Standardization of multiplex PCR for detection of viral uveitis causative agents

Abstract

Purpose : Early diagnosis of infectious uveitis may prevent vision loss, but given the small volume, it is notoriously difficult to test ocular fluid for specific viruses in uveitis. Multiplex PCR is an ideal technique to diagnose viral uveitis, however, sensitivity of commercially available kits is an important concern. This study was aimed to optimize primers to increase sensitivity of multiple real time PCR for diagnosis of two viral uveitis causing agents, herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV).

Methods : In the first steps, we performed uniplex PCR to evaluate the sensitivity of at least four different primers designed to detect each HSV-1 (McKrae strain) and HCMV (AD169 strain). During this step, different pathogen loads of virus were used to test the load dependent sensitivity of primers. After optimization of primers with uniplex PCR, the sequences were further validated at least three times each with multiplex PCR. For this purpose, samples containing different concentrations ranging from low (1x10⁴) to high (1x10⁶) pathogen load mixed in bovine aqueous humor were used. TaqMan probes with intercalated DNA fluorophore (VIC for HSV-1 and FAM for HCMV) primers were used for multiplex PCR validation.

Results : Primer validation resulted in the sequences F: 5’-GCCTTTTGTGTGTGTGTGGG-3’, R: 5’-AGGAAAGAGGAAACAGGCCG-3’ being sensitive to detect low (1x10⁴) and high (1x10⁶) pathogen load of HSV-1 glycoprotein B, while F: 5’-CAAGTGACCGAGGATTGCAA-3’, R: 5’-CACCATGTCCACTCGAACCTT-3’ was sensitive for detecting low (1x10⁴) and high (1x10⁶) pathogen load of HCMV immediate–early protein 1. The same sequences designed with TaqMan probes with intercalated DNA fluorophore primers showed efficient detection of either HSV-1 or HCMV in standalone or mixture of both viruses at different pathogen loads. The cycle threshold values ranged between 19 to 11 and 22 to 14 for low and high pathogen load of HSV-1 and HCMV respectively.

Conclusions : The primers optimized for multiplex PCR were found to be highly sensitive and specific for HSV-1 and HCMV. The results were reproducible and can be used for simultaneous or independent detection of either of the two viruses with the use of only 5 µl of aqueous humor. Further studies will be needed to confirm the efficacy of primers in clinical samples.

This is a 2021 ARVO Annual Meeting abstract.