Patients selection:
This study was conducted over a period of 15 month (from December 2019 to February 2021). We have recruited 132 patients with gastric cancer at the Gastro-Enterology Surgical Center, Mansoura University, Egypt. They have presented initially with gastric symptoms that was confirmed endoscopically and histo-pathologically to be gastric cancer. Healthy participants who had gastric endoscopy procedures and were proved to be free of any gastric pathology and negative for H. pylori infection were recruited as a control group. The epidemiological and clinical data of all participants in the present study were recorded.
We have excluded any participant who fulfilled one or more of the set exclusion criteria that included previous gastric surgery, the use of anti-H. pylori eradication therapy, antibiotics, anti-inflammatory agents, proton pump inhibitors, chemotherapeutic drugs or radiotherapy within one month before the endoscopy procedure.
Samples collection:
A total of 238 stomach biopsies (each of size of 5 mm × 5 mm) were collected by clinicians during the performance of diagnostic gastric endoscopy procedures. Obtained biopsies were stored on ice and immediately transferred to Medical Microbiology and Immunology department, Mansoura University, Egypt for further processing.
A peripheral blood sample of 10 ml was collected under complete aseptic precautions from each study participant for investigating TLR9 gene polymorphisms (TLR9 -1486T/C, rs187084 SNPs).
Isolation of H. pylori from gastric tissue samples:
Collected biopsies were inoculated in sterile tubes with brain heart infusion(BHI) broth (Oxoid- UK), then homogenized by a scalpel on a sterile slide. Homogenized samples were cultured on Colombia agar (Oxoid- UK) plates containing 10% of freshly defibrinated sheep’s blood. Besides, plates were supplemented with amphotericin B (4 mg/L), vancomycin (10 mg/L), polymyxin B (10 mg/L), and trimethoprim (5 mg/L) antibiotics (Oxoid- UK). Cultured plates were incubated under microaerophilic circumstances (Campy pack systems, BBL,Cockeysville, Maryland, USA) at 37°C for 3 days (23).
Culture plates were examined for colonies after 3 days where H. pylori isolates were identified by having small, translucent and round colonies. Further recognition of H. pylori isolates was conducted by Gram stained films followed by biochemical reactions (positive catalase, urease, and oxidase). Suspensions of H. pylori strains were prepared using BHI broth supplemented with 20% glycerol and then kept at -20°C for further analysis (24).
Molecular confirmation of isolated H. pylori strains through amplification of glmM gene.
Whole genomic DNA was obtained from cultured isolates using (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany), in line with the provider’s rules then the resulted DNA was kept −20°C until further completing of laboratory work.
The set of primers used to amplify the targeted gene was mentioned in Table 1 (5). The PCR was carried out in a reaction mixture (25 µL) containing 12.5 µL master mix (Fermentas, Germany), 5 µL DNA, 0.2 µL of each primer (10 pmol) and 7.1 µL nuclease - free water (25).
Table 1
Sequences of sets of primers used
Gene targeted | Sequence | Size of amplified product (bp) | Ref |
glmM | F-5′AAGCTTTTAGGGGTGTTAGGGGTTT3′ R 5′AAGCTTACTTTCTAACACTAACGC3′ | 294 | 5 |
cagA | F-5′GATAACAGGCAAGCTTTTGAGG3′ R-5′CTGCAAAAGATTGTTTGGCAG3′ | 349 | 27 |
sodB | F-5′GCCCTGTGGCGTTTGATTTCC3' R-5′CATGCTCCCACACATCCACC3' | 425 | 21 |
hsp60 | F-5′GCTCCAAGCATCACCAAAGACG3′ R-5′GCGGTTTGCCCTCTTTCATGG3′, | 603 | 21 |
vacA | F-5′CAATCGTGTGGGTTCTGGAGC3′ R-5′GCCGATATGCAAATGAGCCGC3, | 678 | 21 |
vacAs1/s2 | F: 5' ATGGAAATACAACAAACACAC3' R: 5'CTGCTTGAATGCGCCAAAC3' | 259 | 28 |
vacAm1 | F:5'GGTCAAAATGCGGTCATGG3' R: 5'CCATTGGTACCTGTAGAAAC3' | 290 | 28 |
vacAm2 | F: 5'CATAACTAGCGCCTTGCAC3' R: 5'GGAGCCCCAGGAAACATTG3' | 352 | 28 |
TLR9 -1486T/C, rs187084 | 5′TTCATTCATTCAGCCTTCACTCA 3′, 5′ GAGTCAAAGCCACAGTCCACA 3′ | 490 | 30 |
PCR started by an initial denaturation at 94°C for five minutes, then 40 cycles of denaturation for sixty seconds at 94°C, annealing for ninety seconds at 55°C, and extension for 120 seconds at 72°C. The final extension was performed at 72°C for seven minutes (25).
Molecular detection of cagA, sodB, hsp60, and vacA virulence genes of H. pylori using multiplex PCR
Multiplex PCR was undertaken following the coming steps simultaneously: beginning with incubation for 5 minutes at 95°C; then 34 cycles were run, each consisting of one minute at 94°C, then another one minute for primer annealing at 55°C, followed by extension for 60 seconds at 72°C; to be finished with the final extension step at 72°C for ten minutes (26). The reaction volume was 25 µL that contained PCR master mix (Fermentas, Germany), besides 5 µL of DNA, and 0.2 µL of each primer. PCR products were then electrophoresed (27). Standard strain (ATCC26695) was used as a positive control (21). Primer sets used were supplemented in table (1)
Molecular detection of different vacA gene alleles in vacA positive H. pylori isolates (vacAs1/s2, vacAm1 and vacAm2)
PCR was conducted by applying the following cycling parameters: first; 95°C for 5 minutes, second; 35 cycles [95°C for 30 seconds, 54°C for 30 seconds and 72°C for 16, 18 and 21 seconds respectively according to the required allele to be amplified (vacAs1/s2, vacAm1 and vacAm2)]. Then final extension at 72°C for 10 minutes (28). Sets of primers used were listed in table (1).
Genotyping of TLR9 -1486T/C, rs187084 polymorphisms by PCR-RFLP
Genomic DNA was extracted from the obtained buffy coat (leukocyte-enriched fraction of whole blood) by using Gene JET Whole Blood Genomic DNA purification Mini kit (Fermentas Life Sciences, Canada) according to provider’s guidelines then exposed to PCR-RFLP. Blood samples were subjected to centrifugation at 2,500 ⋅g for 10 minutes to release 3 different layers: the upper clear layer containing plasma; the intermediate buffy coat and the bottom layer of concentrated erythrocytes.
PCR reaction was run with thermal cycling conditions of 4 minutes at 95°C then 35 cycles each starting with 30 seconds at 95°C followed by 20 seconds at 60°C, and 30 seconds at 72°C to be ended with final extension for 5 minutes at 72°C to produce a DNA piece of 490 bp (29). The primers used were illustrated in table (1) (30).
Then the DNA products were digested using AflII restriction enzyme (Thermo Scientific, EU, Lithuania) by incubation for three hours at 37°C to yield one of three variants: two fragments of 192 bp, and 327 bp that indicated TT allele, or three fragments of 192 bp, 327 bp, and 490 bp that proved presence of TC allele or an intact PCR fragment of 490 bp indicating presence of CC allele (29).