Evaluation of Four Different DNA Targets in Polymerase Chain Reaction for Detection and Genotyping of Helicobacter pylori
Authors: Abu Salim Mustafa
Polymerase chain reaction (PCR) assays targeting genomic DNA segments have been established for the detection of Helicobacter pylori in clinical specimens. However, the data on comparative evaluations of various targets in detection of H. pylori are limited. Furthermore, the frequencies of vacA (s1 and s2) and cagA genotypes, which are suggested to be involved in the pathogenesis of H. pylori in other parts of the world, are not well studied in Kuwait. The aim of this study was to evaluate PCR assays for the detection and genotyping of H. pylori by targeting the amplification of DNA targets from four genomic segments. The genomic DNA were isolated from 72 clinical isolates of H. pylori and tested in PCR with four pairs of oligonucleotides primers, i.e. ECH-U/ECH-L, ET-5U/ET-5L, CagAF/CagAR and Vac1F/Vac1XR, which were expected to amplify targets of various sizes (471 bp, 230 bp, 183 bp and 176/203 bp, respectively) from the genomic DNA of H. pylori. The PCR-amplified DNA were analyzed by agarose gel electrophoresis. PCR products of expected size were obtained with all primer pairs by using genomic DNA isolated from H. pylori. DNA dilution experiments showed that the most sensitive PCR target was 471 bp DNA amplified by the primers ECH-U/ECH-L, followed by the targets of Vac1F/Vac1XR (176 bp/203 DNA), CagAF/CagAR (183 bp DNA) and ET-5U/ET-5L (230 bp DNA). However, when tested with undiluted genomic DNA isolated from single colonies of all isolates, the Vac1F/Vac1XR target provided the maximum positive results (71/72 (99% positives)), followed by ECH-U/ECH-L (69/72 (93% positives)), ET-5U/ET-5L (51/72 (71% positives)) and CagAF/CagAR (26/72 (46% positives)). The results of genotyping experiments showed that vacA s1 (46% positive) and vacA s2 (54% positive) genotypes were almost equally associated with VaCA+/CagA- isolates (P > 0.05), but with VacA+/CagA+ isolates, S1 genotype (92% positive) was more frequently detected than S2 genotype (8% positive) (P< 0.0001). In conclusion, among the primer pairs tested, Vac1F/Vac1XR provided the best results for detection of H. pylori. The genotyping experiments showed that vacA s1 and vacA s2 genotypes were almost equally associated with vaCA+/cagA- isolates, but vacA s1 genotype had a significantly increased association with vacA+/cagA+ isolates.
Digital Object Identifier (DOI): doi.org/10.5281/zenodo.2643615Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 213
 Warren JR, Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983; 1:1273-1275.
 Moss SF. The clinical evidencelLinking Helicobacter pylori to gastric cancer. Cell Mol Gastroenterol Hepatol. 2016;3:183-191
 Zamani M, Ebrahimtabar F, Zamani V, Miller WH, Alizadeh-Navaei R, Shokri-Shirvani J, Derakhshan MH. Systematic review with meta-analysis: the worldwide prevalence of Helicobacter pylori infection. Aliment Pharmacol Ther. 2018;47:868-876.
 Dhar R, Mustafa AS, Dhar PM, Khan MS, Al Rashidi FJS, Al Shammali AAA, Ali FHAG. Evaluation and comparison of two immunodiagnostic assays for Helicobacter pylori antibodies with culture results. Diagn Microbiol Infect Dis 1998; 30:1 6.
 Cosgun Y, Yildirim A, Yucel M, Karakoc AE, Koca G, Gonultas A, Gursoy G, Ustun H, Korkmaz M. Evaluation of invasive and noninvasive methods for the diagnosis of Helicobacter pylori infection. Asian Pac J Cancer Prev. 2016;17:5265-5272.
 Patel SK, Pratap CB, Jain AK, Gulati AK, Nath G. Diagnosis of Helicobacter pylori: What should be the gold standard? World J Gastroenterol. 2014;20: 12847-12859.
 Sayehmiri F, Kiani F, Sayehmiri K, Soroush S, Asadollahi K, Alikhani MY, Delpisheh A, Emaneini M, Bogdanović L, Varzi AM, Zarrilli R, Taherikalani M. Prevalence of cagA and vacA among Helicobacter pylori-infected patients in Iran: a systematic review and meta-analysis. J Infect Dev Ctries. 2015;9:686-696.
 Li C, Musich PR, Ha T, Ferguson DA Jr, Patel NR, Chi DS, Thomas E. High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay. J Clin Pathol. 1995;48:662-666.
 Song Q, Haller B, Schmid RM, Adler G, Bode G. Helicobacter pylori in dental plaque: a comparison of different PCR primer sets. Dig Dis Sci. 1999;44:479-484.
 Van Doorn LJ, Figueiredo C, Rossau R, Jannes G, van Asbroek M, Sousa JC, Carneiro F, Quint WG.. Typing of Helicobacter pylori vacA gene and detection of cagA gene by PCR and reverse hybridization. J. Clin. Microbiol. 1998;36,1271– 1276.
 Qabandi AA, Mustafa AS, Siddique I, Khajah AK, Madda JP, Junaid TA. Distribution of vacA and cagA genotypes of Helicobacter pylori in Kuwait. Acta Tropica, 2005; 93: 283-288.
 Linpisarn S, Koosirirat C, Prommuangyong K, Suwan W, Lertprasertsuke N, Phornphutkul K. Use of different PCR primers and gastric biopsy tissue from CLO test for the detection of Helicobacter pylori. Southeast Asian J Trop Med Public Health. 2005;36:135-140.
 Mustafa AS, Ahmed A, Abal AT, Chugh T. Establishment and evaluation of a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex. Tubercle and Lung Disease 1995; 76: 336 343.
 Ben Mansour K, Fendri C, Zribi M, Masmoudi A, Labbene M, Fillali A, Ben Mami N. et al. Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients. Ann Clin Microbiol Antimicrob. 2010;9:10.
 Rasmussen LT, Labio RW, Gatti LL, Silva LC, Queiroz VF, Smith Mde A, Payao SL. Helicobacter pylori detection in gastric biopsies, saliva and dental plaque of Brazilian dyspeptic patients. Mem Inst Oswaldo Cruz. 2010;105:326–330.
 Souod N, Kargar M, Doosti A, Ranjbar R, Sarshar M. Genetic analysis of cagA and vacA genes in Helicobacter pylori isolates and their relationship with gastroduodenal diseases in the west of Iran. Iran Red Crescent Med J. 2013;15:371–375.
 Abu-Lubad M, Alzoubi H, Jarajreh D, Sawalqa AA, Bruggemann H, Albataineh E, Aqel A, Al-Zeer M. Analysis of Helicobacter pylori Genotypes Amongst Jordanians' Dental Plaque Samples Gastroenterology Res. 2018;11:46-51.
 Jeyamani L, Jayarajan J, Leelakrishnan V, Swaminathan M. CagA and VacA genes of Helicobacter pylori and their clinical relevance. Indian J Pathol Microbiol. 2018;61:66-69.
 Pormohammad A, Ghotaslou R, Leylabadlo HE, Nasiri MJ, Dabiri H, Hashemi A. Risk of gastric cancer in association with Helicobacter pylori different virulence factors: A systematic review and meta-analysis. Microb Pathog. 2018 May;118:214-219.
 Nejati S, Karkhah A, Darvish H, Validi M, Ebrahimpour S, Nouri HR. Influence of Helicobacter pylori virulence factors CagA and VacA on pathogenesis of gastrointestinal disorders. Microb Pathog. 2018;117:43-48.
 Siddique I, Al-Qabandi A, Al-Ali J, Alazmi W, Memon A, Mustafa AS, Junaid TA. Association between Helicobacter pylori genotypes and severity of chronic gastritis, peptic ulcer disease and gastric mucosal interleukin-8 levels: Evidence from a study in the Middle East. Gut Pathog. 2014;6:41.
 Román-Román A, Martínez-Carrillo DN, Atrisco-Morales J, Azúcar-Heziquio JC, Cuevas-Caballero AS, Castañón-Sánchez CA, Reyes-Ríos R, Betancourt-Linares R, Reyes-Navarrete S, Cruz-Del Carmen I, Camorlinga-Ponce M, Cortés-Malagón EM, Fernández-Tilapa G. Helicobacter pylori vacA s1m1 genotype but not cagA or babA2 increase the risk of ulcer and gastric cancer in patients from Southern Mexico. Gut Pathog. 2017;9:18.