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Updated: Aug 13, 2026
qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue
Published on: July 9, 2014
Detection of cytomegalovirus DNA in paraffin-embedded lung tissue specimens using in situ polymerase chain reaction
Objective:
To develop a possible alternative laboratory diagnostic method, an in situ polymerase chain reaction (ISPCR) for definitive diagnosis of human cytomegalovirus (HCMV) pneumonia in infants.
Methods:
The material involved in this study was formalin-fixed paraffin-embedded lung tissue specimens from 6 infants died of histopathologically diagnosed cytomegalic inclusion body pneumonia (CMIBP) and from 4 infants died of other diseases. When DNA extracts from the specimens of these 10 infants were tested by using a previously applied conventional PCR, the specimens from the 6 infants with CMIBP were positive for HCMV DNA, while those from the other 4 infants were negative. In the ISPCR, biotin-11-dUTP was used for labeling the amplified products and streptavidin, biotin-alkaline phosphatase for in situ color development.
Results:
The results of the study showed specific staining for HCMV DNA in the lung tissue slices from the 6 cases with CMIBP, while no staining was found in the specimens from the 4 infants who died of other diseases. The control ISPCR tests on the HCMV DNA positive specimens without adding either biotin-11-dUTP or Taq DNA polymerase or the primers for HCMV DNA showed negative results. The specific staining for HCMV DNA was seen in bronchial and alveolar epithelial cells, small vascular endothelial cells and leukocytes in the lumens of small blood vessels.
Conclusions:
The results suggested that the ISPCR was able to demonstrate both presence of HCMV DNA in the lung tissues and active HCMV infection; therefore the ISPCR may be applicable in definitive diagnosis of HCMV pneumonia when proper specimens such as lung biopsy material or bronchoalveolar lavage is available for the test.
Insights
In situ polymerase chain reaction (ISPCR) effectively diagnoses human cytomegalovirus (HCMV) pneumonia in infants. This method detects HCMV DNA in lung tissue, confirming active infection for definitive diagnosis.
Area of Science:
- Medical Diagnostics
- Virology
- Pathology
Background:
- Infant pneumonia poses diagnostic challenges.
- Human cytomegalovirus (HCMV) is a significant cause of pneumonia in infants.
- Accurate laboratory diagnosis is crucial for effective treatment.
Purpose of the Study:
- To establish an in situ polymerase chain reaction (ISPCR) as a reliable laboratory diagnostic method.
- To definitively diagnose human cytomegalovirus (HCMV) pneumonia in infants.
- To evaluate ISPCR's utility in detecting HCMV DNA within lung tissues.
Main Methods:
- Utilized formalin-fixed paraffin-embedded lung tissue from infants with and without cytomegalic inclusion body pneumonia (CMIBP).
- Employed ISPCR with biotin-11-dUTP labeling and streptavidin-biotin-alkaline phosphatase for color development.
- Compared ISPCR results with conventional PCR and controls lacking key reagents.
Main Results:
- Specific staining for HCMV DNA was observed in lung tissue from infants with CMIBP.
- No staining was detected in control infants or in negative control ISPCR tests.
- HCMV DNA staining localized to bronchial/alveolar epithelial cells, vascular endothelial cells, and leukocytes.
Conclusions:
- ISPCR successfully demonstrated the presence of HCMV DNA in infant lung tissues.
- The technique indicates active HCMV infection, supporting its use in definitive diagnosis.
- ISPCR is a potentially valuable tool for diagnosing HCMV pneumonia using lung biopsy or bronchoalveolar lavage specimens.
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