The current study aimed to detect the prevalence of Streptococcus pneumoniae by identifying Pneumolysin and determining the gene (ply) using polymerase chain reaction (PCR). The study aimed to highlight the isolation and identification of Streptococcus pneumoniae using morphological, biochemical and Vitek, as well as investigation about pneumolysin phenotypically and molecularly through ply gene and sent the PCR products to sequencing by sanger method. Fifty sputum specimens were collected from patients at AL Salam Hospital, Iben Sina/ Mosul/ Iraq, from August 2021 to March 2022. The isolated bacteria were identified depending on morphology and biochemical properties; Vitek and the ply gene were detected by PCR technique. Five isolates of Streptococcus pneumoniae showed the ability to produce pneumolysin when tested by a double agar layer. When PCR reaction was performed on the Streptococcus pneumoniae pneumolysin gene, the results on gel electrophoresis showed three bands with 238 bp, and the ratio of the presence of the ply gene was 80%. PCR products were then submitted to sequencing by the Sanger method, and the ply sequencing result showed Point mutations that nucleotide and amino acid change with location. Keywords: Streptococcus pneumonia; pneumolysin; ply gene; PCR sequencing.

The current study aimed to detect S.pneumoniae, which is one of the gram-positive bacteria that gives partial α-hemolysis on blood agar media, as well as has the ability to complete blood hemolysis (beta)-hemolysis under anaerobic conditions. Fifty sputum specimens were collected from patients in Ibn sena and Al Salam hospitals in Mosul/ Iraq from august 2021 to March 2022. Five isolates were diagnosed as S.pneumoniae which exhibited an ability to produce hemolysin when phenotypically investigated. Polymerase chain reaction (PCR) technique was used to detect hlys gene Electrophoresis results showed four bands (80% of isolates) with a molecular size of 296 bp the PCR product were then sequenced.