Detection of gyrA and parC Mutations Associated with Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of Oligonucleotide Biochip Technology

Abstract

An oligonucleotide biochip that specifically detects point mutations in the gyrA and parC genes of Neisseria gonorrhoeae was designed and subsequently evaluated with 87 untreated clinical specimens. The susceptibilities of the N. gonorrhoeae strains were tested to determine the prevalence of ciprofloxacin-resistant strains in Anhui Province, People's Republic of China. Conventional DNA sequencing was also performed to identify mutations in gyrA and parC and to confirm the biochip data. The study demonstrates that all of the point mutations in the gyrA and parC genes of N. gonorrhoeae were easily discriminated by use of the oligonucleotide biochip. Fifteen different alteration patterns involved in the formation of ciprofloxacin resistance were identified by the biochip assay. Double mutations in both Ser91 and Asp95 of the GyrA protein were seen in all nonsensitive isolates. Double mutations in Ser91 and Asp95 of GyrA plus mutation of Glu91 or Ser87 of the ParC protein lead to significant high-level resistance to ciprofloxacin in N. gonorrhoeae isolates. The results obtained by use of the oligonucleotide biochip were identical to those obtained by use of DNA sequencing. In conclusion, the oligonucleotide biochip technology has potential utility for the rapid and reliable identification of point mutations in the drug resistance genes of N. gonorrhoeae.

Molecular Characterization of Quinolone-Resistant Neisseria gonorrhoeae Isolates from Brazil

“…A correlation between levels of ciprofloxacin resistance and the location and number of mutations in the gyrA and parC genes has been observed in different studies (21,30,35), although it has not been found in others (19,29).…”

Real-Time PCR Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae in Urine Samples

“…exclusively associated with gyrA mutations (11,12). Furthermore, parC mutations in isolation do not appear to be associated with antimicrobial resistance (19). On the basis of these data and reported estimates of clinically significant resistance for 500 mg of ciprofloxacin ranging between 0.125 and 1.0 mg (10), we restricted our assay to the detection of mutations in the Ser91 region of the gyrA gene.…”

“…More than 99% of QRNG specimens have been shown to have mutations at this site of the gyrA gene (18,19). This method allows for rapid detection of QRNG without complex laboratory techniques or extensive resources.…”

“…In response, methods for detecting QRNG have been developed and reported in the scientific literature that include sequencing (12), microarray biochip (5,13,19), and other molecular techniques (9,11). However, each of these techniques has disadvantages with regard to detecting resistance in certain settings.…”

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

“…The oligonucleotide chips were incubated at 40℃ for 30 min in a moist incubator. After hybridization, the washing procedure was carried out as described in our previous research [9] . The oligonucleotide chip images were obtained with a fluorescent scanner (GenePix 4000B; Axon Instruments, Inc., Calif).…”