Abstract
Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multiplex Genetic Detection System (HMGS) assay was established to simultaneously detect and analyze a set of genes for H. pylori identification, quantification, virulence, and drug resistance by optimizing the singlet-PCR and multiple primers assay. Twenty-one pairs of chimeric primers consisted of conserved and specific gene sequences of H. pylori tagged with universal sequence at the 5′ end were designed. Singlet-PCR assay and multiple primers assay were developed to optimize the HMGS. The specificity of HMGS assay was evaluated using standard H. pylori strains and bacterial controls. Six clinical isolates with known genetic background of target genes were detected to assess the accuracy of HMGS assay. Artificial mixed pathogen DNA templates were used to evaluate the ability to distinguish mixed infections using HMGS assay. Furthermore, gastric biopsy specimens with corresponding isolated strains were used to assess the capability of HMGS assay in detecting biopsy specimens directly. HMGS assay was specific for H. pylori identification. HMGS assay for H. pylori target genes detection were completely consistent with the corresponding genetic background. Mixed infection with different drug-resistant isolates of H. pylori could be distinguished by HMGS assay. HMGS assay could efficiently diagnose H. pylori infection in gastric biopsy specimens directly. HMGS assay is a rapid and high throughput method for the simultaneous identification and quantification of H. pylori, analysis of virulence and drug resistance in both isolated strains and biopsy specimens. It could also be used to distinguish the mixed infection with different resistant genotype strains. Furthermore, HMGS could detect H. pylori infection in gastric biopsy specimens directly.
Highlights
Helicobacter pylori (H. pylori), a micro-aerobic Gram-negative bacteria, is closely associated with a variety of gastroduodenal diseases, such as chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), peptic ulcer diseases (PUD), and gastric carcinoma (GC) (Venerito et al, 2016)
Various detection methods are available in clinical practice, such as culture, histopathology, urea breath test (UBT), rapid urease test (RUT), serology, and PCR, but each has some disadvantages (Wang et al, 2015)
The amplicon sizes for the target genes were as follows (Figures 1A–T): identification gene16S rRNA: 147 bp; quantification gene ureC: 138 bp; internal control gene beta-globin: 199 bp; 10 important virulence genes cagA: 210 bp, dupA: 187 bp, luxS: 242 bp, vacA s1: 299 bp, oipA: 317 bp, vacA m1: 282 bp, vacA m2: 166bp, iceA1: 234 bp, and iceA2: 171bp/277 bp; four main resistance genes 23S rRNA(A2143G): 175 bp/180 bp, rdxA(C148T): 253 bp/258 bp, pbp1A(A1777G): 154 bp/159 bp, and gyrA(C261A/G):306 bp/311 bp, respectively
Summary
Helicobacter pylori (H. pylori), a micro-aerobic Gram-negative bacteria, is closely associated with a variety of gastroduodenal diseases, such as chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), peptic ulcer diseases (PUD), and gastric carcinoma (GC) (Venerito et al, 2016). The analysis of different virulence, gene-mutation related resistance and the bacterial load have been revealed to be very important for effective clinical diagnosis, treatment, and monitoring of H. pylori infection (Kusters et al, 2006; Proença-Modena et al, 2009; Yamaoka, 2010). Various detection methods are available in clinical practice, such as culture, histopathology, urea breath test (UBT), rapid urease test (RUT), serology, and PCR, but each has some disadvantages (Wang et al, 2015). No single method incorporates simultaneous analysis of strain-specific features including identification, virulence characteristics, drug-resistance, quantification, and evaluation of multi-strain infections (Shukla et al, 2011). A rapid, accurate, and high-content quantitative assay is required to directly assist clinical diagnosis in tissue and improve the effectiveness of diagnosis and antibiotic treatment
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