b'There are different ways of approaching specificity in a qPCRHow should clinical laboratories choose between assay. We incorporated probes into our assays to allow forquantitative and qualitative PCR?specific detection of amplified DNA rather than generic amplifiedThis depends on the disease being diagnosed and what the DNA. We could have used a nonspecific dye to detect amplifiedneed is for patient care. While quantitative PCR (qPCR) assays DNA, but that would not tell us much about whether we hadare often used for microbial detection, they are typically not amplified exactly what we were looking for. validated for quantitative detection. Thats because there are very In my view, specificity, whether for an old-school PCR assay orfew microorganisms we have a strong need to quantify and also qPCR, comes down to assay design and validation. Validation isvery few clinically validated quantitative assays. An example of needed to analytically assess specificity, and is just as importanta regularly used quantitative test is HIV viral load measurement; today as it was 3 decades ago. this testing helps guide treatment. Conversely, for many microorganisms, such as Mycobacterium tuberculosis complex, Test developers need to consider what might cross react in thefor example, patients are either infected with the organism or not, assay and be falsely detected. From a microbiologists pointand having a little bit of that organism is no different, technically of view, such an ask can be daunting because there are manyspeaking, than having a lot of it. It would be like being a little microbes we havent yet characterized, named, or sequenced.bit pregnant.On one level, the developer has to think about the disease they are trying to diagnose and on another, the microbesbe theyIn the future, could qPCR assays become more useful pathogens or non-pathogenic microorganismsthat might bein diagnosing other kinds of diseases?present at the site being sampled. Quantifying accurately and precisely using diagnostic assays in Interestingly, nonspecificity can be helpful, if easily recognized.clinical microbiology is harder than people think. Truly quantitative For example, weve had scenarios where weve designed assaysassays usually rely on blood-based specimens because the to detect particular microorganisms but discovered noveldenominator is relatively straightforward: a volume of blood microorganisms that hadnt been known to cause human disease(or plasma) is standard.using our assays. We discovered them through the nonspecificityAt the Mayo Clinic, many PCR assays we use are performed of the assays, but we were able to easily recognize what wason non-blood specimen-types, such as body fluids, urine, happening because of our assay design. Since we were carefulor tissues. For these specimens, quantifying a denominator in how we designed our assays, it was possible to see that thereis more challenging than the situation for blood, and fraught was something being detected that was not what the assayswith variability. Beyond the technical challenge of being able were specifically designed for. to quantify microorganisms in clinical specimens, the clinical question being answered with such quantification would That is obviously not the goal, but it does tell you that we liveneed definition.in a world where we are still learning aboutand discovering There is also the question of the ideal limit of detection of a microorganisms. Some of them are likely to be pathogens we doqPCR assay. It depends on the microorganism being detected. not yet understand.With microorganisms like Mycobacterium tuberculosis complex, the laboratory needs to have the most sensitive assay possible. But there might be other scenarios where the laboratory is looking for microorganisms that could be part of the normal microbiome. In such scenarios, clinical significance may be based on the relative quantity detected.It all depends on what the laboratory is trying to accomplish with the assay. One approach is not necessarily better than another for all applications.8 Molecular testing thermofisher.com/infectiousdisease Contents'