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Enligt "Leaky gut”-hypotesen är ökad translokation av gramnegativa bakterier genom tarmslemhinnan förknippad med neuroimmuna störningar. Denna ökning av permeabiliteten i tarmslemhinnan kan orsakas av störning i tarmfloran efter antibiotikabruk eller sjukdom, vilket kan leda till inflammatoriska processer. Inflammation har sedan tidigare blivit förknippad med allvarlig depressiv störning och självmordsbeteende. Studiens syfte var att utveckla och validera en qPCR-baserad metod för att kunna detektera DNA från tarmbakterier i blod/plasma, som ett tecken på translokering av bakterier. Två primerpar för amplifiering av 16S rDNA utreddes genom observation av PCR-reaktioner med humant och bakteriellt DNA. Det mest optimala primerparets PCR effektiviteten och linjäriteten testades. Metodens funktion kontrollerades sedan med helblod och plasma med tillsats av exogent DNA från E.coli. Den utvecklade qPCR metoden detekterar bakterie DNA i prov med 10 kopior/µl, vilket gör den tillräckligt känslig för detektion av tarmbakterier i blod. / According to the "Leaky gut" hypothesis, increased translocation of gram-negative bacteria through the intestinal mucosa is associated with neuroimmune disorders. The increase of permeability of the intestinal mucosa may be caused by disturbance of the intestinal flora after antibiotic use or disease, which can lead to inflammatory processes. Inflammation has previously been associated with major depressive disorder and suicidal behavior. The purpose of the study was to develop and validate a qPCR-based method for detecting DNA from intestinal bacteria in blod/plasma, as a sign of decreased mucosal integrity. Two different primer pairs, targeting 16S rDNA, were investigated by observing their PCR reactivity with human and bacterial DNA. PCR efficiency and linearity were tested on the most optimal primer pair. The function of the method was then verified with whole blood and plasma with the addition of exogenous DNA from E.coli. The developed qPCR method detects bacterial DNA in samples at 10 copies/µl, making it sufficiently sensitive for detection of intestinal bacterial DNA in blood.

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Molecular diagnostic assays have been developed and utilized to diagnose and to confirm the diagnoses of many plant-parasitic nematodes. We screened several gene sequences of soybean cyst nematode (SCN) [Heterodera glycines, Ichinohen] for their use as molecular markers. A methodology then was developed to use them to detect and quantify the number of H. glycines directly from Mississippi soil. A novel procedure utilizing multiple databases containing nematode DNA and EST sequences was developed to assist in the selection of SCN primers used in the PCR and qPCR assays. In vitro testing demonstrated that the DNA primers and probes developed from the novel procedure for the qPCR assays could accurately detect the presence of SCN. Subsequent testing resulted in a trend of increasing observed numbers of SCN contributing to increasing estimates by qPCR.

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Recent years have seen an explosion of interest in the use of freely available DNA present in aquatic systems, otherwise known as environmental DNA (eDNA), as a tool for monitoring aquatic organisms. However, much remains unknown about the behavior of eDNA over a range of environmental conditions. This is particularly true in high gradient headwater streams, which have received less attention than other types of water bodies. In the summer of 2011, a headwater stream system with well established species distributions was sampled using eDNA techniques. Though species were detected where known to be present, detections also occurred where traditional techniques failed to detect species. This suggests that a cautious approach to positive eDNA detections is advisable. In 2012 a second study was conducted to better understand the dynamics of eDNA concentration in lotic systems. Caged brook trout (Salvelinus fontinalis) were introduced into two otherwise fishless headwater streams, and eDNA samples were collected at evenly spaced intervals downstream of the cage. This was repeated 19 times from mid-summer through autumn, over flows ranging from approximately 1 to 96 l/sec. Quantitative PCR was used to relate DNA copy number to distance from source for each of these 19 sampling events. In all cases, DNA was detectable at 239.5 m from the cage. Increasing flows generally decreased eDNA copy number near the cage but had relatively little effect at downstream sites. Additionally, the presence of leaf biomass during the fall period had the potential to completely erase otherwise high DNA levels.

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