Advantages and disadvantages of detection methods.
Salmonella is one of the leading causes of food-borne illnesses worldwide, and one of the main contributors to salmonellosis is the consumption of contaminated egg, poultry, pork, beef, and milk products. Since deleterious effects of Salmonella on public health and the economy continue to occur, improving safety of food products by early detection of food-borne pathogens would be considered an important component for limiting exposure to Salmonella contamination. Therefore, there is an ongoing need to develop more advanced detection methods that can identify Salmonella accurately and rapidly in foods before they reach consumers. In the past three decades, there have been increasing efforts toward developing and improving rapid pathogen detection and characterization methodologies for application to food products. In this chapter, we discuss molecular methods for detection, identification, and genetic characterization of Salmonella in food. In addition, the advantages and disadvantages of the established and emerging rapid detection methods are addressed here. The methods with potential application to the industry are highlighted in this chapter.
- food-borne pathogens
- rapid detection
- molecular methods
Food-borne disease is one of the major public health problems for the food industry, especially in developing countries . Failure to detect food-borne pathogens may lead to a dreadful effect. The World Health Organization (WHO) reported that in 2010 alone 1.8 million people died from diarrheal diseases, a great proportion of these cases can be attributed to contaminated food and drinking water . The Centers for Disease Control and Prevention (CDC) have estimated that 48 million cases of food-borne illnesses occur in the United States (US) annually, approximately 128,000 cases require hospitalization, and 3,000 cases result in death . The CDC reported that viruses, bacteria, and parasites are major causative agents for food-borne illnesses. Among these, bacterial agents including
Most human salmonellosis cases are associated with consumption of contaminated egg, poultry, pork, beef, and milk products, which are considered one of the most important reservoirs from which
2. Methodologies for detection of
2.1. Culture-dependent methods
Current testing of food samples for the presence of salmonellae can be divided into three steps: (1) detection of pathogen by plate culture, (2) identification of the isolate and its specific serovar designation, and (3) subtyping of the isolate for association with salmonellosis [18, 19]. These methods rely on traditional bacterial culture procedures that apply serial enrichments with increasing selectivity culminating in the isolation of
Due to its sensitivity, with a limit of detection of 1 cfu, this analytical schema is considered as the “gold standard” of regulatory agencies (Figure 1). The disadvantages of this method are as follows. First, it is time-consuming, taking at least a week for isolation and few more days for serotyping and subtyping. The long time frame hampers its application in many food commodities, especially fresh products, before they are consumed or on hold in warehouses while awaiting test results before they spoil. Second, the operation is tedious; the amount of media and numerous plates are required for each sample. The procedures are labor-consuming and necessitate large areas of space, particularly in many sample detections. Finally, the complex ingredients in foodstuffs, such as indigenous microbiota and antimicrobials, make it notably difficult for traditional microbiological methods [11, 26–29].
2.2. Culture-independent methods
Recent advances in technology have made the detection of food-borne pathogens more rapid and convenient, while achieving improved sensitivity and specificity in comparison to conventional methods. These methods employing newer technologies are generally referred as “rapid methods,” which include nucleic acid-based or antibody-based assays that are modified or improved compared to conventional methods [30–35]. These rapid detection methods can be of high value to the food industry by providing several key advantages such as speed, specificity, sensitivity, cost-efficiency, and labor efficiency.
2.2.1. Polymerase chain reaction (PCR)
The largest advance toward faster detection of salmonellae has been in the realm of molecular biology, where polymerase chain reaction (PCR) and quantitative PCR (qPCR) are predominantly being applied as the methods of choice for the detection. Different protocols targeting different specific genes or gene regions specific to salmonellae have been published. Numerous studies have been conducted to detect and characterize
Over the past years, PCR-based methods have advanced to provide high sensitivity for
As we all know, the quality and quantity of target DNA, PCR template, are important factors during the design of a PCR assay. Although well-designed PCR primer and good PCR template can bring high specificity of the target detection, it is still not sufficient to overcome the side effects of PCR inhibitors in samples, such as denatured proteins, organic chemicals, and sucrose. Moreover, the presence of DNA and cells other than those from the targeted organism can affect the efficiency of the PCR methods. To overcome this, an enrichment step is commonly performed to enhance assay sensitivity by ensuring the detection of viable pathogens before PCR reaction. Ferretti et al. reported that PCR with a 6 h nonselective enrichment could detect various
Improvements have also been made on the basic PCR technology as well. In particular, two primary PCR-based methods have emerged over the past several years, such as multiplex PCR and real-time quantitative PCR [47, 48]. The current status of the optimization and development of these PCR applications is summarized in the following.
Multiplex PCR is a modified PCR method that allows for multiple sequence targets to be simultaneously detected within a single reaction. This method has proven useful for the rapid identification of multiple pathogens simultaneously in a given sample. Generally, multiplex PCR amplifies the target samples using multiple primers in a reaction, which can detect and identify several target sequences in
With the appearance of fluorescence technology that endows increased sensitivity (e.g., intercalating dyes such as SYBR Green or labeled probes), the limitations of conventional PCR can be overcome, such as the errors associated with end-point analyses and lack of quantification. The “real-time” aspect of real-time PCR, also referred to as qPCR, technology is linked to its ability to label and cumulatively quantify the generated PCR products at each cycle throughout the ongoing amplification process. The qPCR has been widely used to quantify
|Culture-dependent methods||—Accurate||—Labor and time cost|
|Single and multiplex PCR||—More rapid than culture-based methods (<24 h vs. 5 ~ 7 days)|
—High specificity and sensitivity
—Multiplex PCR (several pathogens at a time)
—Multidetection of several
|—Costs more than culture-based methods and ELISA|
—Difficulty in distinguishing live and dead cells
—Technically can be challenging (optimized PCR condition)
—Enrichment to detect viable cells
—Requires post-PCR processing of products (electrophoresis)
|qPCR||—Not influenced by nonspecific amplification; amplification can be monitored at real time|
—No post-PCR processing of products (gel electrophoresis)
—Rapid cycling (25 min)
—Confirmation of specific amplification by melting curve
—Specific, sensitive, and reproducible
|—Difficulty in multiplex assay|
—Need skilled person and support
—High equipment cost
—Possibility of cross contamination
|Antibody-based method||—More rapid than culture-based methods (2 days vs. 5 ~ 7 days)|
—Can be automated to reduce assay time and manual labor input
—Able to handle large numbers of samples
—More specific than cultural methods
|—Not high sensitivity|
—Difficult to multidetect
—Difficulty to differentiate damaged or stressed cells
—Need to pre-enrichment
—High cross-reactivity with close antigens in bacteria
|Aptamer-based method||—Inexpensive, stable, and can be chemically synthesized than antibody|
—Time saving (2 h vs. 5 ~ 7 days of culture-based methods)
—Automated to reduce manual labor input
—Large numbers of sample detection at one time
—Higher specificity than cultural methods
|—High false-positive results|
—Difficulty in detecting damaged or stressed cells
—Pre-enrichment for production of cell surface antigens
—Possibility of cross contamination
2.2.2. Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA)-based approaches are the most prevalent antibody-based assay for pathogen detection in foods . This immunological approach has been used to detect
2.2.3. Aptamer-based detection assay
Besides antibodies, other biomolecules have been investigated to selectively capture and enrich
Relative to culture-independent detection, researchers have focused on methods to concentrate whole cells within the sample before the pre-enrichment step. The enriched whole
In summary, the mentioned methods here have utility advantages for
In order to meet the current requirement of rapid detection, it is clear that several approaches have emerged including PCR-based, antibody-based, aptamer-based, and other approaches encompassing those stemming from the current genomic era. A clear character of method development direction is moving toward greater automation, cost-saving, and time-saving network integration. It is important to mention that outputs from one approach would serve to strengthen directly or tangentially other approaches. At last, it seems that a suite of tools is emerging for the food safety microbiologist, each with its specific advantages and disadvantages but all with the ability to rapidly and accurately detect
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