Comparison of variability in ‘front end’ sampling and its effect on downstream qPCR & LAMP assay results

Abstract

AbstractMolecular assays and diagnostic tools have become a great necessity in the modern world. The recent COVID-19-19 pandemic highlighted this with molecular diagnostic testing done on a global scale, primarily through PCR and lateral flow testing strips. The use of these techniques was vital during this time and put emphasis on the need for sensitive, specific, and cost effective validated Point-of-case (POC) testing.The significance of the sampling step is key to any successful molecular test. The amount of material that is recovered from a sample controls the sensitivity and reliability of any downstream assay. This project has examined the influence of swab types and buffers on the yields of material for downstream assays, showing an almost 3-fold variation in the amount of sample collected between commonly used swab types. The yield of material extracted from swabs varied with both storage buffer and extraction time, with a chaotrope-based storage buffer showing a statistically significant higher yield when compared to conventional saline, with little effect of incubation times as short as 5 minutes using this buffer. This is important for POC or near-POC testing where rapid extraction is wanted. A primer set consisting of short and long target amplicons was verified to examine the stability of human DNA in a size range from a minimal amplicon size of around 70 bases to 400-500 bases, which covers the range that would be used for molecular testing both for pathogens and the STRs used in forensic human identification. These were used to examine the degradation of DNA samples, both in storage buffers and also in the effect of UV-light that is commonly used for DNA decontamination. This demonstrated that a chaotrope-based buffer can stabilise samples with a minimal reduction in assay sensitivity at up to 60 C for several days. This is significant in showing that cold storage is not needed for samples collected in this way, even in extreme environments. The results of the UV testing showed that UV exposure can be used to remove contamination in assay testing materials, but the exposure times needed led to a significant (several Ct) reduction in sensitivity. A real-world swab sampling experiment showed that skin swabs yielded significantly lower amounts (approx. 5 Ct, circa 100 fold) of human DNA than buccal and nasal swabs, which were similar. However, bacterial loads were around 10 fold (3 Ct) higher for buccal samples than nasal and skin, which were similar. Finally, a comparison was made between qPCR and LAMP testing for a potential LAMP-based diagnostic assay for MRSA to show the potential for downstream assays using both the "gold standard" of qPCR and a loop-mediated isothermal amplification (LAMP)-based assays that has potential use for rapid POC or near-POC testing without the need for expensive equipment. The LAMP method would allow diagnostic capabilities isothermally, so would not need the expensive lab equipment such as a thermal cycler that PCR requires. The LAMP assays designed could be used alongside a validated POC device to allow sensitive and specific POC testing on a global scale for a wide range of pathogenic targets that effect both human and non-humans, such as MRSA or avian flu. This project focusses on an N2 SARS-CoV-2 synthetic sequence as a comparable example, with a small number of preliminary results detailing work on an MRSA target.

Date of Award	9 Jan 2025
Original language	English
Awarding Institution	University of Bedfordshire
Supervisor	Robin Maytum (Supervisor) & Vicki Barlow (Second supervisor)