**4. Rapid and simple detection of** *invA* **gene in** *Salmonella* **spp. by isothermal target probe amplification (iTPA)**

Nucleic acid amplification methods are widely used for detection of food-borne pathogens and the PCR is the most popular and useful method, requires a high precision thermal cycling instrument, which often prevents PCR from being used in routine food pathogen detection by the food industry. Recently, Jung *et al*. (Jung et al. 2010) developed a new highly sensitive and specific isothermal amplification and detection system called isothermal target and probe amplification (iTPA) by employing DNA-RNA-DNA chimeric primers and a FRET probe. The iTPA reaction is done under isothermal conditions between 55 and 65°C using a simple incubator such as a water bath or block heater which is sufficient for amplification. The detection of the fluorescence signal is acquired directly from the amplification reaction tube without any post-amplification handling that reduces the risk of any amplicon-carryover cross-contamination. The iTPA assay is highly specific for the target sequence because the primers and probe recognize five distinct regions on the targeted DNA. The iTPA method is based on a combination of novel ICA (isothermal chain amplification) and FRET cycling probe technology (CPT). In the ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of

M 1 2 3 4 5 6 7 8 9 10 P M N 11 12 13 14 15 16 P

Fig. 12. Detection specificity using the *Salmonella* spp. 674 bp primer in contaminated variety

The minimum detection limit was 10 cells / ml with pure culture, which is far more sensitive than conventional PCR which has detection limit of 10,000 cells / ml. In conclusion, we developed a highly sensitive and specific real-time PCR assay for detection of the five food-borne pathogenic bacteria in food samples. This newly developed assay was successfully used to monitor the dynamics of this novel bacterium in food (Abu *et al*, 2005).

**4. Rapid and simple detection of** *invA* **gene in** *Salmonella* **spp. by isothermal** 

Nucleic acid amplification methods are widely used for detection of food-borne pathogens and the PCR is the most popular and useful method, requires a high precision thermal cycling instrument, which often prevents PCR from being used in routine food pathogen detection by the food industry. Recently, Jung *et al*. (Jung et al. 2010) developed a new highly sensitive and specific isothermal amplification and detection system called isothermal target and probe amplification (iTPA) by employing DNA-RNA-DNA chimeric primers and a FRET probe. The iTPA reaction is done under isothermal conditions between 55 and 65°C using a simple incubator such as a water bath or block heater which is sufficient for amplification. The detection of the fluorescence signal is acquired directly from the amplification reaction tube without any post-amplification handling that reduces the risk of any amplicon-carryover cross-contamination. The iTPA assay is highly specific for the target sequence because the primers and probe recognize five distinct regions on the targeted DNA. The iTPA method is based on a combination of novel ICA (isothermal chain amplification) and FRET cycling probe technology (CPT). In the ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of

food samples

**target probe amplification (iTPA)** 

RNase H, two displacement events occur in the presence of four specially designed primers that lead to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. In the CPT method, a DNA-RNA-DNA chimeric probe is hybridized with the target DNA, and the RNA region of the duplex is specifically cleaved by RNase H. The cleaved probe fragments are disassociated from the target DNA and another intact probe is again hybridized and then cleaved. In the cycling events, a single target DNA molecule results in a large number of cleaved probe fragments, which can be designed to generate fluorescent signals (Fig. 1). In the present study, a sensitive and specific iTPA assay for detecting *Salmonella* spp. in experimentally inoculated food samples was developed.

Fig. 13. The process for the Isothermal target & Probe amplification

#### **4.1 Materials & methods**

#### **[iTPA primers, FRET probe, and reaction conditions]**

The *Salmonella invA* gene (GenBank: EU348369) was used as the target for iTPA primer and probe design. Four primers, two outer and two inner, and one FRET probe which

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 433

medium and then quantified. One picogram of genomic DNA was used as the template. For non-*Salmonella* strains, the genomic DNA was isolated from the overnight culture grown in LB medium and then 2 L of the DNA extraction TE buffer solution was used. Specificity tests were repeated 10 times. To determine iTPA detection limits, serial 10-fold dilutions of a mid-log phase *S. typhimurium* KCTC2515 culture (*ca.* 108 CFU) grown in LB broth were prepared in PBS and quantified using the standard plating method. The detection limit tests were repeated 10 times and the lower limits of detection (CFU per assay) were reported.

Three kinds of foods were used for the study: peanut butters, egg yolk and chicken breasts. Food samples were processed as described in a previous study (Kim *et al*. 2007) with slight modifications. Briefly, A 500 ml Erlenmeyer flask (LB broth 225 ml) containing 25 g of chicken breast was incubated at 37°C overnight and then 9 ml of this solution was transferred into a 10 ml conical tube followed by adding 1 ml of inoculated buffered peptone water of *Salmonella* spp.(1.0 x 109 CFU / ml) to prepare a stock solution. Plastic food bags containing 25 g of chicken breast were inoculated with 1 ml of serial dilutions (1:10 to 1:108) of the stock solution and vigorously mixed using a homogenizer (Pro-media SH-001, ELMEX Ltd., Tokyo, Japan) for about 30 sec to distribute the bacteria followed by adding 225 ml of freshly made LB broth to prepare pre-enriched solutions. The sample preparations for peanut butters and other food samples were the same except that for peanut butters which required an additional washing with a washing solution (0.05% NaOH, 0.5% Tween 20 in PBS buffer solution) due to the high viscosity. 100 L of the preenriched solution was mixed with the washing solution and centrifuged at 10,770 x g for 5 min followed by discarding the supernatant. The pellet was washed with 100% ethanol and then with TE buffer solution twice. The washed pellet was suspended in 200 L of TE buffer solution and heated at 100°C for 10 min in a dry heating block. The crude cell lysate was centrifuged at 10,770 x g for 5 min and an aliquot (2 L) of the supernatant was used for the iTPA assay. For negative samples, the same amount of aliquot (2 L) of uninoculated food samples that had also undergone cultural pre-enrichment was used. The inoculated food sample tests were repeated 10 times and the lower limits of detection

The *Salmonella* spp. *invA*-based iTPA assay, which required only a water bath and the RF-1000 fluorescent reader successfully detected 10 *Salmonella* spp. strains while showing negative results for 40 non-*Salmonella* spp. strains (Table 1), indicating that the *invA-*based iTPA assay was specific for *Salmonella* spp.. The PCR assay using iTPA outer primers yielded amplicons of the expected size (117 bp) for all 10 Salmonella spp. strains. (data not shown) Two sample t-tests were performed for negatives and positives in pure culture. The mean F-score of the negatives was 3.97±0.44 and the mean F-score of the positives was 82.9±6.1 (p≤0.001, data not shown). For a rigorous exclusivity comparison, the positive strains were used at a low concentration of the genomic DNA (1pg, ca. 102 CFU) as the template while the negative strains were used at very high concentration of the genomic DNA (ca. 105 CFU). Neither false positive nor false negative results for the 50 bacterial

**[iTPA testing in experimentally inoculated food samples]** 

(CFU per assay) were reported.

**[Inclusivity and exclusivity of the iTPA assay]** 

**4.2 Results and discussion** 

recognized five distinct regions of the target sequence were designed using the DNASTAR software (Maison, WI). Oligonucleotide sequences and locations of the primers and the probe are shown in Table 1. The DNA primers were synthesized by Genotech (Daejeon, South Korea). The chimeric primers and a FRET probe were synthesized by IDT (San Diego, CA). The iTPA reaction mix in a 20 *μl* volume consisted of the following: 10 mmol / L of Tris-HCl (pH 8.5), 22 mmol / L of MgSO4, 10 mmol / L of KCl, 10 mmol / L of (NH4)2SO4, 0.05 mg ml / L of acetylated BSA, 3 mmol / L of DTT, 0.4 mmol / L of deoxynucleotide triphosphate (dNTP), 0.22 mol / L of each outer primers, 2.2 mol / L of each inner primers, 100 nmol / L of the FRET probe, 5 units of *bst* polymerase (NEB, Ipswich, MA), 5 units of RNase H (Epicentre, Madison, WI), 6 units of RNase inhibitor (Solgent, South Korea), and 2 l l of DNA template (2 l of sterilized water was used for a negative control). The iTPA reaction mix was incubated at 58°C for 60 min in a water-bath and then cooled to room temperature. After a quick spin-down, the reaction tube was inserted into a RF-1000 fluorescent reader (Raplegene, Inc., Sungnam, South Korea) to read the relative RFU (fluorescence relative unit) signal. The RF-1000 fluorescent reader calculates the F-score and it is display on the LCD window. The result was interpreted as a *Salmonella* spp. positive if the F-score was ≥35 or a *Salmonella* spp. negative if the F-score was < 35. This cut-off value was determined using uninoculated food samples that had also undergone cultural pre-enrichment. F-scores of uninoculated egg yolk samples and chicken meat samples were 20±9.5 and 20±7.2 respectively (p≤0.001). The equation used to calculate the F-score is the following:

F-score = [(fluorescence of the sample – fluorescence of the negative control) / fluorescence of the negative control] x 100. Sterilized water was used for the negative control reaction instead of the extracted nucleic acid from inoculated food samples. Since uninoculated food samples may be contaminated, we used sterile water as the negative control.


Table 4. iTPA primers and FRET probe used in this study to detect Salmonella spp.
