Xuan Wang1, Fenggui Yin2, Yuhai Bi1, Gong Cheng3, Jing Li1, Lidan Hou4, Yunlong Li1, Baozhi Yang5, Wenjun Liu6, Limin Yang7. 1. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. 2. College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, China; University of Chinese Academy of Sciences, Beijing 100049, China. 3. Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China; University of Chinese Academy of Sciences, Beijing 100049, China. 4. China Institute of Veterinary Drug Control, Beijing 100081, China; University of Chinese Academy of Sciences, Beijing 100049, China. 5. University of Chinese Academy of Sciences, Beijing 100049, China; College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, China. 6. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; China Institute of Veterinary Drug Control, Beijing 100081, China. 7. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: lmyang@im.ac.cn.
Abstract
BACKGROUND: Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. OBJECTIVES: This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. METHODS: Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. RESULTS: The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. CONCLUSIONS: The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory.
BACKGROUND:Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. OBJECTIVES: This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. METHODS: Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. RESULTS: The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKVclinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. CONCLUSIONS: The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory.
Authors: Ozlem Yaren; Barry W Alto; Kevin M Bradley; Patricia Moussatche; Lyudmyla Glushakova; Steven A Benner Journal: J Vis Exp Date: 2018-03-13 Impact factor: 1.355
Authors: Brett E Etchebarne; Zenggang Li; Robert D Stedtfeld; Michael C Nicholas; Maggie R Williams; Timothy A Johnson; Tiffany M Stedtfeld; Tanja Kostic; Walid T Khalife; James M Tiedje; Syed A Hashsham; Mary J Hughes Journal: Front Microbiol Date: 2017-12-12 Impact factor: 5.640