Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens

Jornal Brasileiro de Patologia e Medicina Laboratorial

Paulo Roberto Veiga Quemelo²  Benedito Antônio Lopes Da Fonseca¹  Danielle Malta Lima¹  Luiz Cesar Peres² 

[1] ,University of São Paulo Ribeirão Preto School of Medicine department of Pathology

关键词: Polymerase chain reaction;    Parvovirus B19;    Amplification;    Reação em cadeia da polimerase;    Parvovírus B19;    Amplicon;   

DOI  :  10.1590/S1676-24442009000200006

来源: SciELO

【 摘 要 】

The polymerase chain reaction (PCR) has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9%) were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.

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