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

Yun-Hsiu Hsu,Wei-Cheng Yang,Kun-Wei Chan

doi:10.3390/ani11020426

Abstract

Simple SummaryIllegal 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.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.

Highlights

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 widely distributed on the island
We describe the first development of the recombinase polymerase amplification (RPA)-lateral flow (LF) assay to detect Formosan
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

Summary

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 widely distributed on the island. 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. A variety of analytic methods have been applied to differentiate animal species of meat, including ELISA [1,2,3,4], immunochromatography [5], and Raman spectroscopy [6,7]

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