Rapid detection and quantification of plasmid‐mediated colistin resistance genes ( mcr‐1 to mcr‐5 ) by real‐time PCR in bacterial and environmental samples

Abstract

Aim The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr‐1 to mcr‐5) by real‐time polymerase chain reaction (RT‐PCR) in diverse matrices. Methods and results The detection limit of two newly designed SYBR Green real‐time PCR assays for mcr‐4 and mcr‐5 and of previously published protocols for mcr‐1 to mcr‐3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n = 1062) and environmental samples (n = 93) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. Conclusions This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. Significance and Impact of the Study Colistin is an antimicrobial commonly used in animals and has recently emerged as a last‐resort treatment in humans. Plasmid‐mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity.

Molecular epidemiology of mcr gene group

“…Другие протоколы используют технологию ПЦР в реальном времени для определения генов группы mcr1 и mcr2 с использованием технологии ПЦРзондов TaqMan® [105,106]. Также представлены основан ные на применении ПЦР в реальном времени с исполь зованием интеркалирующего красителя SYBR® Green методики для определения mcr1 [107][108][109], mcr2 и mcr3 [110], а также мультиплексного определения генов mcr1, mcr2, mcr3, mcr4 и mcr5, дифференциация кото рых происходит благодаря различным точкам плавления ампликонов [111]. Также Borowiak M. и соавт.…”

Current and emerging polymyxin resistance diagnostics: a systematic review of established and novel detection methods

“…Four SYBR Green-based real-time PCR assays have been developed for mcr -1, mcr -2, mcr -3, mcr -4, and mcr -5 detection 92-95 . Bontron et al (2016) designed a SYBR Green-based real-time PCR assay for detection of mcr -1 from cultured bacteria and stools.…”

“…However, in this study all three mcr genes could not be simultaneously detected in one reaction unlike when the Taqman probe was used 94 . A more recent study evaluated the SYBR Green-based real-time PCR method proposed by Li et al (2017) in detecting and quantifying mcr -1 to mcr -3 as well as newly designed assays for mcr -4 and mcr -5 92 . The optimized mcr -1 to mcr -5 PCR assays were validated on bacterial isolates 92 .…”

“…A more recent study evaluated the SYBR Green-based real-time PCR method proposed by Li et al (2017) in detecting and quantifying mcr -1 to mcr -3 as well as newly designed assays for mcr -4 and mcr -5 92 . The optimized mcr -1 to mcr -5 PCR assays were validated on bacterial isolates 92 . The study found that SYBR Green real-time PCR, followed by melting curve analysis, was more efficient in detecting and quantifying mcr -1 to mcr -5 genes in both bacterial isolates 92 .…”

“…The optimized mcr -1 to mcr -5 PCR assays were validated on bacterial isolates 92 . The study found that SYBR Green real-time PCR, followed by melting curve analysis, was more efficient in detecting and quantifying mcr -1 to mcr -5 genes in both bacterial isolates 92 . The described assays detected all five mcr genes with a lower limit of 10 2 .…”