Abstract
Highly sensitive detection of pathogens is effective for screening meat during quarantine inspection and export. The “micro-amount of virion enrichment technique” (MiVET) was recently developed, which is a new method combining virus concentration with immunomagnetic beads and simple RNA extraction with sodium dodecyl benzenesulfonate (SDBS) for the specific and sensitive detection of avian influenza viruses (AIVs). AIV subtypes H3N2 and H4N2 were used to spike the surface of chicken breast meat samples. The modified MiVET protocol was tested by comparing it against three different homogenate preparation conditions, as well as in samples with added α-amylase and collagenase to digest inhibitors. The performance of the modified MiVET was evaluated by real-time RT-PCR assay targeting the matrix gene. Compared with conventional RNA extraction, the modified MiVET reproducibly concentrated AIVs in chicken meat samples with 100–1000-fold improvement by 60 s-hand homogenization. The 30 s- and 60 s-stomacher homogenizations resulted 100-fold and 10–100-fold improvement, respectively. The modified MiVET required < 60 min from homogenate preparation to final RNA elution. Further, use of the modified MiVET also decreased the rate of false-negative results. The modified MiVET is effective for the rapid and highly sensitive detection of AIVs in chicken meat samples, and can be applied to quarantine and export inspection at airports and seaports.
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Acknowledgments
The authors thank Junko Sasaki for technical assistance. This research was supported by the Japan Science and Technology Agency, Grant Number VP30218088304, JSPS KAKENHI, Grant Number JP18K05535, JSPS Bilateral Open Partnership Joint Research Projects and AMED under Grant Number JP19jk0210007.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by RM, YY, KN, FVA, HM, and WY. The first draft of the manuscript was written by WY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Makino, R., Yamazaki, Y., Nagao, K. et al. Application of an Improved Micro-amount of Virion Enrichment Technique (MiVET) for the Detection of Avian Influenza A Virus in Spiked Chicken Meat Samples. Food Environ Virol 12, 167–173 (2020). https://doi.org/10.1007/s12560-020-09425-1
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DOI: https://doi.org/10.1007/s12560-020-09425-1