BMC Microbiology BioMed Central Methodology article Large scale multiplex PCR improves pathogen detection by DNA (original) (raw)
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Journal of Clinical Microbiology, 2002
We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus . This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.
Development of Multiplex PCR Based Assay for the Concurrent Detection of Pathogenic Microorganisms
2015
Objective: Salmonella enterica is an important enteric pathogen which causes gastroenteritis and enteric fever in humans and is widely spread in nature. Outbreaks of Salmonella infections are frequently reported in both developed and developing countries, as this pathogen spreads very rapidly by means of water and the food chain. Methods: A multiplex PCR (mPCR) assay was developed for the detection of multiple Salmonella serotypes in different kind of food products. To increase specificity of this molecular method, three sets of oligonucleotide primer were used to detect the most prevalent salmonella species. Different primer pairs were used for the optimization of the multiplex PCR. The primer pair, ST FW and ST RV, was specific to Salmonella typhi and targeted a randomly selected sequence of 600bp. The primer pair SPFW and SPRV specific to Salmonella paratyphi for a sequence of 800 bp in length. Likewise, the primers pairs SEFW and SERV were designed to amplify a sequence of about...
2014
Salmonella enterica is an important enteric pathogen which causes gastroenteritis and enteric fever in humans and is widely spread in nature. Outbreaks of Salmonella infections are frequently reported in both developed and developing countries, as this pathogen spreads very rapidly by means of water and the food chain. A multiplex PCR (mPCR) assay was developed for the detection of multiple Salmonella serotypes in different kind of food products. To increase specificity of this molecular method, three sets of oligonucleotide primer were used to detect the most prevalent salmonella species. Different primer pairs were used for the optimization of the multiplex PCR. The primer pair, ST FW and ST RV, was specific to Salmonella typhi and targeted a randomly selected sequence of 600bp. The primer pair SPFW and SPRV specific to Salmonella paratyphi for a sequence of 800bp in length. Likewise, the primers pairs SEFW and SERV were designed to amplify a sequence of about 1000bp from S. enter...
Large scale multiplex PCR improves pathogen detection by DNA microarrays
BMC Microbiology, 2009
Background Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. Results To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the...
Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens
2012
sufficient in specifically and simultaneously detecting as few as 10 CFU/ mL of the five pathogens in artificially inoculated food samples after enrichment for 12 h. Finally, each 25-g sample was mixed with 225mL of SEB medium and incubated at 37 o C for 12-h. Then, each1mL of the culture broth was subjected to the multiplex PCR assay as the schematic representation of detection procedure is presented in Figure 3. The assay is reliable, rapid, specific, and robust. Therefore, it can be another tool for the investigation of microbial contamination in raw food and food products, and will also be useful for identifying the sources of food borne out break
Novel Multiplex PCR to Specifically Detect Bacterial Foodborne Pathogens
2013
Bacterial foodborne pathogens prevalent in poultry, especially Escherichia coli, Salmonella spp., Shigella spp., and Listeria monocytogenes, have been reported in many countries including Thailand. Rapid methods for identification and detection of these dominant foodborne pathogens are still required. In our study, multiplex polymerase chain reaction (m-PCR) was developed for detecting multiple bacterial foodborne pathogens. Specific genes for the m-PCR primers were screened and selected. m-PCR targeting the uspA, fimY, ipaH, and prfA gene was used to detect E. coli, Salmonella spp., Shigella spp., and L. monocytogenes, respectively. The optimum conditions for the m-PCR reaction were found to be primer concentrations of 0.02 μM ipaH, 0.036 μM fimY, 0.06 μM uspA,0.12 μM prfA, and 0.4 μM 16S rRNA gene (used as internal control) for at least 10 ng of each bacterial total genomic DNA; and 52 o C was the annealling temperature. The expected PCR products of 884, 489, 422, and 398 bp were ...
BMC Microbiology, 2013
Background: Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified. The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. Results: A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons. With the novel pathogen detection system, fungi, G + and G-bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube.
Gram Type-Specific Broad-Range PCR Amplification for Rapid Detection of 62 Pathogenic Bacteria
1999
Broad-range PCR has proven to be useful for the detection of bacteria. A set of broad-range PCR primers directed against conserved regions in the 16S rRNA gene was designed to specifically amplify either gram- positive or gram-negative bacteria. The gram type-specific broad-range PCR correctly classified all 62 patho- genic species tested. Antibiotic treatment of bacterial infections depends on the species
Journal of Infection, 2013
Objectives: To determine whether systematic testing of faecal samples with a broad range multiplex PCR increases the diagnostic yield in patients with diarrhoea compared with conventional methods and a clinician initiated testing strategy. Methods: 1758 faecal samples from 1516 patients with diarrhoea submitted to two diagnostic laboratories were tested for viral, bacterial, and parasitic pathogens by Fast-Track Diagnostics multiplex real-time PCR kits and conventional diagnostic tests. Results: Multiplex PCR detected pathogens in 530 samples (30%): adenovirus (51, 3%), astrovirus (95, 5%), norovirus (172, 10%), rotavirus (3, 0.2%), Campylobacter jejuni/coli (85, 5%), Salmonella spp. (22, 1%), Clostridium difficile (72, 4%), entero-haemorrhagic Escherichia coli (21, 1%), Cryptosporidium spp. (3, 0.2%), Entamoeba histolytica (1, 0.1%), and Giardia lamblia (59, 3%). In contrast, conventional testing detected a pathogen in 324 (18%) samples. Conclusions: Using a systematic approach to the diagnosis of gastroenteritis improved diagnostic yield. This enhanced detection with PCR was achieved by a combination of improved detection of individual pathogens and detection of pathogens not requested or unable to be tested by conventional tests. This approach also allowed earlier identification for most pathogens and created a workflow which is likely to adapt well for many diagnostic laboratories.