On-site eDNA detection of species using ultra-rapid mobile PCR

Molecular methods, including environmental DNA (eDNA) methods, provide essential information for biological and conservation sciences. Molecular measurements are often performed in the laboratory, which limits their scope, especially for rapid on-site analysis. eDNA methods for species detection provide essential information for the management and conservation of species and communities in various environments. We developed an innovative novel method for on-site eDNA measurements using an ultra-rapid mobile PCR platform. We tested the ability of our method to detect the distribution of silver carp, Hypophthalmichthys molitrix, an invasive fish in Japanese rivers and lakes. Our method reduced the measurement time to 30 min and provided high detectability of aquatic organisms compared to the national observation surveys using multiple fishing nets and laboratory extraction/detection using a benchtop qPCR platform. Our on-site eDNA method can be immediately applied to various taxa and environments.


Introduction
Molecular technologies, such as species identification and gene expression analyses, provide essential information for biological and conservation sciences. However, even with advances in techniques in the last decades, molecular measurements in the laboratory may take a day or more. Ultra-rapid methods from DNA collection to detection are still not well developed (1), especially for environmental DNA (eDNA) analysis, which uses water or soil samples to track the presence of target species (2,3). eDNA analysis is a useful method to investigate the distribution of aquatic and terrestrial organisms (4-6). Approaches using eDNA have provided essential information for ecological management and conservation, facilitating the detection of various kinds of organisms, including endemic, invasive, or parasitic species (2,6,7). eDNA measurements have been mainly performed by quantitative real-time PCR (qPCR, 4-7). However, it is limited to laboratory analysis and laboratory processing can take many hours. These time delays often limit the range of uses for on-site eDNA detection (9,10). Field-portable DNA extraction and PCR platforms offer the potential to change species detection by eDNA on site (8)(9)(10). However, these approaches still take a similar time to laboratory measurements.
Here, we developed a new innovative method for the field processing of eDNA samples and measurements using an ultra-rapid mobile PCR platform (hereafter, mobile PCR) to reduce the measurement time to 30 min and maintain high detectability of aquatic organisms. We demonstrated its on-site use to detect the distribution of silver carp, Hypophthalmichthys molitrix, an invasive fish in Japanese rivers and lakes. We compared the on-site eDNA measurement to the laboratory extraction and detection using a benchtop qPCR platform and the national survey to confirm the performance.

Results
We detected the eDNA of H. molitrix by on-site measurement at 11 out of 15 sites ( In Survey 2, we also detected the eDNA by the laboratory methods using benchtop qPCR at all sites detected by our on-site method (Fig. 1). The relationship between the cycle timing (Ct) of the mobile PCR and eDNA concentration (or Ct) of qPCR was significant ( Fig. 2a, b, LM, p < 0.001). The Ct of mobile PCR was larger than that of qPCR, because the DNA concentration in the field-extracted samples was lower.

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Our ultra-rapid on-site eDNA extraction and measurement method using mobile PCR successfully detected the eDNA of H. molitrix, and analysis took only 30 min. Our method can be applied to many other taxa, including viruses and bacteria and to vertebrates using specific primers. We sampled water from aquatic ecosystems, but the method can be applied to terrestrial systems. For example, Valentin et al. (11) evaluated terrestrial insects on forest leaves by spraying and collecting water. The mobile PCR platform can also perform multiplex PCR for a few independent DNA measurements.
Using multiplex PCR, we can detect species co-existence, for example, for close hostparasites interactions. Therefore, our method has high potential for use with various taxa in different environments, including terrestrial and marine ecosystems.
This ultra-rapid methods can immediately be applied to broad science fields, such as human health (12) and food science (13). For example, Medema et al. (12) detected SARS-CoV-2 RNA from wastewater to evaluate the spread of COVID-19. Our method can be applied to detect RNA viruses, such as SARS-CoV-2, using reverse-transcription qPCR.

Study sites
We conducted field surveys in the Tone River and Lake Kasumigaura: Survey 1: on-site detection only and Survey 2: on-site detection and laboratory measurement. We On-site DNA measurement using mobile PCR We used a primer and probe set to detect H. molitrix (14). We checked the primer specificity for other related species such as carp in Japan using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) and confirmed the specificity for Japanese carp species.
A day before sampling, we made a PCR pre-mix with preliminary mixing of the master mix and primer-probe to bring it on site. Each TaqMan  The PCR conditions were as follows: 95 °C for 15 s, followed by 50 cycles of 95 °C for 3.5 s, and 62 °C for 10 s. In the laboratory, we performed a no-template control (NTC) 8 using DW after the mixture preparation as a regents control. We performed an NTC using DW after all PCR measurements in the day (PCR control).

Laboratory DNA measurement
For Survey 2, we extracted the DNA from the RNAlater-fixed Sterivex filters and purified using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to Miya et al. (15).
We quantified the eDNA using the PikoReal Real-Time PCR (Thermo Fisher Scientific, Waltham, MA, USA). In the laboratory qPCR, we used the same primer-probe set of onsite measurements and the PCR template mix as in our previous studies (4). Each We used a dilution series of 10000, 1000, 100, and 10 copies per PCR reaction (N = 4) for the standard curve using the target DNA cloned into a plasmid. The R 2 values of the standard curves ranged from 0.989 to 0.994 (PCR efficiencies = 93.1−102.0%). We did not detect any positives from the controls for mobile PCR and qPCR, and confirmed no cross-contamination in all eDNA measurements.

Limit of detection (LOD) test
We performed an LOD test for both mobile and qPCR as per the above PCR conditions. We used 1, 2, 4, and 8 copies of the positive control per PCR template with four replicates and detected two copy of the positive control (1/4 replicates). Thus, we determined that the LOD was two copies for both mobile and qPCR.

Distribution data
We obtained a capture survey dataset from the Ministry of Land, Infrastructure, Transport and Tourism, Japan (http://www.nilim.go.jp/lab/fbg/ksnkankyo/index.html). The national fish survey was conducted using multiple fishing gears (casting, gill, and shin net) in 2014. The survey was conducted in three seasons, including spring, summer, and autumn, and H. molitrix was observed in multiple seasons.

Statistical analysis
All statistical analyses were conducted using R ver. 4.0.2.We calculated Cohen's Kappa value to compare the detection probability of H. molitrix distribution between eDNA and the national surveys with the R "irr" package ver. 0.84, with equally weighted data. To 1 0 test the regression between eDNA concentration estimated by qPCR and the Ct of mobile PCR, we performed linear models (LMs) using "lm" function.

Data availability
All data are available in the Supplementary Table S1.