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Multiple primer pairs for polymerase chain reaction (PCR) amplification of human parvovirus B19 DNA
E L Durigon1, D D Erdman, G W Gary
1Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA. 30333.
Abstract:
Human parvovirus B19 is the etiologic agent of erythema infectiosum and transient aplastic crisis in patients with hemolytic anemias and has been associated with fetal death, arthritis, and chronic anemia. Acute B19 infection is best diagnosed by detection of IgM antibodies, whereas the diagnosis of chronic infection often requires the sensitivity of PCR to demonstrate presence of virus over time. To improve our ability to detect B19 DNA by polymerase chain reaction (PCR), we evaluated 19 primers combined into 16 different primer pairs for their ability to detect temporally and geographically diverse B19 isolates. All 16 pairs reacted with all isolates tested but with different sensitivity. Sequence analysis showed few nucleotide changes compared with published sequences. These changes did not explain observed differences in sensitivity between primer pairs. The most sensitive primer pairs detected 350 to 3500 DNA copies after 35 cycles. A second amplification cycle with nested primers improved the sensitivity 100-fold. These 16 primer pairs provide the diagnostic virologist with multiple options for B19 PCR assays.
Insights
Human parvovirus B19 (B19) diagnosis can be improved using polymerase chain reaction (PCR). This study evaluated 16 primer pairs, finding nested PCR significantly enhances B19 DNA detection sensitivity.
Area of Science:
- Virology
- Molecular Biology
- Infectious Diseases
Background:
- Human parvovirus B19 (B19) is linked to erythema infectiosum, aplastic crisis, fetal death, arthritis, and chronic anemia.
- Diagnosing acute B19 infection relies on IgM antibodies, while chronic infection diagnosis often requires sensitive polymerase chain reaction (PCR) for viral DNA detection over time.
Purpose of the Study:
- To enhance the detection sensitivity of B19 DNA using polymerase chain reaction (PCR).
- To evaluate the effectiveness of 16 different primer pairs in detecting diverse B19 isolates.
Main Methods:
- Evaluated 19 primers, combined into 16 primer pairs, for B19 DNA detection via PCR.
- Tested primer pair reactivity and sensitivity against temporally and geographically diverse B19 isolates.
- Performed sequence analysis to identify nucleotide changes and assessed PCR sensitivity in terms of DNA copies detected.
- Investigated the impact of a nested PCR approach on detection sensitivity.
Main Results:
- All 16 primer pairs reacted with tested B19 isolates, but exhibited varying sensitivity levels.
- Sequence analysis revealed minimal nucleotide changes, insufficient to explain sensitivity differences.
- The most sensitive primer pairs detected 350 to 3500 DNA copies after 35 cycles.
- Employing a nested PCR amplification cycle improved sensitivity by 100-fold.
Conclusions:
- The evaluated primer pairs offer diagnostic virologists multiple options for developing B19 PCR assays.
- Nested PCR significantly enhances the sensitivity for detecting B19 DNA, crucial for diagnosing chronic infections.