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@#Abstract: Objective To establish a method for detecting sdaB virulence gene of Streptococcus pyogenes with loop mediated isothermal amplification (LAMP). Methods According to the conserved sequence of Streptococcus pyogenes sdaB gene published in GenBank (GenBank: 69901515), LAMP primers were designed with Primer Explorer V5.0 software. Main components of LAMP reaction system were optimized including of fluorescent dye, MgSO4, betaine, deoxyribonucleosidetriphosphate (dNTP), and Bst DNA polymerase, with the concentration of MgSO4 from 0 mmol/L to 12 mmol/L, betaine from 0 mol/L to 2.4 mol/L, dNTP from 0.2 µmol/L to 2 µmol/L, forward inner primer (FIP) and backward inner primer (BIP) from 0.2 µmol/L to 2 µmol/L respetively, forward outer primer (F3) and backward outer primer (B3) from 0.2 µmol/L to 0.4 µmol/L, Bst DNA polymerase from 0.16 U/µL to 0.96 U/µL, fluorescent dye from 0.2 µmol/L to 2 µmol/L. With the optimized system, the methodological specificity and the minimum detection limit were evaluated on the ABI7500 real-time fluorescent quantitative PCR analyzer, and 13 standard strains including Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus viridis, Enterococcus faecalis, Enterococcus faecium, Neisseria gonorrhoeae, Lactobacillus acidophilus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were detected. Finally, 103 clinical samples were tested. Results The optimized reaction system contained 25 µL reaction mixture, including 0.8 µL of 25 µmol/L fluorescent dye, 1 µL of 100 mmol/L MgSO4, 6 µL of 5 mol/L betaine, 1.4 µL of 25 mmol/L dNTP, 2 µL of 20 µmol/L FIP and BIP, 0.5 µL of 20 µmol/L F3 and B3, 1 µL of 8 U/µL Bst DNA polymerase, and 2 µL of template. After adding deionized water, the mixture was incubated at 63°C for 45 min to complete the reaction. The limit of detection (LOD) was 500 pg/µL. All 12 non-S. pyogenes strains tested were negative. Compared with the culture method, the clinical sensitivity and specificity were 100.0% (16/16) and 96.6% (84/87), respectively, for 103 clinical samples. Conclusions This LAMP assay is reliable for the detection of Streptococcus pyogenes in clinic and is suitable for field detection with good specificity and sensitivity, as well as simply operation.
RESUMEN
The most important gram positive cocci is Streptococcus pyogenes causing pyogenic infections, Group A beta hemolytic streptococci are the most frequently isolated pathogen in pharyngitis among school going children. The objective of the study was to find the prevalence of Group A Streptococcal infection among the Government Higher Primary School Children of an urban community.This cross sectional study was conducted in all the seven Government schools of Ramnagar under the catchment of urban health center of Belgaum city, Karnataka with a sample of 300. The findings showed that the prevalence of beta hemolytic was 30.7%. Majority of children belonged to 5-10 years age group and majority belonged to nuclear family. Children having more number of window in their house, cultured less beta hemolytic (p<0.001).It was concluded that although the several risk factors have been identified, there is a need to do an in-depth study for each risk factor and also further epidemiological studies on these aspects are needed to support the findings of our study.