Oligonucleotide primers used in this work.
In recent years, foodborne illnesses have become the most significant public health issue in both developed and developing countries. The World Health Organization (WHO) reported that in 2010, around 1.8 million people died due to foodborne illness. Therefore, the development of a cost-effective, sensitive, and selective detection method for identifying and monitoring foodborne pathogens is necessary for improved public health. Here, we describe a simple and ultrasensitive colorimetric method for the detection of foodborne pathogens based on HRPzyme-integrated PCR using PC-based ImageJ software. We present insights into different aspects of this method such as the importance of 16S rRNA detection, the modification of traditional PCR primers with a unique functional sequence for generating a color signal, and the application of ImageJ in colorimetric image data acquisition. The performance of the proposed strategy in detecting various foodborne pathogens is comparable to that of the commercial UV-Vis spectrophotometer Tecan Infinite 200 Pro. This detection platform exhibits linearity over wide range, high sensitivity, and high selectivity. The diagnostic capability of this colorimetric system to detect foodborne pathogens was demonstrated with spiked fruit and vegetable samples. This low-cost and effective colorimetric method can be conveniently employed for the analysis of DNA sequences arising from pathogenic bacteria.
- foodborne pathogens
- 16S rRNA
- colorimetric detection
1.1. The importance of pathogenic foodborne pathogens
Food safety is critically important to consumer and public health and to the economic sustainability of the agro-food sector. Due to food poisoning incidents, consumers desire food safety assurances before they purchase food items. Foodborne illnesses, mainly caused by pathogens derived from contaminated water or contaminated and uncooked foods, cause many cases of death due to severe infection [1, 2]. The most widely recognized foodborne diseases are caused by pathogens such as
1.2. General overview of the importance and limitations of recent detection methods for foodborne pathogens
In recent years, a variety of diagnostic approaches, for example immunological methods, such as enzyme-linked immunosorbent assays (ELISAs) with detection limit of 103 cfu mL−1 [6, 7, 8] and molecular biological methods such as polymerase chain reaction (PCR) with detection limit of 102 cfu mL−1 [9, 10, 11] have been employed for the identification of foodborne pathogens. These methods vary in their sensitivity, specificity, cost, and efficacy. Owing to the recent availability of genomic information, molecular-based approaches have garnered considerable attention in terms of the development of molecular diagnostic techniques to detect and characterize pathogens [5, 12]. In the last decade, PCR, which can amplify a small amount of DNA through an amplification process, has been employed as a gold standard method for the molecular diagnosis of nucleic acids. Agarose gel electrophoresis  and real-time measurements  with DNA-binding dyes, such as the SYBR green and EvaGreen dyes, have been employed to detect amplified PCR products. Still, these strategies are expensive, time-consuming, and not user-friendly. Considering these limitations, there is a need to develop advanced methods that can overcome the aforementioned limitations. Thus, several research groups have reported new detection platforms based on molecular beacons  and nanoparticle-tagged probes [15, 16]. However, these advanced methods additionally require fluorescence conjugation or probe thiolation procedures [17, 18, 19, 20], which are also costly and time-consuming. Therefore, a simple, fast, cost-effective, and user-friendly detection platform remains in high demand.
1.3. The introduction of HRPzyme-integrated PCR and its importance
Recently, novel detection platforms have been developed to detect genomic DNA as a target analyte based on colorimetric reactions generated by biocatalysts, such as horseradish peroxidase-mimicking DNAzymes (HRPzymes). HRPzymes consist of a folded structure of a G-quadruplex nucleotide sequence, and they exhibit peroxidase-like activity by folding with a hemin molecule [21, 22]. Importantly, the hemin-HRPzyme folded structure catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) or 2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) in the presence of H2O2 and produces a colored oxidized product [23, 24]. Based on this remarkable property, HRPzyme-based strategies have significantly improved the detection of proteins , small molecules , and heavy metal ions .
Recently, several researchers have reported the use of colorimetric HRPzyme-integrated PCR for the simple and rapid detection of bacteria [28, 29, 30, 31]. This PCR platform can be employed for the simultaneous synthesis of a peroxidase-like DNAzyme using a primer including a complementary DNAzyme sequence. To overcome the need for primer labeling and expensive instrumentation, Cheglakov et al.  developed the HRPzyme-integrated PCR for the visual detection of bacteria. Similarly, Cheng et al.  reported the HRPzyme-based colorimetric PCR for the simple and cost-effective detection of
In the aforementioned studies, researchers utilized a combination of the HRPzyme sequence and a primer to amplify a target gene via PCR amplification. The HRPzyme sequences were generated through PCR amplification of the primer, which is integrated with an anti-HRPzyme sequence. After PCR amplification, in the presence of hemin, the unamplified HRPzyme sequence containing primers forms a catalytic hemin-G-quadruplex structure, which mimics peroxidase activity and produces a colorimetric signal via the oxidation of a peroxidase substrate such as TMB or ABTS. The HRPzyme sequence generated during PCR amplification thus produces an optical signal that can be identified by eyes or with a spectrophotometer. These developed colorimetric protocols have been adopted for the rapid and easy detection of various pathogens by integrating a unique functional sequence with the traditional primer set for the generation of the colorimetric signal. These studies thus demonstrated the broad applicability of a fast, simple, ultrasensitive, and selective detection method for DNA as a target analyte.
1.4. HRPzyme-integrated PCR-based detection of foodborne pathogens using PC-based ImageJ software
We have developed a PCR-based detection platform, termed G-quadruplex-blocking PCR, because the amplification of a specific target blocks the folding of the HRPzyme sequence, inhibiting the peroxidase activity of the HRPzyme. In contrast, in the presence of hemin and the absence of target-specific amplification, the G-quadruplex/hemin complex generates an oxidized substrate by oxidizing ABTS in the presence of hydrogen peroxide (H2O2) [32, 33]. G-quadruplex/hemin complexes can also oxidize different peroxidase substrates such as fluorogenic (e.g., Amplex UltraRed (AUR) and QuantaBlu), electrochemical (e.g., 3-Indoxyl Phosphate and p-aminophenol (PAP)), and luminescent (e.g., luminol), allowing for the combination of the proposed method with fluorometric, electrochemical, and chemiluminescent detection platforms. The developed HRPzyme-integrated colorimetric detection platform allows for color development and provides a simple data analysis tool capable of the detection of specific common foodborne pathogens. Specifically, we used available ribosomal RNA (rRNA) sequence information from microorganisms to design the HRPzyme-integrated PCR assay. The proposed colorimetric detection platform allows the visual detection of food pathogens (even a single cfu per milliliter). In this chapter, we employed sequences of the 16S rRNA, a component of the 30S small subunit of the bacterial ribosome, as a target because several copies of 16S rRNA are present in each bacterium (1 × 103 to 1 × 105 copies) . Further, we demonstrate the integration of this colorimetric HRPzyme-integrated PCR platform with a digital camera and desktop NIH ImageJ software, a simple data analysis tool that is able to measure the gray intensity (32-bit grayscale) of the colored images and further transmit the metadata to a centralized off-site laboratory (Figure 1).
2. Material and methods
2.1. Experimental material
DNA polymerase (
|Pathogens||Target (GenBank number)||Primers (5′3′) (H-F: Forward primer; H-R: Reverse primer)||Product size (bp)|
2.2. Microorganism and culture conditions
Foodborne pathogens, including
2.3. PCR amplification of 16S rRNA sequences specific to foodborne pathogens without genomic DNA isolation
Various concentrations of bacterial cultures were directly employed as template DNA for amplification with gene-specific primer sets (Table 1). Based on our previous report , PCR was performed in a total volume of 50 μL containing 5 μL bacterial culture at various concentrations (0–106 cfu mL−1), 10× PCR buffer (2.5 μL), 25 mM MgCl2 (6 μL), 2.5 mM dNTPs (deoxynucleotides) mix (8 μL), 10× PCR enhancer (5 μL), 25 mM MnSO4 (1 μL), 20 μM forward primer (1 μL), 20 μM reverse primer (1 μL), and Tth DNA polymerase (0.5 μL). The PCR amplifications were performed under the following conditions: initial denaturation at 95°C for 5 min and 60°C for 20 min, then 35 cycles of 10 s at 94°C, 25 s at 60°C, and 10 s for 72°C adding last one cycle of 1 min for 72°C . The PCR amplification was confirmed using 1.5% agarose gel electrophoresis. Then, to get colorimetric signal, PCR product (10 μL) was mixed with 300 μM hemin (5.5 μL), 5.5 mM ABTS (100 μL), 35% H2O2 (0.7 μL), and citrate buffer (1000 μL) with pH 4. After 10 min at room temperature (RT), the absorbance was measured at 410 nm with UV-Vis spectrophotometer (Infinite M2000pro, Männedorf, Switzerland) . All images were taken using a digital camera (Samsung, Seoul, South Korea).
2.4. Spiked sample analysis
Fruit and vegetable samples such as apple, chicory, water dropwort, and white radish samples were purchased from the supermarket, washed with sterile water, and immersed in a plastic bag containing 20% ethanol and 1% lactic acid solution for 10 min. Then, sterilized food samples were cut to specific sizes and inoculated with different concentrations of bacteria, including
2.5. Data acquisition through ImageJ software
ImageJ is a simple, practical, and freely available downloadable program that can be used on any computer with Java 5 or on a virtual machine . Recently, many researchers have employed the ImageJ software to quantify data [37, 38]. The green color intensities produced during the HRPzyme-TMB-H2O2 assay were captured using a digital camera (Samsung, Seoul, South Korea) equipped with a standard 18–55-mm objective lens. Color intensities were measured, quantified, and averaged from three experiments by using the digital camera and ImageJ (Wayne Rasband, National Institutes of Health, Rockville, MD, USA; http://rsb.info.nih.gov/ij). The Δ gray intensity value was obtained by subtracting the average gray intensity of the negative samples from the average gray intensity of the positive samples.
3. Results and discussion
3.1. Principle of HRPzyme-integrated PCR
In this chapter, we established a method for the colorimetric detection of a PCR product generated by HRPzyme-integrated primers, as shown in Figure 1. We modified the forward and reverse primers that contain four regions: a protector sequence, HRPzyme sequence, spacer sequence, and sequence complementary to the 16S rRNA sequence from one of several pathogens. The 16S rRNA sequence was employed as a target for the specific detection of food pathogens. During PCR amplification, cells were first lysed by heating PCR samples containing pathogenic bacteria. Then, Tth DNA polymerase was used to successfully transcribe 16S rRNA into complementary DNA (cDNA), which was further employed as a cDNA template for PCR amplification. The thermostable Tth DNA polymerase is obtained from
3.2. Feasibility study of the designed strategy
In order to demonstrate the feasibility of this concept, we employed 16S rRNA sequences from various foodborne pathogens as target analytes. According to our previous report , this strategy requires two steps for the successful detection of target pathogens: the amplification of target sequences and the subsequent colorimetric signal development. For this reason, we designed 16S rRNA sequence-specific forward and reverse primers containing HRPzyme, protector, and spacer sequences. Under optimized conditions, we confirmed the amplification of 16S rRNA products from
3.3. Sensitivity and selectivity of proposed study
Based on the optimized conditions, we utilized the proposed detection strategy for the analysis of different foodborne pathogens with HRPzyme-integrated primer sets specific to the 16S rRNA sequences of various bacteria, as shown in Table 1. A detailed explanation of the ImageJ-based semiquantitative analysis is illustrated in Figure 3. First, a digital image of the green solution is captured using a digital camera. Then, captured images are converted into 32-bit grayscale utilizing the PC-based ImageJ software, and images are inverted (Figure 3a). Next, spot areas are individually selected, and their gray intensities are measured (Figure 3b). As shown in Figure 3b, the performance of the proposed system in the detection of
Next, we employed this proposed strategy to the detection of different food pathogens. First, we diluted bacteria to different concentrations in the range 0 to 1.0 × 106 cfu mL−1 in 1× PBS buffer. We then directly employed these diluted samples in PCR amplification without first extracting genomic DNA. After PCR amplification, PCR products of different pathogens were used for colorimetric signal generation. As shown in Figure 4a, the electrophoresis gel data reveal the amplification of target gene bands with different concentrations of bacteria. In addition, the results depicted in Figure 4b (grayscale images) show that the color intensity signal decreased with increasing concentrations (number of cfu mL−1) of bacteria. The color intensity data show a linear relationship with the concentration of the target pathogens (Figure 4c). The standardization curve of Δ gray intensity versus the bacterial concentration (cfu mL−1) exhibited good linearity in the range from 1.0 to 1.0 × 106 cfu mL−1 (Figure 4c). We observed clear differences between negative control samples and those containing various concentrations of bacteria. This strategy showed a 10-fold better performance than the gel electrophoresis-based assay. The proposed method could therefore be used in the diagnosis of pathogenic bacteria without needing to first isolate bacterial genomic DNA.
3.4. Detection specificity
In practical application, the specificity of the detection platform is critical. In order to assess the specificity of the proposed detection strategy, we performed a specificity test using different food pathogens. We successfully demonstrated the selectivity of the method for the detection of several bacteria, including
|Detection methods||Detection limit (cfu mL−1)||Detection range (cfu mL−1)||Detection time||Reference|
|Real-time PCR assay based on immunomagnetic separation||1.0 × 101–1.0 × 104||10||1.2 hrs|||
|Immunosensors||1.0 × 104–1.0 × 108||1.0 × 105||1.5 hrs|||
|Antibody-aptamer Sandwich ELISA||1.0 × 103–1.0 × 108||1.0 × 103||<3 hrs|||
|Selective filtration technique combined with antibody–magnetic nanoparticle nanocomposites||2.0 × 101–2.0 × 104||20||45 min|||
|Aptasensor, gold-nanoparticles aggregation||1.0 × 102–1.0 × 107||56||1 hr|||
|Immuno-PCR||1.0 × 103–1.0 × 105||1.0 × 103||4 hrs|||
|Antibody-conjugated magnetic nanoparticles (MNPs)||1.0 × 102–1.0 × 108||100||1 hr|||
|Aptasensor, peroxidase mimics magnetic nanoparticles||Not given||7.5 × 105||1 hr|||
|Aptasensor, gold-nanoparticles aggregation||1.0 × 101–1.0 × 106||10||4 hrs|||
|HRPzyme-Integrated Polymerase Chain Reaction||1.0 × 100–1.0 × 106||1||~1.2 hrs||This study|
3.5. Detection of pathogens from different spiked food samples
A variety of agricultural products have been linked to human illness worldwide. Some agricultural commodities such as fruits and vegetables are more vulnerable to pathogenic bacterial contamination . Therefore, the ability of detection methods to detect pathogens from fresh fruits and vegetables is critical. In this study, we demonstrated the ability of the proposed strategy to detect pathogens among spiked food samples such as apple, chicory, water dropwort, and white radish samples purchased from the local market. Such fruit and vegetable samples contain various biological components  such as proteins, phenols, and polysaccharides that may reduce the reliability of the method. Figure 6 shows the analysis of various food samples spiked with
In this study, we demonstrated the performance of the HRPzyme-integrated PCR-based colorimetric method for the simple, sensitive, and selective detection of 16S rRNA sequences from various food pathogens. The proposed strategy showed several advantages such as label-free, simple and easy procedure, ultrasensitive (detection limit up to single cfu mL−1), highly selective, detection within 70 min, affordable at remote areas, easily integrated into smartphone-based image processor. PCR amplification is carried out with 16S rRNA-specific primers modified at the 5′-end with HRPzyme sequences. After PCR, in the presence of hemin, unamplified primers fold into a G-quadruplex structure, and a colorimetric signal is then generated in the presence of a chromogenic substrate. We successfully detected the presence of foodborne bacteria, including
This research was supported by a grant from the World Institute of Kimchi, funded by the Ministry of Science, ICT and Future Planning (KE1701-5), Republic of Korea, and by the GIST (Gwangju Institute of Science and Technology), Korea, under the Practical Research and Development support program, supervised by the GTI (GIST Technology Institute), Republic of Korea.
Conflict of interest
The authors declare no financial or commercial conflict of interest.