Molecular Methods for Identifying Bacteria in Lower Respiratory Tract Specimens Among Patients With Pneumonia by Multiplex PCR

Pneumonia remains a worldwide health problem with a high rate of morbidity and mortality. Identification of microbial pathogens which cause pneumonia is an important area for optimum clinical management of pneumonia patients and is a big challenge for conventional microbiological methods. The development and implementation of molecular diagnostic tests for pneumonia has been a major advance in the microbiological diagnosis of respiratory pathogens in recent years. Targeted antibiotic selection and more effective de-escalation and improved stewardship for pneumonia patients.

PCR is a simple, yet elegant, enzymatic assay, which allows for the amplification of a specific DNA fragment from a complex pool of DNA.. Only trace amounts of DNA are needed for PCR to generate enough copies to be analyzed using conventional laboratory methods. For this reason, PCR is a sensitive assay.

Each PCR assay requires the presence of template DNA, primers, nucleotides, and DNA polymerase .The DNA polymerase is the key enzyme that links individual nucleotides together to form the PCR product. The nucleotides include the four bases - adenine, thymine, cytosine, and guanine (A, T, C, G) - that are found in DNA. These act as the building blocks that are used by the DNA polymerase to create the resultant PCR product. The primers in the reaction specify the exact DNA product to be amplified. The primers are short DNA fragments with a defined sequence complementary to the target DNA that is to be detected and amplified. These serve as an extension point for the DNA polymerase to build on.There are multiple advantages to PCR. First, it is a simple technique to understand and to use, a nd it produces results rapidly. It is a highly sensitive technique with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. Although PCR is a valuable technique, it does have limitations. Because PCR is a highly sensitive technique, any form of contamination of the sample by even trace amounts of DNA can produce misleading results . In addition, in order to design primers for PCR, some prior sequence data is needed. Therefore, PCR can only be used to identify the presence or absence of a known pathogen or gene