Multiplex PCR for the diagnosis of viral and chlamydial conjunctivitis
Başlık:
Multiplex PCR for the diagnosis of viral and chlamydial conjunctivitis
Yazar:
Elnifro, Elfatah M., author.
ISBN:
9780438043206
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (266 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Adenovirus, herpes simplex virus (HSV) and Chlamydia trachomatis are common causes of keratoconjunctivitis. Prompt and accurate laboratory investigation of these agents is often necessary for infection control and effective patient management. A multiplex polymerase chain reaction (PCR) was developed for rapid, simultaneous detection and differentiation of these aetiological agents. The test was evaluated prospectively against its corresponding uniplex PCRs, vims isolation or a commercial PCR (Amplicor) and immune dot blot (IDB) test for detection of C. trachomatis. The primer pairs ADRJC1/ADRJC2 that target a region of the hexon gene of adenoviruses, YS1/YS2 derived from the polymerase gene of HSV and the primer pair KL1/KL2 for the C. trachomatis plasmid DNA were combined to construct a sensitive and specific multiplex PCR termed the 98-multiplex PCR. Optimisation of the test required adjustment of reaction conditions such as Taq polymerase concentration, magnesium chloride and number of cycles. The multiplex PCR, in presence of single target, produced detection limits of 400 copies of adenovirus DNA, 300 copies of HSV DNA or 100 copies of C. trachomatis plasmid DNA. In a total of 805 eye specimens from 504 patients with keratoconjunctivitis, the multiplex PCR produced identical results to its corresponding uniplex PCRs. The multiplex PCR was significantly more sensitive than virus isolation in detecting adenoviruses or as sensitive as virus isolation for detection of HSV. For C. trachomatis detection, the multiplex PCR produced identical results to those obtained by the Amplicor PCR but both tests were more sensitive and easier to interpret than the IDB test. The multiplex PCR proved sensitive, cost effective and allows detection of agents that are not clinically suspected. In addition, determination and analysis of the nucleotide sequence of the adenovirus PCR products generated by the primer pair ADRJC1/ADRJC2 (140 base pairs) resulted in obtaining restriction endonucleases that produced subgroup- and sometimes serotype-specific restriction patterns. The latter allowed for development of a reliable and specific PCR-based subgenus identification of adenoviruses.
Notlar:
School code: 1543
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