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The page below is a sample from the LabCE course Real-Time PCR. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Microbe Detection

Because real-time PCR can amplify extremely small amounts of genetic material, it can be used to detect and identify minute numbers of microbes in a specimen or in the environment. It can even be used to detect DNA of microorganisms that are nonviable or difficult to grow in culture. Before PCR and other amplification methods, microbes had to be cultured in order to obtain enough genetic material to analyze. Thus, dead microorganisms were almost impossible to identify. Cloning for microbial DNA was the method generally used to garner genetic material from microbes that were hard to grow; this process can be extremely lengthy and time consuming. The use of PCR creates a fast and practical way to replicate microbial genetic material, whether alive or dead.

PCR is also commonly used for microbial fingerprinting in outbreak investigations as a way to confirm transmission or find the outbreak source. One can look for repeats in the genetic sequences by amplifying random polymorphic DNA, yielding PCR products of various sizes. These products can then be run through an electrophoresis gel, creating a band pattern that is known as the DNA fingerprint.

PCR can also be used to fingerprint an entire microbial community in lieu of a specific organism. A community fingerprint is created by amplifying a sequence that is found in all organisms of interest. Community fingerprints can be compared over time to identify stability and variations. PCR has an advantage over culture techniques due to the differences in growth requirements and growth time. With cultures, it is common to miss organisms and determine relative abundance and frequency.