The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. The decreased hands-on time, increased reliability and improved quantitative accuracy of RT-PCR methods have contributed to the adoption of RT-PCR for a wide range of new applications. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. Since the first practical demonstration of the concept real-time PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Real-time PCR offers several advantages over conventional PCR for a diagnostic laboratory, such as increased sensitivity, rapid cycling times, and reduced risk of contamination. [1]

Real-time PCR technology has been widely used in diagnostic. For example, Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic information for cereulide formation, a novel 5' nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay. [2]

Real-time RT-PCR is also the recommended test for confirmation of novel influenza A (H1N1) cases. Currently, novel influenza A (H1N1) virus will test positive for influenza A and negative for H1 and H3 by real-time RT-PCR. If reactivity of real-time RT-PCR for influenza A is strong (e.g. Ct <30) it is more suggestive of a novel influenza A (H1N1) virus. Confirmation as novel influenza A (H1N1) virus by real-time RT-PCR was originally performed only at CDC, but at this time it is available in Haffkine Institute, Mumbai, NIV, Pune, NICD, Delhi and few other laboratories. [3]

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