Designing and Optimizing a Multiplex PCR Method for the Simultaneous Identification of Respiratory Pathogens from Patients in Selected Hospitals of Mashhad

Abstract

Background and Objective: Pneumonia is considered one of the most serious and fatal diseases among children and adults worldwide. Streptococcus pneumoniae and Haemophilus influenzae are pathogenic bacteria causing community-acquired pneumonia. Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila are another group of acute pathogens. The present study aims to identify the mentioned infectious agents through multiplex PCR (Multiplex Polymerase Chain Reaction).

Materials and Methods: This experimental and developmental study used the multiplex PCR method to identify the specific sequences of the genome of prominent pneumonia agents simultaneously. The specific genes selected in this study are LytA for Streptococcus pneumoniae, BexA for Haemophilus influenzae, P1 for Mycoplasma pneumoniae, Mip for Legionella pneumophila, and ompA for Chlamydia pneumoniae. Also, the 16s ribosomal gene was used as a positive control in this study.

Results: In this study, 180 patients with respiratory infections who were referred or hospitalized at Mashhad Hospital were sampled. The results showed that 13 samples were positive. The frequency distribution of Streptococcus pneumoniae with 5 people (2.8%) was the most bacteria isolated. Additionally, Legionella pneumophila bacteria were isolated in two people, and Mycoplasma pneumoniae bacteria and Haemophilus influenzae bacteria were isolated in three people (1.6%). Chlamydia pneumoniae was not isolated. The results revealed that simultaneous identification of Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, and Mycoplasma pneumoniae is possible in one PCR reaction.

Conclusion: This method can be used in the direct and simultaneous identification of these factors in respiratory samples.