In the second round of amplification a series of three internal primer pairs are introduced, producing type-specific amplicons that were easily differentiated based on size upon gel electrophoresis. Nested and multiplex polymerase chain reactions for the identification of bluetongue virus infection in the biting midge. A similar PCR format was used to develop a multiplex PCR for amplification of four different regions of the CMV genome (61). Neurotropic VirusesPCR has proved to be a powerful tool for investigating meningitis and encephalitis caused by a variety of viruses. Surveys of gene families using polymerase chain reaction: PCR selection and PCR drift. PCR products were detected with anti-dinitrophenyl monoclonal antibody and horseradish peroxidase (PCR-enzyme immunoassay). These include PCRs which combined primers to produce a type-specific product size (97, 103) or those that utilized degenerate primers for HPV screening and strain-specific antisense primers for simple typing (56). The former often eliminates nonspecific reactions (particularly production of primer dimers) caused by primer annealing at low temperature (4 to 25°C) before commencement of thermocycling (21). The test could also facilitate the primary classification of unknown virus isolates. Two approaches have been used to address this problem. In heart transplant recipients a hot start nested multiplex PCR was used to evaluate the possible reactivation of HHV-6 and HHV-7 (67). This should provide valuable information for monitoring and treating patients with HPV-related lesions, although the method lacks the capability to define individual HPV types, which limits its usefulness in epidemiological investigations. Clipboard, Search History, and several other advanced features are temporarily unavailable. In the group positive for VZV (28 patients), 16 patients had meningitis and 10 had encephalitis but clinical details were not available for 2 patients. PCR drift is a bias assumed to be due to stochastic fluctuation in the interactions of PCR reagents particularly in the early cycles, which could arise in the presence of very low template concentrations (26, 68); variations in the thermal profiles of a thermocycler, resulting in unequal ramping temperatures; or simple experimental error. In a representative application of multiplex PCR to detect the presence of food toxicants, Kumar et al. In a multiplexing assay, more than one target sequence is amplified by using multiple primer pairs in the same reaction mixture. The other approach is to test sequential specimens and only recommend treatment if positive results persist rather than responding to what may be a transient reactivation of a herpesvirus. The accuracy of this approach was confirmed by examining cervical scrapings from 44 patients. This approach proved to be a rapid, sensitive, and reliable method for the detection and typing of influenza virus and for screening for influenza virus variants, proving capable of identifying new influenza B virus variants (126). The multiplex PCR was used extensively for pathogen identification, Single Nucleotide Polymorphism (SNP) genotyping, mutation analysis, gene deletion analysis, template quantitation, linkage analysis, RNA detection, forensic studies and diet analysis (18, 19). These studies demonstrate the utility of this multiplex RT-PCR for detection of enteroviruses and herpesviruses in CSF samples from patients with various neurological manifestations and the usefulness of the technique in patient management and design of antiviral therapy. This modification allowed a simple simultaneous amplification of both viruses with 100% concordance with its respective uniplex PCRs (47). Multiplex PCR Product Listing Application Overview. Likewise, precautions and methodologies to avoid false-negative results due to reaction failure have to be considered (104). HPVs were detected in samples from 69 (90.8%) of the 76 patients with cervical carcinoma, of which high-risk HPVs accounted for 82.9%. Simultaneous detection of three common sexually transmitted agents by polymerase chain reaction. Thus, the protocol can be used for efficient identification of transplant recipients at risk of clinically significant infection. 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