微生物学

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Isolation of genomic DNA from Mycobacterium species has been a tedious procedure. This can primarily be attributed to thick and waxy cell wall of mycobacteria which hampers lysis of the bacterial cell. We have tested various approaches to isolate mycobacterial DNA and based on this, an optimized protocol is presented here. This protocol involves initial incubation of mycobacteria with lysozyme, followed by SDS-proteinase K treatment to bring about cell disruption. In the case of slowly growing mycobacteria such as BCG (Bacillus Calmette–Guérin) or Mycobacterium tuberculosis (M. tuberculosis), an intermediate step of cell lysis by physical method results in significantly enhanced yield.

Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR).

For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol “Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma” (Vargas-Meneses et al., 2015).