Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system
Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detectio...
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| Published in | Electrophoresis Vol. 37; no. 23-24; pp. 3079 - 3083 |
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| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Germany
Blackwell Publishing Ltd
01.12.2016
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| Subjects | |
| Online Access | Get full text |
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| Summary: | Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug‐resistant Gram‐positive pathogens, we developed a method by using stuffer‐free multiplex ligation‐dependent probe amplification (MLPA) coupled with high‐resolution CE single‐strand conformation polymorphisms (CE‐SSCP) system. We designed three probe sets for genes specific to Gram‐positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram‐positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain‐specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target‐specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug‐resistant Gram‐positive pathogens from sepsis patients’ blood. |
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| Bibliography: | ArticleID:ELPS5972 Korea Health Industry Development Institute - No. H14C2658 istex:BF1AD5FB84B9A30AE56CCE9A4608CC8B72D6E3DD Supplementary Table S1. List of reference strains. Supplementary Table S2. List of clinical isolates. Supplementary Table S3. Separation results based on targeted genes in clinical samples Supplementary Table S4. Sequences of the primers for pre-amplification. Supplementary Figure S1. Overall strategy for developing pathogen identification system based on stuffer-free MLPA-CE-SSCP. After extracting the pathogen DNA, target specific pre-amplification was performed with the extracted genomic DNA from pathogens. PCR reaction was performed using a high-fidelity DNA polymerase kit (New England Biolabs, Ipswich, MA): 10 sec at 98 °C for 1 cycle followed by 35 cycles of 30 sec at 98°C, 30 sec at 53°C, 60 sec at 72 °C, and 1 cycle for 7 min at 72°C. Two stuffer-free MLPA probes (left and right) comprised of target gene specific hybridization sequence and universal primer recognition sites were hybridized to the target region followed by ligation of both probes. Gibbs free energy (ΔG) of each probe sequence was calculated using Mfold software (http://mfold.rna.albany.edu/?q=mfold/) and the probes with ΔG ≥0 were selected. After the ligation step, PCR reaction was performed using universal primers and the ligation product. PCR cycling conditions were as follows: 5 min at 95 °C (initial denaturation), followed by 35 cycles of 30 sec at 95 °C (denaturation), 30 sec at 60 °C (annealing) and 30 sec at 72 °C (extension), and 7 min at 72 °C (final extension). Through CE-SSCP, amplicons of same or similar lengths can be separated based on their sequence characteristics. The mobility of the denatured amplicons is different not by the amplicon size but by sequence characteristics. gDNA, genomic DNA; SSCP, single-strand conformation polymorphisms Supplementary Figure S2. Electropherograms obtained from stuffer-free MLPA-CE-SSCP analysis of MLPA reaction products using single probe sets with genomic DNAs from single pathogens. All MLPA products yielded single peaks and the positions of each peak are relatively distinct. Bottom plot: ROX 500 size standards (Applied Biosystems). X axis, relative migration time (arbitrary units, AU); Y-axis, relative fluorescence intensity ark:/67375/WNG-Q68PJV3Q-R See the article online to view Fig. 1 in colour. These authors have contributed equally to this work. Colour Online ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0173-0835 1522-2683 |
| DOI: | 10.1002/elps.201600372 |