Abstract
Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step “triplex RT-PCR enzyme hybridization” assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection.
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Andrew E S, Scott M R, Katja E, et al. 2007. Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease. J Virol Methods, 143:81–85.
Balamurugan V, Sen A, Saravanan P, et al. 2006. One-step Multiplex RT-PCR Assay for the Detection of Peste des petits ruminants Virus in Clinical Samples. Vet Res Commun, 30:655–666.
CLC bio: CLC Sequence Viewer. http://www.clcbio.com/index.php?id=28.
Conceicao T M, Da P A, Sorgine M H. 2010. A real-time PCR procedure for detection of dengue virus serotypes 1, 2, and 3, and their quantitation in clinical and laboratory samples. J Virol Methods, 163(1): 1–9.
Darja B, Grom J. 2001. Highly sensitive one-tube RT-PCR and microplate hybridisation assay for the detection and for the discrimination of classical swine fever virus from other Pestiviruses. J Virol Methods, 95:101–110.
Elnifro E M, Ashshi A M, Cooper R J, et al. 2000. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev, 13(4): 559–570.
Fang M Y, Jiang L H, Chen C H, et al. 2000. Investigation on the distribution of arbovirus in the south of china by reverse transcription -polymerase chain reactions. Chin J Vector Bio & Control, 11(2): 133–136. (in Chinese)
Galluzzi L, Magnani M, Saunders N, et al. 2007. Current molecular techniques for the detection of microbial pathogens. Sci Prog, 90:29–50.
He J, Kraft A J, Fan J, et al. 2009. Simultaneous Detection of CDC Category “A” DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays. Viruses, 1(3): 441–459.
Iwabuchi I, Mori K, Yamamoto H, et al. 2010. Significance of a simple assay of urine telomerase activity for the detection of bladder cancer. Hinyokika Kiyo, 56(10): 551–557.
Jiang H Y. 2010. Investigation of Japanese Encephalitis virus infection status in the three gorges reservoir area, China. Ji’nan: Shandong university. (in Chinese)
Kumar S, Wang L, Fan J, et al. 2008. Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection. J Clin Microbiol, 46(9): 3063–3072.
Liang G D. 2005. Arbovirus research needs strengthening in China. Chinese J Exp Clin Virol, 19(4): 305–306. (in Chinese)
Liang G D, Wang H Y. 2003. 10 Years review on arbovirus research of China. Chin J Public Health, 19(4): 93–96. (in Chinese)
Liolios L, Jenney A, Spelman D, et al. 2001. Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens. J Clin Microbiol, 39(8): 2779–2783.
Liu H, Gao X Y, Liang G D. 2011. Newly recognized mosquito-associated viruses in mainland China, in the last two decades. Virol J, 8(1): 68.
Li W J, Wang J L, Li M H, et al. 2010. Mosquitoes and mosquito-borne arboviruses in the Qinghai-Tibet Plateau—focused on the Qinghai area, China. Am J Trop Med Hyg, 82(4): 705–711.
Morais B R, Baleotti F G, Ribeiro N R, et al. 2005. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol, 43(2): 696–702.
Palka-Santini M, Cleven B E, Eichinger L, et al. 2009. Large scale multiplex PCR improves pathogen detection by DNA microarrays. BMC Microbiol, 9: 1.
Saxena P, Dash P K, Santhosh S R, et al. 2008. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses. Virol J, 5: 20.
Song H, Wang H Y, Wang P F, et al. 2004. Design and application of M-RT-PCR diagnosis methods for arboviral encephalitis. Chin J Microbiol Immunol, 24(4): 68–74. (in Chinese)
Thompson J D, Gibson T J, Plewniak F, et al. 1997. The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 25(24): 4876–4882.
Wang E, Paessler S, Aguilar P V, et al. 2006. Reverse transcription-PCR-enzyme-linked immunosorbent assay for rapid detection and differentiation of alphavirus infections. J Clin Microbiol, 44(11): 4000–4008.
Wang J W, Fu S H, Wang H Y, et al. 2006. Isolation and identification of Japanese encephalitis in Liaoning Province. Chinese J Exp Clin Virol, 20(1): 61–65. (in Chinese)
Yin Z, Liu J H. 1997. Animal Virology. Beijing: Science Press, p239–244. (in Chinese)
Yu M, Qin E D, Deng Y Q, et al. 2003. Detection of three important arbovirs strains by reverse transcription polymerase chain reaction. Bull Acad Mil Med Sci, 27(4): 262–288. (in Chinese)
Yu Y X. 2005. Global distribution and repeatability of Arbovirus disease. Chinese J Exp Clin Virol, 4(4): 401–407. (in Chinese)
Zhai Y G, Wang H Y, Sun X H, et al. 2008. Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China. J Gen Virol, 89: 1446–1456.
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Foundation items: NIH Grant (2U54AI057160-06); Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (2008SKLID105).
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Dong, D., Fu, Sh., Wang, Lh. et al. Simultaneous detection of three arboviruses using a triplex RT-PCR enzyme hybridization assay. Virol. Sin. 27, 179–186 (2012). https://doi.org/10.1007/s12250-012-3246-9
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DOI: https://doi.org/10.1007/s12250-012-3246-9