title: Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1-4.
abstract: BACKGROUND
  Dengue virus (DENV), the most widely prevalent arbovirus, continues to be a threat to human health in the tropics and subtropics. Early and rapid detection of DENV infection during the acute phase of illness is crucial for proper clinical patient management and preventing the spread of infection. The aim of the current study was to develop a specific, sensitive, and robust reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection and differentiation of DENV1-4 serotypes.
  
  
  RESULTS
  The method detection primers, which were designed to target the different DENV serotypes, were identified by inspection of multiple sequence alignments of the non-structural protein (NS) 2A of DENV1, NS4B of DENV2, NS4A of DENV3 and the 3' untranslated region of the NS protein of DENV4. No cross-reactions of the four serotypes were observed during the tests. The detection limits of the DENV1-4-specific RT-LAMP assays were approximately 10-copy templates per reaction. The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time PCR. The diagnostic rate was 100% for clinical strains of DENV, and 98.9% of the DENV-infected patients whose samples were tested were detected by RT-LAMP. Importantly, no false-positives were detected with the new equipment and methodology that was used to avoid aerosol contamination of the samples.
  
  
  CONCLUSIONS
  The RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis of DENV1-4, especially in hospitals and laboratories that lack sophisticated diagnostic systems.
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. hushengfengsysu@126.com.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. hushengfengsysu@126.com.
authors.forename: Sheng-feng
authors.initials: SF
authors.lastname: Hu
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. limiao2@mail2.sysu.edu.cn.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. limiao2@mail2.sysu.edu.cn.
authors.forename: Miao
authors.initials: M
authors.lastname: Li
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. lanlanzhong74@163.com.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. lanlanzhong74@163.com.
authors.forename: Lan-lan
authors.initials: LL
authors.lastname: Zhong
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. 735768685@qq.com.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. 735768685@qq.com.
authors.forename: Shi-miao
authors.initials: SM
authors.lastname: Lu
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. ll.zailushang@ymail.com.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. ll.zailushang@ymail.com.
authors.forename: Ze-xia
authors.initials: ZX
authors.lastname: Liu
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. pujy1105@163.com.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. pujy1105@163.com.
authors.forename: Jie-ying
authors.initials: JY
authors.lastname: Pu
authors.affiliation: Department of microbiology and immunology, Wenzhou, Medical University, Wenzhou, China. wjs@wmu.edu.cn.
authors.forename: Jin-sheng
authors.initials: JS
authors.lastname: Wen
authors.affiliation: Program of Immunology, Institute of Human Virology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. huangxi6@mail.sysu.edu.cn.|Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China. huangxi6@mail.sysu.edu.cn.|Department of microbiology and immunology, Wenzhou, Medical University, Wenzhou, China. huangxi6@mail.sysu.edu.cn.
authors.forename: Xi
authors.initials: X
authors.lastname: Huang
mesh_terms: D017931:DNA Primers / Q000235:genetics; D003715:Dengue / Q000175:diagnosis* / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D065288:Serogroup; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers