Lang Li1, Jian-An He2, Wei Wang3, Yun Xia2, Li Song1, Ze-Han Chen4, Hang-Zhi Zuo4, Xuan-Ping Tan5, Aaron Ho-Pui Ho6, Siu-Kai Kong7, Jacky Fong-Chuen Loo8, Hua-Wen Li9, Dayong Gu10. 1. School of Public Health, The Second School of Clinical Medicine, Guangdong Medical University, Dongguan, 523808, PR China; Shenzhen International Travel Health Care Center and Shenzhen Academy of Inspection and Quarantine, Shenzhen Customs District, Shenzhen, 518033, PR China. 2. Shenzhen International Travel Health Care Center and Shenzhen Academy of Inspection and Quarantine, Shenzhen Customs District, Shenzhen, 518033, PR China. 3. Department of Laboratory Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Health Science Center, Shenzhen, 518035, PR China. 4. School of Public Health, The Second School of Clinical Medicine, Guangdong Medical University, Dongguan, 523808, PR China. 5. Shenzhen gene-one Biotechnology Co., Ltd., 518000, PR China. 6. Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, PR China. 7. Biochemistry Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, PR China. 8. Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, PR China; Biochemistry Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, PR China. Electronic address: jackyfcloo@cuhk.edu.hk. 9. School of Public Health, The Second School of Clinical Medicine, Guangdong Medical University, Dongguan, 523808, PR China. Electronic address: chineseli@163.com. 10. Shenzhen International Travel Health Care Center and Shenzhen Academy of Inspection and Quarantine, Shenzhen Customs District, Shenzhen, 518033, PR China; Department of Laboratory Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Health Science Center, Shenzhen, 518035, PR China. Electronic address: wanhood@163.com.
Abstract
OBJECTIVE: The nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time- and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples. METHODS: The combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus. RESULTS: High specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5×101 ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5μl sample and the diagnosis could be completed within 2h. CONCLUSIONS: This robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer-probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks.
OBJECTIVE: The nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time- and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples. METHODS: The combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus. RESULTS: High specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5×101 ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5μl sample and the diagnosis could be completed within 2h. CONCLUSIONS: This robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer-probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks.
Authors: Matthew A Lalli; Joshua S Langmade; Xuhua Chen; Catrina C Fronick; Christopher S Sawyer; Lauren C Burcea; Michael N Wilkinson; Robert S Fulton; Michael Heinz; William J Buchser; Richard D Head; Robi D Mitra; Jeffrey Milbrandt Journal: Clin Chem Date: 2021-01-30 Impact factor: 8.327
Authors: Aaron M Jankelow; Hankeun Lee; Weijing Wang; Trung-Hieu Hoang; Amanda Bacon; Fu Sun; Seol Chae; Victoria Kindratenko; Katherine Koprowski; Robert A Stavins; Dylann D Ceriani; Zachary W Engelder; William P King; Minh N Do; Rashid Bashir; Enrique Valera; Brian T Cunningham Journal: Analyst Date: 2022-08-22 Impact factor: 5.227
Authors: Nuttada Panpradist; Qin Wang; Parker S Ruth; Jack H Kotnik; Amy K Oreskovic; Abraham Miller; Samuel W A Stewart; Justin Vrana; Peter D Han; Ingrid A Beck; Lea M Starita; Lisa M Frenkel; Barry R Lutz Journal: EBioMedicine Date: 2021-02-12 Impact factor: 8.143