High-resolution melting-curve analysis of ligation-mediated qPCR;
نویسندگان
چکیده
25 A single-tube method – ligation mediated real-time PCR high-resolution melt analysis 26 (LMqPCR HRMA) – was modified for rapid typing of ESKAPE-pathogens. A 97 % 27 agreement (60/62 isolates) was achieved in comparison to PFGE which indicates that 28 LMqPCR HRMA is a rapid and accurate screening tool to monitor for nosocomial outbreaks. 29 on O cber 1, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Increasing antibiotic resistance in bacterial pathogens presents a great threat to human health 30 and prevention of spread of such pathogens is important (1). The ESKAPE-pathogens (2) 31 (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter 32 baumannii, Pseudomonas aeruginosa and Enterobacter spp.) are especially problematic 33 because of their antibiotic resistance mechanisms, and nosocomial spread (2, 3). By using 34 new strategies for rapid typing of isolates, the possibility to prevent uncontrolled spread of 35 these pathogens is greatly increased. Pulsed field gel electrophoresis (PFGE) has been 36 considered the golden standard for epidemiological typing (4, 5). However, next generation 37 sequencing with whole genome sequencing (WGS) is likely to become the new standard (638 10). Disadvantages for these methods are that they require both special equipment as well as 39 standard protocols and training (4-6, 8-10). Thus, there is a need for a simple and fast typing 40 method enabling real time monitoring of nosocomial outbreaks. Most of already existing 41 molecular typing methods such as multilocus sequence typing (MLST), rep-PCR (such as the 42 commercial platform Diversilab©) and comparative genomic hybridization, although more 43 rapid than PFGE, still suffer from being expensive, species specific, and exhibiting 44 insufficient resolution (11). 45 46 In this study, we further modified ligation mediated PCR (LM PCR) with polyacrylamide gel 47 analysis (12, 13) for use in a single tube, real-time PCR platform with high-resolution melt 48 analysis (LMqPCR HRMA), for rapid epidemiological typing of the multi-resistant ESKAPE49 pathogens. Following optimization, the method was evaluated blindly using a selection of 50 ESKAPE-pathogens, previously defined as being identical, closely related or unrelated by 51 PFGE analysis (definitions in Text S1). 52 53 on O cber 1, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Briefly, LMqPCR HRMA is based on fragmentation by a restriction enzyme (HindIII), 54 followed by ligation of adaptor oligonucleotides to create amplicons. Analysis is done by 55 HRM, resulting in specific melt patterns depending on the number of amplified 56 oligonucleotides, DNA sequence, GC content and length. By altering the denaturation 57 temperature (TD) during PCR the method was optimized for ESKAPE pathogens and 58 performed in duplicates in three separate experiments. For a detailed description of the 59 method see Text S1. A complete list of the isolates, their origin and resistance genes, is 60 presented in Table 1. All isolates had been analysed with PFGE at the Public Health Agency 61 of Sweden (Text S1). A standardized protocol was developed based on several parameters of 62 the melting curve shape to determine if two or more isolates were different within the same 63 run (see Text S1). 64
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