Application of an Improved Micro-amount of Virion Enrichment Technique (MiVET) for the Detection of Avian Influenza A Virus in Spiked Chicken Meat Samples

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-4 2 1 2 3 4stomacher 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.

Development of a simple and highly sensitive virion concentration method to detect SARS-CoV-2 in saliva

“…In our previous study, an immunomagnetic bead method using specific antibodies was successfully used to detect influenza A viruses added to PBS, duck feces, and chicken meat at 10- to 1000-fold sensitive concentration ([ 14 ]; [ 15 ]. Although this method is extremely sensitive, it is not very versatile because it requires the preparation of specific antibodies for each virus species and has the disadvantage that the LOD is reduced when the samples contain many inhibitory substances, such as components of the duck feces and chicken meat.…”

“…However, since the positive samples could be diluted by mixing with negative samples, leading to low virus concentration in the pooled sample, the test can become a false negative if it is below the limit of detection (LOD); hence, accurate detection may not be possible. As a solution, we have developed a method for detecting concentrated viruses in samples through immunomagnetic beads ([ 14 ];[ 15 ]. Still, it is not versatile because it requires specific antibodies for each virus.…”

Development of a simple and highly sensitive method for the concentration and detection of African swine fever virus in oral fluids and its feasibility assessment on raised pigs in Northern Vietnam

“…The immunomagnetic bead method using specific antibodies has been widely used for enrichment of trace viruses (Dhumpa et al ., 2011; Makino et al ., 2020; Yamazaki et al ., 2019). Although this method is very sensitive, it has the disadvantages of requiring the production of antibodies specific for each virus species and of decreasing LOD due to inhibitory substances derived from the sample.…”