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

Abstract

The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.

Keywords: Fluoroquinolone resistance; mismatch amplification mutation assay; polymerase chain reaction; quinolone resistance determining region mutations.

Fig. 1

Principle of the mismatch amplification mutation assay-polymerase chain reaction.

Fig. 2

Illustration of mismatch amplification mutation assay-polymerase chain reaction primers for gyrA (A) and parC (B) mutation detection. The underlined nucleotides are mismatched nucleotides at 3’ end of each mismatch amplification mutation assay primer. The amino acid found in the native protein and the changed amino acid due to point mutation are indicated above the corresponding nucleotide sequences.