Semi-nested Method Research Articles

Validation of a new seminested PCR-based detection method for Legionella pneumophila

We report a new seminested PCR method for detection of Legionella pneumophila based on the simultaneous amplification of a 387 bp fragment of the dotA gene and a 827 bp recombinant fragment that serves as an internal positive control. This new detection system was validated and its specificity and detection limit were determined. Parallel analysis of 90 environmental samples to compare this method, a real-time PCR method and the standard culture isolation method, demonstrated that this seminested method is useful for the study of L. pneumophila.

A single primer pair immunoglobulin polymerase chain reaction assay as a useful tool in fine-needle aspiration biopsy differential diagnosis of lymphoid malignancies.

Different polymerase chain reaction (PCR) assays have proved to be useful tools for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in frozen or paraffin-embedded tissues. However, the usefulness of a single primer pair PCR assay on fine-needle biopsy aspirates has not been proven yet. The authors conducted a wide prospective study of 148 lymph nodes and extranodal lymphoid infiltrates obtained by fine-needle aspiration biopsy (FNAB). The power of a single primer pair PCR amplification of the hypervariable CDRIII region IgH genes was evaluated using a pair of consensus primers. The PCR cytologic results were compared with the final clinicopathologic diagnosis of each case assessed by combining cytologic and/or immunophenotypic data and histologic features or clinical follow-up. Among the 139 cases with an evaluable PCR result, 35 of 40 (87%) B-cell non-Hodgkin lymphomas were detected as a monoclonal band. Monoclonal IgH bands also were detected in two of the five (40%) T-cell lymphomas, two of the seven (29%) Hodgkin lymphomas, and 5 of the 87 (6%) reactive lymphoid disorders. These results are similar to those obtained by other authors using seminested PCR or combining different PCR tests in each sample obtained by FNAB or excisional biopsy. The results of the current study demonstrate the convenience of a single primer pair PCR amplification over seminested methods in terms of lower cost and workload. The existence of PCR false-negative and false-positive results for lymphoma makes it necessary to combine the information obtained by PCR with cytologic and/or immunophenotypic data to optimize the number of lymphoid malignancies correctly diagnosed by FNAB.