In a routine request to exclude relevant viral hemorrhagic fevers in a hospitalized patient we received serum to run PCRs for EBOV, LASV and YFV. Since there are currently about 40 validated in-house RT-PCR assays in our portfolio, not every single assay is subjected to comparison, but some in-house assays are selected exemplarily and compared. According to quality management rules, we always compare a new batch of the basic reaction mix with the old one. These assays are thoroughly validated with this reaction mix. For those viruses, for which there are no commercially available kits and also for confirmation of positive results obtained by using kits, we apply in-house tests using a basic one-step RT-PCR reaction mix from a manufacturer A. One pillar of this diagnostic portfolio is one-step real-time RT-PCR-based detection of viruses causing hemorrhagic fever, like Ebola virus (EBOV), Marburg virus (MARV), Lassa virus (LASV), Yellow fever virus (YFV), and viruses relevant for differential diagnosis. Our division deals with the diagnostics of various highly pathogenic viruses, with real-time PCR as the most popular approach. Here we describe a PCR “case report” with unexpected results in a routine PCR troubleshooting. As long as the results obtained are not different, the new reagent batch is accepted as fully functional. For example, in compliance with quality management, a new batch of a reagent has to be compared with the old one in the same run with the same assay. However, also ready-to-use basic mixes can be subject to a varying PCR performance and have to be controlled for functionality. These ready-to-use mixes have become popular since they contribute to a certain level of standardization and speed up and ease hands-on times. A well-accepted approach to reduce the risk of making an error in preparing the basic PCR mix is the utilization of a ready-to-use mastermix that contains all components except for the primers and the probe(s). The PCR set-up is then started from scratch with new aliquots of components and in most cases the problem is solved. Often problems can be explained by the fact that essential components like Mg 2+ ions or even primers were of poor quality because the expiration date has passed or were unintentionally not added to the reaction mix. The reason for PCR failure is usually rapidly identified. In this context, there is a kind of unwritten law that everyone who has set up PCR reactions by themselves has experienced trouble with amplifications that somehow unexplainably fail. The basic reaction mix can be set up from all mentioned individual components. Components that are common to any PCR and are present in the basic reaction mix are the enzyme for PCR or RT-PCR, the buffer required for the enzymatic reaction, Mg 2+ or other bivalent ions, dNTPs or derivatives, water, and finally as target-specific ingredients primers and probes. The manufacturer and the specifications of PCR components used in in-house PCR assays have to be selected by the user who, in general, relies on his previous experience. For these issues usually in-house assays have to be established and validated by expert laboratories, which can only be as good as it is technically feasible regarding the often low isolate numbers available. This applies for example to the field of neglected as well as new and emerging infectious diseases. However, there are at least as many PCR applications in the field of PCR diagnostics for which there is no real market and unfortunately no commercially available kits are being developed. Ĭonsequently several standardized PCR kits for most clinically important and frequently needed molecular targets have been made commercially available by several manufacturers. In most cases real-time PCR applications have replaced conventional PCR tests due to their obvious benefits, namely speed, increased specificity, (semi-) quantification, reduced work-load, and a minimized risk of carry-over contamination, which is of benefit especially in diagnostic labs. During the past years PCR has become the gold standard for the molecular diagnostics of manifold diseases, such as cancer or infectious diseases.
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