A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision
Recombinant adeno-associated virus (rAAV) is without doubt one of the principal vectors utilized in gene remedy. An correct genome titer is just not solely crucial for medical dosing, but additionally a prerequisite for a lot of analytical assays for AAV product characterization. AAV genome titer is historically decided by qPCR; nonetheless, assay precision is just not optimum regardless of intensive efforts.
Extra not too long ago, droplet digital PCR (ddPCR) emerged as a strong different that gives glorious accuracy and precision. Nonetheless, presently ddPCR is just not as broadly accessible as qPCR and operates at a decrease throughput and the next value. On this paper, we introduce an improved qPCR methodology with two main optimizations: (1) utilizing an AAV reference materials as qPCR commonplace as an alternative of plasmid DNA and (2) implementing a “digestion-free” methodology by including 5% Tween 20 to plain and pattern preparations.
The brand new methodology has been extensively examined with AAV of various serotypes, purification standing, and transgenes encapsidated and was discovered to be extremely correct, exact, and strong. This considerably improved and simplified assay may be simply adopted by researchers within the gene remedy subject and additional automated for high-throughput purposes.
Figuring out septic air pollution publicity routes throughout a waterborne norovirus outbreak – A brand new utility for human-associated microbial supply monitoring qPCR
In June 2017, the Pennsylvania Division of Well being (PADOH) was notified of a number of norovirus outbreaks related to 179 in poor health people who attended separate occasions held at an outside venue and campground over a month interval. Epidemiologic investigations have been unable to determine a single publicity route and due to this fact unable to find out whether or not there was a persistent contamination supply to focus on for publicity mitigation. Norovirus was detected in a contemporary leisure water designated swimming space and a consuming water nicely.
A hydrogeological web site analysis advised a close-by septic leach subject as a possible contamination supply by way of floor water infiltration. Geological characterization revealed a steep dip of the bedrock beneath the septic leach subject towards the nicely, offering a viral transport pathway in a geologic medium not beforehand documented as excessive danger for viral floor water contamination.
The human-associated microbial supply monitoring (MST) genetic marker, HF183, was used as a microbial tracer to reveal the hydrogeological connection between the malfunctioning septic system, consuming water nicely, and leisure water space. Based mostly on environmental investigation findings, venue administration and native public well being officers carried out a sequence of outbreak prevention methods together with discontinuing using the contaminated nicely, issuing a allow for a brand new consuming water nicely, growing transportable rest room and handwashing station availability, and selling correct hand hygiene.
Regardless of the outbreaks on the venue and proof of floor water contamination impacting close by leisure water and the consuming water nicely, no new norovirus circumstances have been reported throughout a big occasion one week after implementing prevention practices. This investigation highlights a brand new utility for human-associated MST strategies to hint hydrological connections between a number of fecal pollutant publicity routes in an outbreak state of affairs. In flip, pollutant supply data can be utilized to develop efficient intervention practices to mitigate publicity and stop future outbreaks related to human fecal contaminated waters.
3D encapsulation and inflammatory licensing of mesenchymal stromal cells alter the expression of frequent reference genes utilized in real-time RT-qPCR
