Abstract
BackgroundEarly detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Surface plasmon resonance (SPR) biosensor is widely used to detect DNA-DNA interaction and offers a sensitive and label-free approach in DNA research.MethodsIn this study, we developed a single-stranded DNA (ssDNA) amplification technique and modified the traditional SPR detection system for rapid and simultaneous detection of mixed infections of four pathogenic microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium tetani and Clostridium perfringens).ResultsWe constructed the circulation detection well to increase the sensitivity and the tandem probe arrays to reduce the non-specific hybridization. The use of 16S rDNA universal primers ensured the amplification of four target nucleic acid sequences simultaneously, and further electrophoresis and sequencing confirmed the high efficiency of this amplification method. No significant signals were detected during the single-base mismatch or non-specific probe hybridization (P < 0.05). The calibration curves of amplification products of four bacteria had good linearity from 0.1 nM to 100 nM, with all R2 values of >0.99. The lowest detection limits were 0.03 nM for P. aeruginosa, 0.02 nM for S. aureus, 0.01 nM for C. tetani and 0.02 nM for C. perfringens. The SPR biosensor had the same detection rate as the traditional culture method (P < 0.05). In addition, the quantification of PCR products can be completed within 15 min, and excellent regeneration greatly reduces the cost for detection.ConclusionsOur method can rapidly and accurately identify the mixed aerobic-anaerobic infection, providing a reliable alternative to bacterial culture for rapid bacteria detection.
Highlights
Detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments
Sequencing confirmed that the four specific sequences after polymerase chain reaction (PCR) amplification were the expected sequences of S. aureus, P. aeruginosa, C. tetani and C. perfringens
Molecular biological methods such as Surface plasmon resonance (SPR) biosensing can detect the specific nucleic acid of bacterial genomes and avoid the difficulties associated with phenotypic changes
Summary
Detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Anaerobic bacterial infection is one of the major causes of death due to the difficulty to identify the bacteria [1,2]. And accurate identification of the pathogenic microorganisms in a co-infection is critical. The identification of pathogenic microorganisms mainly depends on a combination of bacterial culture, morphology, biochemical presentations, and immunological examination. The growth of anaerobic bacteria always requires rigorous culture conditions, and their phenotypic characteristics (e.g., antibiotic sensitivity and biochemical characteristics) are usually unstable and liable to be affected by gene regulation and plasmid loss [4]. Fluorescent quantitative PCR cannot simultaneously discriminate bacteria in mixed infections, despite its potential for relatively accurate quantification. Discrimination among amplification products with similar lengths using electrophoresis is difficult [7]
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