Design of novel specific primers for helicobacter detection with diagnostic utility evaluation of different 16srRNA genus-specific primers: A comparative study

Abstract

Molecular diagnosis of helicobacters by PCR is simpler, more accurate, and feasible compared to other diagnostic methods. Validity and accuracy are highly dependent on the PCR primer design, diffusion time, and mutation rate of helicobacters. This study aimed to design 16srRNA -specific primers for Helicobacter spp. and H. pylori. Application of comparative statistical analysis of the diagnostic utility of the most available 16srRNA genus-specific primers. The new primers were designed using bioinformatics tools (MAFFT MSA and Gblocks command line). A comparative study was applied on nine genus-specific 16srRNA primers in comparison to the ConsH using Insilco and laboratory evaluation. The results demonstrated that the best specificity and sensitivity of the primers designed for this study compared to other primers. The comparative study revealed that the heminested outer/inner primers were the worst. Although H276,16srRNA(a), HeliS/Heli-nest, and Hcom had acceptable diagnostic utility, false positive and false negative results were obtained. Specificity testing on clinical samples indicated a surprising result; that H. pylori was not the sole enemy that we were looking for, but the NPH should be considered as a real risk prognostic for gastric diseases, consequently, a specific diagnosis and treatment should be developed. This study concluded that our designed primers were the most specific and sensitive in comparison with other primers. in addition, Insilco evaluation is not accurate enough for primer assessment and that the laboratory evaluation is mandatory.

Isolation and Identification of Helicobacter spp. From Gastric Biopsies and Autopsies in Human and Animal Gastric Patients

“…Animal samples: 5 samples were collected from Gastric autopsies from Mongrel dogs at the Surgery Department, Faculty of Veterinary Medicine, Cairo University. The samples were collected in sterile cups with sterile saline and transmitted to the Microbiology department, Faculty of Veterinary Medicine, Cairo University (Abdelmalek, et al, 2021) Isolation of H.pylori: Sample processing: The gastric biopsies and autopsies were homogenized by an electric automated homogenizer till all tissue disappeared within the fluid. (Rabiea, et al, 2021).…”

“…PCR:The PCR mixture included 1x master mix (amaROnePCR TM , Cat.No. SM213-0250, GeneDireX, Inc.), 0.25 µmol forward, and reverse primers (ConsH), Forward primer: 5' TCG CTA AGA GAT CAG CCT ATG TCC T 3', Reverse primer: 5' ATT CCA CCT ACC TCT CCC ACA CT 3'.To produce 435bp (Abdelmalek et al 2021), 10 ng DNA, and up to 20 µl nuclease-free water. The amplification cycle for ConsH was as follow: initial denaturation at 94°C/3 minutes, 30 cycles of the following temperatures: 94°C/30 sec, annealing at 60°C/1 minutes, and cyclic extension at 72°C/2 minutes, final extension at 72°C/5 minutes.…”

“…The amplification cycle for ConsH was as follow: initial denaturation at 94°C/3 minutes, 30 cycles of the following temperatures: 94°C/30 sec, annealing at 60°C/1 minutes, and cyclic extension at 72°C/2 minutes, final extension at 72°C/5 minutes. Gel electrophoresis of the PCR products was performed in 1.5% agarose (Vivantis, cat.no: pco701) against a 100 bp DNA ladder (Vivantis, cat.no:NL1405) (Abdelmalek et al 2021).…”