Abstract: Objective To establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method
based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of
rotavirus, astrovirus, norovirus and sapovirus. Methods Primers were designed according to the conserved genome sequence
of the 4 viruses and the homologous tail sequences were added to the 5’ end. The multiplex RT-PCR system was constructed
by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed
primers. The specificity, stability and sensitivity of the method were evaluated systematically. Results The 4 multiplex RT-PCR
methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4
diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92,
9.6 and 48 pg per reaction, respectively. Conclusion The established HAND system-based multiplex RT-PCR assay allows
simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for
application in general medical laboratories.