Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection ofCampylobacter jejuniand Mutation ingyrAGene Related to Fluoroquinolone Resistance

Abstract

Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples

“…A MAMA-PCR was used to detect mutations at four major QRDRs ( gyr A 83, gyr A 87, par C 80 and par C 84). It has been used to detect point mutations in gyr A and par C regions in fluoroquinolone-resistant bacterial pathogens282930. The point mutation S83L at gyr A 83 was the most commonly observed change3132 followed by mutation at gyr A 87.…”

Rapid screening of point mutations by mismatch amplification mutation assay PCR

“…Due to the lack of 3′-5′ exonucleolytic proofreading activity in Taq polymerase, the mismatch between the primer’s 3′ terminal base and the template will stop the PCR (Tindall 1988 ). Given this, MAMA PCR was well established and experimented in the late 1980s in the detection of point mutation of several disease conditions (Cui et al 2016 ; Deekshit et al 2019 ; Santhosh et al 2017 ). The MAMA PCR technique is also widely used for the detection of point mutations in the quinolone resistance determining regions (QRDRs) of fluoroquinolone-resistant bacterial pathogens (Deekshit et al 2019 ; Kakuta et al 2020 ; Ota et al 2022 ).…”

Mismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens

“…Over the years, many methods have been developed to detect such mutations34. The MAMA-PCR technique is also widely used for the detection of point mutations in the quinolone resistance determining regions (QRDRs) of fluoroquinolone-resistant bacterial pathogens5678.…”