Applications in Qualitative PCR

The nPCR systems are currently widely employed in both qualitative, simple[3,4] or multiplex,[5-13] and quantitative1-14,15-1 diagnostic tests, as well as in PCR systems employed in the identification of viruses.[16-19] Regarding the above mentioned qualitative nPCRs, the primer design allows to detect from 10 to 100 genome copies per microliter of any adenovirus[4] or lyssavirus,[3] and can be performed from a variety of samples including cerebro-spinal fluid (CSF), urine, stools, respiratory swabs, conjunctival swabs, or brain necropsies. These nPCRs can be included in multiplex systems, especially adenovirus nPCR,[12] as the range in hybridization temperatures and biochemical conditions is very high.

The first nested multiplex PCR for detection and typing of herpesviruses (HSV-1 and -2, VZV, CMV, HHV-6, and EBV) was applied to CSF from patients with meningitis, encephalitis, and other clinical syndromes.[5] This assay was further modified to include a reverse transcription step and primer pairs to detect enterovirus cDNA.[6,7] Utilizing equimolar concentrations of primers aligning the 3' ends with one of two consensus regions within the herpesvirus DNA polymerase gene and the 5' ends with the related or nonrelated sequences of each agent to be amplified, the first round of amplification yielded a 194-bp fragment indicating the presence of herpesvirus. The second round of amplification utilizing primer mixtures contained nonhomologous and type-specific primers selected from different regions of the aligned DNA polymerase genes of human herpesviruses producing a product with a different size for each related virus. These studies demonstrate the utility of this multiplex RT-nPCR

for the 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. Additionally, a multiplex nPCR method was developed for the simultaneous detection and typing of all human lymphotropic herpesviruses described to date, including EBV, CMV, HHV-6, variants A and B, HHV7, and HHV8.[8]

A multiplex nPCR was also developed for differentiating simultaneously the DNA of polyomaviruses JC, BK, and SV40.[9] In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV, and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different sizes for each related virus. Cerebrospinal fluid was examined from AIDS patients with clinical and neurora-diological evidence of progressive multifocal leukoence-phalopathy and from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by hemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease. Other multiplex nPCR systems described allow the simultaneous detection of parainfluenza virus types 1 to 4,[10] measles virus, rubella virus, and parvovirus B19[11] or phlebovirus.[13]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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