nPCR-HMA found 2 MOI from 8 clinical samples (25%), which convinced the potential of the method for genotyping and showing a high MOI found in P. falciparum in Thailand.   The validation of the developed nPCR-HMA quality revealed a high precision, with CV (5.59%). In addition, tests also showed the same amount of band presented (100%) and also showed the same allelic no. (83.33%). The experiment of HMA between MAD20 reference DNA and K1, which is a single, strain breeding, found only 1 heteroduplex. In addition, the tests showed the test of MAD20/MAD20 or K1/K1 HMA does not have a heteroduplex band, but they presented a homoduplex band only. Our results demonstrated that a developed nPCR-HMA for the Pfmsp1 gene has a good accuracy and precision of allelic detection.

Conclusion
We found that the established nPCR-HMA is a rapid and reproducible method that can detect heteroduplexes derived from nPCR amplicons for a PfMSP1 block 2 target site. It can discriminate the P. falciparum allelic variants to be 9 allelic variants from 8 clinical isolates. This is clearly confirmed because each sample tested was on the same run of polyacrylamide gel electrophoresis. Moreover, we can classify the allelic variants by MI in a high reproducibility with a good accuracy. The results demonstrated that the established nested PCR-HMA is effective to use for screening Pfmsp1 allelic variations and indicated that the P. falciparum from clinical isolates has a high genetic diversity. These results also showed a high prevalence of the mix allelic infection.

Data Availability
The data used to support the findings of this study are included within the article.