Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

Abstract

Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ؎ 0.05°C for invA, 85.56 ؎ 0.28°C for ipaH, and 89.21 ؎ 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10 4 CFU g ؊1 ; however, the limit of detection was 10 3 CFU g ؊1 . This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens. Infectious diarrhea is a global health problem that is still responsible for thousands of deaths worldwide, especially in children (1). It can be classified based on its clinical presentation as one of two syndromes-noninflammatory or inflammatory diarrhea (2). Among cases of inflammatory diarrhea, Shigella is the most common cause, followed by Campylobacter and Salmonella (3). These invasive organisms primarily target the lower bowel; they invade the intestinal mucosa to induce an acute inflammatory reaction and activate cytokines and inflammatory mediators (4).Because the time frame in which treatment choices must be made is short and the conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. We searched for DNA sequences that were highly conserved between the different species of each genera, and we selected the following genes as targets, invA (invasion A gene) for Salmonella spp., ipaH (invasion plasmid antigen H) for Shigella spp., and 16S rRNA for Campylobacter spp., to develop a fluorescence-based real-time PCR procedure to simultaneously identify these enteropathogens. In this method the post-PCR products are identified based on melting-point curve analysis. We have also standardized the technique to quantify these bacteria directly from stool samples. MATERIALS AND METHODSBacterial strains. A total of 147 enteropathogenic strains (Table 1) were analyzed, including clinical isolates representative of Salmonella spp., Shigella spp., and Campylobacter spp., as well as other enteropathogens. These clinical strains had previously been identified based on serology, biochemical assays, and real-time PCR for the diarrheagenic Escherichia coli (5). In add...

Association Between Shigella Infection and Diarrhea Varies Based on Location and Age of Children

“…1 Multiple studies have evaluated molecular detection methods to estimate the prevalence of Shigella-positive stools, but many of these studies examined only diarrheal cases and/or were not community based. [2][3][4][5][6][7][8] Molecular methods range from qualitative polymerase chain reaction (PCR) to multiplex PCR to an absolute quantitative measure using quantitative (q)PCR; most use the invasion plasmid antigen-H (ipaH) gene as a molecular marker. These studies consistently report an increased prevalence of Shigella-positive stools and a lower limit of detection compared with conventional culture techniques.…”

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

“…and L. monocytogenes demonstrated sensitivity of 10 3 CFU mL À1 in spiked nonfat dried milk (Singh et al, 2012). Barletta et al (2013) have developed an assay utilising nonspecific dye SYBR Green for detection of Campylobacter, Salmonella and Shigella in stool samples, which included DNA extraction step and showed the detection limit of 10 3 CFU per gram of spiked faeces. The inclusion of pre-enrichment step increased the sensitivity of melting curvebased assays in food samples by several logs and improved the reaction performance up to few CFU mL À1 (Singh et al, 2012).…”

“…(Kawasaki et al, 2010;Suo et al, 2010;Russo et al, 2014). There are few recent reports on qPCRbased assays addressing Campylobacter, Salmonella and Shigella in food (Barletta et al, 2013;Russo et al, 2014). Attempts to increase the target numbers in qPCR assays led to development of protocols assessing higher number of pathogens simultaneously in a single reaction.…”

Targeted and Highly Multiplexed Detection of Microorganisms by Employing an Ensemble of Molecular Probes

“…(iv) In theory, real-time PCR (RT-PCR) is promising as a fast and inexpensive technology to diagnose infectious agents. However, successful applications of RT-PCR have concentrated on only three bacteria (36), five bacteria (37), or six pathogens (38) and, recently, a 10-species biofilm model (39). It seems unlikely that fluorescence resonance energy transfer coupled with the melting temperatures of the amplicons will succeed in highly multiplexed identification and detection assays.…”