OPTIMIZATION OF ANNEALING TEMPERATURE AND PRIMER CONCENTRATION OF CYTOCHROME B (CYT B) GENE FOR PIG DNA DETECTION WITH REAL-TIME PCR METHOD

Background: One company in Indonesia has developed a pig DNA detection kit by designing primers with the real-time Polymerase Chain Reaction (PCR) method using the cytochrome b (cyt b) gene. It is necessary to optimize the PCR process to optimize pig DNA detection, including annealing temperature and primer concentration, which can increase sensitivity, specificity, and precision. Purpose: This research aims to determine the optimum annealing temperature and primer concentration for the detection of pig DNA using cyt b gene. Method: In this research, the extracted sample isolates were subjected to 12 treatments with 2 repetitions. Optimal data analysis was based on the lowest Cycle Threshold (CT) value in the amplification curve. Result: Out of a total of 24 samples, an increase in the CT value was observed at annealing temperatures of 57 °C, 59 °C, and 60 °C compared to 58 °C, across various primer concentrations. The primer concentrations with the lowest CT values were successively found to be 0.4 μM, 0.3 μM, and 0.2 μM. Conclusion: The results of the research that has been conducted indicate that the optimal annealing temperature for detecting pig DNA using the cyt b gene in this research is 58 °C, and the optimal concentrations of forward and reverse primers are 0.4 μM.

Introduction

Heat-cured acrylic resin is the most commonly used denture base material. Heat-cured acrylic resin, known as acrylic resin, has experienced thermal activation polymerization (Anjai et al., 2018(Anjai et al., 2018)). This material has a gingiva like texture and color, making it aesthetically pleasing, affordable, and easy to manipulate and repair. However, heat-cured acrylic resin is also porous and tends to absorb fluid (Naini, 2015(Naini, 2015)).

Denture bases in contact with saliva formAcquired Dental Pellicle(ADP) and generate plaque formation, which increases the accumulation of microorganisms in the oral cavity, especiallyCandida albicans. This condition results in denture stomatitis (Dama, 2013(Dama, 2013); Wirayuni, 2017(Wirayuni, 2017)). Periodic cleaning using a denture cleanser is essential to prevent this possibility. However, chemical denture cleansers are reported to increase the surface roughness of heat-cured acrylic resin due to its acid content. Studies suggest using natural or herbal ingredients to minimize this drawback (Dewi et al., 2020(Dewi et al., 2020)).

Edamame (Glycine max L. Merril) is a soybean from the legume group, harvested at the peak of ripening before reaching the hardening period. This plant is originally from Japan. Edamame contains essential compounds such as flavonoids, saponins, isoflavones, alkaloids, and tannins, which have antifungal and antibacterial properties (Abd-Alla et al., 2019(Abd-Alla et al., 2019); Abdel-Hadya et al., 2019(Abdel-Hadya et al., 2019)). A previous study proved that edamame extracts at a concentration of 50 mg/ml could inhibit the growth ofCandida albicans(Igboabuchi and Ilodibia, 2018(Igboabuchi & Ilodibia, 2018); Kristiana et al., 2022(Kristiana et al., 2022)). In addition, other studies also show that 50% of edamame extracts effectively inhibit the number of Streptococcus mutans colonies (Devi et al., 2023(Devi et al., 2023)). However, flavonoid compounds in edamame in contact with heat-cured acrylic resin might cause an increase in the surface roughness of heat-cured acrylic resin.

Surface roughness is crucial for dentures since it affects their quality and longevity and induces bacteria adhesion. The ideal cleanser should keep the mechanical and physical characteristics of the denture base resin the same after extended usage (Porwal et al., 2017(Porwal et al., 2017); Sharma et al., 2017(Sharma et al., 2017)). For reference, the surface roughness of a heat-cured acrylic resin denture base is acceptable if the value is not more than 0.2 μm (Onwubu and Mdluli, 2022(Onwubu & Mdluli, 2022); Vinagre et al., 2023(Vinagre et al., 2023)).

The surface roughness of heat-cured acrylic resin dentures generally increases as dimensional changes because of the duration of use (Arafa, 2016(Arafa, 2016)). Those changes might happen during intraoral use. Ningsih et al. (2020)(Ningsih et al., 2020)suggested that after 1 to 3 years of wear. If adjusted to the contact time between the edamame extract and the acrylic resin plate for 15 minutes in short-term soaking), the usage period of one year is equivalent to four days, and three years is equal to a soaking time of 11 days (Arruda et al., 2015(Arruda et al., 2015)). Thus, we chose those periods to analyze the possibility of 50% of edamame extracts changing surface roughness on prolonged use and whether it is still clinically tolerable.

Material and Method

Specimen preparation

Twenty four heat-cured acrylic resin specimens were fabricated according to the manufacturer's guidelines using conventional flasking and pressure-pack techniques. The acrylic resin was placed in a square-shaped cuvette with a size of 10 x 10 x 2 mm (based on the American Dental Association (ADA) specification No. 12). The polymerization process of the resin was carried out by placing the flask in boiling water at 100^oC for 20 minutes. The cuvette was left until it reached room temperature before opening. Subsequently, the acrylic sample was smoothed with 500 number scouring paper until the surface was flat and smooth. Then, the acrylic resin specimens were immersed in distilled water for 48 hours and in artificial saliva for one hour.

Preparation of edamame bean extract

The variant of edamame that we used in this research is MF 116 (PT Mitratani Dua Tujuh, Jember, Indonesia). First, edamame beans were dried in an oven at 50^oC for one day. The dried edamame seeds were then ground into powder and sieved. Subsequently, maceration was carried out using 70% ethanol solvent at a 1 : 5 (gr/ml) ratio for three days. The obtained filtrate was then evaporated using a rotary evaporator (Siddiq, 2016(Siddiq, 2016)). Finally, 50% edamame bean extract was obtained by diluting 1.5 ml extract in 1.5 ml distilled water.

Immersion process

Twenty-four specimens of acrylic resin (n = 6/group) were distributed into two control groups and two treatment groups, namely A1 and A2 (immersion in distilled water for four days and 11 days, respectively), and B1 and B2 (immersion in 50% edamame bean extract for four and 11 days, respectively). Duration of four and 11 days for immersion was equivalent to using a denture cleanser solution for one year and three years, respectively.

The measurement of surface roughness (Ra)

The surface roughness of the samples before and after treatment was measured using a surface roughness tester TR 220. Measurements are taken on one side of the sample on three different lines. Each sample's average surface roughness (Ra) was then noted and analyzed.

Statistical analysis

The average

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