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The Capital Region of Denmark - a part of Copenhagen University Hospital
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Nonaplex PCR using Cliffhanger primers to identify diarrhoeagenic Escherichia coli from crude lysates of human faecal samples

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  1. Lipidomics of human adipose tissue reveals diversity between body areas

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  2. Dyslipidemia at diagnosis of childhood acute lymphoblastic leukemia

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  3. Risk for development of inflammatory bowel disease under inhibition of interleukin 17: A systematic review and meta-analysis

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  4. Newborn body composition after maternal bariatric surgery

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  1. Multicenter evaluation of the QIAstat-Dx respiratory panel for detection of viruses and bacteria in nasopharyngeal swab specimens

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  2. Multicenter evaluation of the new QIAstat Gastrointestinal Panel for the rapid syndromic testing of acute gastroenteritis

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  3. Concerns regarding the validity of the conclusion in a recently published paper on Roche Liat implementation

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  4. Ceftriaxone-resistant Salmonella enterica serotype Typhi in a pregnant traveller returning from Karachi, Pakistan to Denmark, 2019

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Sensitive, probe-based detection of multiple DNA targets is limited by the competitive reannealing of the antiparallel duplex DNA helix with the complementary DNA strand. To address this, we developed Cliffhanger primers, which create single-stranded DNA overhangs on PCR amplicons while simultaneously increasing the multiplex PCR efficacy and allowing PCR amplification using crude lysates of human faecal samples. A multiplex PCR that targeted eight genes from diarrhoeagenic Escherichia coli plus an internal control was performed and compared to a routine method that consisted of culture followed by multiplex PCR with fragment length separation. A total of 2515 clinical faecal samples from patients with diarrhoea were tested using both methods, and there was a significant increase in clinical sensitivity and negative predictive value with the Cliffhanger method for seven out of eight genes. All Cliffhanger-only positive samples were confirmed by Sanger sequencing of the PCR amplicon. Notably, the Cliffhanger method reduced the total sample turn-around time in the laboratory from 20 hours to 6 hours. Hence, use of Cliffhanger primers increased assay robustness, decreased turn-around time and increased PCR efficacy. This increased the overall clinical sensitivity without the loss of specificity for a heavily multiplexed PCR assay.

Original languageEnglish
JournalPLoS One
Volume13
Issue number6
ISSN1932-6203
DOIs
Publication statusPublished - 1 Jun 2018

ID: 54693792