Nonaplex PCR using Cliffhanger primers to identify diarrhoeagenic Escherichia coli from crude lysates of human faecal samples
| Autor: | Flemming Scheutz, Uffe Vest Schneider, Gorm Lisby, Nikolaj Dam Mikkelsen, Alice Friis-Møller |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Male Oligonucleotides lcsh:Medicine Artificial Gene Amplification and Extension medicine.disease_cause Pathology and Laboratory Medicine Polymerase Chain Reaction Biochemistry law.invention chemistry.chemical_compound Feces Sequencing techniques law Nucleic Acids Medicine and Health Sciences DNA sequencing lcsh:Science Polymerase chain reaction Sanger sequencing Multidisciplinary Nucleotides Amplicon Bacterial Pathogens Experimental Organism Systems Medical Microbiology symbols Prokaryotic Models Female Pathogens Research Article Diarrhea Escherichia 030106 microbiology Gastroenterology and Hepatology Biology Research and Analysis Methods Microbiology 03 medical and health sciences symbols.namesake Signs and Symptoms Model Organisms Enterobacteriaceae Reannealing Diagnostic Medicine Complementary DNA Multiplex polymerase chain reaction medicine Escherichia coli Genetics Humans Molecular Biology Techniques Molecular Biology Microbial Pathogens DNA Primers Bacteria lcsh:R Gut Bacteria Dideoxy DNA sequencing Organisms Biology and Life Sciences DNA Molecular biology chemistry lcsh:Q Multiplex Polymerase Chain Reaction |
| Zdroj: | PLoS ONE PLoS ONE, Vol 13, Iss 6, p e0199766 (2018) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
| Nepřihlášeným uživatelům se plný text nezobrazuje | K zobrazení výsledku je třeba se přihlásit. |