Multiplexed assays have greater clinical sensitivity
There are two kinds of sensitivities: analytical sensitivity, and clinical sensitivity. Analytical sensitivity is all about copy numbers: How many copies would your test need to give a positive result? The more sensitive an assay is, the less copy it will require to yield a positive result. Clinical sensitivity is all about the patient: when a patient is sick with a disease, how often could your assay give an answer? People often get these two kinds of sensitivity mixed up. They would say, “when I compare a multiplexed assay, its sensitivity is not as good as a regular real-time PCR.” ...
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Reinventing PCR: Who?
Why us? why we were the lucky ones that developed the new multiplex PCR strategies? The Polymerase Chain Reaction was invented more than 20 years ago, the inventor, Kary Mullis, won Nobel Price in 1993. I believe that the reason why so many people failed to make multiplexing work is because we all trusted the TM (melting temperature) too much. During early years of PCR, some cleaver engineers developed software programs to aid the primer design. And at the core of many of these software, is the prediction of TM. It makes sense, for a pair of primers (or many ...
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Reinventing PCR: How?
To solve the three problems mentioned before, we came up with this multiplex PCR strategy called tem-PCR (for target enriched multiplex PCR). We use at least two pairs of nested primers for each amplification target, so if there is 10 targets we want to amplify together (multiplex), there will be total of 40 primers (4x10). The inside primers for each target have a tag (a short DNA sequence) that is shared by all the target, and it can be recognized by a pair of primers that we call “superprimers”. The “trick” is that all these target specific nested primers were ...
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Reinventing PCR: What?
What are the challenges for developing multiplex PCR assays? The three major difficulties are: incompatibility, high background, and poor reproducibility. The first problem (incompatibility) makes assay development very difficult; the second problem (high background) makes automation very difficult; and the third problem (poor reproducibility) makes regulatory approval very difficult. These are the reasons why we still do not see many multiplex molecular assays on the market. First, the primer incompatibility issue. Each targets need one pair of primers to amplify. Yet each primer has its own “optimal” condition (temperature, pH, iron concentration, etc). To make two primers work together is ...
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Reinventing PCR: Why?
Why PCR need to be reinvented? Because it can not multiplex, of course! (Or should we learn from Clinton and say, “it is the multiplexing, stupid!”) When PCR was invented some 20 years ago, it can only amplify one target at a time. And it remain so for so many years. People tried to do multiplex PCR, but it is difficult. For 2-3 targets, it is fine, but more than 3, it become increasingly impossible. Why we need multiplex? Because many of the diseases, as we know it, are defined by symptoms. By sharing symptoms, diseases appears identical to physician's ...
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iCubate at the 15th Annual AMP meeting
iCubate Reveal our platform for the first time at the 15th Annual AMP meeting in Orlando. Please visit us at booth 1523 ...
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