Browsing by Author "Kitchin, Natasha"

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    Genomic resource development for the South African scallop, Pecten sulcicostatus
    (Stellenbosch : Stellenbosch University, 2016-03) Kitchin, Natasha; Rhode, Clint; Roodt-Wilding, Rouvay; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Although South Africa boasts a rich biodiversity, many South African species, especially marine species, remain uncharacterised. Many of these species show great potential for commercial use, in particular for the aquaculture of endemic species for high-value, specialised markets. Only once these species have been identified and genetically characterised can the feasibility of aquaculture be evaluated. ‘Scallop’ refers to species of marine bivalve molluscs in the family Pectinidae, although it may also refer to species in other closely related families within the superfamily Pectinoidea. There are 29 scallop species in the waters surrounding South Africa, and of these, Pecten sulcicostatus has been identified as a candidate species for aquaculture. Non-destructive sampling is necessary for studies on genetic fitness and population structure to not be hindered by the death of the study individuals. DNA extraction methods using non-destructive sample tissue have not been developed for scallops, therefore this study compared the use of various tissue types in DNA extraction, allowing for the development of an effective non-destructive DNA extraction method for P. sulcicostatus using tentacle tissue and mucus swabs. This study also allowed for the development of an effective DNA extraction method for use on dried or degraded tissue, which will, in turn, allow for the use of opportunistic and historic samples in future studies on P. sulcicostatus. Despite the potential commercial value of P. sulcicostatus, no genetic resources are available for this endemic species. The development of genetic markers will assist in future studies on the genetic composition of this species as well as the genetic constitution of P. sulcicostatus populations along the South African coast – factors which are important for the formulation of effective genetic management strategies. This study therefore aimed to develop genetic markers for P. sulcicostatus and to conduct preliminary analyses using these markers to demonstrate their usefulness for future studies, which will assist in the establishment of a sustainable aquaculture industry. This study allowed for the optimisation of a fragment of the 16S rRNA gene which was successfully used to determine intra- and interspecific genetic diversity and shed light on the evolutionary relationship of five Pecten species. A set of 10 microsatellite markers was developed for P. sulcicostatus using cross-species amplification from Pecten maximus, a sister species to P. sulcicostatus, with a success rate of 50%. The set of microsatellite markers was successfully applied to generate genetic diversity data, which, in future studies, could be used to evaluate the extent of intra- and interpopulation genetic partitioning and variation. This study also provided the first reduced genome sequences for P. sulcicostatus, with over 7.3 million reads. The use of two bioinformatic approaches aided in the identification of 55 putative microsatellite markers as well as 2 530 putative SNPs. Although currently limited, this study marks the first step towards providing genetic information to assist in the development of genetic management strategies within the context of establishing a sustainable aquaculture industry for this endemic species. The genetic resources developed in this study could be used in various downstream applications such as genetic diversity assessment, population structure inference, linkage studies as well as marker assisted selection. In future, the microsatellite markers and SNPs developed in this study could also be continuously used to monitor genetic diversity as the species is subjected to aquaculture.
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    Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping
    (Nature, 2017) Venter, Rouxjeane; Derendinger, Brigitta; De Vos, Margaretha; Pillay, Samantha; Dolby, Tanya; Simpson, John; Kitchin, Natasha; Ruiters, Ashley; Van Helden, Paul D.; Warren, Robin M.; Theron, Grant
    Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >“low” Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.
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    Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate secondline genotypic drug susceptibility testing and spoligotyping
    (Springer Nature, 2017-11-01) Venter, Rouxjeane; Derendinger, Brigitta; De Vos, Margaretha; Pillay, Samantha; Dolby, Tanya; Simpson, John; Kitchin, Natasha; Ruiters, Ashley; Van Helden, Paul D.; Warren, Robin M.; Theron, Grant
    ENGLISH ABSTRACT: Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >“low” Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.