Acoustically driven faecal DNA extraction and qPCR

Larson, Eloise (2018) Acoustically driven faecal DNA extraction and qPCR. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.

Abstract

Johne’s disease, caused by Mycobacteria avium subsp. paratuberculosis (MAP), plagues cattle and dairy farmers worldwide. Infected animals suffer from chronic granulomatous enteritis, reduced fertility, decline in milk production and emaciation. It is spread through colostrum, milk and faeces. Johne’s disease has also become strongly associated with human Crohn’s disease due to the similarity in symptoms and the presence of MAP in samples taken from Crohn’s disease patients. Currently there is not cure for Johne’s disease, therefore, routine testing and isolation or culling of diseased animals are measures used to prevent infection.
At present, the gold standard test for MAP is by faecal culture, which can take up to 4 months to reach a diagnosis. The low-cost ELISA test has a shorter diagnosis duration, however, it uses blood or milk samples for testing which do not correlate with the bacterial shedding in faecal samples. As a result of this mismatch in bacterial shedding between blood, milk and faeces, ELISA testing lacks in both sensitivity and specificity to MAP when compared to faecal culture and PCR tests. PCR can be sensitive and specific for MAP testing, although it is currently the most expensive test for MAP detection within the UK. Reducing the cost of PCR testing was one of the motivating factors during the development of this assay and device.
In this thesis, surface acoustic waves (SAW) have been used to create, develop and test a diagnostic device and the assay for Johne’s disease. SAW is becoming an increasingly popular tool in the field of diagnostic devices due to its multi-functionality which allows for many pieces of laboratory equipment to be consolidated into one device. In using SAW there was no longer a need for laboratory equipment usually used to perform a DNA extraction because SAW could be used to mix, heat and move the sample droplet. Traditional, laboratory-based faecal DNA extraction was adapted for use with SAW. To date, faecal DNA extraction using SAW has not been published. The SAW-driven, droplet-based assay developed during this project used 90% less DNA extraction reagents than the traditional tube-style method. Due to the thermal resistant nature of MAP it is particularly difficult to lyse during DNA extraction. The newly created SAW faecal DNA extraction was used to retrieve and clean DNA sufficiently for successful PCR to follow. In the context of faecal samples, this was particularly challenging due to the PCR inhibitors found in these complex samples. To investigate the effectiveness of the SAW DNA extraction, K-10 strain of genomic MAP DNA, MAP cell cultures and pre-tested bovine faecal samples were tested to prepare the MAP DNA for amplification and detection. DNA extraction was followed by SAW PCR. The sensitivity and specificity of the SAW PCR was ensured by using both IS900 and F57 target sequences. IS900 is repeated 17 times in the MAP genome thereby providing sensitivity down to 102 CFU/g. F57 is only repeated once therefore providing specificity. Specificity of the assay was further improved by using TaqMan probes to quantify the PCR. In order to keep this PCR-based assay stable in the absence of cold-chain storage, the disaccharide sugar trehalose was added to the PCR reagents and freeze dried to determine its ability to maintain performance. These experiments enhanced the future portability of this assay. It was found that reagents maintained activity for at least 41 days after freeze drying and this duration is expected to be extended.
During this thesis, a newly developed acoustically driven DNA extraction and qPCR was integrated onto one device which had the capability of detecting as few as 5 MAP genomes. This novel proof-of-concept research, lays the foundation for an acoustically driven portable device for use on faecal samples and in this case, for the detection of Johne’s disease.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Surface acoustic wave device, MAP, Johne's disease, faecal, qPCR, DNA extraction.
Subjects: Q Science > QR Microbiology
S Agriculture > SF Animal culture > SF600 Veterinary Medicine
T Technology > T Technology (General)
Colleges/Schools: College of Science and Engineering > School of Engineering > Biomedical Engineering
Supervisor's Name: Cooper, Professor Jonathan
Date of Award: 2018
Embargo Date: 11 June 2020
Depositing User: Mrs Eloise Larson
Unique ID: glathesis:2018-30625
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 12 Jun 2018 14:43
Last Modified: 25 Jul 2018 08:36
URI: http://theses.gla.ac.uk/id/eprint/30625

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