Related Experiment Video
Updated: Oct 8, 2025
Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
Published on: October 23, 2011
Optimization and validation of two multiplex qPCR assays for the rapid detection of microorganisms commonly invading
Andrew D Winters1, Roberto Romero2, Emma Graffice3
1Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, MD, and, MI, Detroit, MI, USA; Perinatal Research Initiative in Maternal, Perinatal and Child Health, Wayne State University School of Medicine, Detroit, MI, USA; Department of Biochemistry, Microbiology, and Immunology, Wayne State University School of Medicine, Detroit, MI, USA.
Abstract:
Microbial invasion of the amniotic cavity (MIAC) leading to infection is strongly associated with adverse pregnancy and neonatal outcomes. Limitations of current diagnostic assays to detect MIAC rapidly and accurately have hindered the ability of obstetricians to identify and treat intra-amniotic infections. We developed, optimized, and validated two multiplex quantitative polymerase chain reaction (qPCR) assays for the simultaneous detection and quantification of microbial taxa commonly associated with MIAC. The first assay allows for the quantification of general bacterial and fungal loads in amniotic fluid and includes a human reference gene to allow for assessing the integrity of clinical samples and the DNA extraction process. The second assay allows for the detection and quantification of four specific bacterial taxa commonly associated with MIAC: Ureaplasma spp., Mycoplasma hominis, Streptococcus agalactiae, and Fusobacterium nucleatum. The qPCR assays were validated by using both microbial isolates and clinical amniotic fluid samples. The assays were further validated by comparing qPCR amplification results to those from the microbial culture and bacterial 16S rRNA gene sequencing of amniotic fluid. Both assays demonstrated high reproducibility and are sensitive and specific to their intended targets. Therefore, these assays represent promising molecular diagnostic tools for the detection of MIAC. Most importantly, these assays may allow for administration of timely and targeted antibiotic interventions to reduce adverse perinatal outcomes attributed to intra-amniotic infections.
Insights
New quantitative polymerase chain reaction (qPCR) assays accurately detect microbial invasion of the amniotic cavity (MIAC). These rapid diagnostic tools aid in timely treatment, potentially improving pregnancy and neonatal outcomes.
Area of Science:
- Obstetrics and Gynecology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Microbial invasion of the amniotic cavity (MIAC) is linked to poor pregnancy and neonatal outcomes.
- Current diagnostic methods for MIAC lack speed and accuracy, delaying treatment.
- Effective diagnostics are crucial for managing intra-amniotic infections.
Purpose of the Study:
- To develop and validate multiplex quantitative polymerase chain reaction (qPCR) assays for detecting microbial invasion of the amniotic cavity (MIAC).
- To enable simultaneous quantification of general bacterial/fungal loads and specific bacterial taxa associated with MIAC.
- To provide rapid and accurate diagnostic tools for intra-amniotic infections.
Main Methods:
- Developed two multiplex qPCR assays: one for general microbial loads and a human reference gene, and another for specific bacteria (Ureaplasma spp., Mycoplasma hominis, Streptococcus agalactiae, Fusobacterium nucleatum).
- Validated assays using microbial isolates and clinical amniotic fluid samples.
- Compared qPCR results with microbial culture and 16S rRNA gene sequencing.
Main Results:
- Both qPCR assays demonstrated high reproducibility, sensitivity, and specificity.
- Assays successfully quantified general microbial loads and detected specific bacterial pathogens.
- Validation confirmed the accuracy of qPCR compared to traditional methods.
Conclusions:
- The developed multiplex qPCR assays are promising tools for rapid and accurate MIAC detection.
- These assays can facilitate timely and targeted antibiotic interventions.
- Improved diagnostics for MIAC may lead to reduced adverse perinatal outcomes.
