Nick Adams for your continued support over the years – it has meant a lot to me. Thanks to Megan Pask for your organization and endless knowledge and kindness - you made my time here much easier. Thank you to all the other members of the Haselton Lab for all your help and friendship.
Thanks to my family and friends for supporting me during these crazy two years.
The Global Health Importance of More Versatile Viral Diagnostics
Cases of co-infection with chikungunya and dengue virus in the same patient have been reported in several countries (10); what is believed to be the first case of co-infection with all three viruses was reported in Colombia in 2016 (11). While molecular methods for detecting infection with specific viruses have been in use for years (12), these techniques have not been utilized to their full potential in the developing world.
Current Diagnostic Techniques Do Not Address the Needs in Low Resource Settings
The primary methods of diagnosis for Zika recommended by the CDC are a test for the detection of antibodies to Zika and a test for the detection of Zika virus nucleic acids in the patient's blood or urine (16). Antibodies are formed as the body tries to fight a virus in the days and weeks after infection, making the former method less useful for early-stage detection. Viral nucleic acid sequences can be found in the patient during infection, making this method much more successful.
A "very low viral load" for dengue virus, meanwhile, was considered to be 1 RNA copy per µL of the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany (17). According to the WHO, the average viral load in a review of several studies of Zika infections is on the order of 10 RNA copies per µL in blood and 1 RNA copy per µL in serum (18). While early tests showed it could be very effective in developing countries, tests in tropical areas report high failure rates due to high ambient temperatures, erratic power supplies, and dust entering the system (20).
Diagnostics Based on Polymerase Chain Reaction Require a Controlled Environment . 4
Current PCR machines are entirely dependent on accurate control of the temperature of the reaction over long periods of time. Precise temperature control is crucial for achieving the conditions required to melt double-stranded DNA or anneal primers to target strands; without this control, amplification can be prevented despite the presence of the target sequence. In programming PCR machines, technicians assume the double- or single-stranded state of the nucleic acids at specific temperatures.
In this approach, the addition of fluorescently labeled strands of L-DNA corresponding to the primers used to amplify the D-DNA target allows the melting and annealing states of the DNA to be monitored (Figure 1B). During cooling (red), the Texas Red channel is used to monitor annealing of the primers. 7 Red annealing probe and the BHQ target probe were added to the reactions at 100 nM with 20 nM of the HEX melting probe.
Adaptive PCR Must Be Modified for Multiplexed Detection of Viral RNA
Methods
- Oligonucleotide Design
- LabVIEW Reverse Transcription Program
- Single Fluorescence Channel Heating and Cooling Program
- LabVIEW Multiplex Program
- Limit of Detection of RNA Targets on Adaptive RT-PCR
The molecular beacon was designed with a quencher and a fluorophore at each end so that as the temperature rises to the desired TM of 50 °C, the hairpin pulls apart and the fluorescence increases in a sigmoidal curve (see Section 3.1). Monitoring the fluorescence of the beacon at the point where it changes most significantly with temperature enables detection of small temperature fluctuations and thus precise control of the heating element. After the prescribed dwell time, the system was programmed to switch to adaptive cycling PCR as previously reported (24).
Temperature holding was tested seven times over three days to ensure consistency of system decision making and temperature range (see Section 3.1). To allow the use of three of the four available fluorescence channels on the Adaptive PCR instrument for amplification detection, the annealing and melting L-DNA probes were measured on the same color channel. Therefore, the two plateaus are distinct from each other and can be distinguished by the program using a Gaussian fit, as in the original adaptive PCR iteration (24).
The same progression is used during the cooling cycle—LabVIEW fits the data from the first plateau, that of the molten probe annealing, and then ignores it to fit the second, more important plateau, which shows the annealing of the baking probe. The original adaptive PCR cycling program was modified to allow simultaneous detection of multiple PCR target fluorophores. Depending on which channels are set to "ON", Qiagen ESElog devices will detect the selected colors in order and record up to three points.
Different fluorophores were chosen for each of the three probes used in the chikungunya, dengue and Zika assay so that they can all be detected separately in the reaction. Data from triplicate experiments were averaged and normalized by adjusting fluorescence values to a scale of 0-100 units. Each L-DNA probe was added at 50 nM and L-DNA complements were added at 125 nM.
Relative concentrations were chosen to create derivatives of fluorescence signal of approximately the same order of magnitude so that neither the annealing nor the melting probe signal could bleed into or overpower the other.
Results and Discussion
- Optically-Controlled Reverse Transcription Step
- Single-Channel Cycling and Multiplex PCR
- Limit of Detection of the Chikungunya, Dengue, and Zika Assay
- Multiplexed Reaction Specificity
However, the fluorescence slope and melting plateaus of the molecular beacon and the melting of the L-DNA primer sequences overlapped greatly, preventing differential signal detection. While this is the same as the fluorophore conjugated to the Zika probe, the concentration of the molecular beacon is low enough that the signal is negligible compared to the signal produced by Zika amplification. To confirm the accuracy of the molecular beacon-based retention step, a thermocouple was inserted into the reaction tube for seven 30-min retention trials (Figure 4).
The periodicity of the temperature data is a result of the control mechanism - the system periodically switches between heating and cooling when the fluorescence value fluctuates. The amount of time required to cool below the fluorescence set point is directly related to the ambient temperature in the room, as the cooling condition is the absence of a heater. However, this required the use of two of the four available thermal circulation channels.
The two probes can be monitored in the same channel because the melting and annealing points of the primers (~60°C) and targets (~87°C) are distinct and the melting and annealing curves do not overlap (Figure 5B). Interestingly, the melting and annealing curves were initially slightly more difficult to distinguish during the cooling phase of the cycle. This appeared to be a result of the slower kinetics of annealing, which relies on diffusion for binding of the two oligonucleotides.
The middle plateau at each part of the cycle represents the melting of the primers and annealing of the template strands. The concentrations of the melting and annealing probes were optimized to ensure that an equal signal was detected from each. The LabVIEW program causes the Qiagen ESELog devices to detect from each of the three channels at the end of each cooling cycle, displaying the data from each channel in separate graphs.
Each of the RNA virus targets was tested at 5,000 copies/reaction in both the RotorGene and Adaptive PCR. Each of the concentrations of Zika virus was clearly distinguishable from the others (Figure 7C), meaning that the viral loads of 10 copies/µL blood and 1 copy/µL serum (18) are both detectable. All three viruses were amplified within one cycle in combination with one or both of the other targets.
Conclusions
Future Directions
- Affordable
- User-Friendly
- Rapid and Robust
- Equipment-Free and Deliverable
We also performed some preliminary tests using a lyophilizable RT-PCR kit, in which a master mix of all necessary reagents can be produced and separated into individual reaction volumes, and then freeze-dried until use. These tubes then only need to be rehydrated with the desired patient sample and placed in the Adaptive PCR device. The adaptive PCR system is more robust than standard PCR because it overcomes the challenges of several contaminants known to inhibit proper thermal cycling (24).
The speed of the test can be greatly improved by including another reaction tube. However, the speed of the reaction was accompanied by inaccurate detection of annealing and melting conditions, which reduced the consistency and efficiency of the reaction. Improving the device to be smaller and more portable would meet both of the last two INSURANCE criteria.
The device is expected to be about the size of a shoebox and include a slot mechanism for inserting the reaction tube. Clinics in low-resource settings simply need to have a supply of lyophilized reactions for each of the desired tests and a device that allows them to select the test they want to perform, insert the sample, and press “Run.” Such a system could be delivered to desired groups with some external funding and would significantly increase the level of diagnostic care for those in resource-poor settings.
Oliveira Melo AS, et al. 2016) Zika virus intrauterine infection causes fetal brain abnormalities and microcephaly: tip of the iceberg. 2016) Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay. Wu G & Zaman MH (2012) Low-cost tools for diagnosing and monitoring HIV infection in resource-poor settings.
Laue T, Emmerich P and Schmitz H (1999) Detection of dengue virus RNA in patients after primary or secondary dengue infection using TaqMan Automated. CDC (DENV-1-4 Real-Time RT-PCR Assay for Detection and Serotype Identification of Dengue Virus. All data can be found in Haselton Lab Dropbox in Gates Phase 1.5 > Temperature Probes > Data > Adaptive RT-PCR CDZ Data ( Erin's thesis).
Illustration of L-DNA and Adaptive PCR
The Current Adaptive PCR Prototype
Molecular Beacons for Temperature Holding
Thermocouple-Monitored Molecular Beacon Holds
Fluorescence as a Function of Time for Double- vs. Single-Channel Cycling
Comparison of RotorGene and Adaptive PCR
Limit of Detection of Chikungunya, Dengue, and Zika Viruses
Amplification of PCR Targets in Combination at 50 Copies/Reaction
