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Title: Improving Detection of Adenovirus Infections Following Hematopoietic Stem Cell Transplants
Authors: Da Silva, Bernardo
Advisors: Flint, Jane
Department: Molecular Biology
Class Year: 2017
Abstract: Human adenoviruses (HAdVs) pose a significant health burden for immunocompromised individuals following hematopoietic stem cell transplantation (HSCT). Accurate monitoring of HAdV loads is critical for identifying patients at risk for disseminated infection. Quantitative PCR (qPCR) is the method of choice for HAdV load-quantification, and existing protocols predominantly rely on primers that target the penton, hexon, and or/fiber HAdV genes. Arguably the most commonly used primers in the clinical field are those originally proposed by Heim et al., which target the hexon gene. To date, 71 types of HAdVs have been identified, yet no assay has been validated for the quantitative amplification of all HAdVs. Here, we investigate the potential for a one-reaction qPCR assay requiring only one pair of consensus primers to quantify all HAdVs reliably. Previous studies have identified the E1A and DNA polymerase (POL) genes as promising candidates. Sequence analyses of the E1A, POL, penton, hexon, and fiber genes of all 71 HAdVs revealed that the POL and hexon genes are the most suitable targets for pan-HAdV amplification. We developed two consensus primer pairs, one targeting the POL gene and the other the hexon gene, to detect all HAdVs. The ability of these primers to quantify different HAdVs was evaluated using qPCR with five HAdV serotypes. The primers developed by Heim et al. were also included in these tests assays. All three primer sets failed to accurately amplify the HAdVs tested with similar sensitivity and are therefore not suitable for pan-HAdV amplification. We speculate that that the limited yet considerable degree of heterogeneity across the POL and hexon genes of all HAdV species prevents the design of a one-reaction, one-primer set assay for pan-HAdV quantification. Future studies should consider species-specific primers and reactions to establish sensitive and reliable protocols for quantitative detection of HAdV infections following HSCT.
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Molecular Biology, 1954-2019

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