Simultaneous detection of 13 viruses involved in meningoencephalitis using a newly developed multiplex PCR Mag-array system.

Abstract

The early detection and identification of pathogens in central nervous system viral infections associated with neurological disease increases the survival rate. However, the limitations of current diagnostic methods contribute to a lack of proper diagnosis in 62% of patients. Therefore, a robust method for detecting multiple viruses in a single reaction with high specificity, throughput, and speed is required. A multiplex PCR Mag-Array (MPMA) system was developed that integrates three strategies: chimeric primer design, temperature switch PCR, and MagPlex-TAG techniques. The MPMA was used to amplify 13 target viral sequences simultaneously, with plasmids containing specific viral sequences as standard samples. To evaluate its clinical performance, 177 cerebrospinal fluid (CSF) samples were tested. The MPMA system presented high specificity and efficiency in detecting a control panel of 13 plasmids. Among 177 CSF samples, consistent results were achieved for 19 samples pre-tested using a commercial kit. Viral pathogens were found in 28/138 undiagnosed samples, with herpes simplex viruses (HSV-1 and HSV-2) being predominant. The 20 non-infectious samples revealed negative results. Compared to sequencing methods, sensitivity for detecting HSV-1 and HSV-2 was 100% and 98.78%, respectively, and specificity was 100% and 98.22%, respectively. A robust MPMA system that can simultaneously and reliably detect 13 meningoencephalitis-associated viruses with high specificity, throughput, and speed has been developed.