Optimized protocols for the detection of porcine circovirus 2 DNA from formalin-fixed paraffin-embedded tissues using nested polymerase chain reaction and comparison of nested PCR with in situ hybridization.
Optimized DNA extraction method and nested polymerase chain reaction (PCR) were developed for the detection of porcine circovirus 2 (PCV2) from formalin-fixed, paraffin-embedded tissues. Conventional PCR, nested PCR, and in situ hybridization methods were also compared for the detection of PCV2 in archival tissues. A method based on xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for PCR analyses reliably and consistently. Twenty-six (70%) of the 37 tissues examined gave positive results with conventional PCR, whereas all the 37 tissues gave positive results using the nested PCR. A distinct positive signal for PCV2 was detected in spleen and lymph node from all the 37 pigs by in situ hybridization. The nested PCR and in situ hybridization could be applied successfully to archival tissues for the detection of porcine circovirus 2 DNA.
Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Kyounggi-Do, South Korea.
J
J
Kim
C
C
Chae
D000818:Animals; D018173:Circoviridae Infections / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis*; D005557:Formaldehyde; D017403:In Situ Hybridization; D008198:Lymph Nodes / Q000821:virology; D016612:Paraffin Embedding / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013154:Spleen / Q000821:virology; D013552:Swine; D013553:Swine Diseases / Q000821:virology*; D016707:Tissue Fixation / Q000379:methods
D004279:DNA, Viral; D005557:Formaldehyde