Use of dd<scp>PCR</scp> in experimental evolution studies

Koch, Hanna; Jeschke, Alina; Becks, Lutz

doi:10.1111/2041-210x.12467

Cited by 15 publications

(19 citation statements)

References 41 publications

(61 reference statements)

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“…After optimisation of the ddPCR conditions, our assay proved to be very sensitive to measuring FA of MUT/WT alleles in faecal larval cultures from infected sheep, irrespective of the DNA concentrations (0.1–132.5 ng/μL). This is basically in agreement with findings in other studies quantifying the frequencies of rare alleles ( Taylor et al, 2015 ; Koch et al, 2016 ; Uchiyama et al, 2017 ).…”

Section: Discussionsupporting

confidence: 93%

Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus

Elmahalawy

Halvarsson

Skarin

et al. 2018

International Journal for Parasitology: Drugs and Drug Resistan

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Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT). Initially, we designed dyf-7 primers from a worldwide collection of adult Haemonchus contortus DNA. With the sequence data, we created a haplotype network. We then optimised and used the same sets of primers and probes in a droplet digital PCR (ddPCR) assay for precise quantification of dyf-7 allele frequencies in pre- and post-anthelmintic treatment faecal larval cultures. The fractional abundance (FA) of the mutant SNP was within the range 7.8 and 31%. However, the FA was generally stable in samples collected from the same farms, even though they were obtained on different occasions up to 25 months apart. There was also no indication that the level of IVM resistance as measured by the faecal egg count reduction test was higher on farms with high FA. Furthermore, by comparing FA in samples from the same farms pre- and post-IVM treatment, we found no evidence of a correlation between dyf-7 and level of IVM resistance. Based on these results, dyf-7 is not a suitable marker for field testing of IVM resistance in H. contortus.

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Section: Discussionsupporting

confidence: 93%

Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus

Elmahalawy

Halvarsson

Skarin

et al. 2018

International Journal for Parasitology: Drugs and Drug Resistan

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“…In channel 2, where fluorescence is emitted with the help of the MT probe carrying a HEX™ fluorescence-producing molecule, a second band is seen at ∼4000 AU. Koch et al (2016) observed a similar occurrence of the second band in channel 2 and concluded that it was likely due to the lower efficacy with which secondary dyes, such as HEX™ and VIC ® are detected. However, upon further discussion with BioRad technicians, it was suggested that these lower-specificity bands represent amplified DNA being bound to by the wrong probe (i.e.…”

Section: Discussionmentioning

confidence: 80%

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR

Baltrušis

Halvarsson

Höglund

2018

International Journal for Parasitology: Drugs and Drug Resistan

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Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the β tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.

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“…On the other hand, QPCR is advantageous when comparing more than two organisms because the currently available BioRad DDPCR system can only multiplex two fluorescent dyes (Koch et al . ). DDPCR is often, but not always, both more sensitive and more expensive per sample than QPCR (Kim et al .…”

Section: Discussionmentioning

confidence: 97%

“…DDPCR is also advantageous in assays of unculturable organisms because it does not require a standard curve of DNA quantity to cycle threshold. On the other hand, QPCR is advantageous when comparing more than two organisms because the currently available BioRad DDPCR system can only multiplex two fluorescent dyes (Koch et al 2016). DDPCR is often, but not always, both more sensitive and more expensive per sample than QPCR (Kim et al 2014;Nathan et al 2014;Yang et al 2014).…”

Section: Recommendations For Future Researchersmentioning

confidence: 99%

Measuring microbial fitness in a field reciprocal transplant experiment

Boynton

Stelkens

Kowallik

et al. 2016

Molecular Ecology Resources

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Microbial fitness is easy to measure in the laboratory, but difficult to measure in the field. Laboratory fitness assays make use of controlled conditions and genetically modified organisms, neither of which are available in the field. Among other applications, fitness assays can help researchers detect adaptation to different habitats or locations. We designed a competitive fitness assay to detect adaptation of Saccharomyces paradoxus isolates to the habitat they were isolated from (oak or larch leaf litter). The assay accurately measures relative fitness by tracking genotype frequency changes in the field using digital droplet PCR (DDPCR). We expected locally adapted S. paradoxus strains to increase in frequency over time when growing on the leaf litter type from which they were isolated. The DDPCR assay successfully detected fitness differences among S. paradoxus strains, but did not find a tendency for strains to be adapted to the habitat they were isolated from. Instead, we found that the natural alleles of the hexose transport gene we used to distinguish S. paradoxus strains had significant effects on fitness. The origin of a strain also affected its fitness: strains isolated from oak litter were generally fitter than strains from larch litter. Our results suggest that dispersal limitation and genetic drift shape S. paradoxus populations in the forest more than local selection does, although further research is needed to confirm this. Tracking genotype frequency changes using DDPCR is a practical and accurate microbial fitness assay for natural environments.

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Use of ddPCR in experimental evolution studies

Cited by 15 publications

References 41 publications

Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus

Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR

Measuring microbial fitness in a field reciprocal transplant experiment

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