Multiplex PCR: optimization and application in diagnostic virology.

PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.

PCR and restriction endonuclease analysis for rapid identification of human adenovirus subgenera.

Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.

Multiplex polymerase chain reaction for diagnosis of viral and chlamydial keratoconjunctivitis.

PURPOSE: To develop a multiplex polymerase chain reaction (PCR) for the detection of adenovirus, herpes simplex virus, and Chlamydia trachomatis in conjunctival swabs. METHODS: Oligonucleotide primers for detection of the 3 agents were combined in one reaction and evaluated for optimal performance using control DNAs of adenovirus type 2, herpes simplex virus, and C. trachomatis plasmid. The multiplex PCR was evaluated prospectively against its corresponding uniplex PCRs, virus isolation, Chlamydia Amplicor PCR, and an immunoassay technique (immune dot blot test) in a total of 805 conjunctival swabs from patients with suspected viral and chlamydial keratoconjunctivitis. RESULTS: The multiplex PCR was as sensitive as uniplex PCRs for the detection of the agents in clinical specimens. In the prospective study, 48 of 49 (98%) clinical specimens were positive for adenovirus by the multiplex PCR compared with 26 of 49 (53%) by adenovirus isolation. For herpes simplex virus detection, the multiplex PCR had a sensitivity of 92% (34/37) compared with 94.5% (35/37) by cell culture. The multiplex PCR produced identical results to the Amplicor PCR (21/21; 100%) compared with 71% (15/21) by the immune dot blot test. CONCLUSIONS: With clinical specimens the multiplex PCR was as sensitive as its respective uniplex PCRs but more sensitive than adenovirus isolation and as sensitive as herpes simplex virus isolation or C. trachomatis Amplicor PCR. It has the potential to replace several diagnostic tests with consequent savings in cost. The test also reduces the risk of misdiagnosis by the clinicians.

Polymerase chain reaction for detection of Chlamydia trachomatis in conjunctival swabs.

AIMS/BACKGROUND: Ocular Chlamydia trachomatis infection in the west occurs as opthalmia neonatorum, acquired from the mother, or adult paratrachoma which is also associated with current genital tract infection. Accurate rapid laboratory diagnosis facilitates management, but the relative merits of antigen detection or DNA amplification tests are unresolved. METHODS: A polymerase chain reaction (PCR) test was developed which amplified part of the plasmid shared by all the serovars of C trachomatis. Conjunctival swabs were tested using an in house immune dot-blot test (IDBT) for chlamydial lipopolysaccharide antigen, a commercial direct fluorescent antibody (DFA) test for chlamydial elementary bodies, and the PCR (DNA extracted using guanidinium lysis buffer). RESULTS: The PCR achieved a detection limit of 100 plasmid copies (10 elementary bodies). In a combined retrospective and prospective clinical evaluation, the PCR and IDBT gave identical results with 21 positive and 57 negative eye swabs. However, interpretation of the DFA test required meticulous examination of the stained smear, sometimes by two microscopists. CONCLUSIONS: The PCR is likely to play an increasing role in the diagnosis of ocular C trachomatis infection because of its excellent sensitivity and specificity.