Papers by Keyword: Multiplex PCR

Abstract: nfectious microbial pathogens constitute the largest cause of morbidity and mortality worldwide. Early diagnosis and rapid infection control measures can lead to improved outcomes, earlier discharges and reduced nosocomial infections. Conventional diagnostic methods for infectious diseases such as microscopy, culture, and immunological methods, in most cases, are not universally applicable, less sensitive and could take from days to months to complete depending on the pathogen. Molecular assays based on nucleic acids such as polymerase chain reaction (PCR) have improved the sensitivity, specificity and turn-around time in diagnostic microbiology laboratories. These tests are particularly important to detect very low levels of pathogens in clinical samples, and for organisms that have long half-lives or are non-culturable. However, individual molecular tests are available for only a limited number of the more common infectious agents. Moreover, infectious disease events arising from novel pathogens or genetic variants have significantly increased, recently, for which, routine diagnostic methods are not yet available. Therefore, molecular methods and technologies capable of detecting multiple pathogens in a single test have become available over the last few years. Although, these methods are based on the conventional nucleic acid amplification and hybridization chemistry, enhanced multiplexing capability has been achieved through innovations in nucleic acid labeling techniques, and post-amplification analytic methods and instrumentation. The availability of these test kits brought a new level of convenience to the physicians ordering practices, and to the laboratory personnel, as they require very little hands on time. However, these tests are yet unaffordable to many laboratories, and in many cases, the sensitivity is poor compared to that of single-target, real-time PCR assays. Looking into the future, the revolutionary, next generation sequencing (NGS) technology is now being considered as a potential method for rapid identification of hundreds of pathogens, in an unbiased manner, with a single test that could significantly benefit patients who are critically ill with undiagnosed disease.

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) transmission between pigs and humans can be a dangerous infection source for the community. To control and prevent such disease, MRSA research in healthy pigs has been conducted in the US and Holland. This paper reports the first such study in Vietnam. This study aims to optimize the method for investigating MRSA carriage in tonsil and nasal swabs of healthy pigs. Potential samples were selected rapidly by Multiplex PCR (M-PCR) assay with 3 primers (Staph 756, mecA, Sa442) and then combined with conventional methods to detect and pick up MRSA strains. These strains were grouped based on genome pattern by pulsed field gel electrophoresis (PFGE). This study showed that we successfully isolated MRSA and the rate of MRSA carriage samples was 3.63%, much lower than that reported in the USA (49%) and Holland (39%), however the USA and Holland studies investigated pigs from industrial scale piggeries, whereas our study investigated pigs from small farms, which is the usual approach to pig farming in Vietnam. The MRSA patterns identified and associated MRSA strains were different from identified MRSA strains in other countries. This research provides important information about the carriage rate of MRSA in pigs in the Western province of Vietnam and demonstrates the importance of ongoing investigations in this area.

Abstract: To establish a rapid, sensitive and specific multiplex PCR method for the simultaneous detection of Staphylococcus aureus, Salmonella spp, and listeria monocytogenes. Three pairs of primers have been designed according to the Staphylococcus aureus nuc gene, Salmonella spp IpaB gene, listeria monocytogenes inlA gene. Orthogonal experimental design was used to determine Multiplex PCR amplification system for Food-borne Bacterial Pathogens of four factors (Taq DNA polymerase, Mg²⁺, dNTP and primers) from four levels; three DNA fragments of 210bp,280bp and 476bp were amplified. The specificity and the sensitivity of this method was valued. Template was prepared using FTA filter; the three food-borne Bacterial Pathogens were simultaneously detected by the multiplex PCR technology which have been designed; The sensitivity of this method was 3.0×10²cfu/mL for Staphylococcus aureus, 2.0×10²cfu/mL for Salmonella spp, and 3.5×10²cfu/mL for listeria monocytogenes. This method lies on its accuracy, rapidity and efficiency in the diagnosis, so it could be a useful method for the simultaneous detection of the three species of bacteria in food.