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The reaction mix contains sulfurylase, luciferase, APS and luciferin in addition to classical PCR reagents. In pyrosequencing, a linear amplification of the template is performed and the synthesis of the reverse complement strand can be monitored online. Since Sanger sequencing is well established but still comes along with some drawbacks, like the inability to sequence extremely short PCR amplicons often used in diagnostic PCR, and still some hours to obtain a result, pyrosequencing has evolved to be a promising alternative. Alternatively, the PCR product can be sequenced to obtain information on such polymorphisms. With carefully designed probes, this can be achieved thanks to characteristic single nucleotide polymorphisms (SNPs) using methods such as fluorescence curve melting analysis. Particularly in this setting, a subsequent typing of the pathogen may be required for the completion of the diagnosis. These stretches can be either selected to be highly pathogen-specific or to encompass a whole group, genus or family of pathogens. Today the identification of infectious pathogens is usually based on the PCR-amplification and detection of stretches of the pathogen’s genome. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A–F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software.
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Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap.
#FINDING SNP USING BIOEDIT SOFTWARE#
Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens.