Bacterial detection and quantification

Rapid detection by real-time PCR has been an important advantage where early diagnosis and appropriate antibiotic therapy are vital for survival, and traditional methods are often time-consuming. For instance, the early detection of bacterial DNA in the blood of critically ill patients with traditional culture diagnostic is still technically difficult (Cursons et al., 1999). Real-time PCR assays have been developed for quantification of different bacteria, including Chlamydia pneumoniae in human atherosclerotic plaques (Ciervo et al., 2003), intestinal bacterial populations (Ott et al., 2004) and Streptococcus pneumoniae in nasopharyngeal secretions (Saukkoriipi et al., 2004). Helicobacter pylori is considered to be the major causative agent of gastritis in acute or chronic forms and an important factor for etiology of peptic ulcer and gastric cancer. Real-time PCR technique based on the amplification of a fragment of the 23S rRNA gene has been developed (Lascols et al., 2003). This system aimed at detecting H. pylori directly from gastric biopsy specimens, quantifying the bacterial density and testing the susceptibility of the strain to clarithromycin. Realtime PCR also proved its high sensitivity for assessment of CagA protein, which is one of the main virulence factors of H. pylori, as a diagnostic tool for the pathogenity of H. pylori infection (Yamazaki et al., 2005). The detection of H. pylori colonization has been performed using a sensitive and specific quantitative test based on the amplification of the fragment 26-kDa Helicobacter-specific gene by real-time PCR (SYBR® Green I) and sensitivity of the developed system is reported as five bacterial cells per PCR sample (Mikula et al., 2003)

The protection of populations against a bioterrorism act has become a major concern for the governments. For such threats, a rapid, specific and sensitive detection of the microbial agent is of paramount importance. Yersinia pestis, is enzootic in many countries and the causative agent for bubonic plague, can be viewed as a potential biological weapon, according to its ability to cause high rates of morbidity and mortality in humans, with fatality rates of 50-100% if untreated (Brubaker, 1991). The intentional dissemination of plague would most likely occur by aerosolization, causing fulminant pneumonia in exposed individuals (Loiez et al., 2003). Out of 11 species of Yersinia, only three are pathogenic for humans. This may cause false identification in commercially available biochemical identification systems (Tomaso et al., 2003). With recently developed real-time PCR systems for detection, identification and quantification of Yersinia pestis

(Tomaso et al., 2003; Loiez et al., 2003), it is possible to achieve sensitive and reliable results in rapid diagnosis of the pneumonic plague which is the form of plague most likely to result from a biological warfare attack.

Real-time PCR has also improved the detection of Bacillus anthracis, another potential biological terrorism tool. Bacillus anthracis is etiologic agent for zoonotic disease anthrax, and recognized as a potential agent of bioterrorism. Because of non-specific early symptoms of disease Bacillus anthracis infection and diagnosing anthrax in humans is difficult (Makino and Cheun, 2003). Identification of the organism in cultures, clinical specimens and environmental samples are essential for ensuring that patients infected with B. anthracis are treated as soon as possible (Ryu et al., 2003). Therefore, detecting and monitoring anthrax spores in the environment, especially in the air, can improve prognosis due to earlier initiation of therapies and prevent spread of the infection. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis but it takes 24 to 48 hours or longer (Bell et al., 2002). Conventional PCR-based techniques were considered as a useful tool for detection of Bacillus anthracis but they had limitations. Rapid-cycle real-time PCR instruments, like LightCycler®, can be used for reliable and fast detection without carryover contamination (Makino and Cheun, 2003; Bell et al., 2002).

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