Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

Control Charting Genomic Data

Background Control charting is routine in the quality assurance of traditional clinical laboratory testing. Genomic tests are not typically managed by control charting. We examined control charting to monitor the performance of a clinical next-generation sequencing (NGS) assay. Methods We retrospectively examined 3 years of control material (NA12878) data from clinical genomic epilepsy testing. Levey-Jennings plots were used to visualize changes in control material depth of sequencing coverage in genomic regions of an epilepsy genomic panel. Changes in depth of coverage were correlated with changes in the manufactured lot of capture probe reagent. Depth of coverage was also correlated between quality control material and clinical samples. Results Fifty-seven sequencing runs of NA12878 were analyzed for 1811 genomic regions targeting 108 genes. Manufactured probe lot changes were associated with significant changes in the average coverage of 537 genomic regions and the lowest coverage of 173 regions (using a critical cut-off of P < 5.52 x 10−6). Genomic regions with the highest sensitivity to lot-to-lot variation by average sequencing depth of coverage were not the same regions with the highest sensitivity by lowest sequencing depth of coverage. Levey-Jennings plots displayed differences in genomic depth of coverage across capture probe reagent lot changes. There was moderate correlation between the changes in depth of sequencing across lot changes for control material and clinical cases (r2 = 0.45). Conclusions Genomic control charting can be used routinely by clinical laboratories to monitor assay performance and ensure the quality of testing.

A triple functional sensing chip for rapid detection of pathogenic Listeria monocytogenes

Here a triple functional sensing chip was created for L. monocytogenes detection by integrating three biomarkers (Listeriolysin O (LLO) at protein level, hly gene at genetic level, and acetoin at metabolic level). Liposome encapsulated catechol was used for LLO detection via LLO pore-forming ability. hly gene was specifically captured by using a thiolated capture probe on nanoporous gold (NPG). As an electroactive label, methylene blue was embedded in double-stranded structures to generate an electrochemical signal for hly detection. Combined with the electrocatalysis of NADH by NPG, the acetoin detection was achieved by measuring the consumption of NADH as a cofactor under acetoin reductase catalysis. Importantly, the L. monocytogenes detection results obtained by detecting three biomarkers using the chip can be mutually verified, which reduces the probability of false positives based on a single marker. Moreover, the detection time was reduced to about 90 min, making it a rapid and reliable tool for L. monocytogenes detection.

Graphitic Carbon Nitride as an Amplification Platform on an Electrochemical Paper-Based Device for the Detection of Norovirus-Specific DNA

Norovirus is one of the leading causes of gastroenteritis, acute vomiting, intense diarrhoea, acute pain in the stomach, high fever, headaches, and body pain. Conventional methods of detection gave us very promising results but had disadvantages such as low sensitivity, cost ineffectiveness, reduced specificity and selectivity, etc. Therefore, biosensors can be a viable alternative device which can overcome all setbacks associated with the conventional method. An electrochemical sensor based on oxidized graphitic carbon nitride (Ox-g-C3N4) modified electrochemical paper-based analytical device (ePAD) was fabricated for the detection of norovirus DNA. The synthesized Ox-g-C3N4 nanosheets were characterized by field emission scanning electron microscopy (FESEM), X-ray Diffraction (XRD), UV-Vis spectroscopy and X-Ray Photoelectron Spectroscopy. The capture probe DNA (PDNA) modified electrodes were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). These two characterization techniques were also employed to find the optimal scan rate, response time and temperature of the fabricated sensor. The fabricated biosensor showed a limit of detection (LOD) of 100 fM. Furthermore, the specificity of the reported biosensor was affirmed by testing the response of capture probe DNA with oxidized graphitic carbon nitride (PDNA/Ox-g-C3N4) modified ePAD on the introduction of a non-complimentary DNA. The fabricated ePAD sensor is easy to fabricate, cost effective and specific, and requires a minimum analysis time of 5 s.

A potential reusable fluorescent aptasensor based on magnetic nanoparticles for ochratoxin A analysis

An aptasensor for the detection of ochratoxin A (OTA) in environmental samples was developed. It displayed high sensitivity and good selectivity. Factors such as specific binding between a FAM (5-carboxyfluorescein)-labeled aptamer (f-RP) and OTA, and a magnetic property of a streptavidin magbeads-modified capture probe (bm-CP) resulted in aptasensor’s linear relationship between fluorescence intensity and the concentration of OTA. This characteristic is present at the OTA concentration ranges from 0.100 μM to 25.00 μM with a LOD (limit of detection) of 0.0690 μM. The bm-CP can be reused through melting, washing and magnetic separation, which contributes to cost reduction. In addition, the proposed method is simple and detection process is fast. The aptasensor can be used in real samples.

SOX-17 Gene Sequence Complementation on Silica-alumina Nanocomposite-modified Dielectrode Surface for Analyzing Gastric Cancer Progression

BackgroundGastric cancer is as the gastrointestinal issue, the second most death-cause complication worldwide. The survival rate of gastric cancer is lesser due to diagnosing it at the advanced stage. SRY-box containing gene 17 (SOX-17) expression and methylation participate a crucial role in the gastric cancer. MethodsIn this research, capture probe modified interdigitated electrode was used to quantify the SOX-17 gene target sequence. To improve the detection, IDE sensing surface was physically-modified by silica-alumina (Si-Al) nanocomposite. Through the biotin-streptavidin strategy, capture probe was immobilized on the surface and complemented by the target sequence. ResultsDoubled the level of capture probe immobilization was noticed on the Si-Al modified surface. From 1 aM concentration of target sequence was detected in the presence of Si-Al nanocomposite, while it was reached 10 aM in the absence, shows ten-folds difference. In addition, higher level of current changes was registered with all the concentrations of target sequence. Control experiments with single, triple and complementary sequences of target were done and there is no significant changes in current were recorded in the substituted sequences, representing the specific detection of target SOX-17 gene sequence. ConclusionThe detection method is shown with nanocomposite-modified IDE surface helps to recognize the gastric cancer effectively.