[Multiplex Real Time PCR for Atypical Respiratory Pathogens]

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Table of Contents

CHAPTER: LITERATURE REVIEW5

Introduction5

Multiplex Real-Time PCR5

Real-Time PCR6

Community-acquired pneumonia6

Causes of Community Acquired Pneumonia7

Major Causes7

Legionella pneumophila7

Diagnosis and Treatment8

Mycoplasma pneumoniae9

Treatment and Diagnosis10

Chlamydophila pneumoniae11

Treatment and Diagnosis11

Pathogenesis of Community Acquired Pneumonia12

Microbiology and Etiology13

REFERENCES16

Chapter: Literature Review

Introduction

Respiratory diseases have had a major impact on the overall health of cattle and continue to be of great importance even today. Many of the diseases that have been shown to impact the respiratory tract of cattle have been grouped into an overall category known as bovine respiratory disease (BRD) complex. This includes shipping fever syndrome, mucosal disease, enzootic calf pneumonia, acute respiratory distress syndrome, hemorrhagic syndrome, and atypical interstitial pneumonia (Gogorza, 2006, 209). Pathogens that have been implicated in the causation of this complex include microbial and viral pathogens. Although there is a large list of pathogens that contribute to BRD, the clinical signs of infection are very similar. Typical signs include rapid respiration, anorexia, nasal and/or ocular discharge, depression, fever, interstitial pneumonia and reproductive failure (Gogorza, 2006, 212).

Multiplex Real-Time PCR

Multiplexing is the use of multiple primers to allow amplification of multiple templates within a single reaction. The term multiplex real-time PCR is more commonly used to describe the use of multiple fluorogenic oligoprobes for the discrimination of multiple amplicons (Saliki, 2004, 69). As the number of fluorophores has increased, this method has become much more common because of its ability to differentiate multiple amplicons in a single reaction. So far it has been used for the detection and typing of different BVDV strains grown on Madin-Darby bovine kidney (MDBK) cells (Saliki, 2004, 74).

Real-Time PCR

In contrast to conventional RT-PCR, Real-Time RT-PCR allows one to visualize the amplification of the amplicon as it progresses in real time. This approach has provided great insight into the kinetics of the reaction. The monitoring of accumulating amplicon in real time has been made possible by the labeling of primers, probes or amplicon with fluorogenic molecules. Real-time has gained wider acceptance because of its improved rapidity, sensitivity, reproducibility, and the reduced risk of carry-over contamination (Young, 2006, 218). Increased speed is largely the result of reduced cycle times, removal of post-PCR detection procedures, and the use of fluorogenic labels and sensitive methods for detecting their emissions. Disadvantages of using real time in comparison to conventional PCR include the inability to monitor amplicon size without opening the system, the incompatibility of some platforms with some flurogenic chemistries, and the relatively restricted multiplex capabilities of current applications. In addition, start up cost is high and may be prohibitive to low through ...