Bushmeat Species Identification: Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow (LF) Strip for Identification of Formosan Reeves’ Muntjac (Muntiacus reevesi micrurus)

Simple Summary Illegal hunting of wild animals and the consumption of bushmeat are recognized not only as a threat to biodiversity, but also as a risk for transmitting zoonotic diseases. Illegal sales of meat products from Formosan Reeves’ muntjac (Muntiacus reevesi micrurus) is a growing issue in Taiwan, bringing forth the demand for a fast and cost-effective technique for meat species identification. In this study, a new recombinase polymerase amplification combined with a lateral flow strip to identify Formosan Reeves’ muntjac in meat products was described. This method only requires minimal sample preparation and an isothermal heating process. The result can be interpreted by the naked eye within 30 min. The system we designed efficiently detected a variety of meat products, and no cross-reactions were observed with other animal species. This simple assay provides a sensitive and specific method to identify bushmeat sources in various meat products, which holds the potential for on-field application in the future. Abstract The identification of animal species of meat in meat products is of great concern for various reasons, such as public health, religious beliefs, food allergies, legal perspectives, and bushmeat control. In this study, we developed a new technique to identify Formosan Reeves’ muntjac in meat using recombinase polymerase amplification (RPA) in combination with a lateral flow (LF) strip. The DNA extracted from a piece of Formosan Reeves’ muntjac meat was amplified by a pair of specific primers based on its mitochondrial cytochrome b gene for 10 min at a constant temperature ranging from 30 to 45 °C using RPA. Using the specific probe added to the RPA reaction system, the amplified products were visualized on the LF strip within 5 min. The total operating time from quick DNA extraction to visualizing the result was approximately 30 min. The RPA-LF system we designed was efficient when using boiled, pan-fried, roasted, stir-fried, or stewed samples. The advantages of simple operation, speediness, and cost-effectiveness make our RPA-LF method a promising molecular detection tool for meat species identification of either raw or variously cooked Formosan Reeves’ muntjac meat. It is also possible to apply this method to identify the meat of other wildlife sources.


Introduction
Formosan Reeves' muntjac (Muntiacus reevesi micrurus), a subspecies of Muntiacus reevesi, classified and protected as a rare and valuable species in Taiwan before 2019, is now widely distributed on the island. Due to its increasing population, the Taiwan government relaxed the restriction of the Wildlife Conservation Act, allowing Taiwanese Indigenous people to hunt Formosan Reeves' muntjacs for cultural or ritual purposes only. However, Formosan Reeves' muntjacs are sometimes sold to restaurants for business. The suspected meats sold in restaurants were delivered to a professional laboratory for source identification, but this time-consuming analysis usually causes an economic loss to the restaurant if the distrained meat products were eventually defined as legal meats. This issue demands a fast, simple, and cost-effective identification technique for wildlife conservation officers to identify meat sources on-site.
To our knowledge, RPA has not been applied to the identification of wild animal meat. In this study, we describe the first development of the RPA-LF assay to detect Formosan Reeves' muntjac DNA (frmRPA-LF). The cytochrome b gene was chosen as the target for the frmRPA-LF. We also evaluated the difference in the performance of the frmRPA-LF between crude DNA extract and purified DNA extract, as well as among DNA samples extracted from meat processed by different cooking methods.

Sample Collection
All wild animal meat samples, including Formosan Reeves' muntjac, Formosan serow, masked palm civet, Formosan macaque, and Formosan pangolin were provided by the Forestry Bureau of the Council of Agriculture, Executive Yuan, and the Institute of Wildlife Conservation at the National Pingtung University of Science and Technology. Other meat samples, such as pork, beef, chicken, salmon, ostrich, lamb, tilapia, and rabbit, were obtained from local markets. All of the meat samples were frozen and stored at −20 • C until used.

Preparation of Cooked Meat
The Formosan Reeves' muntjac meat was diced into 7 pieces of 3 cm × 3 cm × 1 cm, and then cooked until ready to consume. Each piece of meat was cooked in one of the following 7 different cooking methods: boiling, roasting, pan-frying, stewing, stir-frying with soy sauce, stir-frying with barbecue sauce, and stir-frying with sesame oil. For boiling, a diced sample was boiled for 5 min in 1000 mL of water with half a teaspoon of salt. A convection oven was used to roast the Formosan Reeves' muntjac meat, mixed with garlic, pepper, and salt at 180 • C for 10 min. For pan-frying, the diced sample was fried in a stainless-steel pan at 170 • C for 5 min. For stewing, the sample was braised in a stainless-steel cooking pot with 500 mL of water, 100 mL of red wine and half a teaspoon of salt for 20 min. Three other pieces of meat samples were stir-fried separately for 10 min in soy sauce, barbecue sauce, or sesame oil. All the cooked meat samples were rinsed with water before DNA extraction procedures.

Preparation of Purified Genomic DNA
Purified genomic DNA was extracted from the meat samples using the DNeasy ® Blood & Tissue Kit (QIAGEN, Valencia, CA, USA). Animal muscle tissue, weighing approximately 10-15 mg, was first ground in a 180 µL Buffer ATL. Then, Proteinase K was added into Animals 2021, 11, 426 3 of 11 the mixture, preceding incubation at 56 • C for approximately one hour until complete digestion of the tissue. The following extraction procedures were performed according to the manufacturer's protocols: a total volume of 200 µL of DNA mixture was eluted and 5 µL of the product was then used in the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) to estimate the quantity of genomic DNA.

Preparation of Crude Genomic DNA
To shorten the diagnostic time, a single-step extraction technique of UniversAll Tissue Extraction Buffer (Yeastern Biotec, Taipei, Taiwan) was tested for its applicability. For each of the 8 cooked and uncooked Formosan Reeves' muntjac samples, a 2 mm cube was placed into a 1.5 mL Eppendorf tube and mixed with 100 µL of the extraction buffer. The cube of muscle tissue was then ground with a disposable polypropylene pellet pestle. The pestle was gently rotated back and forth manually for 30 s until the meat sample was well-crushed. The genomic DNA was obtained and ready to use after incubating at room temperature for 10 min. Although the required temperature for UniversAll Tissue Extraction Buffer, according to the instructions, was 37 • C, the samples we extracted under room temperature in this study yielded a sufficient amount of DNA for the following tests (see Table 1). The grinding procedure may facilitate the DNA extraction process and make the incubation at room temperature work as efficiently as incubating at 95 • C.

Primer Design
Full cytochrome b sequences of Formosan Reeves' muntjac (accession number: EF035447) and common food animals were downloaded from GenBank and aligned on the software DNAStar (DNASTAR, Madison, WI, USA). Primers were designed using the software Primer3 Input (http://bioinfo.ut.ee/primer3/) and NetPrimer (http://www.premierbiosoft. com/netprimer/) for initial primer screening. According to the TwistAmp DNA Amplification Kits Assay Design Manual, the primer size is best between 30 and 36 nucleotides and its GC content between 20% and 70%. The range of the primer Tm value is set between 50 to 100. Primer dimers, hairpins, and self-annealing are prone to cause artifacts and thus should be avoided. Reverse primers were labeled with biotin for the lateral flow strip analysis. The expected size of the amplification product is best between 100 and 200 bp, in order to shorten the amplification time.

Probe Design
For the utilization of the lateral flow detection system, we designed a labeling probe. The 5 end of the probe was labeled with carboxyfluorescein (FAM), while the 3 end was blocked by a three-carbon (C3) spacer. Then, the THF (tetrahydrofuran) site was placed 30 bp from the 5 end of the probe, replacing one nucleotide. At least 15 bp should be added to the 3 end after the THF residue. There is no fixed instruction describing the best location for a probe, but the possibility of dimer artifacts should still be avoided.

Lateral Flow Analysis
Lateral flow assays were performed following the instructions provided by the TwistAmp nfo Kit (TwistDx, Cambridge, UK). Briefly, 2 µL of the amplified products were diluted in 98 µL of the HybriDetect Assay Buffer (Milenia Biotec, GieBen, Germany). After properly mixing by pipetting, 10 µL of the diluted products were loaded on the sample pads of the Milenia HybriDetect 1 strips (Milenia Biotec, Giessen, Germany). The strips were directly placed into the HybriDetect Assay Buffer until the whole strip fully absorbed the buffer. Finally, the results were visualized by the naked eye. The reaction was considered positive when both the sample and control lines were visible. If only the control line was visible, the test was interpreted as negative.

Detection Limit
The detection limit for the meat cooked in different ways was further tested and assessed. In addition to frozen raw meat, 7 common Taiwanese cooking methods including boiling, pan-frying, roasting, stewing, and stir-frying in soy sauce/sesame oil/barbecue sauce were chosen for sensitivity estimation. Besides cooking methods, the difference between purified and crude DNA extracted from cooked and raw meats was also examined to estimate the detection limit of the frmRPA-LF. The concentration of each extracted genomic DNA used for the detection limit estimation are listed in Table 1.

Primer and Probe Selection
As a preliminary experiment, three sets of primer pairs targeting a unique region on mitochondrial cytochrome b gene were initially designed and tested without probes (data not shown). The primer pair we selected to use for this study was designed to yield an amplified product of 193 bp in length. A hybridization probe designed for the LF strip gave a final primer-probe combination product of 128 bp. The primers and probe used in our study are shown in Table 2 and Figure 1. sessed. In addition to frozen raw meat, 7 common Taiwanese cooking methods including boiling, pan-frying, roasting, stewing, and stir-frying in soy sauce/sesame oil/barbecue sauce were chosen for sensitivity estimation. Besides cooking methods, the difference between purified and crude DNA extracted from cooked and raw meats was also examined to estimate the detection limit of the frmRPA-LF. The concentration of each extracted genomic DNA used for the detection limit estimation are listed in Table 1.

Primer and Probe Selection
As a preliminary experiment, three sets of primer pairs targeting a unique region on mitochondrial cytochrome b gene were initially designed and tested without probes (data not shown). The primer pair we selected to use for this study was designed to yield an amplified product of 193 bp in length. A hybridization probe designed for the LF strip gave a final primer-probe combination product of 128 bp. The primers and probe used in our study are shown in Table 2 and Figure 1.

Optimization of frmRPA-LF Reaction
The assay conditions were optimized by performing the frmRPA-LF reaction at various incubation temperatures and amplification times. As shown in Figure 2A, after 20 min of incubation, the temperature settings ranging from 30 to 45 • C gave visually positive bands on the LF strip, although a slightly fainter band was observed at 30 • C. The results suggest that the frmRPA-LF reaction would not require strict temperature control, although the temperature settings at 30 • C and lower might not lead to optimal reaction, compared to the settings at 35 to 45 • C. Furthermore, optimization of the reaction time of the frmRPA-LF was carried out at a constant temperature of 37 • C by varying the incubation time from 5 to 30 min. As shown in Figure 2B, although the positive reaction was visible after 5 min of incubation, amplification for 10 min and longer may ensure a better quality of the LF strip signal.
suggest that the frmRPA-LF reaction would not require strict temperature control, although the temperature settings at 30 °C and lower might not lead to optimal reaction, compared to the settings at 35 to 45 °C. Furthermore, optimization of the reaction time of the frmRPA-LF was carried out at a constant temperature of 37 °C by varying the incubation time from 5 to 30 min. As shown in Figure 2B, although the positive reaction was visible after 5 min of incubation, amplification for 10 min and longer may ensure a better quality of the LF strip signal.

Cross-Reactivity Analysis
To evaluate the specificity of the frmRPA-LF assay, cross-reactions were tested using the purified genomic DNAs of both wild and food animals. Only the Formosan Reeves' muntjac strip displayed a positive reaction, and no such signals were observed on the other species (Figure 3).

Cross-Reactivity Analysis
To evaluate the specificity of the frmRPA-LF assay, cross-reactions were tested using the purified genomic DNAs of both wild and food animals. Only the Formosan Reeves' muntjac strip displayed a positive reaction, and no such signals were observed on the other species (Figure 3).

Detection Limit
The influences of different DNA extraction methods and meat preparations on our frmRPA-LF were evaluated using purified and crude DNA extracts from variously cooked and raw Formosan Reeves' muntjac meat. The DNA concentrations of both purified (Figure 4A) and crude extracts ( Figure 4B) are shown in Figure 4. DNA extracted from Formosan Reeves' muntjac meat gave positive results regardless of the type of meat preparations, from raw to all different cooking methods. The results indicate that our frmRPA-LF may be able to detect DNA extracted from both raw and cooked meat and would not be affected by spices, sauce, oil, or different cooking methods.

Detection Limit
The influences of different DNA extraction methods and meat preparations on our frmRPA-LF were evaluated using purified and crude DNA extracts from variously cooked and raw Formosan Reeves' muntjac meat. The DNA concentrations of both purified ( Figure 4A) and crude extracts ( Figure 4B) are shown in Figure 4. DNA extracted from Formosan Reeves' muntjac meat gave positive results regardless of the type of meat preparations, from raw to all different cooking methods. The results indicate that our Animals 2021, 11, 426 7 of 11 frmRPA-LF may be able to detect DNA extracted from both raw and cooked meat and would not be affected by spices, sauce, oil, or different cooking methods.

Detection Limit
The influences of different DNA extraction methods and meat preparations on our frmRPA-LF were evaluated using purified and crude DNA extracts from variously cooked and raw Formosan Reeves' muntjac meat. The DNA concentrations of both purified (Figure 4A) and crude extracts ( Figure 4B) are shown in Figure 4. DNA extracted from Formosan Reeves' muntjac meat gave positive results regardless of the type of meat preparations, from raw to all different cooking methods. The results indicate that our frmRPA-LF may be able to detect DNA extracted from both raw and cooked meat and would not be affected by spices, sauce, oil, or different cooking methods.

Discussion
Illegal bushmeat consumption may lead to the transmission of zoonotic diseases to humans and threaten wildlife biodiversity [29]. The Taiwan Wildlife Conservation Act prohibits hunting wild animals without authorized permission. Still, wildlife is often hunted and sold to restaurants illegally, giving rise to the demand of an available on-site diagnostic tool to identify meat species in food, quickly and accurately. In this study, we demonstrated a new technique of the isothermal RPA in combination with a lateral flow detection to identify Formosan Reeves' muntjac meat in raw meat and various meat preparations.
A variety of techniques analyzing protein composition have been utilized to identify meat origin [1,5,6,30]. However, heat and pressure processing may cause protein denaturation, posing problems of possible misdiagnosis [31]. Antibodies against heat-stable biomarkers have also been developed for meat species detection and are available as an option; however, these antisera are known to show cross-reaction among different mammalian species [32]. DNA-based detection methods provide a reliable choice for meat species identification, due to DNA's heat stability and abundance in tissue [33]. We chose the mitochondrial cytochrome b DNA sequence as the target gene because it has a couple of advantages over genomic DNA sequences. First, mitochondrial DNA is present in as many as 2500 copies in each cell, making it a rich amplifiable source of gene detection. Second, the variation of cytochrome b sequence also facilitates species discrimination [34].
When developing a new frmRPA-LF technique, the top priority was to prevent crossreaction with other meat species from happening. Therefore, we aligned the cytochrome b sequences of food animals and wild animals, often sold illegally in restaurants, in order to determine a unique target region for the frmRPA-LF assay. The second priority was to optimize the reaction, so we analyzed the optimal temperature and time. The optimal amplification temperature was evaluated using six constant temperature settings. Positive LF signals were observed when performing the frmRPA-LF at temperatures ranging from 30 to 45 • C (Figure 2A). However, the positive reaction when amplified at 30 • C was weaker in comparison to higher temperature settings. This may be due to the decreased RPA enzyme kinetics caused by the low temperature, which can be compensated with a longer reaction time [35]. The broad range of incubation temperatures of the frmRPA-LF makes it possible to be applied on-site at room temperature, in a water bath, or with body heat. This advantage also makes RPA superior to traditional polymerase chain reaction (PCR) methods because it eliminates the need for a time-consuming thermocycling process. Recent publications have reported that isothermal nucleic acid amplification methods such as helicase-dependent amplification and loop-mediated isothermal amplification may work at a constant temperature ranging from 60 to 65 • C [36,37]. Compared to the high temperature in such isothermal amplification methods, RPA can be operated at a lower constant temperature and does not require strict temperature control. Both the advantages of less energy requirement and stable amplification at different temperature settings facilitate the field applicability of frmRPA-LF. In the present study, the time required for amplification to yield a detectable level of positive reaction on the LF strip was 5 min ( Figure 2B), which is significantly faster than the recommended reaction time (20 min) by the TwistAmp ® nfo Kit Quick Guide. RPA incubation times for 5 min or even less have also been reported in previous studies [38][39][40]. The length of incubation time might be associated with the initial number of DNA copies [20]. Considering that there are thousands of copies of mitochondrial DNA per cell [41], the number of DNA copies might also contribute to the highly efficient amplification of frmRPA-LF. Ten minutes of amplification time was decided in order for the frmRPA-LF assay to compensate for the low reaction rate caused by the low temperature. After optimizing the reaction conditions, the specificity of our frmRPA-LF was evaluated using meat samples from five wild animals and eight food animals. The results from the cross-reaction assay showed that our frmRPA-LF method detected specifically the meat samples from Formosan Reeves' muntjac, and no cross-reaction with other species was observed (Figure 3).
To examine the detection limit, the frmRPA-LF assay was performed using raw meat and variously cooked meat samples. As Figure 4 illustrates, cooking methods including boiling, stir-frying with sauces and sesame oil, pan-frying, roasting, and stewing did not affect the detection of DNAs extracted from Formosan Reeves' muntjac meat. This indicates that both seasoning and heating before DNA extraction from the meat sample would not affect the amplification of the frmRPA-LF reaction. Some studies also reported that pork, chicken, lamb, and goat cooked at 120 • C can be identified by PCR, with the exception of horse meat [42][43][44]. In 2006, Arasan and his colleagues tested the influences of different cooking methods including boiling, roasting, pressure cooking, and panfrying on the accuracy of PCR. The animal species of origin of all the meat samples were successfully determined, with the exception of charred meat samples. The charred meat failed the PCR reaction presumably due to DNA degeneration by excessive heating; thus, it was suggested to avoid sampling the charred portions. The authors also compared the influence of different sizes of PCR products on the results and concluded that smaller-sized products are superior to the larger ones, since DNA can be broken down easily during the cooking process at a high temperature [45]. In our frmRPA-LF design, the sizes of the first amplification products (193 bp) and primer-probe hybridization products (128 bp) were both below 200 bp. This not only shortened the amplification time, but also avoided the misdiagnosis due to DNA destruction. Moreover, the results from the evaluation of efficiency of crude DNA extracts showed that crude DNA extracts worked as well as purified DNA (Figure 4b). The use of crude DNA extracts can simplify the frmRPA-LF assay and reduce the total cost.

Conclusions
We successfully developed a novel frmRPA-LF assay to identify Formosan Reeves' muntjac meat in raw and cooked meat samples. It is a fast, simple, sensitive, cost-effective, precise, and potentially applicable technique. The frmRPA-LF only requires minimal sample preparation and an isothermal heating process. Within 30 min, the results can be interpreted