Development of Polymerase Chain Reaction assay protocol for assessment Salmonella sp. in cow raw milk

Salmonella sp. is a pathogenic bacterium that may associated with acute diarrhoea in human. These bacteria may be transmitted in a variety of ways, including consumption of contaminated cow raw milk. Salmonella sp. is troublesome to assessment due to methodological restrictions. The aim of this study was to development the protocol of Polymerase Chain reaction (PCR) assay for Assessment Salmonella sp. in cow raw milk. This method is comprised of (1) biochemical assay and (2) bacterial DNA purification from Selenite Cystine Broth and XLD Xylose Lysine Deoxycholate culture using Wizard® Genomic DNA Purification Kit followed by PCR detection. The biochemical assay is divided into several stages, namely bacterial isolation, Gram staining and conventional biochemical tests. The PCR optimization was done with Salmonella sp. The oligonucleotide primer invasion protein (invA) gene (F: 5’-TCGTCATTCCATTACCTACC-3’; R: 5’-AAACGTTGAAAAACTGAGGA-3’) were used for targeting the diagnosis of Salmonella at the genus level. In biochemical tests, Salmonella sp. results showed posit if result of catalase, oxidase, citrate, TSIA, lactose-sucrose-mannitol fermentation, urea, and methyl-red. Conversely, negative result for Voges-Proskauer. PCR protocol consist of 30 PCR cycles with initial denaturation at 94°C for 45 seconds, denaturation at 94°C for 20 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 15 seconds and finally at 72°C for 2 minutes. The conventional method detection results obtained as many as 9 positive samples Salmonella sp. and the PCR method obtained 7 positive samples Salmonella sp. In conclusion, our results indicate that the developed protocol would be utilized as a routine analysis for monitoring cow raw milk contamination and the protocol of the PCR technique provides results at a handful of time required by the biochemical assay (24 hours compared to 2–3 days).


Introduction
Typhoid fever pathogens are bacteria caused by Salmonella sp.Salmonella typhi is one of the harmful species.The World Health Organization (WHO) reports that in 2018, there were an estimated 21 million cases of typhoid fever worldwide, with 128,000 to 161,000 deaths per year.The majority of these cases originated in South Asia and Southeast Asia.According to surveys conducted in major hospitals in Indonesia, typhoid fever is an endemic public health issue in that country.It affects an average of 500/100,000 people on an annual basis, with a death rate of about 5% [1].
Salmonella sp. is a member of the Enterobacteriaceae family of bacteria.It is a Gram-negative, straight rod-shaped, motile, anaerobic, facultative, with O antigens (somatic antigens), H (flagella antigens), and Vi (K capsule) antigens.Salmonella sp.lives in both the human and animal digestive tracts.Food and beverages, including cow's milk, can spread Salmonella sp.[2][3].
Humans are exposed to Salmonella sp.through food and drink.These bacterial cells can persist and produce toxins in the intestinal mucosa.Salmonella contamination with a sizable number of 10 Million cells in this poison causes disease [4].Foodborne illnesses can be brought on by drinking water contaminated with Salmonella sp.Consuming contaminated food or drink can result in foodborne illnesses.Salmonella sp.contamination of cow's milk probably starts in the teat glands and filter material [5].By merely washing hands with water and allowing bacteria to stick to milking hands, non-sterile milking hands have the potential to be contaminated [6].
When compared to molecular-based identification, a simple culture method for detecting Salmonella is more cost-effective, according to earlier research by Bell et al [7] and Awang et al [8].For confirmation and identification, it takes a while and involves several steps.Compared to the culture technique, the PCR (Polymerase Chain Reaction) method is more sensitive and yields more encouraging results.While traditional methods based on enrichment techniques run the risk of missing Salmonella genetic sequences on selective media, PCR can resolve this risk and determine whether the sequences are present or absent [8].
Salmonella sp. is typically found in foods and beverages, and its presence is typically detected using selective media cultures, specifically SCB media (Selenite Cystine Broth) and XLD media (Xylose Lysine Deoxycholate).tests for sugar (lactose, sucrose, and mannitol), oxidase, urease, MR VP, and citrate.The conventional approach using standard culture media has limitations.It takes too long and necessitates a laborious process, making a laboratory with high biosafety and low sensitivity necessary.PCR is one of the many DNA-based pathogen detection techniques that have been developed.Fast processing times, brief processing times, and high sensitivity are all benefits of PCR [6].
Therefore, the objective in this study was to development a PCR assay protocol for the evaluation of Salmonella spp. in raw cow's milk.This procedure includes a biochemical test, followed by the purification of bacterial DNA from Selenite Cystine Broth and XLD Xylose Lysine Deoxycholate culture using a Wizard® Genomic DNA Purification Kit, and PCR detection.Salmonella sp. in food and beverage samples should be diagnosed with the aid of the results obtained.

Sample collection
Random sampling was used for the sampling process, and there were specific inclusion and exclusion criteria, including fresh cow's milk, purchases from roadside vendors or farms, plastic packaging, and raw milk, as well as purchases from supermarkets, packaging in cups or carton boxes, and ripe milk.Nine-tee samples from raw cow milk were collected from different seller located in Surabaya, Indonesia.

Culture-based method
Samples of cow's milk were isolated in 1 mL of Buffered peptone water using a micropipette and put into a test tube then incubated at 37℃ for 24 hours then continued with the isolation step in test tubes with Selenite Cystine Broth (SCB) media and Xylose Lysine Deoxycholate agar (XLD agar) media petri dishes, then incubated at 37℃ for 24 hours.Colonies suspected of being Salmonella bacteria at the isolation stage were purified by growing the colonies on XLD media by taking the colonies using an ose needle and then scraping them onto the XLD media.The purification results were incubated at 37℃ for 24 hours [9].The identification of isolates was carried out in three phases [10]; cultural characterization, morphological characterization, biochemical characterization.The purified colonies were then subjected to Gram staining, Biochemical Tests (Methyl red-Voges Proskauer Test, Citrate Test, TSIA Test, Urea Test, Sugar Fermentation Test, Catalase Test, Oxidase Test).The positive control used was Salmonella sp.

PCR-based method
DNA isolation was carried out by culturing bacteria in Nutrient Agar (NA) media and then incubating at 37℃ for 24 hours.DNA extraction and purification were carried out according to the protocol of a commercial manual kit (Wizard® Genomic DNA Purification Kit).To determine DNA concentration and purity, 2 µL of each DNA sample was tested using a spectrophotometer with a wavelength of 260 nm and 280 nm and by 0.8% agarose gel electrophoresis [11].
The primer used to amplify the target DNA was the invA gene primer with a size of 119 bp with the following base sequence Forward: 5' TCGTCATTCATTACCTACC 3'; and Reverse: 5' AAACGTTGAAAAACTGAGGA 3' [13].PCR reactions were made in a total volume of 50 µL.The components of the PCR reaction were 5 µL primer F, 5 µL primer R, 25 µL Go Taq PCR mix, 5 µL Nuclease free water, and 10 µL DNA template.The resulting mixture is then homogenized and spinner.Amplification was carried out by PCR cycles with initial-denaturation at 94℃ for 45 seconds, denaturation at 94℃ for 20 seconds, annealing at 57℃ for 15 seconds, extension at 72℃ for 15 seconds and finally at 72℃ for 2 minutes, with cycle amplification of 30 times.Amplified PCR products were visualized on 2% agarose gel and stained with Ethidium Bromide (EtBr).DNA bands were observed under ultraviolet light.

Data interpretation and analysis
The results obtained will be matched with some literature to find out whether Salmonella is present in the sample or not.If the results are in accordance with the literature, the sample is positive for Salmonella.The results of a positive reaction are indicated by looking at the pattern of DNA banding that is formed whether there is a change or not with a DNA marker.After recording the two methods, an evaluation will be made regarding the comparison of the suitability of the results between conventional and PCR methods.Data were analyzed using IBM SPSS Statistics 22 (IBM corporation) with the Will Coxon test which is a non-parametric test used to measure the differences between 2 groups of paired data.

Culture-based method
Bacteria isolated 19 samples of pure cow's milk.Following the planting of samples on peptone media, SCB and XLD media were used.All 19 inoculated samples underwent a color change on the SCB medium.Table 1 presents colony characteristics of XLD media.On XLD media, the following sample was introduced.

PCR -based method
The bacterial isolates were grown on NA media for the next step, namely DNA isolation.DNA was extracted by using Wizard® Genomic DNA Purification Kit according to the manufacturer's protocol.
Then the results of DNA isolation were tested qualitatively by electrophoresis and quantitatively tested by spectrophotometer.The DNA samples were then proceeding to PCR.The final step in the PCR method is DNA amplification by PCR using a specific primer for the InvA gene with forward primer 5'-ACAGTGCTCGTTTACGACCTGAAT-3' and reverse primer 5'AGACGACTGGTACTGATCGATAAT-3' minutes.The method was development method from previous methods [11].The PCR condition consist of denaturation 94℃ for 30 seconds, annealing 56℃ for 30 seconds, extension 72℃ for 2 minutes, final extension 72℃ for 10 minutes.Figure 1 revealed that seven isolates that contained InvA genes for amplification of the 119 bp fragment.
The invA gene plays a role in causing illness in humans.The gene is located in the Salmonella pathogenicity island (SPI-R) area which has an operon that functions as a repository for genetic information.The invA gene in Salmonella sp is located on the chromosome which is capable of producing proteins that can provide invasive properties to invade epithelial cells.The invA gene is the main gene in the operon invABC that encodes the protein found in Salmonella's inner membrane and necessary for invading the host's intestinal epithelial cells.[15].The use of the PCR method with the InvA gene is mostly used in diagnostic laboratories.The PCR examination technique with the invA gene is very sensitive and specific in detecting salmonella.The invA gene contains specific sequences for the genus Salmonella and is considered an international standard for its characterization [16].After this stage, electrophoresis of PCR results was continued using agarose gel with a concentration of 1.5% and a DNA marker that served as a reference to determine the size of the amplified DNA.The positive PCR results were obtained in samples 1, 4, 5, 8, 9, 11, and 12.In conventional PCR, which detects the pathogen, is known to distinguish between closely related species using species-specific primers [17].Therefore, primer design is essential.To obtained a favorable result from PCR, reaction parameters may occasionally need to be optimized [18].The goal of the current study was to development PCR assay using the gene sequences of Salmonella sp. and to develop a method for simultaneous detection of Salmonella sp.

Data analysis
The research was carried out by taking samples and examining samples using two different methods, namely the conventional method and the PCR method.Table 4. presents the results of the identification of Salmonella sp. using the culture method and the PCR method.A number of PCR-based assays have been devised to identify Salmonella sp. from raw cow milk's isolates.The culture-based method requires at least 27 hours for the incubation of microbes on agars and also requires more laboratory space and incubators.The PCR method requires less than 2 hours for DNA extraction and 2 hours for PCR.This indicated that PCR can be used when target microbes are difficult to detect using cell-culture techniques, but it can only be used when the DNA sequences of the target microbes are known.Several gene target for Salmonella sp detection could be invA, bcfD (a conserved fimbria operon gene), phoP (adaptation to low Mg 2+ environments and the control of acid resistance genes), siiA (inner membrane proteins with one and three transmembrane (TM) helices), etc [19].Our development of PCR assay for the detection of Salmonella sp.enables the identification in raw cow milk's sample.In many countries, Salmonella sp.infections are increasingly common [20], so it was important to development this assay.Our PCR assays found it was able to correctly identify Salmonella sp.isolates that had previously been classified culture-based method.Although our assay was able to positively identify Salmonella sp. from extracted DNA from eight samples, we acknowledge there is a lack of additional primers in our collection.A further limitation is that our experiments were performed using DNA extracted from purified cultures and therefore need improvisation using mixed culture.Further optimization of our PCR assay is planned for working directly on sampling from raw cow milk.Our PCR assay should directly plug in to obtain the lineage level resolution as direct assays are developed for clinical use in the future.

Conclusions
Our findings demonstrate that once the presence of bacteria has been established by both methods, culture-based estimates can be used as reasonably trustworthy indicators of Salmonella sp.However, the culture-based method may falsely report the absence of Salmonella sp.Therefore, we propose that development of development of PCR assay protocol for assessment Salmonella sp. in cow raw milk.We do advise authors to consider the statistical techniques created specifically for this circumstance, though, when there are grounds for thinking that culture-based assays or PCR-based methods are significantly left-censored.

Figure 1 .
Figure 1.PCR amplification of Salmonella sp.invA gene primers positive invA gene amplification for isolates (Duplo for each sample); C: Salmonella sp.; M: 1 kb DNA ladder.The DNA band at 119 bp shows marker presence, as visualized by gel electrophoresis using 2% agarose.

Table 1 .
Results and Colony Characteristics of Xylose Lysine Deoxycholate (XLD) Agar [14]biochemical tests carried out in this study included the citrate test, coagulase test, catalase test, oxidase test, Triple Sugar Iron agar (TSIA) test, methyl red and Voges-Proskauer tests (MR-VP test), and carbohydrate fermentation test (sucrose, lactose, and mannitol).Biochemical tests aim to determine the physiological properties of bacteria and the metabolic processes of bacterial cells.According to the SNI 2897:2008 standard[14], Salmonella sp showed positive catalase, negative oxidase, unable to ferment sucrose and lactose, mannitol fermentation occurred, positive citrate test, unable to hydrolyze urea enzyme, positive TSIA, positive MR, negative VP.The results of Gram staining presented in Table2and biochemistry test in Table3.

Table 4 .
Comparison of presence Salmonella sp. in cow raw milk using the culture method and PCR.