Establishment of Multiplex Solid-Phase Strip PCR Test for Detection of 24 Ocular Infectious Disease Pathogens.

PURPOSE

To establish and evaluate a new multiplex solid-phase strip polymerase chain reaction (strip PCR) for concurrent detection of common ocular infectious disease pathogens.

METHODS

A new multiplex strip PCR was established to detect 24 common ocular infectious disease pathogens: herpes simplex virus (HSV) 1, HSV2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) 6, HHV7, HHV8, human T-cell lymphotropic virus (HTLV)-1, adenovirus, Mycobacterium tuberculosis, Treponema pallidum, Propionibacterium acnes (P. acnes), bacterial 16S ribosomal RNA (rRNA), Candida species (Candida sp.), C. glabrata, C. krusei, Aspergillus, Fusarium, fungal 28S rRNA, Toxoplasma (T. gondii), Toxocara, Chlamydia trachomatis (C. trachomatis), and Acanthamoeba. Strip PCR was tested with a negative control (distilled water) and standard positive control DNA. Cutoffs of quantification cycle (Cq) values were determined with noninfectious ocular samples to avoid false-positives caused by contamination with P. acnes, bacterial 16S, and fungal 28S from reagents and ocular surfaces. A pilot study to evaluate the strip PCR was performed using infectious ocular samples (aqueous humor, vitreous, cornea, and tears) by strip PCR and previously developed capillary-type multiplex PCR and quantitative real-time PCR (qPCR).

RESULTS

Strip PCR was verified with negative and positive controls. Strip PCR rapidly detected HSV1, HSV2, VZV, EBV, CMV, HHV6, HHV7, HTLV-1, adenovirus, P. acnes, bacterial 16S, Candida sp., C. glabrata, Aspergillus, fungal 28S, T. gondii, C. trachomatis, and Acanthamoeba in patient samples. The sensitivity was comparable to that of qPCR.

CONCLUSIONS

Our novel strip PCR assay is a simple, rapid, and high-sensitivity method for detecting ocular infectious disease pathogens.