Multiplex PCR for detection of MCR genes in clinical fecal samples

¹ School of Public Health, Fujian Medical University, Fuzhou 350801, China
² Key Laboratory of Molecular Epidemiology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
³ Institute of Bioengineering, Chuangdong Academy of Science, Guangzhou 510316, China
⁴ Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 10083, China

^* Corresponding author: hushuangfang@126.com

Abstract

Plasmid-mediated colistin-resistance genes have been reported worldwide in recent years. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect transferable colistinresistance genes (mcr-1 to mcr-6) in Enterobacteria for clinical laboratory purposes.The authors first designed six new primer pairs to amplify mcr-1 to mcr-6 gene products to achieve stepwise separation of amplicons between 87 to 216 bp,then divided these primers into two subgroups with the assistance of a pair of universal primers for the detection of currently described mcr genes and their variants in Enterobacteria. The protocol was validated by testing 29 clinical isolates of Escherichia coli of human origin, each well characterised and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. It was able to detect mcr-3 and mcr-4 as singletons or in combination. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance in limited resources conditions, and this method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance.