Detection of Salmonella typhimurium ATCC 14028 in Powder Prepared Traditional Medicines Using Real-Time PCR

The detection of Salmonella typhimurium ATCC 14028 using real-time PCR on powdered traditional medicinal products was carried out in the microbiology and molecular biology testing laboratory of the Food and Drug Administration in Gorontalo. This research aims to provide a reference for alternative testing methods in testing the products of traditional powder preparations on the market. The sample consisted of 10 traditional powder preparations spiked with positive control of S. typhimurium ATCC 14028 phase 2. The method used in the study was real-time PCR analysis using the SYBR® Green method, while DNA isolation using the direct PCR method. Data analysis was performed by analyzing the sample's melting temperature (Tm) curve and comparing it with positive control. The results showed that S. typhimurium ATCC 14028 was detected in samples at an average Tm value of 84.18°C, with ranges of 84.0-84.5°C. For positive control, the Tm value was at 85.2°C, while for the negative control, the Tm value was not detected. Based on these data, it can be concluded that S. typhimurium ATCC 14028 in traditional medicine products powder preparations can be detected using real-time PCR.


INTRODUCTION
Traditional medicines are ingredients in the form of the plant, animal, mineral substances, galenic preparations, or mixtures of these ingredients, which have been used for treatment from generation to generation and can be applied following the prevailing norms in society 1,2 . So far, the method of detecting pathogenic bacteria in traditional medicinal products is still using conventional test method guidelines using selective media. However, the development of science and technology has led to several other alternatives in detecting pathogenic bacteria, one of which is using molecular techniques 5, 6 . Molecular analysis to identify pathogenic bacteria using real-time has advantages when compared to

Detection of Salmonella typhimurium ATCC 14028 in Powder Prepared
Traditional Medicines Using Real-Time PCR

Materials
The materials used in this study were samples of traditional medicine powder, tryptic soy broth (TSB) and agar (TSA) enrichment media, xylose lysine deoxycholate (XLD) and brilliant green agar (BGA) selective media, and a PCR kit with QuantiNova SYBR® Green (Qiagen).

Sample criteria and sampling period
The main criteria for the sample used in this study was a traditional medicine powder consisting of several traditional medicines for shooting pain or "pegal linu" and herbal medicine for women's health. Sampling was carried out at markets and pharmacies in Gorontalo City from October to December 2020.

Sample preparation
The sample consisted of 10 types of traditional medicinal powder preparations spiked with positive control S.

Real-time PCR analysis
Melt curve analysis was carried out using real-time PCR

Positive control
The

Negative control
The negative control used was no template control (NTC), a master mix combined with primer and nucleic acid-free water. The total negative control volume was 10 μL, consisting of 1 μL forward primer, 1 μL reverse primer, 3 μL RNase free water, with the rest was SYBR® Green master mix 14 .

Specificity
Specificity was carried out by replacing the target bacterial DNA with other bacterial DNA to see whether the non-target DNA was amplified or not. In addition, another function of the specificity test was to examine whether the primer used was specific or not.

Limit of detection
The limit of detection (LOD) was carried out by replacing the sample DNA template with a positive control diluted 10 times. This step aims to determine the sensitivity of the tool in detecting low concentrations of target DNA.

Data analysis
Data analysis was carried out by seeing the amplification results of the melting temperature (Tm) value, the melting point at the temperature at which the melt occurred, and comparing the melting point for the positive control 14 .

Isolation on selective media
The isolation process on selective media began with including as a source of nutrition for amino acid or polypeptide raw materials, maintaining the osmotic balance of the media, sources of carbohydrates that will be needed in the fermentation process, as well as an acidbase indicator. While in XLD media, Salmonella has a translucent round shape with a black spot in the middle.
The color change is caused by the fermentation of glucose by Salmonella into organic acids such as lactic, acetic, and formic acids, resulting in a decrease in pH 19 . Xylose lysine deoxycholate is a non-autoclave medium whose components will be damaged if harvested at temperatures above 100°C. Xylose carbohydrates in XLD play a role in the fermentation process to condition the pH of the media to become acidic, thereby increasing the pH to become alkaline. Meanwhile, decarboxylate enzymes produce amines or diamines and carbon dioxide by breaking down amino acid groups. Xylose lysine deoxycholate media is a selective medium for S.
typhimurium because it has sodium deoxycholate, an inhibitor for Gram-positive bacteria 18 .

Real-time PCR analysis
Real-time PCR analysis was performed using a qualitative test method using SYBR® Green, and the results were presented in Table I  The results of real-time PCR amplification using the qualitative analysis method SYBR® Green were shown

DATA AVAILABILITY
None.