Multiplex Accelerated PCR System for One-Step Helicobacter pylori cagA + Genotypes Detection: A Guide for Clinical Testing.

Helicobacter pylori cagA + genotype is a leading risk factor for gastric cancer development making accurate identification and timely eradication of H. pylori critical to deadly gastric cancer prevention. Traditional clinical diagnostic methods, including conventional in vitro culture, histological examination, and (13/14)C-urea breath test methods, could only identify the presence of H. pylori, but these means are not capable of identification of cagA + strains. Herein, we firstly built a multiplex detection system based on novel accelerated PCR that could realize one-step detection of as low as 20 copies of H. pylori 16S rDNA and cagA genes within 30 min. In addition, this novel system performed strong anti-jamming capacity, and exhibited that it could specifically differentiate H. pylori cagA- and cagA + genotypes co-existence with other 4 kinds of gastrointestinal pathogens. Furthermore, this one-step system showed remarkable performance on rapid H. pylori infection diagnosis and cagA + genotypes identification in clinical gastric mucosa samples. Specifically, it outperformed histological examination in terms of accuracy and was superior to conventional PCR and DNA sequencing in terms of efficiency. This rapid, sensitive, and reliable H. pylori detection and identification system would break the limitation of traditional methods and realize H. pylori infection diagnosis and cagA + genotypes identification.

Accelerated cycling PCR: A novel tool for rapid, sensitive and specific detection of single-nucleotide mutation within 30 min.

Rapid detection of single-nucleotide mutations (SNMs) has played a vital role for point-of-care testing. We herein first introduced accelerated thermal cycling into conventional allele-specific qPCR (AS-qPCR), named accelerated cycling PCR (AC-PCR) to achieve rapid and sensitive detection of SNM. It could simultaneously detect 10 copies of H. pylori DNA and identify its clarithromycin-resistance genotype within 30 min, and showed 100-fold enhanced specificity than AS-qPCR. Therefore, AC-PCR shows great potential in clinical diagnosis for drug-resistance mutation or genotyping analysis.

Establishment of a TaqMan-MGB probe multiplex real-time PCR system for one-step levofloxacin and clarithromycin resistant Helicobacter pylori detection.

Due to the abuse of antibiotics, the prevalence of antibiotic resistant Helicobacter pylori strains continues to increase. Therefore, antibiotic resistance assessment is now essential in addition to general H. pylori diagnosis in medical institutions to fulfill clinicians administering effective antibiotic regimens. However, the conventional antibiotic resistance assessment methods, such as in vitro antibiotic susceptibility test and E-test, are skilled-staff dependent and time-consuming. The aim of this study was to establish an easy-operating TaqMan-MGB probe multiplex real-time PCR system for one-step detection of levofloxacin and clarithromycin resistance mutations with concurrent H. pylori infection diagnosis. Through the optimization of primers, probes and reaction buffers, this proposed system could accurately distinguish the recombinant plasmids with different mutation markers. More importantly, the diagnosis results of this detection system exhibited excellent consistence with the gold standard of gastric biopsy and Sanger sequencing on the detection of H. pylori infection and relevant antibiotic resistant strains, the Kappa values of which all exceeded 0.90. In addition, the results of this detection system could also be applied for the prevalence statistics of antibiotic resistance patterns for patients by age, gender and geographical location. This simple and rapid system should be beneficial for clinicians issuing personalized treatments according to the patient's H. pylori strains and avoid the abuse of antibiotics.