Development of conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens

Abstract

Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.

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“…In a bid to combat the laborious time that we spend on the post amplification using gel electrophoresis, to increase both sensitivity and specificity while paying more attention to the reliability of the test result, real time Polymerase Change Reaction (real time-PCR) was required [8,13,15]. Real time PCR is a very powerful and rapid nucleic acid amplifying devices that is also simple to use.…”

“…This simply means that both the amplification and analysis occur together. [13,14,15] In contrast to the conventional PCR, either Deoxyribonucleic acid (DNA) dyes or the fluorescent probes are among the reagents mixture added before running the machine. Since there is no need to transfer samples from a thermocycler to gel electrophoresis reading, (Samples are automatically analyzed and the results are shown on the computer screen).…”

“…Sensitivity is the ability to detect the smallest of amount of DNA, cDNA or RNA for a reaction and serial dilution (e.g. 2 X 102 to 2 X 1010) is used to determine the sensitivity of PCR assay [13]. qPCR has achieved a milestone in the field of water monitoring.…”

“…Among these NATs, PCR still stood distinct. On top of its ability in rapidly detecting pathogenic DNA, PCR tests are also highly sensitive and specific [8,11,12,13].…”

Etiological profile of early neonatal bacterial sepsis by multiplex qPCR

“…The qPCR amplification reactions followed the protocol described by Anbazhagan et al [14] with some modifications. In a final total of 25 µL reaction, 3 pmol of each primer, 10 ng genomic DNA from clinical specimens, 12.5 µL SYBR Green PCR Master Mix (Invitrogen Life Technologies, Carlsbad, USA), and deionized water were included.…”