Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections

Abstract

Background Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. Methods Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. Results The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. Conclusion The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

Improved Canker Processing and Viability Droplet Digital PCR Allow Detection of Erwinia amylovora Viable Nonculturable Cells in Apple Bark

“…Although narrow, the obtained dynamic range with the QX200 ddPCR fits the results reported using other dPCR platforms [ 32 , 33 , 34 , 35 , 36 ]. The dynamic range of most commercially available dPCRs is of four orders of magnitude, mainly because of the limited sample partitioning capacity of the current dPCR platforms.…”

Digital and Absolute Assays for Low Abundance Molecular Biomarkers

“…Surface-capture platforms are particularly suitable for this multiplexing technique, as multicolor single-molecule fluorescence microscopy is well-developed and widely accessible, and diverse classes of optical molecular barcodes have been developed. , As an example, the Nanostring nCounter can distinguish and quantify >500 genes in the same sample using bar-coded fluorescent tags . Small degrees of multiplexing have also been achieved in digital assays based on solution partition including ddPCR and Simoa. , These readouts use spectrally distinct sensors or fluorescent beads for each target, which is intrinsically limited compared with more information-dense codes of molecular probes that include both ratiometric intensities and spatial ordering.…”

“…16 Small degrees of multiplexing have also been achieved in digital assays based on solution partition including ddPCR and Simoa. 27,32 These readouts use spectrally distinct sensors or fluorescent beads for each target, which is intrinsically limited compared with more informationdense codes of molecular probes that include both ratiometric intensities and spatial ordering.…”

Development and Preliminary Application of a Droplet Digital PCR Assay for Quantifying the Oncolytic Herpes Simplex Virus Type 1 in the Clinical-Grade Production

“…DdPCR has been proved to be a sensitive, repeatable, and rapid method used in research [ 23 ] and field studies [ 24 ]. As a result, ddPCR has been substantially applied into the quantification of the gene copy number [ 25 ], clinical diagnoses of various microorganisms’ infections [ 26 , 27 ], industrial production [ 28 ], and a variety of research as well [ 29 , 30 ]. DdPCR is a more mature dPCR technology that has been widely used in medical investigations and clinical applications [ 31 , 32 ].…”