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Primers frequently used for detecting the t(14;18) major breakpoint also amplify Epstein-Barr viral DNA
G H Segal1, M Scott, T Jorgensen
1Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville.
Abstract:
We observed a potentially misinterpretable polymerase chain reaction (PCR) amplification product generated with standard primers used to detect the major breakpoint region (mbr) of chromosomal translocation t(14;18). This unexpected phenomenon was initially detected during attempts to transform follicular lymphomas in vitro with Epstein-Barr virus (EBV). Additional studies were performed using the EBV-producing cell line MCUV5, cell lines from EBV-transformed normal B-lymphocytes, and an excised lymph node from a patient with documented EBV-associated infectious mononucleosis. These samples consistently produced a 167-base pair product, which was indistinguishable from a t(14;18) lymphoma product when viewed on ethidium bromide-stained gels. Through DNA sequencing and gene bank analysis, the product was identified as a portion of the EBV genome. A mbr-specific 20-base oligonucleotide probe was able to discriminate between true translocations and the EBV-related amplifications. These results underscore the importance of employing a specific detection system, and comprehensively screening primers when working with PCR.
Insights
Standard polymerase chain reaction (PCR) primers detecting t(14;18) translocations can yield false positives due to Epstein-Barr virus (EBV) DNA. Specific probes are crucial for accurate detection in lymphoma diagnostics.
Area of Science:
- Molecular biology
- Oncology
- Virology
Background:
- The t(14;18) chromosomal translocation is a hallmark of follicular lymphoma.
- Polymerase chain reaction (PCR) is commonly used to detect this translocation.
- Epstein-Barr virus (EBV) is associated with certain B-cell lymphoproliferative disorders.
Purpose of the Study:
- To investigate an unexpected PCR amplification product observed during EBV-related studies.
- To determine the origin of a non-specific PCR product mimicking the t(14;18) translocation.
- To assess methods for distinguishing true t(14;18) translocations from artifactual amplifications.
Main Methods:
- Utilized PCR with standard primers for the major breakpoint region (mbr) of t(14;18).
- Analyzed samples including EBV-producing cell lines and EBV-infected B-lymphocytes.
- Performed DNA sequencing and gene bank analysis to identify the PCR product.
- Employed a specific mbr-oligonucleotide probe for differential detection.
Main Results:
- A 167-base pair PCR product was consistently generated from EBV-infected samples.
- This product was indistinguishable from a true t(14;18) translocation product on ethidium bromide-stained gels.
- DNA sequencing identified the artifactual product as originating from the EBV genome.
- A specific mbr probe successfully differentiated EBV-derived products from actual translocations.
Conclusions:
- Standard PCR primers for t(14;18) can produce false-positive results due to EBV coinfection.
- Comprehensive primer screening and specific detection probes are essential for accurate molecular diagnosis.
- This finding highlights the need for careful interpretation of PCR results in the context of viral associations.