J Bacteriol Virol.  2009 Mar;39(1):41-51. 10.4167/jbv.2009.39.1.41.

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

• Oh SA
• Kim MS
• Jang SY
• Kim SJ
• Lee JI
• Lee GC
• Lee CH

• KMID: 1474179
• DOI: http://doi.org/10.4167/jbv.2009.39.1.41

Abstract

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.

Keyword

Enteric viruses; RT; PCR; Multiplex

MeSH Terms

Collodion
Disease Outbreaks
DNA
DNA Viruses
DNA, Complementary
Enterovirus
Gastroenteritis
Hepatitis A virus
Hip
Humans
Norovirus
Poliovirus
Polymerase Chain Reaction
Reverse Transcription
RNA Viruses
Rotavirus
Sensitivity and Specificity
Viruses
Collodion
DNA
DNA, Complementary

Figure

• 

  Figure 1. Detection of human enteric viruses by multiplex RT-PCR. DNA or RNA was extracted from a mixture of 8 different human enteric viruses. cDNA was synthesized from RNA sample by RT reaction using specific primers for HRV or random 9-mer. PCR was performed with specific primer sets for each virus (lane 2, AdV; lane 3, AstV; lane 4, PV; lane 5, CoxA; lane 6, CoxB; lane 7, HAV; lane 8, HRV; lane 9, NorV). Multiplex PCR was performed by using combinations of primer sets for two viruses (lane 10, EntV+HAV; lane 11, AstV+NorV; lane 12, AdV+HRV) or four viruses (lane 13, EntV+HAV+AstV+NorV). Lane 1, size marker. AdV, Adenovirus; AstV, Astrovirus; CoxA, Coxsackievirus A; CoxB, Coxsackievirus B; HAV, Hepatitis A virus; HRV, Human rotavirus; NorV, Norovirus; PV, Poliovirus; EntV, Enterovirus, PV+CoxA+CoxB. Arrows indicate the DNA bands corresponding to the expected PCR products.

• 

  Figure 2. Suggested protocol of the RT-PCR with minimum hands-on steps to detect human enteric viruses from an unknown sample. AdV, Adenovirus; AstV, Astrovirus; EntV, Enterovirus, CoxA+CoxB+PV; HAV, Hepatitis A virus; HRV, Human rotavirus; NorV, Norovirus.

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