Rapid and simultaneous detection of Salmonella, Shigella, and Staphylococcus aureus in fresh pork using a multiplex real-time PCR assay based on immunomagnetic separation
Introduction
The incidence of food-borne infections has markedly increased over the last 20 years, with nearly a quarter of the population at higher risk for illness today (Omiccioli, Amagliani, Brandi, & Magnani, 2009). It is estimated that in the United States, each year food borne illnesses affect 6 to 80 million people, causing up to 9000 deaths and costing about 5 billion dollars (Balaban & Rasooly, 2000). Three major food-borne pathogens affecting people worldwide are Salmonella sp., Shigella sp., and Staphylococcus aureus. The incidence of Salmonella infections in humans constitutes the most frequently recorded cause of diarrheal illnesses in almost all industrialized countries. A majority of cases of human salmonellosis are due to the consumption of contaminated poultry, pork, and eggs (Kotzekidou, 2013). Salmonella leads to the second highest number of foodborne infections in the United States (Singh & Mustapha, 2013). Shigella spp. are exquisitely fastidious gram-negative organisms that annually cause an estimated 164.7 million cases of shigellosis worldwide, resulting in 1.1 million deaths (Thiem et al., 2004). A recent study reported clinical estimates indicating that S. aureus has caused more than 94,000 serious infections and more than 18,000 deaths in the United States since 2005 (Schmelcher et al., 2012).
Conventional assays for these three pathogens in common use are based on microbiological techniques. Even though these methods are effective for the recognition of microorganisms, they are rather time-consuming, requiring several days (at least 90 h) before the results are obtained. Thus, these tests cannot quickly provide accurate data for many emergency events. PCR-based methods have been found suitable for detection of pathogens in foods because of their rapid and simple application (Chen et al., 2012, Fan et al., 2007, Kotzekidou, 2013, Yang et al., 2011a). Real-time PCR (RT-PCR) allows accurate, automated, and quantitative detection of different microorganisms without the need to open the tubes for electrophoresis after amplification, thus reducing the risk of cross contamination (Elizaquível, Gabaldón, & Aznar, 2011). In addition, RT-PCR has been proven to be cost-effective on a per-run basis, when implemented in a high-throughput laboratory (Martell et al., 1999). Multiplex PCR can simultaneously amplify more than two gene sequences in the same reaction, and recently, it has been used to detect a variety of pathogens (Elizaquível et al., 2011, Garrido et al., 2012, Kim et al., 2006).
Immunomagnetic separation (IMS) uses magnetic beads coated with specific antibodies (IMBs) to capture target pathogens and subsequently separate them from the complex in a simple magnetic field. By sequestering the IMB-pathogen conjugates, interfering and inhibiting materials can be removed by washing. The IMS method has been proven to be an effective way to separate various types of pathogens (Jothikumar et al., 1998, Nou et al., 2006, Taha et al., 2010, Wang et al., 2007, Xiao et al., 2007, Zhao et al., 2008).
Thus, a technique that combines IMS with RT-PCR could be rapid, highly specific, and highly sensitive. This technique could be widely used in the food safety field (Fedio et al., 2011, Fusco et al., 2011, Garrido et al., 2012, Hagren et al., 2008, Lucero Estrada et al., 2012, Yang et al., 2011b).
The objective of this study was to develop a method for rapid, simultaneous detection of Salmonella sp., Shigella sp., and S. aureus from fresh pork within 8 h that offers the opportunity for a quick response in an emergency when these bacteria are detected. The limit of detection (LOD) of this method was determined by detecting these pathogens in spiked samples. Furthermore, the sensitivity, specificity, and accuracy of this method were compared with those of the widely used culture method.
Section snippets
Standard strains and culture media
The 37 bacterial strains used in this study are listed in Table 1. The culture media including nutrient broth medium (NB, Oxoid, UK), modified nutrient broth (MNB), xylose lysine deoxycholate agar (XLD, Oxoid, UK) and Baird–Parker agar medium (BP, Oxoid, UK).
Preparation and specificity verification of immunomagnetic beads (IMBs)
Uncoated magnetic beads (particle diameter, 180 nm, AllMag, Shanghai, China) were washed three times with 10 mM phosphate-buffered saline (PBS) containing Tween 20 (PBST, pH7.4). Then freshly prepared 5 mg/mL
Specificity of IMBs
The high capture rate was obtained for specificity of three types IMBs, 95.93% for Salmonella sp., 96.25% for Shigella sp., and 97.97% for S. aureus, with very low nonspecific capture rates (Table 4).
Specificity of primers and probes for multiplex RT-PCR
The specificity of the primers and probes was also tested with other bacterial strains (Table 1). Table 1 shows that PCR amplification was only observed with target strains and that all the non-target strains were RT-PCR negative, indicating the high specificity of the primers and probes.
Standard curve
To
Discussion
In recent years, IMS-RT-PCR has become a popular tool for identifying pathogens because of its sensitivity, specificity, and time-efficiency (Birce and Griffiths, 2005, Hagren et al., 2008, Garrido et al., 2012). In this study, an IMS-multiplex RT-PCR assay was successfully applied to detect Salmonella, Shigella, and S. aureus in spiked fresh pork and commercial samples.
The presence of RT-PCR inhibitors in fresh pork samples, the medium components, and the residual components from the DNA
Conclusions
A rapid and simultaneous assay using IMS-multiplex RT-PCR was developed to detect Salmonella, Shigella, and S. aureus in fresh pork. This method with a low LOD (<10 CFU/g) has high (>99%) SE, SP, and AC for Salmonella, Shigella, and S. aureus in comparison with the currently widely used culture methods. Furthermore, this method can significantly reduce the time (less than 8 h) of detection for these pathogens in laboratories, which offers the potential for a quick response in an emergency when
Acknowledgments
This work was financially supported by the Beijing Municipal Science & Technology Commission of the Beijing Nova program (China) through research grant 2008B31.
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2022, Food ControlCitation Excerpt :At the same time, we established a local BLAST library to compare our newly discovered targets and previously reported targets and found that the novel uncovered targets showed 100% presence in the target Staphylococcus species while partial presence in the target Staphylococcus species for the reported target. The detailed results revealed that S. aureus targets (nuc, sac, kan, tuf, femB, entB, femA, and mecA) were 58.60%–97.64% (Brakstad et al., 1992; Capurro et al., 2009; Jonas et al., 2002; Ma et al., 2014; Mehrotra et al., 2000; Song & Ko, 2008; Wilson et al., 1991), S. epidermidis targets (recN, serp0107, gseA, and atlE) were 94.79%–100% (Ikeda et al., 2004; Iorio et al., 2011; Kilic & Basustaoglu, 2011; Liu et al., 2006), and S. haemolyticus targets (mvaA and Shae) were 74.04%–95.60% (Iwase et al., 2007; Schuenck et al., 2008). Therefore, the detection targets obtained in this study for the four common pathogenic Staphylococcus spp. by pangenome analysis display a better specificity to meet the needs of food safety and clinical testing.