Detection of Monkeypox Virus DNA in Airport Wastewater, Rome, Italy

Environmental surveillance can be a complementary tool for detecting pathogens circulating in communities. We detected monkeypox virus DNA in wastewater from Italy’s largest airport by using real-time PCR assays targeting the G2R region and F3L and N3R genes and sequencing. Wastewater surveillance can be quickly adapted to investigate emerging threats.


Comparison of Primers and Probe Sets
To select the primer and probe sets providing better real-time PCR results, a 10 −2 dilution of the Monkeypox DNA Slovenia ex Gran Canaria, Ref-SKU: 005N-04716 provided by EVAg was tested as control, using PCR IDs 1002, 1003, 1004, 1005, 1008, 1016 (Table 1).
For the sake of comparison, all primer/probe sets were assayed in the same conditions: real-time reactions were prepared in 25 μL volume using the TaqPath BactoPure Microbial Detection Master Mix (Thermo Fisher Scientific, https://www.thermofisher.com), 500 mmol of each primer, 250 nmol of each probe, and 5 μL of sample DNA. Amplification conditions included an initial activation at 95°C for 2 min, and 45 cycles of 10 s at 95°C and 30 s at 60°C.

Global (EVAg)
To standardize material for the optimization and performance characterization of the  Tenfold dilutions of the DNA stock were prepared in molecular grade Tris-EDTA buffer dilutions of 10 −2 , 10 −3 , 10 −4 and 10 −5 were tested in quadruplicate by ddPCR by using the QX200 system (Bio-Rad, https://www.bio-rad.com) and the ddPCR Supermix for probes kit without deoxyuridine triphosphate (dUTP) (BioRad). The reaction mixture included: 10 μL ddPCR supermix, primers 500 nmol, probes 250 nmol, and nuclease-free water to a final volume of 20 μL. Primers and probes were those described for PCR nos. 1003, 1004, and 1005. Droplets were generated as recommended by the manufacturer and amplification was performed on a 9600 GenAmp thermalcycler (Applied BioSystems) as follows: 95°C for 10 min, followed by 94°C for 30 s and 60°C for 60 s (40 cycles), and by a final stage at 98°C for 10 min. Results were acquired using the Bio-Rad QX200 Droplet Reader and QX Manager Standard Edition version 1.2 to provide absolute quantification of the target sequence (Appendix Table 2).
Because 2 copies of the G2R region are in MPXV genome due to its location at the ITR Samples were then tested with the 3 PCR assays by using the following reagents: AgPath-ID One-Step RT-PCR Reagents (Applied Biosystems) for PCR 1003, TaqPath BactoPure Microbial Detection Master Mix (Thermo Fisher Scientific) for PCR 1004, and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) for PCR 1005.
All reactions were prepared in 25 μL volume using 500 mmol of each primer, 250 nmol of probe and 5 μL of sample DNA. AgPath-ID reactions included 1 μL/reaction of enzyme mix and 1.67 μL of detection enhancer. Reactions were run on a QuantStudio 12K Flex (Applied Biosystems).
The following amplification conditions were used to obtain Cq values (Appendix Table   3). For AgPath-ID One-Step RT-PCR Reagents, an initial reverse transcription inactivation step at 95°C for 5 min, followed by 45 cycles of 10 s at 95°C and 30 s at 60°C. Reactions were run on a QuantStudio 12K Flex (Applied Biosystems), and graphically summarized results (Appendix Figure).
Based on Cq values, in all PCR assays, better amplifications were achieved at a probe concentration of 500 nmol and slightly better results were obtained with a primer concentration of 800 nmol. Although, in the primer, the differences with other concentrations were minimal (often >1 ΔCq). Therefore, concentrations of 500 nmol of probe and 800 nmol of primers were used for the analysis of the environmental samples.

LOD50 on Target in Wastewater (Monkeypox DNA Diluted in Nucleic Acid Extracted from Wastewater Samples)
To assess the sensitivity of the real-time PCR assays in the condition of use (i.e., detecting monkeypox virus in wastewater samples), the LOD50 of each reaction was also calculated, using the same approach described above, by testing the target monkeypox DNA diluted in nucleic acid extracted from wastewater samples, which include potential inhibitors of the polymerization reaction.
We prepared 2-fold dilutions of the standardized EVAg Monkeypox DNA in molecular grade TE buffer pH 8.0 starting from the 10 −4 dilution (74 copies/μL). Each dilution was then used to spike 1:10 proportion nucleic acid extracted from wastewater samples collected from urban wastewater treatment plants during November-December 2021, prior to emergence of monkeypox virus in Italy. Spiked samples were tested in 8 replicates with PCR 1003 (F3L), PCR *Results are expressed as no. positive/no. analytic replicates. LOD50, limit of detection for which the probability of detection is 50%; LOD95, limit of detection for which the probability of detection is 95%.