Molecular Approach for Screening and Identification of Food Containments using Colony PCR

Food is the primary cause for diseases in humans and carries high risk pathogens. Assessment of the safety in foods is needed to validate the presence of pathogenic bacteria. We used colony PCR for this approach to detect foodborne pathogens such as Escherichia coli , Lactobacillus and Bacillus cereus . Suitable primers were selected based on specific gene 1040 for Escherichia coli , gene S2 for Lactobacillus , and gene NVF for Bacillus cereus . Agarose gel electrophoresis is used for the detection of amplified products against a suitable marker. ImageJ is used for DNA band analysis, enabling precise quantification, normalization, and statistical comparisons. These studies have established a promising role in the detection of pathogens in various environmental samples. The insights gained from this study may serve as the foundation for rapid detection of foodborne diseases in the food industry.


INTRODUCTION
Food technologists are currently showing their interest in molecular methods as a powerful tool for identifying and detecting contaminants in food specimens. 1Colony PCR Analysis is one of the widely used techniques that utilizes the principles of polymerase chain reaction (PCR) to observe contaminants at the molecular level. 2,3owerful screening techniques are necessary as demand for the safety and integrity of food products has shot up due to the expansion of the food business globally. 4Colony PCR is costeffective for detecting bacterial contamination because it directly amplifies DNA from colonies, avoiding expensive steps like DNA extraction and purification required in traditional methods.This reduces overall expenses while maintaining accuracy in identifying contaminants. 5One of the main important concerns for food manufacturers, agencies, and customers is quality and safety. 6acteria, fungi, viruses, toxins, and chemical residues pose hazardous health risks and economic implications. 7Conventional detection methods consume a lot of time and as well as money with hard labor work, which may lack sensitivity, specificity, or the capacity to effectively detect emerging pathogens. 8,9Furthermore, these techniques may not support rapid detection, identification, and delay in responses which can increase risk factors in public health. 10n response to these challenges, molecular techniques have transformed food safety by offering sensitive, specific, and rapid means of detecting and identifying contaminants. 11Analysis by colony PCR in food safety involves a broad spectrum of contaminants and food pathogens such as Salmonella, Escherichia coli, Listeria, Lactobacillus, Bacillus cereus and Staphylococcus due to their prevalence in foodborne infections. 12,13ungal contaminants also exhibit health effects such as Aspergillus, Penicillium, and Fusarium species, which produce mycotoxins, are also investigated. 14,15Colony PCR offers rapid, sensitive and diverse solutions for detecting and identifying various contaminants in foods. 16,17With the advent increase in use of molecular approach methods in the field of molecular biology and recombinant technology, screening of contaminants in colony is possible with the use of primers in bacterial culture. 18This method offers quick identification of pathogens and the results are promising among the other methods in sensitivity and specificity. 19

Preparation of food samples
Food samples were collected from the canteen and supermarkets of the KL University campus, Andhra Pradesh, India.The food specimens were collected in 5 ml screw cap tubes.Sample is collected carefully and sealed with parafilm until it is spread over agar plates.The list of food samples is shown in Table 1.

Identification of food contaminants
Samples were collected in vials, and double-distilled water was added to dilute them.Following dilution, the samples were streaked onto LB agar plates, which were then incubated for 24 hours.After incubation, bacterial growth was observed, and colonies were collected into PCR tubes, to which master mix was added. 20acterial identification is accomplished using primers designed for specific genes at specific temperatures 56°C for Escherichia coli, 55.4°C for Lactobacillus and 54°C for Bacillus cereus which facilitate DNA amplification.

Characterization of contaminant by AGE
The samples after PCR were taken out and loaded into 1% agarose gel after the electrophoresis the gel was observed under gel doc. 21

RESULTS AND DISCUSSION
The assessment of specific bacterial strains in food samples was conducted by observing bands in gel electrophoresis.The presence of bands serves as an indicator of successful primer annealing to bacterial genomic DNA, resulting in amplification.This process allows for the identification of specific bacteria targeted by each primer set.Conversely, the  absence of bands in certain gels suggests the non-existence of the targeted bacteria in those food samples.This absence might be attributed to the presence of other bacterial strains not covered by the three different primers designed for the specific bacteria.The gel electrophoresis results are visually represented in Figure 1.For quantitative analysis of the amplified bands, ImageJ software was utilized to measure the band areas, and the corresponding values are presented in Table 2.A graphical representation of the relationship between food sample and the area of the bands is shown in Figure 2.This graphical analysis offers a comprehensive insight into the bacterial composition across various food samples, emphasizing differences in band areas and facilitating the identification of specific bacterial strains.The graph indicates that E. coli exhibits higher intensity compared to Lactobacillus and Bacillus cereus.Bacillus cereus demonstrates the lowest intensity among the three bacterial strains.

CONCLUSION
In this research endeavour, colony PCR was conducted on a total of 26 food samples suspected of contamination.The DNA extraction and amplification process were conducted using PCR for 35 cycles.To verify the presence of PCR amplicons, 12 µl of the PCR product was loaded onto a 1% agarose gel.This investigation introduces a rapid method for detecting contaminants in food samples.However, for enhanced accuracy and reliability, it is advisable to optimize further and validate the colony PCR technique, particularly when dealing with larger sample sets.Additional efforts towards optimization and validation can contribute to the robustness and effectiveness of the colony PCR method in identifying contaminants, thereby strengthening its utility in food safety and quality assurance protocols.

Figure 2 .
Figure 2. Graphical representation of food samples with bacteria vs area of the bands obtained.Blue line represents E. coli, orange line represents Lactobacillus and grey line represents Bacillus cereus

Table 2 .
The dataset comprises information regarding food samples and their corresponding band areas

Table 1 .
List of food samples collected from various sources