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Supporting InformationInstrument-Free Point-of-Care Molecular Detection ofZika VirusJinzhao Songa, Michael G. Mauka, Brent A. Hackett b, Sara Cherry b, Haim H. Baua andChangchun Liua,*aDepartment of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia,Pennsylvania 19104, USAbDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia,Pennsylvania 19104, USA*Corresponding authorDr. Changchun LiuDepartment of Mechanical Engineering and Applied MechanicsUniversity of Pennsylvania210 Towne Building220 South 33rd St.Philadelphia, Pennsylvania 19104-6315, USAPhone: (215)746-2848E-mail: lchangc@seas.upenn.eduS‐1
S1. RT-LAMP Primer DesignTable S1. Flavivirus sequences used for alignment in this ngue 1Dengue 2Dengue 3Dengue ation source(Host: human)CountryYear ofisolation-Serum---------Fetus’ brain autopsySalivaUrineAnminiotic liquidCerebrospinal fluidSalivaSerumSerumPlasma-Cell 012002201120142014S‐2Year 42013201620152015
Figure S1. Alignment of the sequences of 19 Americas ZIKV isolates, 4 different subtypes dengue, and 2 chikungunya virus isolates from Brazil.S‐3
Figure S2. Alignment of the sequences of 19 Americas ZIKV isolates. Arrows indicate the positions of the various primers along the targetsequence. R A, G.S‐4
S2. RT-qPCR assays protocolTotal RNA was extracted with Trizol reagent (Invitrogen). cDNA was generatedfrom random hexamers to prime reverse transcription reactions using M-MLV reversetranscriptase. Quantitative PCR (qPCR) was performed with the cDNA using PowerSYBR Green PCR Master Mix and a StepOne Plus RT-PCR system (AppliedBiosystems). Reactions were initially run at 95 C for 10min, then 40 cycles of 95 C for20 sec, 52 C for 30 sec, and 72 C for 30 sec. The following primer sequences were used:(Fwd)-TCCCTTATAGGGCACAGACC, (Rev)-GACCCTTCCTCACCCAAGTA.Table S2. Mean cycle threshold (CT) values of the RT-PCR assay for zika virus detectionZika virus(PFU/reaction)5.E 045.E 035.E 025.E 015.E 005.E-01Log Zika 9897-0.30103Mean cyclethreshold rminedFigure S3. The mean cycle threshold (CT) values (in cycles) of the RT-PCR assay as a functionof PFU (n 3).S‐5
S3. Field detection of zika virus with minimal instrumentationFigure S4. Rapid, electricity-free field-testing for ZIKV in saliva with our chemically-heatedcup. The temperature is regulated with phase change material.S‐6
transcriptase. Quantitative PCR (qPCR) was performed with the cDNA using Power SYBR Green PCR Master Mix and a StepOne Plus RT-PCR system (Applied Biosystems). Reactions were initially run at 95 C for 10min, then 40 cycles of 95 C for 20 sec, 52 C for 30 sec, and 72 C for 30 sec. The following primer sequences were used:
