A non-optical multiplexed PCR diagnostic platform for serotype-specific detection of dengue virus
Introduction
In a laboratory, reverse-transcription PCR (rt-qPCR) based on optical detection can be done on a single sample in a 96-well format. With different primer sets in each well, large number of targets can be screened with one single sample. Such large-library multiplexed PCR cannot be applied to point-of-care (POC) applications, because of the bulkiness of well-loading robotics and optical instruments. One potential solution is to do multi-target detection in a single or a small number of PCR reactors with multiple primer sets. However, with the large DNA and primer population in an untreated heterogeneous POC sample, interfering reactions like primer dimerization and mispriming will significantly reduce the sensitivity and selectivity of an optically based single-PCR reactor platform. Both reactions compete with the target-amplification reaction to reduce the latter’s yield (sensitivity). Molecular beacon type optical reporters cannot differentiate between the target amplicons and the products of these interfering reactions (selectivity). Even if these selectivity/sensitivity issues are overcome, it is challenging to select proper fluorophores with appropriate spectral bandwidth to avoid spectral emission overlap and output signal interference. This limits optics-based PCR platforms to the detection of no more than five targets simultaneously for POC applications [1].
Recently, electrochemical sensor arrays, particularly ion-sensitive field-effect transistor (ISFET) arrays, have been suggested as a replacement to the optical sensor to allow multiplexed PCR in a single PCR reactor [[2], [3], [4]]. By functionalizing oligo probes complementary to the target, it is hoped that only the amplified targets will be detected, after they hybridize with the probes to form duplexes, even in the presence of interfering reactions. However, electrochemical sensors that detect charge or electron transfer reactions to or from ions have their own sensitivity and selectivity issues. Due to Debye screening of the target charge and current, its signal is sensitive to the pH, the ionic strength and the multitude of factors that control the length of the hybridized duplexes: the duplex conformation, the pairing sequence location on the target and the lengths of the dangling tails [3,5,6]. Such factors are difficult to control for POC samples without extensive pretreatment. Given that PCR reactions change the pH significantly, the pairing sequence location can be arbitrary and non-specific hybridization are quite ubiquitous, electrochemical sensor arrays have not enabled multiplexed PCR. A most recent attempt is to carry out multiplexed PCR in low ionic strength such that the Debye length is sufficiently large to accommodate the hybridized target, regardless of the duplex conformation, the pairing sequence location and length of the dangling tails that bracket the pairing sequence [7]. However, screening of electrostatic repulsion between annealing nucleic acids becomes weak at low ionic strengths very low PCR yield results. The yield is so low that it cannot reach the necessary sensitivity for early screening of pathogens from bio-samples.
Clearly, a viable POC multiplexed PCR platform has not been reported. Instead of the electrochemical sensor array, we use an array of ion-selective membrane (IEM) sensors [8]. We demonstrate, for the first time, the ion-depletion action of the ion-selective membrane promotes the selectivity and sensitivity of the sensor without sacrificing the yield of the PCR reaction, which is still carried out in the usual 50 mM ionic strength of the PCR buffer. Ours is the first POC multiplexed PCR platform that can identify the serotype of a Dengue virus in a heterogeneous blood sample filled with the RNAs of other vector-borne viruses.
We validate our POC multiplexed PCR platform with an important POC application: sensitive and selective detection of specific dengue serotype in blood with viral RNAs from other vector-borne diseases, including those from the other serotypes with similar sequences. Dengue virus (DENV) is one of the rapidly spreading global health problems. It is a common tropical infection in more than 120 counties, including Southeast Asia, Africa, Central and South America, and the Western Pacific [9]. Some 2.5 billion people or one third of world population are at a risk from dengue infection at any given time [10]. World Health Organization (WHO) currently estimated over 50–100 million infections worldwide every year [11,12]. Dengue is caused by a virus of the Flaviviridae family and exists as four genetically closely related serotypes DENV (1–4) [13,14]. DENV is transmitted by infected mosquito vectors, mainly Aedes aegypti, and can cause symptoms such as high fever for 4–7 days with headache and joint pain [15,16]. However, infections due to other arboviruses such as Zika virus (ZIKV), Yellow Fever virus (YFV) and Chikungunya virus (CHIKV) also show similar symptoms, making it very difficult to identify DENV based on symptoms [[17], [18], [19], [20], [21]]. Despite the absence of an effective DENV treatment, an early diagnosis can improve clinical outcomes and is crucial for monitoring disease outbreaks. Specifically, as the severity of the disease varies by serotype and history of past infection, early identification of DENV serotype is clinically important.
Immunoassay techniques, although rapid and accurate, typically can be used only after the body has begun to mount an immunological response to the foreign pathogen through antibody production and are hence also ineffective for rapid epidemic control [22,23]. Moreover, antibody-based assays suffer from cross-reactivity, especially in identifying species within different flavivirus genus, such as ZIKV or different DENV serotypes.18 Multiplexed PCR in a single reactor that is sufficiently sensitive and selective remains the most viable solution.
In our new IEM sensor array-based platform, serotype-specific oligoprobe is attached to each sensor of the multiplex IEM sensor module for selective capture and identification of the amplified DENV serotype products. When an electric field is applied across an IEM, external concentration polarization occurs across the membrane. The depletion side is almost ion-free and hence controls the ion current through the membrane. A limiting current regime develops at a sufficiently high voltage such that the differential resistance is almost infinite. At higher voltages, an overlimiting current is observed when an electroconvective instability sets in [8,24]. When IEMs are functionalized with oligo probes on the depletion side, the duplexes formed after hybridization with the oligos cannot be depleted. Consequently, the hybridized targets produce a large shift in the non-equilibrium ionic current that results a shift in current-voltage curve (CVC) in the overlimiting current of the IEM [8,25]. Moreover, the vortices of the electroconvective instability in the over-limiting region also serves to shear off non-specifically bound targets whose bonds with the probes are weaker. We further enhance the selectivity with a hydrodynamic shear on our IEM biochip.
Importantly, the overlimiting current only occurs after ions have been depleted from the membrane surface, thus significantly increases the Debye length to enhance both the sensitivity and selectivity of the sensor, much like the recent work on low-ionic strength electrochemical sensing [7]. The sensitivity is enhanced because the presence of a small number of charged targets (and their counterions) in the ion-depleted region can produce a large change in the current. Selective because the Debye thickness is large (>30 nm) in the ion-depleted region so the current signature is not sensitive to the length, conformation of the duplex and the location of the pairing sequence. The hybridized target molecules hence do not protrude beyond the Debye layer. The platform also uses hydrodynamic shear to shear off non-specifically adsorbed non-targets from the membrane surface, thus further enhancing the detection selectivity of the platform and minimizing any false positive detection. Additionally, the platform does not require extensive sample pretreatment and expensive lab equipment. It uses a simple off-chip chitosan modified filter paper-based dip and rinse technique to remove PCR inhibitors and isolates the negatively charged target DENV RNAs from the lysing buffer (TRIzol™ reagent) treated RNA spiked plasma sample for downstream amplification of different DENV serotypes. Hence, the POC platform has the potential to be used as epidemiologic surveillance tool by identifying the presence of DENV serotypes from a large population of potentially infected people in a low-resource setting. The multiplex target-specific probes allow the platform to be scaled up so that a large number of targets can be identified with a one-step procedure from a single sample, although we will only demonstrate its potential with 4 targets—the 4 serotypes of Dengue.
In this paper, we present a low-cost non-optical integrated on-chip PCR-based diagnostic platform coupled with a multiplexed ion exchange membrane (IEM) sensor for analysis of the PCR amplified products for the detection of four DENV (1–4) serotypes. A serotype-specific oligoprobe is attached to each sensor of the multiplex sensor module for selective capture and identification of the amplified DENV serotype products. When IEM captures and hybridizes with the target, it produces a change in non-equilibrium ionic current that results a shift in current-voltage curve. Without any sample pretreatment, this platform uses a simple off-chip chitosan modified filter paper based dip and rinse technique to remove PCR inhibitors and isolates the negatively charged target DENV RNAs from the lysing buffer. Yet, it demonstrates higher selectivity and sensitivity against all other PCR-based technologies. Hence, the platform has the potential to be used as epidemiologic surveillance tool by identifying the presence of DENV serotypes from a large population of potentially infected people in a low-resource setting.
Section snippets
Sample preparation
All DENV serotypes were obtained from Naval Medical Research Center (NMRC) and the following strains are used: WP-74 (DENV-1); OBS8041 (DENV-2); CH53489 (DENV-3) and H241 (DENV-4). The isolated ZIKV RNA was received from Emory University and PRVABC59 strain is used. The extracted DENV and ZIKV RNA were then spiked into the human blood plasma sample (ZenBio Inc., NC, USA). All oligonucleotides including primers and probes were obtained from Integrated DNA Technologies, Inc.
Chitosan modification of Fusion 5 filter paper
Chitosan (medium
Platform layout
The visual representation of the integrated microfluidic platform is shown in Fig. 1. The assay protocol involves three steps and takes ∼90 min to detect four DENV serotypes from 1 mL of DENV RNA spiked in plasma sample. Step 1, the DENV RNA is extracted upstream from TRIzol™-chloroform treated RNA spiked plasma sample using a chitosan-modified filter paper-based dip and rinse extraction technique (10 min). Step 2, the extracted RNA filter paper is then mixed with PCR reagents and introduce in
Conclusion
We have successfully developed a multiplexed and turn-key integrated on-chip PCR microfluidic platform for rapid extraction of DENV RNA from human plasma for selective detection of all four DENV serotypes in a single chip with a single PCR reactor that contains all the primer sets. A chitosan-modified filter paper was used to extract nucleic acids of different DENV serotypes. The dip and rinse technique can isolate template DENV RNAs from as low as 100 copies per mL of RNA spiked human plasma.
Declaration of Competing Interest
The authors declare no competing nonfinancial interests but do declare the following competing financial interests. Both S. Senapati and H.-C. Chang hold some stock in AgenDx Biosciences Inc., a startup biotechnology company that has licensed some parts of the proposed sensing platform.
Acknowledgements
The authors would like to thank the The Far Eastern Group for financial support. The authors would also like to thank Dr. Muktha S. Natrajan, Emory University for providing the Zika virus strain.
Ze Yin received his pH.D. degree in Physic Electronics from the College of Electronic Science and Engineering at Jilin University, China in 2017. After that, he worked as a postdoctoral research associate at the University of Notre Dame, USA, for the development of microfluidics and medical diagnostic devices. He is currently working as a postdoctoral researcher at Texas A&M University, USA. His research interests are in discovering and designing new optical nanomaterials, combining them with
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Ze Yin received his pH.D. degree in Physic Electronics from the College of Electronic Science and Engineering at Jilin University, China in 2017. After that, he worked as a postdoctoral research associate at the University of Notre Dame, USA, for the development of microfluidics and medical diagnostic devices. He is currently working as a postdoctoral researcher at Texas A&M University, USA. His research interests are in discovering and designing new optical nanomaterials, combining them with Nanophotonics/Nanoplasmonic/Microfluidic devices for bio-applications.
Zeinab Ramshani received her pH.D. degree from the Department of Electrical and Computer Engineering at Western Michigan University, USA, in 2017. She has been working as a post-doctoral research associate in Center for Microfluidics and Medical Diagnostics, University of Notre Dame, USA, since 2017. Her research interests have focused on piezoelectric devices for fluid manipulation, chemical/ bio electrical sensing technologies and microfluidic platforms.
Jesse Waggoner received his M.D. from the School of Medicine, Duke University, in 2006. He did his residency training in Internal Medicine at Duke University, and then completed Fellowship in Infectious Diseases at Stanford University. Currently he is an assistant professor at Emory University in the Department of Medicine, Division of Infectious Diseases. His current research focuses on the development, implementation and evaluation of improved diagnostics for pathogens important to the global health community. He has developed a number of molecular tests for viral pathogens such as dengue virus, chikungunya virus, and Zika virus as well as assays for malaria and leptospirosis.
Benjamin Pinsky is a pathologist in Stanford, California and is affiliated with Stanford Health Care-Stanford Hospital. He received his medical degree from University of Washington School of Medicine in 2007 and has been in practice between 6–10 years. Dr. Pinsky's research interests include the development and application of molecular assays for the diagnosis and management of infectious diseases.
Satyajyoti Senapati received his M.S. from Gauhati University, Assam and pH.D. (2006) from National Chemical Laboratory, Pune, India. He is Assistant Research Professor at Chemical and Biomolecular Engineering at University of Notre Dame. His research focuses on the development of integrated devices and sensors to identify different biomolecules of pathogens. He also actively participates in technology transfer efforts in conducting market research as well as in developing functioning prototypes, portable instruments and the biochip manufacturing process. His invented technologies are licensed to 3 biotech startups.
Hsueh-Chia Chang received his B.S. from Caltech (1976) and pH.D. from Princeton (1980). He holds the Bayer Chair of Chemical Engineering at Notre Dame. Prof Chang won the Frenkiel Award from the American Physical Society, the lifetime achievement award from the American Electrophoresis Society and is an APS Fellow. He was honored as a Distinguished Fellow of the UK Royal Society of Engineering for his work on electrokinetics. More than half of his 60 PhD/postdoc students hold faculty positions on all 6 continents. His technologies are being commercialized by 3 startups. He is the author of Electrokinetically Driven Microfluidics and Nanofluidics and is the founding editor of the Journal of Biomicrofluidics.