Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)

Abstract

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

Keywords: COVID diagnosis; Polypurine reverse-Hoogsteen hairpin; SARS-CoV-2 RNA detection; electrochemical magnetoassay; fluorescent microarray; thermal lateral flow.

Figure 1

Design of the PPRH CC1 capture and reporter probes for the detection of the viral RNA.

Figure 2

Binding of PPRHs CC1 and CC3 to DNA and RNA probes corresponding to SARS-CoV-2. Increasing amounts of PPRH-CC1 or PPRH-CC3 were incubated with 500 ng of 6-FAM-labeled ssDNA or ssRNA probes corresponding to replicase (top panel) or spike (bottom panel) sequences. Hp-Sc6 was included as a non-targeting control PPRH.

Figure 3

Scheme of the microarray system developed to validate the CC pairs designed for detecting SARS-CoV-2 viral RNA. It comprises a glass surface onto which DNA capture probes (PPRH or duplex) are chemically bounded. These capture probes printed on the glass slide hybridize selectively with the corresponding synthetic target and this binding is reported through the addition of a second probe, which allows the detection since it is coupled to a fluorophore. Thus, the principle of the system is based on the complementarity base pairing between the probes and the corresponding target nucleotide sequence.

Figure 4

Microarray system designed to test simultaneously (multiplex) all three CC oligonucleotide pairs assayed in both formats (PPRH and duplex clamp). (A): Scheme of the chip organization and the concentrations used to build standard curves for each CC pair are shown together with a fluorescence image of a microarray well. (B): Calibration curve obtained for the CC1 pair and the corresponding analytical parameters of this sigmoidal curve obtained in a multiplex microarray assay (RFU: relative fluorescence units) are also plotted. All standard curves obtained were designed to record at least 3 spot replicates for each concentration value.

Figure 5

Scheme of the thermal lateral flow system and the analytical procedure. 1—Sample addition to a solution containing gold nanoprisms and the capture oligonucleotide labeled with biotin; 2—preincubation; 3—preincubated sample is added to the TLF strip, leaving the samples flow 15 min; 4—sample drying at 37 °C for 15 min; 5—test developing by NIR laser irradiation.

Figure 6

Results obtained with the thermal lateral flow system. (A): Profile of ipeak^® (IUL S.A. commercial lateral flow reader) quantification of spiked samples with different concentrations of targets (from 0 to 5 nM) in UTM using capture sequences duplex-CC1, CC2, and CC3 duplex and PPRH-CC1 and CC2. Results with PPRH-CC3 sequence are not shown in this graph because NPRs aggregation was observed during the testing assay. Grey level intensity measurements were obtained from the LF reader to determine the value of LOD of the thermal lateral flow system. Standard deviation of three replicates carried out consecutively is drawn as error bars. (B): Image obtained from the back part of the LF strips (thermosensitive material) after the laser irradiation using a 1064 nm laser. The strips were loaded with spiked samples in UTM from Biocomma with different concentrations of DNA, from 0–5 nM, to determine the visual LOD of the system.

Figure 7

Scheme of the electrochemical biosensor platform and the analytical procedure: 1—sample addition to a solution containing functionalized MNPs and the detection oligonucleotide labeled with HRP enzyme; 2—hybridization assay on the MNP surface; 3—pretreated sample added to the electrochemical device; 4—steps taken place in the electrochemical device, including MNP flow, trapping and concentration, and electrochemical detection.

Figure 8

(A): Current response profiles to different concentrations of the target DNA CC1 sequence in hybridization buffer using the MNPs modified with PPRH—CC1. (B): Analytical signal values recorded with the three different capture probes, CC1, CC2, and CC3, in a duplex and PPRH format for different concentrations of the corresponding oligonucleotide target DNA sequences in hybridization buffer. (C): Increase in response relative to the blank response (in %) recorded in the UTM from Biocomma with the PPRH—CC1, CC2, and CC3, respectively, for three of the tested oligonucleotide concentrations. Standard deviation of three replicates carried out consecutively is drawn as error bars.

Figure 8

(A): Current response profiles to different concentrations of the target DNA CC1 sequence in hybridization buffer using the MNPs modified with PPRH—CC1. (B): Analytical signal values recorded with the three different capture probes, CC1, CC2, and CC3, in a duplex and PPRH format for different concentrations of the corresponding oligonucleotide target DNA sequences in hybridization buffer. (C): Increase in response relative to the blank response (in %) recorded in the UTM from Biocomma with the PPRH—CC1, CC2, and CC3, respectively, for three of the tested oligonucleotide concentrations. Standard deviation of three replicates carried out consecutively is drawn as error bars.

Figure 9

Testing of six real samples provided by the Biobank of the Aragon Health System (3 negatives and 3 positives by PCR: sample #1, Ct 35; sample #2, Ct 19 and sample #3, Ct 30) in UTM from Biocomma with the PPRH-CC1 as capture sequence using the two developed biosensor devices. (A): Profile of ipeak^® (IUL S.A. commercial lateral flow reader) quantification (grey intensity level measurements, right side) of the results obtained with the TLF together with the signal obtained for the blank and control of 0.001 nM of target DNA CC1 sequence and the image obtained from the back part of the LF strips (thermosensitive material, left side) after the laser irradiation using a 1064 nm laser used. This image was used to measure the grey level intensity to develop the results of the thermal lateral flow assay. (B) Analytical signal values obtained by the electrochemical biosensor platform, together with the signal obtained for the blank and control of 0.41 nM of target DNA CC1 sequence. Standard deviation of three replicates carried out consecutively is drawn as error bars.