Development and evaluation of a meat mitochondrial metagenomic (3MG) method for composition determination of meat from fifteen mammalian and avian species.

BACKGROUND: Bioassessment and biomonitoring of meat products are aimed at identifying and quantifying adulterants and contaminants, such as meat from unexpected sources and microbes. Several methods for determining the biological composition of mixed samples have been used, including metabarcoding, metagenomics and mitochondrial metagenomics. In this study, we aimed to develop a method based on next-generation DNA sequencing to estimate samples that might contain meat from 15 mammalian and avian species that are commonly related to meat bioassessment and biomonitoring. RESULTS: In this project, we found the meat composition from 15 species could not be identified with the metabarcoding approach because of the lack of universal primers or insufficient discrimination power. Consequently, we developed and evaluated a meat mitochondrial metagenomics (3MG) method. The 3MG method has four steps: (1) extraction of sequencing reads from mitochondrial genomes (mitogenomes); (2) assembly of mitogenomes; (3) mapping of mitochondrial reads to the assembled mitogenomes; and (4) biomass estimation based on the number of uniquely mapped reads. The method was implemented in a python script called 3MG. The analysis of simulated datasets showed that the method can determine contaminant composition at a proportion of 2% and the relative error was < 5%. To evaluate the performance of 3MG, we constructed and analysed mixed samples derived from 15 animal species in equal mass. Then, we constructed and analysed mixed samples derived from two animal species (pork and chicken) in different ratios. DNAs were extracted and used in constructing 21 libraries for next-generation sequencing. The analysis of the 15 species mix with the method showed the successful identification of 12 of the 15 (80%) animal species tested. The analysis of the mixed samples of the two species revealed correlation coefficients of 0.98 for pork and 0.98 for chicken between the number of uniquely mapped reads and the mass proportion. CONCLUSION: To the best of our knowledge, this study is the first to demonstrate the potential of the non-targeted 3MG method as a tool for accurately estimating biomass in meat mix samples. The method has potential broad applications in meat product safety.

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei.

BACKGROUND: Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. METHODS: A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. RESULTS: Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. DISCUSSION: In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.