Reduced volume PCR amplification using AmpFℓSTR ® Identifiler ® kit

Iyavoo, Sasitaran; Cummings, Steve; Wolejko, Agata; Furmanczyk, Dagmara; Graham, Richard; Myers, R. J.; Haizel, Thomas

doi:10.1016/j.fsigss.2015.09.157

Cited by 5 publications

(4 citation statements)

References 4 publications

(5 reference statements)

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“…The cell‐free lysates were used as genomic DNA for all polymerase chain reactions optimized in reduced volumes (8–19 μl), simplex (sPCR) and multiplex PCR (mPCR) using Master cycler ProS Eppendorf, Germany (Iyavoo et al, ; Olsen et al, ). All primer sets used in this study were synthesized from Europhins (Operon, USA), and stock/working solutions were prepared in Tris‐EDTA buffer (Ali et al, ).…”

Section: Methodsmentioning

confidence: 99%

The unravelled Enterococcus faecalis zoonotic superbugs: Emerging multiple resistant and virulent lineages isolated from poultry environment

Hasan

Ali

Rehman

et al. 2018

Zoonoses and Public Health

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This study aimed to investigate the zoonotic potential by virtue of phylogenetic analysis, virulence and resistance gene profiles of Enterococcus faecalis originating from poultry environment. The ERIC, BOX and RAPD PCR analysis showed the clustering of E. faecalis strains (n = 74) into five groups (G1-G5) and fifteen sub-clusters (B1-B15), which share 50%-80% similarities with ATCC E. faecalis and clinical strains of human infection. E. faecalis strains harboured seven enterocins genes including ent1097 (85%), entB (84%), enterolysinA (51%), entSEK4 (51%), entL50 (31%), entA (25.7%) and ent1071 (14.9%). The highest prevalence of gelE-sprE (90%), lip-fl (90%) followed by cylL (62%), hyl (60%), katA (16%) and cylA (5.4%) was observed in poultry isolates. The fsr operon and gelE-sprE was co-associated in 66.2% strains. E. faecalis also harboured biofilm and endocarditis-associated genes, including efaAfs (97%), ebp-pilli (ebpABC and srtC 69.9%-80%), asa1 (71%), agg (55%), ace (54%) and esp-Tim (3%). Despite all found sensitive to vancomycin, 98.6% strains were multi-drug resistant to five to twelve tested antimicrobials. An increased-level of resistance (≥32 μg/ml) was observed to ampicillin (8.1%), meropenem (21.6%), chloramphenicol (73.4%), erythromycin (90.5%), tetracycline (100%) and high-level resistance to kanamycin (79.7%) and gentamicin (52.7%). The multi-drug resistant E. faecalis (MDRe.f) were carried pbp4 (90%), tetL (90%), tetM (70%), ermB (81%), cat (52.7%), acc6-aph2 (58.1%), aaph(3)-III (49.9%), gyrA (97%) and parC (98%) genes. Moreover, these MDRe.f were also harboured, hospital-associated marker IS16 (58%) and pheromone responsive genes, that is ccf (88%), cpd (74%), cob (62%) and eep (66%). Thus, regardless of the distinct phylogenetic background of E. faecalis of poultry origin, ATCC E. faecalis and clinical strains of human origin, we found major similarities in virulence, resistance gene profiles and mobile genetic elements (IS16 and pheromone responsive plasmids), supporting the zoonotic/reverse zoonotic risk associated with this organism.

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Section: Methodsmentioning

confidence: 99%

The unravelled Enterococcus faecalis zoonotic superbugs: Emerging multiple resistant and virulent lineages isolated from poultry environment

Hasan

Ali

Rehman

et al. 2018

Zoonoses and Public Health

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“…This entails a waste of reagents that could otherwise be used to analyse many more samples, thus reducing the costs per sample. It has been reported that the reduction in PCR volumes reduces costs, 1–7 but it seems that this also involves a benefit in the analysis of degraded or low template DNA samples, for which it has been observed that reduction in PCR volume improves the quality of the resulting profiles. 2,8…”

Section: Resultsmentioning

confidence: 99%

“…In the case of the typing of short tandem repeats (STRs) by capillary electrophoresis (CE), it has already been demonstrated that reduction of the polymerase chain reaction (PCR) volume to half or one third of its original volume makes it possible to increase the number of samples analysed and limit kit expenses simultaneously, without affecting the ability to obtain a good quality DNA profile. 1–8 One way of reducing the cost of NGS, at least in part, could therefore be to reduce library preparation reaction volumes.…”

Section: Introductionmentioning

confidence: 99%

Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation

Turrina

Leo

2021

Med Sci Law

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The introduction of next generation sequencing (NGS; also known as massively parallel sequencing) technology in the field of forensic genetics has been welcomed by the scientific community, above all because it complements the weaknesses of capillary electrophoresis (CE) in the analysis of genetic markers, such as single nucleotide polymorphism (SNP) typing. However, one of the main obstacles to its adoption does not seem to be the cost of the instrumentation, but rather the cost of the NGS library preparation kits. With the aim of reducing the cost of library preparation without compromising the quality of the results, we tried to scale down reaction volumes for the first two polymerase chain reactions in the amplification and enrichment phases of the targeted loci of library preparation using the ForenSeq™ DNA Signature Prep kit. We used 1 µL templated DNA input to a concentration of 1 ng/µL, instead of the 5 µL at 0.2 ng/µL recommended by the manufacturer. Our findings indicate that reduction of the library preparation volume using the ForenSeq™ DNA Signature Prep kit did not interfere with the quality and reproducibility of the DNA profiles obtained and can help lower the overall cost of NGS.

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“…In total 40 buccal swabs from three routine casework batches were extracted and amplified with the current accredited procedures [3,4] employing QIAamp ® DNA Mini Kit (Qiagen ® , UK) and AmpFℓSTR ® Identifiler ® PCR Amplification Kit (Thermo Fisher Scientific ® , UK) at the old premises together with the blank, positive and negative controls for each batch.…”

Section: Sample Selection Dna Extraction and Pcr Amplificationmentioning

confidence: 99%

Validation of ABI 3500xL Genetic Analyzer after decommissioned and recommissioned at new premises

Iyavoo

Draus-Barini

Haizel

2017

Forensic Science International: Genetics Supplement Series

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This validation was aimed to demonstrate that there is no effect on the Applied Biosystems ® 3500xL Genetic Analyzer (Thermo Fisher Scientific ® , UK) after decommissioned and recommissioned at new premises. Same sample sets were tested on the ABI 3500xL Genetic Analyzer at both old and new premises using current accredited procedures. The results showed genotyping concordance which means using the ABI 3500xL Genetic Analyzer at the new premises provides no risk regarding the accuracy, precision and reproducibility of the results and also showed a very good performance on sensitivity, specificity, repeatability and good profile quality based on the assessment of locus peak balance and peak height.

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Reduced volume PCR amplification using AmpFℓSTR ® Identifiler ® kit

Cited by 5 publications

References 4 publications

The unravelled Enterococcus faecalis zoonotic superbugs: Emerging multiple resistant and virulent lineages isolated from poultry environment

The unravelled Enterococcus faecalis zoonotic superbugs: Emerging multiple resistant and virulent lineages isolated from poultry environment

Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation

Validation of ABI 3500xL Genetic Analyzer after decommissioned and recommissioned at new premises

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