Abstract
Helicobacter pylori (H. pylori) is a kind of bacteria which can colonize on the stomach mucosa. Previous ntudies showed that it is closely associated with gastric diseases. H. pylori could be cured by using the triple therapy, which is composed of two antibiotics and a proton pump inhibitor and it has been comment used to eradicate H. pylori in routine practice. However, some strains of H. pylori were found which could express resistance to one kind of the antibiotics: quinolones. The Epsilometer test and Kirby-Bauer disk diffusion susceptibility test are the two presently used methods to confirm the resistance to antibiotics after treatment. However, these methods are relatively time-consuming and the culture result is strongly affected by the procedure of sample collection, transportation and storage. Therefore, a new method is in great need to prevent the artificial influence of sample collection, transportation and storage and shorten the experimental time as well. To detect the resistance of quinolones, besides phenotypic tests, some molecular diagnostic methods were reported recently. Some point mutations in the gyrase gene were found to express a resistance to quinolones. A mutation resulting in the change of a single amino acid substitution at any one or both of the two different sites in the gyrase A subunit affects the ability of the drug in binding to it. Polymerase chain reaction (PCR) and subsequent detection are conventional molecular diagnostic techniques for confirmation of the resistance to antibiotics. Single nucleotide polymorphism polymerase chain reaction (SNP-PCR) is a relatively new method to distinguish the single point mutations from a normal gene by applying a pair of specific primers without using complicated sequencing processes. Nevertheless, this diagnostic process requires relatively expensive and bulky apparatus and is labor-intensive. In this study, an integrated microfluidic system was developed to detect the resistance to quinolones in H. pylori by using the molecular diagnostic technique of SNP-PCR. The microfluidic system could combine the entire experimental procedure including sample pre-treatment, PCR/SNP-PCR, and optical detection on a single chip/system. Besides, more than one reaction chambers were designed on a single chip to detect multiple samples. The process could be performed within 1 hour with detection limits of 100, 102, and 102 bacterial cells for the detection of H. pylori and two different single nucleotide polymorphism site (mutation site) strains. Being accompanied by the advantages of microfluidic systems, the study fits clinical needs and may be promising for fast diagnosis of H. pylori and its drug-resistance.