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.
Subject(s)
Food Microbiology/methods , Influenza A virus/isolation & purification , Influenza in Birds/virology , Meat/virology , Poultry Diseases/virology , Virology/methods , Animals , Chickens , Food Microbiology/instrumentation , Influenza A Virus, H3N2 Subtype/classification , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/isolation & purification , Influenza A virus/classification , Influenza A virus/genetics , Virion/classification , Virion/genetics , Virion/isolation & purification , Virology/instrumentationABSTRACT
Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a condition that threatens the sustainability of the livestock industry. A fluorescent loop-mediated isothermal amplification (fLAMP) assay targeting BLV env sequences was developed and used to evaluate 100 bovine blood samples. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay took 65 min, while the fLAMP assay took 8 min to 30 min from the beginning of DNA amplification to final judgement with a comparable limit of detection. The fLAMP is a potential tool for the rapid and simple diagnosis of BLV infection to supplement ELISA testing and can be used by local laboratories and slaughterhouses without special equipment.
