Molecular-based methods that have been used to detect and identify bacteria include nucleic acid sequence-based amplification (NASBA), QP-replicase, and PCR, including the following modifications: real-time or quantitative, reverse transcriptase, nested, and multiplex (see Chapter 7 for explanations of these methods). Product detection is accomplished by a variety of methods including Southern blot hybridization (see Chapter 6), agarose gel electro-phoresis (see Chapter 5), PCR (see Chapter 7), sequenc ing (see Chapter 10), enzyme immunoassay, dot blot hybridization (see Chapter 6), and restriction enzyme analysis (see Chapter 6).
Real-time PCR, or quantitative PCR (qPCR), is used increasingly for detection of infectious agents as it provides the sensitivity of PCR with more information than is available from conventional PCR. The quantitative capability of qPCR allows distinction of subclinical levels of infection (qualitatively positive by conventional PCR) from higher levels with pathological consequences. Furthermore, qPCR programs can be designed to provide closed-tube sequence or typing analysis by adding a melt curve temperature program following the amplification of the target (Fig. 12-2). Like conventional PCR, qPCR can be performed on DNA extracted directly from clinical specimens, including viral, bacterial, and fungal pathogens.
Design of a qPCR method requires selection of a target gene unique to the specimen or specimen type for which primers and probes can be designed. The DNA-specific dye, SYBR Green, can be used in place of probes if the amplicon is free of artifacts such as misprimes or primer dimers (see Chapter 7). Probe types used most often include fluorescent energy transfer hybridization probes and hydrolysis (TaqMan) probes. The require-
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