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qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue
Published on: July 9, 2014
Real-time polymerase chain reaction detection of Neisseria meningitidis in formalin-fixed tissues from sudden deaths
Amparo Fernández-Rodríguez1, Belén Alcalá, Roberto Alvarez-Lafuente
1Microbiology Laboratory, Instituto Nacional de Toxicología y Ciencias Forenses, c) Luis Cabrera no 9, 28002 Madrid, Spain. a.fernandez@mju.es
Abstract:
Accurate identification of meningococcal sudden deaths is needed to avoid underestimation of the true incidence of the disease. This study analyzed the usefulness of a real-time polymerase chain reaction (PCR) protocol using MGB (3'-minor groove binder) probes to detect Neisseria meningitidis in formalin-fixed paraffin-embedded tissues from sudden deaths where a meningococcal fulminating infection was suspected. The protocol included detection of meningococcal DNA (ctrA gene), multiplex B/C PCR serogrouping (siaD gene), and rapid confirmation of PCR products by microcapillary electrophoresis. Sixty-nine tissues from 15 culture-confirmed meningococcal sudden deaths were analyzed (positive cases). Validation studies were performed. In each positive case, both the ctrA and the B/C siaD genes were detected. The ctrA was detected in 81.2% of the samples, whereas the serogroup (B or C) was identified in 44.9% of them. Therefore, this protocol may improve nonculture diagnosis and case ascertainment in meningococcal disease deaths, particularly when formalin-fixed tissues are the only available specimen.
Insights
This study shows a real-time polymerase chain reaction (PCR) protocol effectively detects Neisseria meningitidis DNA in preserved tissues. This method aids in accurately identifying meningococcal disease deaths, especially when traditional cultures fail.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Forensic Pathology
Background:
- Accurate identification of meningococcal sudden deaths is crucial for understanding disease incidence.
- Traditional culture methods may fail in diagnosing fatal Neisseria meningitidis infections from preserved tissues.
Purpose of the Study:
- To evaluate a real-time PCR protocol for detecting Neisseria meningitidis in formalin-fixed paraffin-embedded tissues.
- To improve non-culture diagnosis and case ascertainment in suspected meningococcal disease deaths.
Main Methods:
- Developed and validated a real-time PCR protocol using MGB probes for Neisseria meningitidis detection.
- Included detection of meningococcal DNA (ctrA gene) and multiplex B/C PCR serogrouping (siaD gene).
- Utilized microcapillary electrophoresis for rapid confirmation of PCR products.
Main Results:
- The ctrA gene was detected in 81.2% of positive cases.
- Serogroup B or C was identified in 44.9% of positive cases.
- The protocol successfully detected meningococcal DNA in culture-confirmed fatal cases.
Conclusions:
- The real-time PCR protocol is a valuable tool for diagnosing meningococcal disease in formalin-fixed tissues.
- This method enhances case ascertainment for sudden deaths suspected to be caused by Neisseria meningitidis.
- The protocol offers a reliable alternative when culture-based methods are not feasible.
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