Primers and probes
Primers and probes targeting HSV1, HSV2, and HPeV were reported previously. Primer-probe set targeting highly conserved 5’ untranslated region (5’UTR) of EV was designed from an alignment of GenBank sequences using Primer Premier 5.0 (Primer Biosoft International, USA). The specificity and performance of all primers and probes were evaluated by BLAST analysis and synthesized by Sangon, China. EV probe was labeled with a fluorophore of FAM. HSV1 was labeled with VIC. HPeV and HSV2 were labeled with FAM and VIC. The sequences of the primers and probes and their optimized concentrations were given in Table 1.
Table 1
Sequences of primers and probes
|
|
Concentration, nmol/L
|
|
Primer/probe
|
Sequence (5’-3’)
|
Simplex ddPCR
|
Quadruplex ddPCR
|
Ref
|
EV-fora
|
GGTGTGAAGAGCCTATTGAGCT
|
400
|
400
|
In house
|
EV-revb
|
GAAACACGGACACCCAAAGTAGT
|
400
|
400
|
|
EV-probe
|
FAM-TCCTCCGGCCCCTGAATGCGGCTAATCC-BHQ1
|
200
|
200
|
|
HSV2-for
|
CGCTCTCGTAAATGCTTCCCT
|
400
|
400
|
[23]
|
HSV2-rev
|
TCTACCCACAACAGACCCACG
|
400
|
400
|
|
HSV2-probe
|
FAM-CGCGGAGACATTCGAGTACCAGATCG-BHQ1
|
—
|
100
|
|
|
VIC- CGCGGAGACATTCGAGTACCAGATCG-BHQ1
|
200
|
100
|
|
HPeV-for
|
GTGCCTCTGGGGCCAAAAG
|
400
|
400
|
[24]
|
HPeV-rev
|
TCAGATCCATAGTGTCGCTTGTTAC
|
400
|
400
|
|
HPeV-probe
|
FAM-ACGGGTACCTTCTGGGCATCCTTCG-BHQ1
|
200
|
50
|
|
|
VIC-ACGGGTACCTTCTGGGCATCCTTCG-BHQ1
|
—
|
150
|
|
HSV1-for
|
CGGCCGTGTGACACTATCG
|
400
|
400
|
[23]
|
HSV1-rev
|
CTCGTAAAATGGCCCCTCC
|
400
|
400
|
|
HSV1-probe
|
VIC-CCATACCGACCACACCGACGAAC-BHQ1
|
200
|
400
|
|
aForward primer |
bReverse primer |
Test materials, nucleic acid extraction, and reverse transcription
EV strain and positive clinical specimens of HSV1/2 and HPeV confirmed by sanger sequencing were used for assay development and performance evaluation. The viral RNA and DNA were simultaneously extracted and purified with a kit on an automated extraction machine both from Tianlong, China. Reverse transcription was performed then on T100 thermal cycler (Bio-Rad, USA) with a mixture of 8μl of EV or HPeV nucleic acid, 4μl of 5×PrimeScritBuffer, 2μl of Random 6mers, 1μl of PrimeScript RT Enzyme Mix I, and 5μl of nuclease-free water (TaKaRa, Japan). The reaction was carried out at 37°C for 30min and 85°C for 5s.
Simplex and quadruplex ddPCR
The QX200 Droplet Digital PCR System (Bio-Rad, USA) was used for the assay development of simplex and quadruplex ddPCR. For simplex ddPCR, the reaction mixture consisted of 10 µL 2× ddPCR Supermix for probes (No dUTP), a pair of specific primer-probe set including 1 µL primers and 1 µL probes at a final concentration of 400 and 200nM, respectively, 2 µL cDNA/DNA, and 6 µL nuclease-free water to 20 µL.
As the QX200 ddPCR System has only two detection channels, two targets at most can be quantified within a single reaction in general settings. In this study, we adopted the strategy of probe ratio-based multiplexing to develop a quadruplex ddPCR assay. Based on the simplex ddPCR, 4 sets of primers and probes were added in one single reaction to develop the quadruplex ddPCR. For EV, 200 nmol FAM-labeled probe was added; for HSV1, 400 nmol VIC-labeled probe was added; for HSV2, 100 nmol FAM-labeled and 100 nmol VIC-labeled probe were added; for HPeV, 50 nmol FAM-labeled and 150 nmol VIC-labeled probe were added. All the primers were added at a final concentration of 400nM. A reaction mixture of 20μL was composed of 10μL 2×ddPCR Supermix for probes (No dUTP, Bio-Rad), 1μL primer mix, 1μL probe mix, 2μL cDNA/DNA, and 6μL nuclease-free water. The 20μL reaction mix and 70μL droplet generation oil were added to a droplet generation cartridge (DG8, Bio-Rad) to generate approximately 20,000 nanoliter-sized droplets in a droplet generator (QX200 system, Bio-Rad). Then 40μL emulsion made of water-in-oil droplets was transferred to a 96-well plate and sealed with a pierceable aluminum foil using a PX1 PCR plate sealer (Bio-Rad) at 180°C for 5 seconds. Samples were amplified in the T100 Thermal Cycler (Bio-Rad) according to the following steps: 96°C for 10min (enzyme activation), 40 cycles of 98°C for 30s (denaturation) and 55°C for 60s (annealing), and 60°C for 2min. The amplified droplets were detected in a Droplet Reader (Bio-Rad).
Performance evaluation of quadruplex ddPCR
The cDNA/DNA mixture of four targets from EV stock and positive clinical specimens confirmed by Sanger sequencing after reverse transcription was used to evaluate the performance of the Quadruplex ddPCR assay. The copy number of each target was determined by simplex ddPCR and then diluted to the corresponding concentrations we need.
The analytical sensitivity or limit of detection (LOD) was assessed using dilutions of the cDNA/DNA mixture in DNase- and RNase-free water with concentrations of 50, 10, 5, and 2 copies per reaction. Each concentration was detected in 20 replicates. The LOD for each target was defined as the lowest dilution where at least 19 replicates still produced a positive result [25]. Based on the LOD assessment, the limit of quantification (LOQ) was defined as the lowest concentration at which the CV (coefficient of variation) of all replicates was below 25% [25].
Another group of 10-fold serial dilutions of nucleic acid mixture throughout 2000 to 2 copies per reaction was used to evaluate the dynamic range. Each concentration was run in 3 replicates.
For the limit of blank (LOB), 10 NGS-negative (next-generation sequencing) CSF samples and 4 no-template controls (NTC) were used. The number of positive droplets ≥LOB was considered positive.
The analytical specificity of the assay was determined by performing cross-reactivity tests in other pathogens susceptible to CNS infections as well including influenza virus, adenovirus, respiratory syncytial virus, rotavirus, Streptococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853), Streptococcus pneumoniae (ATCC49619), Escherichia coli (ATCC25922), Staphylococcus epidermidis, Staphylococcus hominis, group B streptococcus, Staphylococcus haemolyticus, Klebsiella pneumoniae and Acinetobacter baumannii (clinical isolation strains identified by mass spectrometry).
Validation of quadruplex ddPCR using clinical specimens
A total of 97 CSF samples from patients suspected of viral CNS infections were collected for assay validation from May 2020 to December 2021 and stored at -70℃ before use. These samples along with 10 HPeV-positive stool samples and 2 HSV2-positive secretion samples were simultaneously detected by qPCR/RT-qPCR and ddPCR methods. The commercial qPCR/RT-qPCR kits (HSV1 from Bai Tai, HSV2 from Da An, EV from Mo Le, and HPeV from Shuo Shi, China) were used to detect these clinical samples following the manufacturer's instructions on an Applied Biosystems 7500 system (Thermo Fisher, USA). The results of the two methods were compared. This study was approved by the ethical committee of the Children’s Hospital of Fudan University (2021276).
Data analysis
The results and data of ddPCR were generated and analyzed by QuantaSoft software version 1.7.4.0917 or QuantaSoft™ Analysis Pro version 1.0.596 when templates were relatively high (Bio-Rad). Linear regressions were done using the GraphPad Prism7 software and CV for LOQ was calculated by Excel.