Amplification of Herpes simplex type 1 and Human Herpes type 5 viral DNA from formalin-fixed Alzheimer brain tissue
John D Rodriguez1, Donald Royall, Luke T Daum
1Department of Biology, The University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249, USA.
Abstract:
It is known that nucleic acids from formalin-fixed tissues are not nearly as good templates for DNA amplification as those extracted from fresh tissues. However, specimens stored in most pathologic archives are initially fixed in formalin. The possibility of an infectious etiology of several diseases including Alzheimer's underscores the usefulness of archived tissue in assessing the association of infectious agents with specific pathology. In this report, we describe in detail a method resulting in robust amplification of HSV1 and Human Herpes type (HHV) 5 viral DNA targets using formalin-fixed Alzheimer brain frontal and temporal tissue as source of amplification template. Herpes simplex type 2 viral DNA was not detected in the limited samples examined in this study. Amplicons were verified by sequence analysis. Brain tissue stored in formalin longer than 1 year prior to post-formalin-fixation analysis gave rise to significantly shorter amplicons consistent with the observation that template DNA integrity decreases significantly with increasing time of storage in formalin. Thus, this report should be useful in PCR-based investigations assessing the regional presence of viral DNAs in formalin-fixed brain tissue.
Insights
This study details a method for robustly amplifying herpes simplex virus type 1 (HSV1) and human herpesvirus 5 (HHV-5) DNA from formalin-fixed Alzheimer
Area of Science:
- Neuropathology
- Virology
- Molecular Biology
Background:
- Nucleic acids from formalin-fixed tissues yield poor templates for DNA amplification compared to fresh tissues.
- Pathologic archives primarily store formalin-fixed specimens, limiting their use in infectious etiology studies.
- Investigating infectious agents in diseases like Alzheimer's disease requires methods for analyzing archived tissues.
Purpose of the Study:
- To describe a method for robust DNA amplification of viral targets from formalin-fixed Alzheimer brain tissue.
- To assess the feasibility of using archived formalin-fixed brain tissue for PCR-based viral detection.
- To evaluate the impact of formalin storage duration on DNA integrity and amplicon length.
Main Methods:
- Developed and detailed a protocol for DNA extraction and amplification from formalin-fixed Alzheimer brain tissue.
- Targeted amplification of Herpes Simplex Virus type 1 (HSV1) and Human Herpesvirus 5 (HHV-5) DNA.
- Verified amplified DNA sequences through sequence analysis.
Main Results:
- Achieved robust amplification of HSV1 and HHV-5 viral DNA targets from formalin-fixed Alzheimer brain tissue.
- Herpes Simplex Virus type 2 DNA was not detected in the limited samples analyzed.
- DNA integrity decreased with longer formalin storage, resulting in significantly shorter amplicons.
Conclusions:
- The described method enables robust PCR-based detection of specific viral DNA in formalin-fixed Alzheimer brain tissue.
- Longer formalin storage duration negatively impacts DNA integrity, affecting amplicon length.
- This technique is valuable for investigating the presence of viral DNA in archived neuropathologic specimens.
