Fecal samples collection and reference bacterial strains
Children under 2 years old with diarrhea symptom suspected to gastroenteritis (n = 163) in Qazvin, Iran, during December 2019 to February 2020 were referred to the central laboratory of Qods Children Hospital of Qazvin for microbiological investigation. As previously described by WHO Laboratory investigations manual of Enteric infections for faecal sample preparation, all suspected specimens were collected in disposable and clean containers then examined macroscopically and microscopically for color, mucus, consistency, and red blood cells [14]. All specimens were kept at 4 °C prior to bacterial isolation procedure. We also used S. sonnei ATCC 25931; S. flexneri ATCC 12022; and S. boydii ATCC 12030, purchased from Pasteur institute of Iran (Pasteur institute, Tehran, Iran), as Shigella reference strains in this study. All reference strains were activated by inoculation in Bovine Heart Infusion (BHI) broth and incubation at 37 °C overnight.
Shigella isolation and species identification
All specimens were initially cultured on MacConkey agar and Salmonella-Shigella agar (SSA) media (Promedia, Spain) incubated aerobically for 24 h at 37 °C. Non-lactose-fermenting and H2S-negative colonies on MacConkey agar and SSA were isolated for biochemically identification. Biochemical tests consisted of Urease, Triple Sugar Iron (TSI), Motility, Siminous Citrate, and Indole tests. Biochemically confirmed colonies were further serologically analyzed for Shigella species identification. Serological identification was carried out using slide-agglutination test with DIFCO Shigella species specific antisera (Difco Lab, Michigan, USA) as the gold standard assay [15].
Genomic DNA extraction
Bacterial isolates and the reference strains were inoculated into LB broth then incubated at 37 °C overnight. After centrifugation of the incubated LB broth tubes at 6000 RPM for 10 min, the cell palette in each centrifuge tube were subjected to genomic DNA extraction employing Sinaclon gram-negative DNA extraction Kit (Sinaclon, Iran) according to the instructions described by the manufacturer. The quantity and quality of the extracted genome were evaluated using NanoDrop Spectrophotometer (Thermo Scientific, USA). Before PCR-HRM, concentration of all DNA templates was adjusted to 50 ng. µL-1.
RAPD and ERIC-PCR coupled with HRM assay
In this study, we used the single random oligonucleotide primer 5`-CGC GTG CCA G-3` for RAPD-PCR and species identification of isolated Shigella strains [16]. Each reaction tube contained 20 µL of final volume, including 10 µL of 2X HotStart EvaGreen PCR-HRM master mix (Solis BioDyne Co, Estonia), 0.5 µL of RAPD primer (20 pM), 1 µL of DNA template (50 ng), and distilled deionized water to the final reaction volume. RAPD-PCR coupled with HRM assay was performed using Rotor-Gene 6000 real-time PCR instrument (Corbett, Australia) as follows: initial denaturation at 95 °C for 5 min; 35 cycles of 95 °C for 1 min, 36 °C for 1 min, and 4 min at 72 °C. Then HRM was performed between 70 and 95 °C with continuous ramping 0.1 °C s-1. Fluorescence of melting profile was normalized between 80 and 95 °C by Rotor-Gene 6000 series software version 1.7 (Corbett, Australia) then normalized and difference melting curves were obtained for each isolate.
For ERIC-PCR amplification two primers have been used including ERIC1R 5`-ATG TAA GCT CCT GGG GAT TCA C-3` and ERIC2 5`-AAG TAA GTG ACT GGG GTG AGC G-3` [16]. Both RAPD and ERIC primers used in this study previously were employed by researchers for DNA fingerprinting of enterobacterial pathogens. ERIC-PCR was performed in 20 µL final reaction volume containing 10 µL of 2X HotStart EvaGreen PCR-HRM master mix (Solis BioDyne Co, Estonia), 1 µL of each primer (10 pM), 1 µL of DNA template, and distilled deionized water to 20 µL. Rotor-Gene 6000 real-time PCR instrument (Corbett, Australia) also was employed for ERIC-PCR-HRM procedure. Thermal cycling program was initial denaturation step at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 1 min, and extension at 65 °C for 8 min. Afterwards, HRM was carried out from 70 to 95 °C with 0.1 °C s-1 ramping. Also, normalized and difference melting curves finally were achieved from the Rotor-Gene 6000 series software by fluorescence normalization of melting profile between 80 and 95 °C.
Data analysis
All difference curves obtained from Rotor-Gene 6000 series software were exported to Excel (Microsoft Office Excel software, Microsoft Corp., Redmond, USA) files then they were used as the input data of analysis. Principal components analysis (PCA) was employed for analysis and data categorization as a dimension reduction method by SPSS software version 23.0 (SPSS, Inc., Chicago, USA). Also, sensitivity and specificity of the methods have been evaluated using receiver operating characteristic (ROC) curve analysis by SPSS. All measurements and analysis were carried out in triplicates.