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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.
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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.
Table 1.
The list of viruses and their specific primers used in this study
| Virus | Primer | Sequence (5′→3′) | Reference | Detection limita |
|---|---|---|---|---|
| AdV | hexAA1885 hexAA1913 | GCCGCAGTGGTCTTACATGCACATC CAGCACGCCGCGGATGTCAAAGT | 15 | 5.3 |
| Adhex-1 Adhex-2 | GCCACCGATACGTACTTCAGCCTG GGCAGTGCCGGAGTAGGGTTTAAA | 1 | 4 | |
| AstV | MON340 Astman-2 | CGTCATTATTTGTTGTCATACT TCGCTTCATACATCAAACCC | 1, 16 | 6 |
| MON340 MON348 | CGTCATTATTTGTTGTCATACT ACATGTGCTGCTGTTACTATG | 16 | 4 | |
| Cox | UC52 UC53 | CAAGCACTTCTGTTTCCCCGG TTGTCACCATAACCAGCCA | 17 | 4 (CoxA) 5 (CoxB) |
| EcV | Primer 1 Primer 3 | CAAGCACTTCTGTTTCCCCGG ATTGTCACCATAAGCAGCCA | 18 | – |
| EV1 EV2 | AAGCACTTCTGTTTCC CATTCAGGGGCCGGAGGA | 19 | 5 | |
| EntVb | E2 E1 | TCCGGCCCCTGAATGCGG CACCGGATGGCCAATCCAAT | 20 | 6 |
| Primer 1 Primer 3 | CAAGCACTTCTGTTTCCCCGG ATTGTCACCATAAGCAGCCA | 18 | 5.5 (CoxB)~6 (CoxA) | |
| HAV | 16A 6A | GTTTTGCTCCTCTTTATCATGCTATG GGAAATGTCTCAGGTACTTTCTTTG | 21 | 6.7 |
| HAVC-L HAVC-R | CAGCACATCAGAAAGGTGAG CTCCAGAATCATCTCCAAC | 22 | 2 | |
| HBV | 409 703A | CATCCTGCTGCTATGCCTCATCT CGAACCACTGAACAAATGGCACT | 21 | – |
| HRV | aBT1 RVG9 | CAAGTACTCAAATCAATGATGG GGTCACATCATACAATTCT | 23 | 5.7 |
| Con3 Con2 | TGGCTTCGCCATTTTATAGACA ATTTCGGACCATTTATAACC | 24 | 4 | |
| MV | MV1 MV2 | TTAGGGCAAGAGATGGTAAGG GTTCTTCCGAGATTCCTGCCA | 25 | – |
| MuV | MuV first F MuV first R | ATGTAATTAATGCCAACTGCAAGG GAAAAGCTTATATCTAACGATGGG | 26 | – |
| NV-GIIF1 NV-GIIR1 | GGGAGGGCGATCGCAATCT CCRCCIGCATRICCRTTRTACAT | 27 | 4 | |
| NorV | JV12 JV13 | ATACCACTATGATGCAGATTA TCATCATCACCATAGAAAGAG | 28, 29 | 4 |
| MUN2-1F MUN2-1R | GATTACTCTCGGTGGGATTCA GGGGTCCTTCAGTTTTGTCA | This study | 4 | |
| NorV | MUN2-2F MUN2-2R | GGTGGGATTCAACACAACAG GTTCTCCGCAGGAAAGTCAG | This study | 5 |
| MUN2-3F MUN2-3R | GGCACCGTGGTCACACTACT TGGTTTTCTTGGCAGCTTCT | This study | 3 | |
| PV | PVPCR2 2A | GTCAATGATCACAACCCAC AAGAGGTCTCTATTCCACAT | 30, 31 | 6.4 |
Table 2.
Comparison of specific and random primers in reverse transcription reactions to detect RNA enteric viruses by RT-PCR
| Viruses | Detection limit (-log10 dilution) | ||
|---|---|---|---|
| Specific primer | Random 6-mer | Random 9-mer | |
| AstV | 6 | 5 | 5 |
| CoxA | 4 | 4 | 5 |
| CoxB | 5 | 5 | 6 |
| HAV | 6.7 | 5 | 6 |
| HRV | 5.7 | 1 | 2 |
| NorV | 5 | 5 | 5 |
| PV | 6.4 | 5.5 | 6 |
Table 3.
Comparison of universal primers to detect enteroviruses by multiplex PCR
| Virus | Detection limit (-log10 dilution) | ||||
|---|---|---|---|---|---|
| Controla | RT primer | Universal primers for PCR | |||
| EV1/EV2 | E1/E2 | Primer1/3 | |||
| Random 6-mer | 4.2 | 4.7 | 5 | ||
| Random 9-mer | 6 | 6 | 4 | ||
| PV | 6.4 | EV1/EV2 | 5 | − | − |
| E1/E2 | −b | 6 | − | ||
| Primer1/3 | − | − | 5.7 | ||
| Random 6-mer | 5 | 6 | 5.5 | ||
| Random 9-mer | 4.5 | 6.3 | 5 | ||
| CoxA | 4 | EV1/EV2 | 5 | − | − |
| E1/E2 | − | 6 | − | ||
| Primer1/3 | − | − | 6 | ||
| Random 6-mer | 4.25 | 6.25 | 5 | ||
| Random 9-mer | 5 | 6.7 | 5 | ||
| CoxB | 5 | EV1/EV2 | 5 | − | − |
| E1/E2 | − | 6 | − | ||
| Primer1/3 | − | − | 5.5 | ||
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