Development of a novel dengue virus serotype-specific multiplex real-time reverse transcription-polymerase chain reaction assay for blood screening

BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)‐1, ‐2, ‐3, and ‐4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion‐transmitted cases...

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Published in	Transfusion (Philadelphia, Pa.) Vol. 56; no. 12; pp. 3094 - 3100
Main Authors	Tezuka, Kenta, Kuramitsu, Madoka, Okuma, Kazu, Nojima, Kiyoko, Araki, Kumiko, Shinohara, Naoya, Matsumoto, Chieko, Satake, Masahiro, Takasaki, Tomohiko, Saijo, Masayuki, Kurane, Ichiro, Hamaguchi, Isao
Format	Journal Article
Language	English
Published	United States Blackwell Publishing Ltd 01.12.2016 Wiley Subscription Services, Inc
Subjects	Blood Safety Dengue - diagnosis Dengue - prevention & control Dengue - transmission Dengue fever Dengue Virus - genetics Genes Humans Multiplex Polymerase Chain Reaction Polymerase chain reaction Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction RNA, Viral - analysis RNA, Viral - blood Serogroup Transfusion Reaction
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Abstract	BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)‐1, ‐2, ‐3, and ‐4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion‐transmitted cases have also been reported after the use of blood and plasma products in DENV‐endemic countries. The aim of this study was to develop a novel multiplex reverse transcription–polymerase chain reaction (RT‐PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS Large‐scale oligonucleotide screening was performed to obtain DENV‐specific primers and probes using a variety of DENV clinical isolates. A multiplex RT‐PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype‐specific multiplex RT‐PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION This established serotype‐specific multiplex RT‐PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion‐transmitted DENV infection.
AbstractList	BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)‐1, ‐2, ‐3, and ‐4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion‐transmitted cases have also been reported after the use of blood and plasma products in DENV‐endemic countries. The aim of this study was to develop a novel multiplex reverse transcription–polymerase chain reaction (RT‐PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS Large‐scale oligonucleotide screening was performed to obtain DENV‐specific primers and probes using a variety of DENV clinical isolates. A multiplex RT‐PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype‐specific multiplex RT‐PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION This established serotype‐specific multiplex RT‐PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion‐transmitted DENV infection. BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection. Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening. Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection. BACKGROUNDDengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening.STUDY DESIGN AND METHODSLarge-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated.RESULTSA number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL.CONCLUSIONThis established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection.
Author	Kurane, Ichiro Hamaguchi, Isao Saijo, Masayuki Shinohara, Naoya Kuramitsu, Madoka Okuma, Kazu Tezuka, Kenta Nojima, Kiyoko Araki, Kumiko Satake, Masahiro Takasaki, Tomohiko Matsumoto, Chieko
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References_xml	– reference: Kutsuna S, Kato Y, Moi ML, et al. Autochthonous dengue fever, Tokyo, Japan, 2014. Emerg Infect Dis 2015;21:517-20. – reference: Muñoz-Jordán JL, Collins CS, Vergne E, et al. Highly sensitive detection of dengue virus nucleic acid in samples from clinically ill patients. J Clin Microbiol 2009;47:927-31. – reference: Martina BE, Koraka P, Osterhaus AD. Dengue virus pathogenesis: an integrated view. Clin Microbiol Rev 2009;22:564-81. – reference: Waggoner JJ, Abeynayake J, Sahoo MK, et al. Comparison of the FDA-approved CDC DENV-1-4 real-time reverse transcription-PCR with a laboratory-developed assay for dengue virus detection and serotyping. J Clin Microbiol 2013;51:3418-20. – reference: Domingo C, Niedrig M, Teichmann A, et al. 2nd International external quality control assessment for the molecular diagnosis of dengue infections. PLoS Negl Trop Dis 2010;4. pii: e833. – reference: Tambyah PA, Koay ES, Poon ML, et al. Dengue hemorrhagic fever transmitted by blood transfusion. N Engl J Med 2008;359:1526-7. – reference: Stramer SL, Linnen JM, Carrick JM, et al. Dengue viremia in blood donors identified by RNA and detection of dengue transfusion transmission during the 2007 dengue outbreak in Puerto Rico. Transfusion 2012;52:1657-66. – reference: Sabino EC, Loureiro P, Esther Lopes M, et al. Transfusion-transmitted dengue and associated clinical symptoms during the 2012 epidemic in Brazil. J Infect Dis 2016;213:694-702. – reference: Linnen JM, Vinelli E, Sabino EC, et al. Dengue viremia in blood donors from Honduras, Brazil, and Australia. Transfusion 2008;48:1355-62. – reference: WHO Guidelines Approved by the Guidelines Review Committee. Dengue: guidelines for diagnosis, treatment, prevention and control: new edition. Geneva: World Health Organization; 2009. – reference: Pozzetto B, Memmi M, Garraud O. Is transfusion-transmitted dengue fever a potential public health threat? 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Snippet	BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)‐1, ‐2, ‐3, and ‐4, which are transmitted to humans... Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by... BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans... BACKGROUNDDengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by...
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SubjectTerms	Blood Safety Dengue - diagnosis Dengue - prevention & control Dengue - transmission Dengue fever Dengue Virus - genetics Genes Humans Multiplex Polymerase Chain Reaction Polymerase chain reaction Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction RNA, Viral - analysis RNA, Viral - blood Serogroup Transfusion Reaction
Title	Development of a novel dengue virus serotype-specific multiplex real-time reverse transcription-polymerase chain reaction assay for blood screening
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