Abstract
Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.
Highlights
S. aureus is one of the most adaptable human pathogens causing a wide variety of diseases in humans ranging from localized skin and soft-tissue infections to more serious disease due to disseminated infection including septicemia, pneumonia, endocraditis, deep-seated tissue abscesses, osteomyelitis and meningitis [1]
Real-time PCR assays based on platforms such as the ABI 7500 sequence detection system, more commonly referred to as the Taqman, have steadily become more established for the detection and identification of a variety of organisms including clinically important bacteria such as C. jejuni [4] [5], Listeria monocytogenes [6] and Salmonella species [7]
A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established
Summary
S. aureus is one of the most adaptable human pathogens causing a wide variety of diseases in humans ranging from localized skin and soft-tissue infections to more serious disease due to disseminated infection including septicemia, pneumonia, endocraditis, deep-seated tissue abscesses, osteomyelitis and meningitis [1]. Real-time PCR assays based on platforms such as the ABI 7500 sequence detection system, more commonly referred to as the Taqman, have steadily become more established for the detection and identification of a variety of organisms including clinically important bacteria such as C. jejuni [4] [5], Listeria monocytogenes [6] and Salmonella species [7]. The probe is designed to hybridise an internal region of the PCR product and is labelled at the 5’ end with a fluorescent reporter dye and at the 3’ end with a quencher dye. During PCR amplification the hybridised probe is hydrolysed by the 5’ nuclease activity of Taq polymerase, separating the reporter dye from the quencher. An increase in reporter dye fluorescence is a direct result of target amplification. Repeated PCR cycles result in an increase in fluorescence corresponding to amplification of the target PCR product, which is detected and recorded by the system
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