Biosensors and Bioelectronics 86 (2016) 420–425

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

http://d0956-56

n CorrE-m

journal homepage: www.elsevier.com/locate/bios

Attomolar Zika virus oligonucleotide detection based onloop-mediated isothermal amplification and AC susceptometry

Bo Tian a, Zhen Qiu a, Jing Ma b, Teresa Zardán Gómez de la Torre a, Christer Johansson c,Peter Svedlindh a, Mattias Strömberg a,n

a Department of Engineering Sciences, Uppsala University, The Ångström Laboratory, Box 534, SE-751 21 Uppsala, Swedenb Department of Immunology, Genetics and Pathology, Uppsala University, The Rudbeck Laboratory, SE-751 85 Uppsala, Swedenc Acreo Swedish ICT AB, Arvid Hedvalls backe 4, SE-411 33 Göteborg, Sweden

a r t i c l e i n f o

Article history:Received 28 April 2016Received in revised form16 June 2016Accepted 28 June 2016Available online 29 June 2016

Keywords:Zika virusLoop-mediated isothermal amplificationMagnetic nanoparticlesBrownian relaxationAC susceptometer

x.doi.org/10.1016/j.bios.2016.06.08563/& 2016 The Authors. Published by Elsevie

esponding author.ail address: mattias.stromberg@angstrom.uu.s

a b s t r a c t

Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such asreverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zikaoutbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia isonly about one week after symptom onset. By combining loop-mediated isothermal amplification(LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zikavirus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMPreagents including the analyte and biotinylated primers, and their hydrodynamic volumes are drama-tically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changesof the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used toquantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zikavirus oligonucleotide in 20% serum with a total assay time of 27 min, which can hopefully widen thedetection window for Zika viremia and is therefore promising in worldwide Zika fever control.& 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Zika virus (ZIKV), the causative agent of the infectious diseaseZika fever, is a positive-sense RNA virus that belongs to the familyFlaviviridae, genus Flavivirus, which is related to dengue fever,yellow fever, Japanese encephalitis and West Nile fever (Kunoet al., 1998; Sikka et al., 2016). Until recently, Zika fever was con-sidered mild and self-limited and therefore has received less at-tention (Haug et al., 2016; Petersen et al., 2016). However, it hasrecently been found that ZIKV infection could trigger Guillain-Barré syndrome (Cao-Lormeau et al., 2016; Broutet et al., 2016);and could also be associated with microcephaly fetal death (Fauciand Morens, 2016; Brasil et al., 2016). ZIKV is mainly transmittedby day-time active female Aedes aegypti and Aedes albopictusmosquitoes (Sikka et al., 2016), but can also be transmitted sexu-ally (Foy et al., 2011; Venturi et al., 2016; Oster et al., 2016; D’Or-tenzio et al., 2016), from mother to the fetus (Calvet et al., 2016;Melo et al., 2016; Schuler-Faccini et al., 2016), and potentiallythrough blood transfusion (Musso et al., 2014; Marano et al., 2016).The recent outbreaks of ZIKV have drawn the world's attention

r B.V. This is an open access articl

e (M. Strömberg).

towards this relatively unstudied virus, particularly for its asso-ciation with the increased number of newborns presenting mi-crocephaly reported in Brazil (Campos et al., 2015; Rodriguez-Morales, 2015; Heukelbach et al., 2016).

The incubation period of Zika fever ranges from three to twelvedays after the bite of an infected mosquito, which is similar toother infections caused by flaviviruses (Goeijenbier et al., 2016;Sikka et al., 2016), and the clinical signs persist for two to sevendays (Korzeniewski et al., 2016). Laboratory diagnostics of ZIKV arebased mainly on the analysis of serum, by using viral RNA and/orantibody based detection methods (Haug et al., 2016; Al-Qahtaniet al., 2016). Due to serological cross-reactivity among flavivirusesin IgM-antibody based assays, diagnostics based on moleculartesting are considered more reliable (Haug et al., 2016). The cur-rent prevalent molecular detection method is reverse-transcrip-tion polymerase chain reaction (RT-PCR), which can detect 320copies/μL (approximately 530 aM) ZIKV RNA in a total assay time(excluding the time for RNA extraction) of about 90 min (Fayeet al., 2008; Faye et al., 2013; Musso et al., 2015). However, sinceviremia decreases over time, RT-PCR based methods should beperformed during the first seven days after symptom onset (Hauget al., 2016; Buathong et al., 2015), when the ZIKV loads are re-ported at the level of approximately 1 fM in body fluid samples(Lanciotti et al., 2008; Gourinat et al., 2015; Barzon et al., 2016);

e under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

B. Tian et al. / Biosensors and Bioelectronics 86 (2016) 420–425 421

after that, reliable results can only be provided by labor-intensiveserologic testing (Z four days after symptom onset, and IgM an-tibodies persist from two weeks to several months) (Petersenet al., 2016). Therefore, more sensitive and reliable molecular de-tection methods are required to meet the challenge of ZIKV dailydiagnoses on blood donators, pregnant women and travelers fromareas with ongoing ZIKV transmission.

Among the reported isothermal molecular detection techni-ques that have the potential to replace traditional PCR/RT-PCR,loop-mediated isothermal amplification (LAMP) has become anexcellent option due to its high amplification efficiency, specificity,low cost, time-saving protocol and the ability of amplifying non-denatured DNA samples (Notomi et al., 2000; Tomita et al., 2008).In our previous work (Tian et al., 2016b), we reported a LAMPbased optomagnetic biosensor, which could detect 10 aM syntheticNewcastle disease virus DNA in 30 min. It has been demonstratedthat Bst 3.0 polymerase could amplify both RNA and DNA se-quences under identical conditions, and the LAMP-optomagneticbiosensor was verified by performing tests on clinical RNA samples(vaccine and tissue specimens) (Tian et al., 2016b). The LAMP-optomagnetic assay was performed in a three-step procedure in-cluding amplification, labeling and read-out. However, due to theoptical influence of the Mg2P2O7 precipitation, the LAMP-opto-magnetic detection procedure required opening of the reactiontube after the LAMP reaction, which greatly increased the risk ofcarryover contamination (Tomita et al., 2008).

Here, we present a simple and homogeneous ZIKV detectionmethod that combines LAMP and a magnetic AC susceptibilityreadout method based on the response of the magnetic nano-particle (MNP) system, and the three step procedure is reduced toa two-step procedure. Streptavidin-coated 100 nm magnetic na-noparticles were mixed with LAMP reagents including biotinylatedinner primers prior to the reaction. During the LAMP reaction, thehydrodynamic volume of the MNPs dramatically increased due tobinding between the streptavidin groups on the particles and thebiotinylated amplicons. Mg2P2O7, a by-product produced in asuccessful LAMP reaction (Mori et al., 2001), was also involved inthe increase of the hydrodynamic volume of the MNPs. The in-crease in hydrodynamic volume of the MNPs resulted in a Brow-nian relaxation frequency shift to lower frequency which wassubsequently measured by a portable AC susceptometer (Astalanet al., 2004). An increased target oligonucleotide concentrationresulted in a larger frequency shift to lower frequency. The LAMP-AC susceptometer biosensor achieves a limit of detection (LOD) of1 aM synthetic ZIKV oligonucleotide with a total assay time of27 min. The same sensitivity was achieved when performing thetest in 20% serum samples. From these results we can hopefullywiden the detection window of ZIKV viremia and contribute to theZika fever control.

2. Materials and methods

2.1. Reagents

Bst 3.0 polymerase, isothermal amplification buffer II andMgSO4 were purchased from New England Biolabs (Ipswich, UK).dNTP mix was purchased from Thermo Fisher Scientific (Waltham,USA). Fetal bovine serum, D-biotin and MQ water were purchasedfrom Sigma-Aldrich. Streptavidin modified 100 nm MNPs (multi-core magnetic beads containing clusters of small single domainparticles, 10 mg/mL, 6�1012 particles/mL) were purchased fromMicromod (Rostock, Germany). For the serum sample detection,10 nM synthetic ZIKV oligonucleotide was serial diluted with 20%fetal bovine serum.

2.2. Primer design

RNA-dependent-RNA-polymerase (NS5) sequences of 51 ZIKVstrains from the outbreak in Easter Island in 2014 (GenBank ac-cession number: KM078929-KM078979) (Tognarelli et al., 2016)were compared and a 230 bp highly conserved region (Table S1)was chosen as the target gene for primer design. LAMP primers(inner primer pair FIP/BIP, outer primer pair F3/B3 and loop primerpair LF/LB) were designed using Primer Explorer version 4 (https://primerexplorer.jp/e/), and the sequences are shown in Table S1.The inner primer FIP was labeled with a biotin group at the 5′-end.The target sequence and six primers were synthesized by In-tegrated DNA Technologies (Coralville, USA).

2.3. LAMP reaction

A 100 μL reaction mixture contained 200 μg/mL MNPs (strep-tavidin-coated MNPs or biotin-blocked MNPs), 1.6 μM inner pri-mers, 0.2 μM outer primers, 0.4 μM loop primers, 1� isothermalamplification buffer II (containing 2 mM MgSO4), 6 mM MgSO4

(8 mM in total), 32 U Bst 3.0 polymerase, 1.4 mM dNTPs and 40 μLof analyte solution (synthetic ZIKV oligonucleotide). The mixturewas incubated at 69 °C (metal bath) for 12 min

2.4. AC susceptometry measurement

After incubation, the reaction tube was mounted in a portablecommercial AC susceptometer, DynoMag

s

(Acreo Swedish ICT AB,Göteborg, Sweden), to measure the frequency-dependent AC sus-ceptibility. Sixteen data points were measured in the frequencyrange of 4–100 Hz, and the readout time was 15 min. The fre-quency-dependent out-of-phase component of the susceptibility,χ″, was recorded. The position of the χ″ peak in frequency is de-fined as the Brownian relaxation frequency fB, which is given by

πη= ( )f k T V/ 6B B h , where k TB is the thermal energy, η is the dynamicviscosity and Vh is the hydrodynamic volume of the relaxing entity.

2.5. Optimization of LAMP reaction

Agarose gel electrophoresis analysis was employed to evaluatethe influence of different reaction temperatures (61, 63, 65, 67, 69and 71 °C) and different inner primer concentrations (0.4, 0.8 and1.6 μM). Synthetic ZIKV oligonucleotide at a concentration of 1 pMwas used as analyte, and the Mg2P2O7 precipitations were re-moved by centrifugation after 12 min amplification. To find anoptimum biotinylated inner primer to streptavidin-MNP ratio thatcould balance the reaction efficiency and the blocking effectcaused by the excess of biotinylated primers, the inner primerconcentration vs. streptavidin-MNP concentration was evaluatedin the AC susceptometer by amplifying 1 fM synthetic ZIKVoligonucleotide.

2.6. Scanning electron microscopy measurement

After the LAMP reaction, the MNPs (streptavidin-coated MNPsor biotin-blocked MNPs) suspended in the reaction mixture wereseparated by a magnetic stand, washed twice by 50 μL MQ waterand resuspended in 25 μL MQ water. A 100 μL control reactionmixture (positive LAMP reaction without MNPs) was centrifuged,washed twice with 50 μL MQ water and resuspended in 25 μL MQwater to collect the Mg2P2O7 precipitation. The morphology andnanostructure of the three samples (volume-amplified streptavi-din-coated MNP aggregates, volume-amplified biotin-blockedMNP aggregates and Mg2P2O7 precipitation) were characterized byscanning electron microscopy (SEM, Zeiss-1530) using an in-lens

B. Tian et al. / Biosensors and Bioelectronics 86 (2016) 420–425422

detector for secondary electrons and the microscope was operatedat 3 kV electron beam accelerating voltage.

3. Results and discussion

3.1. AC susceptometry detection and setup

An illustration of the LAMP based susceptometry biosassay ispresented in Fig. 1. Synthetic ZIKV oligonucleotides are mixed withstreptavidin-MNPs and other LAMP reagents including biotiny-lated inner primers. Due to the binding of amplicons (and/orMg2P2O7) during the LAMP reaction, the hydrodynamic volume ofMNPs increases, which results in a shift in fB to lower frequency.

3.2. Optimization of LAMP reaction

Agarose gel electrophoresis analysis showed that the optimumreaction temperature was 69 °C (Fig. S1A). At this temperature theLAMP reaction produced a maximum amount of amplicons andtherefore this temperature was employed in the following tests.Although Fig. S1B shows that the reaction efficiency can be im-proved by increasing the concentration of inner primers, it may beanticipated that the higher amount of biotinylated inner primerwould lead to a smaller fB shift, since the polymerase wouldpreferentially amplify the free inner primers instead of primersthat are already bound to MNPs (according to the supplier,200 μg/mL streptavidin-MNPs can be saturated with approxi-mately 0.02 μM biotin groups). Therefore, the shift in fB wasevaluated with respect to the inner primer concentration vs.streptavidin-MNP concentration. As shown in Fig. S2, detection of1 fM synthetic ZIKV oligonucleotides gave the largest fB shift with1.6 μM inner primers and 200 μg/mL MNPs.

Considering that the biotin-streptavidin reaction is much fasterthan the LAMP reaction, the streptavidin groups on the surface ofMNPs can be saturated by biotinylated primers before the primersbeing amplified. Moreover, polymerases that bind to the surface ofMNPs contribute more to the MNP volume amplification. There-fore, a higher MNP concentration results in a lower amount ofpolymerases on each MNP, and consequently in less amplicons oneach MNP (Fig. S2). The amplification time was also optimized andthis was done by turbidity-based naked-eye detection (data notshown). Due to the high amplification efficiency and the end-pointdetection format, signals of positive samples reach the saturationlevel easily and are then not useful for quantification. Therefore, alonger reaction time results in a narrower dynamic detectionrange. An amplification time of 12 min was chosen to achieve

Fig. 1. Illustration of the LAMP based susceptometry assay. Two independent steps, incubation; target oligonucleotides are amplified and the product/byproduct interacts withAC susceptometer and measured (for clarity, the excitation coil is shown with a section

rapid attomolar target detection without narrowing the dynamicdetection range.

3.3. Quantitative synthetic ZIKV oligonucleotide detection

Serial dilutions of synthetic ZIKV oligonucleotides, rangingfrom 1 pM to 100 zM, were amplified in presence of streptavidin-MNPs and analyzed by the AC susceptometer; the spectra of χ″ areshown in Fig. 2A, showing that fB shifts to lower values with in-creasing analyte concentration. Three independent measurementswere performed for each analyte concentration. Since we focusmore on the peak position, the χ″ spectra were not normalizedwith respect to χ′∞ (high-frequency value of the in-phase com-ponent of the volume susceptibility) as done in previous work(Zardán Gómez de la Torre et al., 2011). The typical spectra of thethree independent measurements are shown in this work, and fBare extracted from each measurement for the following analysis.

Interestingly, the lowest fB observed in this work is 7.6 Hz(Fig. 2A, 1 pM target sequence), which is much lower than ob-served in previous work (approximately 36 Hz, 1 pM target se-quence) (Tian et al., 2016b). The frequency peak obtained by theoptomagnetic setup is not exactly equal but very close to the fB(Donolato et al., 2015; Tian et al., 2016a). The difference betweenthe protocol used in this work and the one used in previous workis that in the previous work the biotinylated amplicons were in-cubated with MNPs after the LAMP reaction. Therefore, we hy-pothesize that the much larger shift in fB when using this newprotocol is explained not only by binding of MNPs to amplicons,but also by the Mg2P2O7 precipitation that is formed during theLAMP reaction and deposited onto the surface of the MNPs.

To verify this hypothesis, we first investigated whether thebiotin-blocked streptavidin-MNPs could be volume-amplified byincubating with already formed biotinylated amplicons or Mg2P2O7

precipitation. The biotinylated amplicons and the Mg2P2O7 pre-cipitation were separated by centrifugation after the LAMP reac-tion and incubated for 15 min at room temperature with biotin-blocked MNPs, respectively. Fig. S3 shows that neither the bioti-nylated amplicons nor the precipitated Mg2P2O7 could obviouslychange fB of the biotin-blocked MNPs. This means that (1) thestreptavidin groups on the MNPs were completely blocked; (2) thebiotin-blocked MNPs have no interaction with the already formedLAMP products and byproducts; and (3) the viscosity is not ob-viously changed by the LAMP reaction.

Next we investigated whether biotin-blocked MNPs could bevolume-amplified during the LAMP reaction. The biotin-blockedMNPs were mixed with LAMP reagents and the analyte, and re-acted with serial dilutions of synthetic ZIKV samples; the AC

cubation and measurement, are included in the illustration from left to right. In-MNPs during the LAMP reaction. Measurement; the reaction tube is mounted in thein the middle removed to reveal the pickup coil).

Fig. 2. Typical χ″ spectra for the indicated concentrations of synthetic ZIKV oligonucleotide. (A) Streptavidin-MNP based LAMP-AC susceptometry analysis. (B) Biotin-blockedMNP based LAMP-AC susceptometry analysis.

B. Tian et al. / Biosensors and Bioelectronics 86 (2016) 420–425 423

susceptibility results are shown in Fig. 2B. A frequency shift isobserved for ZIKV concentrations ≥ 100 aM, and the lowest ob-served fB is about 20 Hz (1 pM analyte). This result clearly de-monstrates that MNPs have an interaction with the Mg2P2O7

precipitation during its formation. This interaction was furtherconfirmed by scanning electron microscopy (SEM).

The dose-response curves of fB vs. ZIKV oligonucleotide con-centration are shown in Fig. 3 for triplicate measurements. Thecut-off value was calculated as the average signal of the blankcontrol samples minus three standard deviations, and the LOD wasdefined as the lowest ZIKV oligonucleotide concentration whosevalue is below the cut-off. The LOD and dynamic detection rangeof streptavidin-MNP based LAMP-AC susceptometry analysis were1 aM and 1–103 aM, respectively. For the biotin-blocked MNPbased analysis, MNPs were volume-amplified only by the inter-action with Mg2P2O7, and the sensitivity was reduced: an LOD of

Fig. 3. Dose-response curves for synthetic ZIKV oligonucleotide detected bystreptavidin-MNP based (black circles) and biotin-blocked MNP based (red squares)LAMP-AC susceptometry analysis. Error bars indicate one standard deviation basedon three independent measurements. Cut-off values are indicated by the horizontallines. (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

32 aM was achieved. The average coefficients of variation in thedynamic detection range of streptavidin-MNP based and biotin-blocked MNP based analysis were 9.75% and 8.95%, respectively.

3.4. Scanning electron microscopy study

By using SEM, we further investigated the hypothesis that MNPcan be volume-amplified by the deposition of Mg2P2O7 during theLAMP. The MNPs were separated, washed and collected after theLAMP reaction for SEM analysis (1 pM target analyte). Due to therigorous separation and washing procedure, the aggregates at-tracted by the magnetic stand are considered to be MNPs withstrongly attached amplicons (for streptavidin-MNPs) and/orMg2P2O7 precipitation (for both streptavidin-MNPs and biotin-blocked MNPs). Fig. 4 shows the morphology and nanostructuresof the collected volume-amplified MNP aggregates, as well as theprecipitated Mg2P2O7 that were collected from the control LAMPreaction without MNPs. Compared to the regular nanostructures ofthe Mg2P2O7 precipitation (Fig. 4C), magnetic aggregates based onvolume-amplified streptavidin-MNPs (Fig. 4A) and volume-am-plified biotin-blocked MNP (Fig. 4B) contain a dense mass of ir-regular Mg2P2O7 nanostructures. The existence of these irregularMg2P2O7 nanostructures or Mg2P2O7 fragments implies that MNPsare involved in the formation of Mg2P2O7 nanostructures and havea strong interaction with them. It is thus confirmed that theMg2P2O7 precipitation occurs on the MNP surface during the LAMPreaction, and results in an additional shift of fB. This phenomenonalso implies that other magnetic nanostructures can possibly serveas labels of LAMP products without surface functionalization,which can be further utilized to establish Mg2P2O7 depositionbased biosensors.

3.5. Spiked serum sample detection

Since blood analysis is a routine work in ZIKV diagnosis andcontrol, serum samples were tested by the proposed streptavidin-MNP based LAMP-AC susceptometry assay. The matrix effects ofserum can slow down the amplification and reduce the sensitivityof the biosensor. After evaluation of several serum concentrations,it was found that serum concentrations lower than 20% have noobvious impact on the sensitivity of proposed system. Therefore,synthetic ZIKV oligonucleotide samples were spiked into 20%serum to simulate five times diluted pure serum samples, and

Fig. 4. Scanning electron microscopy images of volume-amplified streptavidin-MNP aggregates (A), volume-amplified biotin-blocked MNP aggregates (B) and precipitatedMg2P2O7 nanostructures (C).

Fig. 5. 20% serum sample measurements. (A) Typical χ″ spectra for the indicated concentrations of synthetic ZIKV oligonucleotide. (B) Dose-response curve of streptavidin-MNP based LAMP-AC susceptometry analysis. Error bars indicate one standard deviation based on three independent measurements. The cut-off value is indicated by thehorizontal line.

B. Tian et al. / Biosensors and Bioelectronics 86 (2016) 420–425424

analyzed by the streptavidin-MNP based LAMP-AC susceptometrysystem. The spectra and dose-response curve of 20% serum de-tection are shown in Fig. 5. While the LOD (1 aM synthetic ZIKVoligonucleotide) is preserved for the 20% serum samples, the dy-namic detection range (approximately four orders of magnitude,from 1 aM to 104 aM) is larger than for samples without serum(approximately three orders of magnitude, Fig. 3). We attribute theextended dynamic detection range to the matrix effects of theserum, which can slow down the reaction but not influence theequilibrium (the lowest fB is 7.9 Hz, nearly the same as for thesample without serum). The average coefficient of variation in thedynamic detection range was 7.73% for 20% serum detection. Theresult shows that detection on 20% serum samples can achieve anLOD of 1 aM with a total assay time of 27 min (12 min LAMP and15 min readout), which is two orders of magnitude more sensitiveand faster than the reported RT-PCR based ZIKV detection.

3.6. Specificity evaluation

The specificity (selectivity) of the proposed streptavidin-MNPbased LAMP-AC susceptometry assay was investigated by con-sidering four types of synthetic NS5 gene from relevant viruses(Dengue virus, GenBank accession number: EF595819; Yellow fe-ver virus, GenBank accession number: AY541441; Japanese en-cephalitis virus, GenBank accession number: FJ515937; and WestNile virus, GenBank accession number: AY187015) as negativecontrols (synthesized by Integrated DNA Technologies). The syn-thetic oligonucleotides were spiked into 20% serum at a con-centration of 1 pM. Results presented in Fig. S4 show that theresponses from the negative controls are similar to the blankcontrol, indicating a high specificity of the proposed method.

4. Conclusion

This work presents a first report of a LAMP-AC susceptometryassay for rapid and highly sensitive quantitative ZIKV detection.

B. Tian et al. / Biosensors and Bioelectronics 86 (2016) 420–425 425

The proposed detection system recognizes 1 aM synthetic ZIKVoligonucleotide in 27 min. The assay has a dynamic detectionrange of 1 aM-10 fM targets in 20% serum sample. Although weonly detected synthetic Zika virus DNA, we have demonstrated in aprevious paper that the enzyme (Bst 3.0 polymerase) could am-plify both RNA and DNA sequences under identical conditions, andperforms well when detecting clinical RNA samples (vaccine andtissue specimens) (Tian et al., 2016b). This method is approxi-mately two orders of magnitude more sensitive and faster than thereported RT-PCR based ZIKV serum detection (Faye et al., 2008;Faye et al., 2013), and is thus a promising method for ZIKV diag-nosis and control. In future work, a heating unit will be integratedinto the AC susceptometer in order to automate and simplify thedetection procedure.

Acknowledgments

This work was financially supported by Swedish ResearchCouncil Formas (Project nos. 221–2012-444, 221–2014-574 and2011–1692).

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2016.06.085.

References

Al-Qahtani, A.A., Nazir, N., Al-Anazi, M.R., Rubino, S., Al-Ahdal, M.N., 2016. J. Infect.Dev. Ctries. 10 (3), 201–207.

Astalan, A.P., Ahrentorp, F., Johansson, C., Larsson, K., Krozer, A., 2004. Biosens.Bioelectron. 19 (8), 945–951.

Barzon, L., Pacenti, M., Berto, A., Sinigaglia, A., Franchin, E., Lavezzo, E., Brugnaro, P.,Palu, G., 2016. Eur. Surveill. 21 (10), 2–6.

Brasil, P., Pereira Jr., J.P., Raja Gabaglia, C., Damasceno, L., Wakimoto, M., RibeiroNogueira, R.M., Carvalho de Sequeira, P., Machado Siqueira, A., Abreu de Car-valho, L.M., Cotrim da Cunha, D., Calvet, G.A., Neves, E.S., Moreira, M.E., Ro-drigues Baiao, A.E., Nassar de Carvalho, P.R., Janzen, C., Valderramos, S.G.,Cherry, J.D., Bispo de Filippis, A.M., Nielsen-Saines, K., 2016. N. Engl. J. Med.

Broutet, N., Krauer, F., Riesen, M., Khalakdina, A., Almiron, M., Aldighieri, S., Espinal,M., Low, N., Dye, C., 2016. N. Engl. J. Med. 374 (16), 1506–1509.

Buathong, R., Hermann, L., Thaisomboonsuk, B., Rutvisuttinunt, W., Klungthong, C.,Chinnawirotpisan, P., Manasatienkij, W., Nisalak, A., Fernandez, S., Yoon, I.K.,Akrasewi, P., Plipat, T., 2015. Am. J. Trop. Med. Hyg. 93 (2), 380–383.

Calvet, G., Aguiar, R.S., Melo, A.S., Sampaio, S.A., de Filippis, I., Fabri, A., Araujo, E.S.,de Sequeira, P.C., de Mendonca, M.C., de Oliveira, L., Tschoeke, D.A., Schrago, C.G., Thompson, F.L., Brasil, P., Dos Santos, F.B., Nogueira, R.M., Tanuri, A., de Fi-lippis, A.M., 2016. Lancet Infect. Dis.

Campos, G.S., Bandeira, A.C., Sardi, S.I., 2015. Emerg. Infect. Dis. 21 (10), 1885–1886.Cao-Lormeau, V.M., Blake, A., Mons, S., Lastere, S., Roche, C., Vanhomwegen, J., Dub,

T., Baudouin, L., Teissier, A., Larre, P., Vial, A.L., Decam, C., Choumet, V., Halstead,S.K., Willison, H.J., Musset, L., Manuguerra, J.C., Despres, P., Fournier, E., Mallet,

H.P., Musso, D., Fontanet, A., Neil, J., Ghawche, F., 2016. Lancet 387 (10027),1531–1539.

D’Ortenzio, E., Matheron, S., de Lamballerie, X., Hubert, B., Piorkowski, G., Maquart,M., Descamps, D., Damond, F., Yazdanpanah, Y., Leparc-Goffart, I., 2016. N. Engl.J. Med.

Donolato, M., Antunes, P., Bejhed, R.S., Zardán Gómez de la Torre, T., Østerberg, F.W.,Strömberg, M., Nilsson, M., Strømme, M., Svedlindh, P., Hansen, M.F., Vavassori,P., 2015. Anal. Chem. 87 (3), 1622–1629.

Fauci, A.S., Morens, D.M., 2016. N. Engl. J. Med. 374 (7), 601–604.Faye, O., Faye, O., Diallo, D., Diallo, M., Weidmann, M., Sall, A.A., 2013. Virol. J. 10,

311.Faye, O., Faye, O., Dupressoir, A., Weidmann, M., Ndiaye, M., Alpha Sall, A., 2008. J.

Clin. Virol. 43 (1), 96–101.Foy, B.D., Kobylinski, K.C., Chilson Foy, J.L., Blitvich, B.J., Travassos da Rosa, A.,

Haddow, A.D., Lanciotti, R.S., Tesh, R.B., 2011. Emerg. Infect. Dis. 17 (5), 880–882.Goeijenbier, M., Slobbe, L., van der Eijk, A., de Mendonca Melo, M., Koopmans, M.P.,

Reusken, C.B., 2016. Neth. J. Med. 74 (3), 104–109.Gourinat, A.C., O’Connor, O., Calvez, E., Goarant, C., Dupont-Rouzeyrol, M., 2015.

Emerg. Infect. Dis. 21 (1), 84–86.Haug, C.J., Kieny, M.P., Murgue, B., 2016. N. Engl. J. Med.Heukelbach, J., Alencar, C.H., Kelvin, A.A., De Oliveira, W.K., Pamplona de Goes

Cavalcanti, L., 2016. J. Infect. Dev. Ctries. 10 (2), 116–120.Korzeniewski, K., Juszczak, D., Zwolinska, E., 2016. Int. Marit. Health 67 (1), 31–37.Kuno, G., Chang, G.J., Tsuchiya, K.R., Karabatsos, N., Cropp, C.B., 1998. J. Virol. 72 (1),

73–83.Lanciotti, R.S., Kosoy, O.L., Laven, J.J., Velez, J.O., Lambert, A.J., Johnson, A.J., Stanfield,

S.M., Duffy, M.R., 2008. Emerg. Infect. Dis. 14 (8), 1232–1239.Marano, G., Pupella, S., Vaglio, S., Liumbruno, G.M., Grazzini, G., 2016. Blood

Transfus. 14 (2), 95–100.Melo, A.S.O., Malinger, G., Ximenes, R., Szejnfeld, P.O., Sampaio, S.A., de Filippis, A.

M.B., 2016. Ultrasound Obstet. Gynecol. 47 (1), 6–7.Mori, Y., Nagamine, K., Tomita, N., Notomi, T., 2001. Biochem. Biophys. Res. Com-

mun. 289 (1), 150–154.Musso, D., Nhan, T., Robin, E., Roche, C., Bierlaire, D., Zisou, K., Yan, A.S., Cao-Lor-

meau, V.M., Broult, J., 2014. Eur. Surveill. 19 (14), 6–8.Musso, D., Roche, C., Nhan, T.X., Robin, E., Teissier, A., Cao-Lormeau, V.M., 2015. J.

Clin. Virol. 68, 53–55.Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N.,

Hase, T., 2000. Nucleic Acids Res. 28 (12), E63.Oster, A.M., Brooks, J.T., Stryker, J.E., Kachur, R.E., Mead, P., Pesik, N.T., Petersen, L.R.,

2016. Morb. Mortal. Wkly. Rep. 65 (5), 120–121.Petersen, L.R., Jamieson, D.J., Powers, A.M., Honein, M.A., 2016. N. Engl. J. Med. 374

(16), 1552–1563.Rodriguez-Morales, A.J., 2015. J. Infect. Dev. Ctries. 9 (6), 684–685.Schuler-Faccini, L., Ribeiro, E.M., Feitosa, I.M.L., Horovitz, D.D.G., Cavalcanti, D.P.,

Pessoa, A., Doriqui, M.J.R., Neri, J.I., Neto, J.M.D., Wanderley, H.Y.C., Cernach, M.,El-Husny, A.S., Pone, M.V.S., Serao, C.L.C., Sanseverino, M.T.V., Embry, B.M.G.S.Z.,2016. Morb. Mortal. Wkly. Rep. 65 (3), 59–62.

Sikka, V., Chattu, V.K., Popli, R.K., Galwankar, S.C., Kelkar, D., Sawicki, S.G., Stawicki,S.P., Papadimos, T.J., 2016. J. Glob. Infect. Dis. 8 (1), 3–15.

Tian, B., Bejhed, R.S., Svedlindh, P., Strömberg, M., 2016a. Biosens. Bioelectron. 77,32–39.

Tian, B., Ma, J., Zardán Gómez de la Torre, T., Bálint, Á., Donolato, M., Hansen, M.F.,Svedlindh, P., Strömberg, M., 2016b. (submitted for publication).

Tognarelli, J., Ulloa, S., Villagra, E., Lagos, J., Aguayo, C., Fasce, R., Parra, B., Mora, J.,Becerra, N., Lagos, N., Vera, L., Olivares, B., Vilches, M., Fernandez, J., 2016. Arch.Virol. 161 (3), 665–668.

Tomita, N., Mori, Y., Kanda, H., Notomi, T., 2008. Nat. Protoc. 3 (5), 877–882.Venturi, G., Zammarchi, L., Fortuna, C., Remoli, M.E., Benedetti, E., Fiorentini, C.,

Trotta, M., Rizzo, C., Mantella, A., Rezza, G., Bartoloni, A., 2016. Eur. Surveill. 21(8), 2–5.

Zardán Gómez de la Torre, T., Mezger, A., Herthnek, D., Johansson, C., Svedlindh, P.,Nilsson, M., Strømme, M., 2011. Biosens. Bioelectron. 29 (1), 195–199.