Development of RT-PCR Method to Detect Various Human Enteric Viruses

Human enteric viruses are one of the major causes of acute gastroenteritis outbreaks. A rapid and precise detection of virus is critical for prompt diagnosis. For this purpose, nucleic acid-based techniques such as reverse transcription (RT)-PCR have been developed. Although RT-PCR is a rapid, specific and sensitive method to detect virus, many steps or reactions are required, especially when various types of viruses are targeted. In this study, we developed a quick and effective method to detect human enteric viruses with a few reactions. Our candidate viruses were as follows: one DNA virus (adenovirus: AdV) and seven RNA viruses including poliovirus (PV), coxsackievirus A (CoxA) and B (CoxB), human rotavirus (HRV), hepatitis A virus (HAV), norovirus (NorV), and astrovirus (AstV). With this amount of samples, theoretically, a total of fifteen biomolecular reactions have to be performed, which include seven RT reactions and eight subsequent PCR with specific primers in each case. Specific primers, enterovirus universal primers, and random primers were applied independently to compare the outcomes of RT and PCR steps in each viral sample. We found that random 9-mer is ideal for the RT reactions of RNA viruses with negligible differences in sensitivity and specificity of viral detection except HRV. Hence, HRV cDNA generated by HRV-specific primer and AdV DNA were amplified in a single tube by duplex PCR. The cDNAs generated by RT using random 9-mers were divided into two reaction tubes without losing sensitivity: one duplex PCR detects enteroviruses (PV, CoxA, CoxB) and HAV, the other detects NorV and AstV. In conclusion, it is possible to detect eight enteric viruses with a substantially reduced number of reactions, which are composed of five reactions, two RT and three PCR reactions.

Development of UPSR Primer Design Program for Efficient Detection of Viruses

PCR is a rapid and sensitive method for detection of viruses from clinical samples and good primers are essential for successful PCR. However, high mutation rate of viral genomes often results in failure in detecting viruses, and there have been attempts to develop primers from multiple viral sequences. Thus, we developed a program called Universal Primers Score Ranking (UPSR) which generates primers from multiple sequences and ranks the quality of primers automatically. The feasibility of the UPSR program was tested using hepatitis B viruses (HBV) isolated from Korean patients. UPSR generated primer candidates with quality score ranks according to two T(m) values. We found that T(m2) values calculated based on the thermodynamics of nearest neighboring bases were better correlated with actual detection rate of HBV from patients' sera. The primer with number 1 rank by T(m2) values detected more samples than any other primers designed by UPSR, commercial primer, or other reference primers suggested by previous literatures. Thus, UPSR proved to be easy and useful to design primers from multiples sequences in detecting viruses.