The direct boil RT-mPCR: A simple and rapid method for detection of foot-and-mouth disease virus genome in clinical samples without nucleic acid extraction

Abstract

Foot-and-mouth disease (FMD) is a highly contagious, vesicular viral disease of transboundary importance. Simple, rapid and economical diagnosis of FMD virus (FMDV) plays a critical role in the implementation of suitable measures to control the spread of the disease. RNA extraction is the essential step that allows for rapid diagnosis of FMDV through PCR. Although, the commercial RNA extraction kits are broadly used owing to their robustness and efficiency, these kits are often expensive and labour-intensive. In this study, boiling in distilled water of clinical samples containing FMDV to release the viral RNA was tested as an alternative to the commercial RNA extraction kit. A FMDV serotype differentiating agarose gel electrophoresis-based one step RT-multiplex PCR targeting the VP1 coding sequences of FMDV serotypes O, A and Asia1 was used as read out. Results were compared with extracted RNA by use of a commercial RNA extraction kit. Various FMDV containing samples, including cell culture isolates, tongue epithelial suspension, blood and pooled-milk samples collected from FMD-affected animals were used in this study. Simple boiling of samples without additional purification steps can be used as an alternative RNA preparation method to detect FMDV genome in cell culture and tongue epithelial suspensions. However, the boiling method may not be applicable for detection of FMDV genome in infected blood and milk samples.