The Potential of grxB Gene for Detection of C. sakazakii in Infant Formula Milk Using Real-Time Polymerase Chain Reaction

. Cronobacter sakazakii is one of the bacteria that causes food poisoning that contaminates infant formula. This pathogen causes necrotizing enterocolitis, sepsis


Introduction
Microorganisms contamination in food is a major public health issue because of the emergence of foodborne pathogens [1]. The bacterial pathogen Cronobacter sakazakii is frequently detected in infant formula and is causes food poisoning as well as illnesses like necrotizing enterocolitis, bacteraemia, meningitis, and septicaemia. Infants with the infection have reported case fatality rates between 40 and 80 percent [2]. Therefore, there is an urgent need to develop fast and accurate methods for detecting foodborne pathogens, especially in detecting C. sakazakii in food to ensure food safety. The culturing method is one of the conventional methods used to detect pathogenic bacteria in food products. However, using this method requires a relatively long time and is not specific for detecting pathogenic bacteria. real-time PCR, a molecular-based technique, can be used to identify pathogenic bacteria in food, including C. sakazakii a [3,4]. * Corresponding author: muktiningsih@unj.ac.id The UNJ Salmonella Team has developed and produced specific primers as target genes for the identification of 9 foodborne pathogenic bacteria in prior research. One of them is a primer pair that has been successful in amplifying a piece of the 95 bp fim-C gene fragment using real-time PCR to quickly and accurately detect the pathogenic bacterium Salmonella typhi [5]. Using real-time PCR, this study aims to develop a fast, specific, and sensitive method for detecting C. sakazakii bacteria with grxB target genes. In our previous study, we optimized the ideal annealing temperature for the grxB gene at 60°C with a size of 151 base pairs [6].

Preparation of Culture Sample.
Tryptic Soya Agar (TSA) media (Merck) was used to culture Cronobacter sakazakii ATCC 29544 (Microbiologist, Minnesota) for 18 hours (overnight culture). After incubation, one colony was inoculated into Tryptic Soy Broth (TSB) and incubated for 18 hours at 37°C and 150 rpm in an incubation shaker (YIHDER LM-400D). The spread plate method was used to grow bacteria on TSA, and it was incubated at 37°C for 18 hours. The dilution of the bacteria was 10 -5 -10 -7 .

DNA isolation
One milliliter of C. sakazakii culture stock from TSB was added to the microtube, then centrifuged (Sorval Legend Micro 17R) at 5000 x g for five minutes. Then, using the Geno Plus Genomic DNA Extraction Miniprep System (Viogene) in accordance with the manufacturer's instructions, gram-negative bacterial DNA from C. sakazakii bacteria was isolated from pellets. The A260/A280 ratio and DNA concentration were measured using a nanodrop spectrophotometer (Nanovue Plus) to ensure DNA was successfully isolated.

Optimization annealing temperature of grxB primer pairs
To determine the ideal temperature for the annealing phase, where the primer grxB is attached, gradient PCR (Takara PCR Thermal Cycler) was used. The PCR assay was performed with a final volume of 25 μL containing 1 μL of pure C. sakazakii DNA isolate, 1 μL of forward and reverse primers, 12.5 μL of Master Mix (NZYTaq), and 9.5 μL of Nuclease Free Water (NFW) contains. The gradient PCR assay protocols were as follows: initial denaturation at 95°C, followed by 40 cycles of denaturation at 95°C, annealing at a temperature between 53 and 62°C, and an extension step at 72°C. The final extension at 72 °C completed the PCR amplification process.

Confirmation assay of Primer grxB using real-time PCR
The assay was performed using a qPCR magnetic induction cycler (biomolecular system) and the total volume used for the reaction mixture was 20 μL. The preparation used consisted of 10 µL of Master Mix SYBR green dye (Smobio), 1 µL each forward and reverse primer, 1 µL C. sakazakii DNA isolate template, and 7 µL NFW. In addition, one reaction was also used for Non-Template Control (NTC) as a negative control and negative control containing NFW and Master Mix. Amplification was carried out for 40 cycles with predenaturation at 95°C, denaturation at 95°C, followed by annealing at 60°C, extension at 72°C and final extension at 72°C.

Specificity and sensitivity assay.
Several non-target bacteria, including Vibrio parahaemolyticus, Vibrio alginolyticus, Listeria monocytogenes, Salmonella typhi, Yersinia enterocolitica, Staphylococcus aureus and klebsiella pneumoniae, used to assess the specificity of the designed grxB primer pair. Each reaction mixture containing target and non-target bacterial DNA samples received a pair of grxB primers. The concentration of each bacterial isolate was equalized to 50 ng/ µL. Based on the amplification curve and the corresponding melting curve, the real-time PCR curve findings will be examined. Multilevel dilution sensitivity testing was done on DNA template isolates of the bacteria. The amplification curves and standard curves represent the outcomes of this sensitivity test.

Cultivation of C. sakazakii bacterial
Bacteria on Tryptic Soy Agar (TSA) showed the formation of yellow colonies on the surface of TSA media. The temperature range for C. sakazakii bacterial growth is 6-47 °C, with the ideal temperature for growth being 41-45 °C and the minimal growth temperature being 5.5-8.0 °C. On TSA media, it can also produce yellow pigments. The yellow colonies on the TSA, each measuring between 1-2 mm and 2-3 mm, revealed that the bacteria produced were in fact C. sakazakii bacteria [7].

DNA Analysis
The nanodrop spectrophotometer was used for the DNA isolate quantification test. The wavelength ratio of A260nm/A280nm revealed the purity of the DNA isolate. The isolation results were characterized by agarose gel electrophoresis using a 10.000 bp ladder as shown in Fig. 1. The result showed the DNA bands isolated from pure cultures of C. sakazakii (lane 1-4) appeared at a position higher than the marker size of 10.000 bp, it can be assumed that the results obtained are in accordance with the size of the whole genome sequence of Cronobacter sakazakii ATCC 29544 which is 4,511,265 bp [8]. DNA purity and concentration were measured using nanodrop (Nanovue plus) with a result as described in Table 1. The wavelength of A260nm is used because the absorbance of DNA will be optimum as well as protein will absorb maximally at the wavelength of A280nm. The requirements for the purity of a good DNA isolate are 1.8-2.0. There are impurities in the genetic material if it is less than 1.8 or greater than 2.0 [9]. Based on Table 1 it can be assumed that the pure culture results of isolated C. sakazakii produce good purity.

Optimization of grxB Primer Pairs Annealing Temperature
The stages in specific gene amplification by PCR are denaturation, annealing and extension. The annealing stage is an important step because it affects the efficiency of the PCR process [10]. DNA amplification by PCR requires a pair of primers (forward and reverse) to limit the area to be amplified.
[11]. Smudge-free intact DNA bands have good density and brightness, indicating good amplification results [12]. based on our previous research, it has been found that the optimal annealing temperature for a pair of grxB primers is at 57°C to 61°C. This temperature range will be used in the real-time PCR procedure.

Confirmation assay of Primer grxB
The molecular method is more efficient, more accurate, and more sensitive than the culture method for detecting C. sakazakii. In this study, we used real-time PCR to develop a detection method. real-time PCR data are the number of PCR cycles required to reach a given level of fluorescence (the "cycle threshold" Ct) [13]. A confirmation assay was carried out to find out that the grxB primer could recognize the target bacteria by amplifying the grxB DNA in the target bacteria C. sakazakii. The grxB gene primer with a concentration of 2 pmol was used at this stage with the target bacteria C. sakazakii which had a concentration of 53 ng/µL. Confirmation assay resulted from that grxB-f and grxB-r primer pairs can amplify C. sakazakii DNA at Ct 11,90 and 12,10, as shown in Fig. 2A. The melting curve Fig. 2B. of C. sakazakii was at 85,77°C and 85,82°C. Non template control amplified ct 36 with the differences 24 cycles and different melt curve with low peak at 86,18 °C. The results of NTC amplification considered as non-target as evidenced by the difference in Ct/cycle distance between C. sakazakii and NTC target bacteria samples >10 cycles [14]. Furthermore, the melting curve results showed no mispriming. This means that the primers amplified only the target DNA as indicated by the formation of one peak [15].

Specificity and Sensitivity assay
The grxB primer pair was tested with non-target bacteria and showed good results, as shown in Fig. 3A. There are differences in ct values up to 12-24 different cycles between target and non-target bacteria. If the ct value between target and non-target bacteria has a range of 10 different cycles, then the non-target bacteria is considered as a negative control. [16]. C. sakazakii bacteria and non-target bacteria have a different melting curve ( Fig. 3B and Table 2). From the Ct value and the melting curve, it can be concluded that the grxB primer is specific for detecting C. sakazakii bacteria. The sensitivity test aims to obtain a standard curve so that the Limit of Detection (LoD) of primer grxB can be identified in detecting C. sakazakii bacterial DNA. LoD is a parameter used to describe the sensitivity of an analytical method [17]. C. sakazakii isolate was diluted as much as five times of dilution. This assay gave the results that C. sakazakii DNA template can still detect at a lower concentration of 0,003392 ng/µL or 3,392 pg/µL at 24,06 with LoD 0,0045 CFU/mL ( Fig. 4 and Table 3). The standard curve is obtained by plotting the Ct value (y axis) against the transformation concentration of the target nucleic acid dilution (x axis). Standard curve has a regression value of R 2 = 0,9964 and equation y = -3,132 x + 14,93.

Confirmation Test on Food Samples with real-time PCR
The grxB primer pair can amplify C. sakazakii DNA in artificially contaminated formula milk samples at Ct 13 to 14 (Fig. 5A). The appearance of a sigmoid curve in infant formula samples indicates that C. sakazakii is able to live and reproduce in formula milk samples so that bacterial DNA can be recognized with real-time PCR. C. sakazakii contamination in infant formula has been attributed to biofilms, which are communities of microorganisms that attach to biological or abiotic surfaces. C. sakazakii can form biofilms on food, food processing surfaces and equipment leading to crosscontamination [18]. The melting curve in Figure 5B shows that the pure culture sample and the artificially contaminated infant formula have nearly the same melting temperature. Real-time PCR results on artificially contaminated infant formula are presented in the table 4. These results can be concluded that this method can be developed as a detection model kit to detect C. sakazakii bacteria quickly, sensitively, and specifically and shows accurate results.

Conclusion
This research has successfully developed a detection method that is rapid, specific and accurate through realtime PCR method using a primer pair grxB for the C.
sakazakii bacteria in infant formula milk. C. sakazakii DNA with a concentration of 53 ng/µL could be amplified by the grxB gene primer pair with a Ct value of 12 and a Tm value of 85.7ºC. The grxB primer that we have designed is specific for detecting the target C. sakazakii bacteria and can detect C. sakazakii bacteria as low as 0.0045 CFU/mL or the equivalent of a sample DNA concentration of 3.392 pg/µL at Ct 24.06. Furthermore, the results of this study will be used for the development of a detection method for C. sakazakii in formula milk samples in validation tests to improve the detection kit that is being developed.